WO2021137134A1 - Isothiocyanate and isoselenocyanate compounds - Google Patents

Isothiocyanate and isoselenocyanate compounds Download PDF

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Publication number
WO2021137134A1
WO2021137134A1 PCT/IB2020/062485 IB2020062485W WO2021137134A1 WO 2021137134 A1 WO2021137134 A1 WO 2021137134A1 IB 2020062485 W IB2020062485 W IB 2020062485W WO 2021137134 A1 WO2021137134 A1 WO 2021137134A1
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Prior art keywords
group
isothiocyanate
general formula
compound
isoselenocyanate
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PCT/IB2020/062485
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French (fr)
Inventor
Carmela FIMOGNARI
Andrea MILELLI
Piero SESTILI
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Alma Mater Studiorum - Università di Bologna
Università Degli Studi Di Urbino "Carlo Bo"
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Publication of WO2021137134A1 publication Critical patent/WO2021137134A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the present invention relates to a new family of isothiocyanate and isoselenocyanate compounds suitable to be used as antitumour drugs, theranostic agents with combined properties of tumour di- agnosis and treatment, and as fluorescent probes having also chemopreventive, diagnostic and an- titumour properties.
  • theranostics is an emerging strategy with enormous potentialities, since the technologies and de- velopment abilities of the diagnostic field are increasingly applied to improve the efficiency and cost-effectiveness of the finding, of the development and of the marketing of drugs.
  • the term theranostics designates the development of diagnostic tests directly connected with the application of specific therapies.
  • Theranostics represents a combinatory diagnosis and a therapeutic approach to cancer, aimed at removing multi-step procedures, hence reducing treatment delays and improv- ing the patient’s treatment. It offers several benefits, including better diagnosis, specific administra- tion of drugs, reduction of toxic effects on normal tissues and the like (Palekar-Shanbhag et al. , 2013).
  • the probe should be able to penetrate the outer lipid/phospholipid membrane at a relatively high speed while preserving integrity and performance at a cellular level, should feature an intracellular localization profile which may be observed with a microscope and should target a specific organelle while preserving cell viability and proliferation as well as membrane permeability.
  • the available fluorescent dyes which are able to label/target specific organelles, such as LysoTrackerTM, ER-TrackerTM and MitoTrackerTM allow specific functions of the corresponding or- ganelles to be monitored and can be used at low concentrations for any experimental approach (Perry et al. , 2011).
  • the chloromethyl derivatives of fluorescent probes based on ros- amine which are currently used as mitochondrial probes, are lipophilic and cation- ic compounds which can electrophoretically accumulate in mitochondria in reply to changes of the mitochondrial membrane potential (Scorrano et al., 1999).
  • the reactive chloromethyl groups may form covalent bonds with SH groups of mitochondrial proteins. This prevents them from being released even if mitochondria depolarize (Scorrano et al., 1999).
  • mole- cules which are localized in mitochondria are based on europium (III) and terbium (III) complexes of heptadentate ligands bearing azaxanthone or azathioxanthone (Kielar et al., 2008; Law et al., 2009; Murray et al., 2008). Co-colouring experiments with these complexes showed that mitochon- dria fusion with lysosomes occurred only after significantly long incubation times (> 4 hours of in- cubation) (Manning et al., 2006).
  • Z is selected from: S, Se;
  • Y is selected from: O, NH or S;
  • R, R’ which may be identical to or different from each other, are selected from the group compris- ing:
  • alkyl with 1 to 10 carbon atoms preferably alkyl selected from the group com- prising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ; cycloalkyl with 3 to 6 carbon atoms, preferably cycloalkyl selected from the group comprising: heterocycloalkyl with 4 to 5 carbon atoms, comprising 1 or 2 heteroatoms selected from N and O, preferably heterocycloalkyl selected from the group comprising: aryl with 6, 12 or 18 carbon atoms, preferably aryl selected from the group comprising: heteroaryl with 4 to 5 carbon atoms, comprising 1 heteroatom selected from N and O, preferably heteroaryl selected from the group comprising:
  • R is selected from the group comprising:
  • alkyl with 1 to 10 carbon atoms, preferably alkyl selected from the group com- prising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ;
  • X is selected from the group comprising:
  • n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; -CH 2 CH 2 O-CH 2 CH 2 -; wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • V is selected from the group comprising: -(CO)- or -(CH 2 ) n - wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and R''' is selected from the group comprising:
  • cycloalkyl selected from the group comprising: aryl selected from the group comprising: heteroaryl selected from the group comprising: or isomers or pharmaceutically acceptable salts thereof;
  • tumours solid tumours, liquid tumours, leu- kaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • tumours solid tumours, liq- uid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (I) to a subject, a patient such as a human being or an animal, needing the same;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • Z is selected from: S, Se;
  • R and R’ which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH 2 ; alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ; cycloalkyl selected from the group comprising: heterocycloalkyl selected from the group comprising: aryl selected from the group comprising: heteroaryl selected from the group comprising:
  • R is selected from the group comprising:
  • alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ;
  • X is selected from the group comprising:
  • n is: 1 , 2, 3, 4 or 5 or 6; wherein n is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
  • n 1 , 2, 3 or 4;
  • V is selected from the group comprising: -(CO)- or -(CH 2 ) n - wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
  • cycloalkyl selected from the group comprising: aryl selected from the group comprising: heteroaryl selected from the group comprising: or isomers or pharmaceutically acceptable salts thereof;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (II) to a subject, a patient such as a human being or an animal, needing the same;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • Z is selected from: S, Se;
  • R and R’ which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH 2 ; alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ; cycloalkyl selected from the group comprising: heterocycloalkyl selected from the group comprising: aryl selected from the group comprising: heteroaryl selected from the group comprising:
  • R is selected from the group comprising:
  • alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ;
  • X is selected from the group comprising:
  • n 1 , 2, 3, 4 or 5 or 6;
  • n 1 , 2, 3 or 4;
  • V is selected from the group comprising: -(CO)- or -(CH 2 ) n - wherein n is: 1 , 2, 3, 40 5; and R''' is selected from the group comprising:
  • cycloalkyl selected from the group comprising: aryl selected from the group comprising: heteroaryl selected from the group comprising: or isomers or pharmaceutically acceptable salts thereof;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • tumours solid tumours, liq- uid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (III) to a subject, a patient such as a human being or an animal, needing the same;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • Z is selected from: S, Se;
  • R and R’ which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH 2 ; alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ; cycloalkyl selected from the group comprising: heterocycloalkyl selected from the group comprising: aryl selected from the group comprising: heteroaryl selected from the group comprising:
  • R is selected from the group comprising:
  • alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ;
  • X is selected from the group comprising:
  • n is: 1 , 2, 3, 4 or 5 or 6; wherein n is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
  • n 1 , 2, 3 or 4;
  • V is selected from the group comprising: -(CO)- or -(CH 2 ) n - wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
  • cycloalkyl selected from the group comprising: aryl selected from the group comprising: heteroaryl selected from the group comprising: or isomers or pharmaceutically acceptable salts thereof;
  • tumours solid tumours, liquid tumours, leu- kaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • tumours solid tumours, liq- uid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (IV) to a subject, a patient such as a human being or an animal, needing the same;
  • isothiocyanate and/or isoselenocyanate compounds of general formula (IV) for use as chemopreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
  • Figure 1 shows through graphs the cytotoxicity of the theranostic (TN) compounds according to the present invention, identified by code TN82 ⁇ 6-((6-isothiocyanatohexyl)oxy)-9-(o-toluoyl)-3H- xanthen-3-one ⁇ and by code TN46 (6-(2-(2-isothiocyanatoethoxy)ethoxy)-9-(o-toluoyl)-3H-xanthen- 3-one ⁇ , respectively, and identified inside the description of the present invention; in actual fact, cells of human t-cell acute lymphoblastic leukaemia (Jurkat) were treated with TN82 (circles), with TN46 (squares) (panel A) or with sulforaphane (SFR, insert) for 1 h and then cultured for an addi- tional 72 h in a medium without the compounds under study (according to the present invention); human lymphoblastoyd cells (TK6) were treated for 4 h
  • Cytotoxicity was determined by means of (Trypan Blue) dye exclusion tests or by means of analyses of intracellular alkaline es- terase (MUH assay). * p ⁇ 0.05; ** p ⁇ 0.01 ; **** p ⁇ 0.0001 versus control cultures (untreated cells).
  • Figure 2 shows through graph the cytotoxicity of the reference compounds, whose synthesis is re- ported, identified with code RF27 (said reference compound: 6-((6-aminoexyl)oxy)-9-(o-toluoyl)-3FI- xanthen-3-one, being the prodromal compound of the theranostic compound TN82) and with code RF42 (said reference compound: 6-(2-(2-aminoethoxy)ethoxy)-9-(o-toluoyl)-3FI-xanthen-3-one, be- ing the prodromal compound of the theranostic compound TN46), respectively, and identified inside the description of the present invention and on Jurkat cells (A) treated for 24 h with the compounds under study (according to the present invention). ** p ⁇ 0.01 ; **** p ⁇ 0.0001 versus control cultures (untreated cells).
  • Figure 3 shows through graphs the data confirming the apoptosis of Jurkat cells due to the com- pounds according to the present invention, such as TN82 and TN46, which induce apoptotic cell death.
  • Panel A TEM (transmission electron microscope) picture of cells which were treated for 1 h with TN82 and cultured for an additional 5 h in a medium without the compounds under study (according to the present invention). The pres- ence of apoptotic cells (ap) and secondary necrotic cells (n) is remarked.
  • the dashed lines in B-D refer to the values obtained in control cells (untreated cells). * p ⁇ 0.05; ** p ⁇ 0.01 ; *** p ⁇ 0.001 ; **** p ⁇ 0.0001 versus control cultures (untreated cells).
  • Figure 4 shows through graphs and picture the data confirming that the compounds according to the present invention, such as TN82 and TN46, induce DNA single-strand breakage in Jurkat and TK6 cells.
  • the cells were treated with TN82 (open circles) or with TN46 (squares) for 1 h and im- mediately submitted to analysis to assess the presence of DNA strand breaks through fast halo as- say.
  • Panels A and B show the extent of DNA damage in Jurkat cells (A) and in TK6 cells (B). The extent of DNA single-strand breakage is expressed as nuclear diffusion factor (NDF).
  • NDF nuclear diffusion factor
  • Figure 5 shows through graph the data confirming that the reference compounds RF27 and RF42 do not induce DNA single-strand breakage in Jurkat cells.
  • the cells were treated with RF27 (cir- cles) or RF42 (squares) for 1 h and immediately submitted to analysis to assess the presence of DNA strand breaks through fast halo assay.
  • the extent of DNA single-strand breakage is ex- pressed as nuclear diffusion factor (NDF).
  • Figure 6 shows through graphs the data confirming the correlation between DNA damage and cell proliferation inhibition.
  • the Jurkat cells were treated with TN82 (A) or TN46 (B) at concentrations of 0 - 2 - 4 - 6 ⁇ M for 1 h and immediately checked for DNA damage or incubated for 72 h in a culture medium without the compounds under study (according to the present invention) and counted in order to determine their proliferation.
  • Figure 7 shows through graph the data relating to the formation of micronuclei (MN) induced by treatment with TN82 or TN46.
  • the TK6 cells were treated with increasing concentrations of TN82 (white bar graphs) or TN46 (black bar graphs) for 4 h, left to proliferate for an additional 20 h in a culture medium without the compounds under study (according to the present invention) and ana- lysed to measure the MN number.
  • the dashed line refers to the baseline of MN measured in con- trol cells (untreated cells).
  • the graph also shows the number of MN induced by mitomycin C (0.8 ⁇ g/ml) and vinblastine (2 ⁇ g/ml) used as positive controls. * p ⁇ 0.05; ** p ⁇ 0.01 ; *** p ⁇ 0.001 ; **** p ⁇ 0.0001 versus control cells (untreated cells).
  • Figure 8 shows through graph the data confirming the RNA damage induced by TN82 or by TN46 in Jurkat cells.
  • the cells were treated with TN82 or with TN46 at concentrations of 0 - 6 - 12 - 18 ⁇ M for 24 h.
  • Figure 9 shows through graphs the data confirming that the compounds according to the present invention, such as TN82 and TN46, are cytotoxic and genotoxic under metabolically limiting condi- tions (4°C).
  • Jurkat cells were treated with TN82 (circles) or TN46 (squares) for 1 h at 4°C and left to proliferate for an additional 72 h in a medium without the compounds under study (according to the present invention) (A) or immediately checked for DNA damage through fast halo assay (B).
  • the panels C and D show the correlation between DNA damage and the corresponding cytotoxic re- sponses. ** p ⁇ 0.01 ; **** p ⁇ 0.0001 versus control cells (untreated cells).
  • Figure 10 shows through graphs the data relating to the effects of ortho-phenanthroline (o-phen) and rotenone (rot) on the cytotoxic and genotoxic activity of TN82 and TN46.
  • the cells were treat- ed with TN82 (6 ⁇ M) or with TN46 (4 ⁇ M) for 1 h, or with sulforaphane (SFR, 30 ⁇ M for 3 h), in the presence or in the absence of ortho-phenanthroline (3 ⁇ M) or rotenone (1 ⁇ M); the cells were im- mediately checked for DNA damage through fast halo assay (A) or for cytotoxic activity after 48 h of growth in a medium without the compounds under study (according to the present invention) (B).
  • A fast halo assay
  • B cytotoxic activity after 48 h of growth in a medium without the compounds under study (according to the present invention)
  • Figure 11 shows through graphs the data relating to the determination of GSFI levels in Jurkat cells treated with TN82 (A) or with TN46 (B) 0 - 2 - 4 - 6 ⁇ M for 1 - 3 - 6 - 24h. Influence of higher (C, D) or lower (E, F) intracellular GSH levels on cell proliferation.
  • Jurkat cells were pre-treated with NAC 5 mM or with BSO 0,2 mM for 24 h, treated with TN82 6 ⁇ M or with TN46 4 ⁇ M for 1 h and left to proliferate for 48-72 h in a medium without the compounds under study (according to the present invention).
  • Figure 12 shows through graphs the date relating to the determination of intracellular GSH levels and the formation of DNA single-strand breakage in Jurkat cells which were pre-treated or were not pre-treated with NAC 5mM or with BSO 0.2 mM for 24 h and which were thereafter treated with TN82 or with TN46 (B) 0 - 2- 4 - 6 ⁇ M for 1 h: determination of intracellular GSH levels (A) and for- mation of DNA single-strand breakage (B and C).
  • Figure 13 shows through graphs the data confirming that the compounds according to the present invention, such as TN82 and TN46, are cytotoxic on blasts isolated from leukaemia patients.
  • the blasts taken from peripheral blood (A) or bone marrow (B) were treated with the TNs for 24 h.
  • Figure 14 shows through graphs the data confirming that the compounds according to the present invention, such as TN82 and TN46, are not cytotoxic on human lymphocytes of peripheral blood at any of the tested concentrations after 4 h of treatment; after 24 h of treatment, TN82 is not cytotox- ic at any of the tested concentrations, whereas TN46 is cytotoxic at the highest tested concentra- tion (32 ⁇ M). Lymphocytes were treated at different concentrations of TN82 or TN46 (4-32 ⁇ M) for 4 h (A) or for 24 h (B).
  • Figure 15 shows a series of pictures confirming the use of the compounds according to the present invention, such as TN82, as biomedical probe, particularly as fluorescent probe.
  • Intracellular locali- zation of TN82 Microscopic analysis of cervix adenocarcinoma cells (HeLa) transfected with red fluorescent protein (RFP) and treated with subtoxic concentrations (1 ⁇ M) of TN82.
  • Two pictures representing the co-localization of TN82 left) and RFP (centre) were selected to perform the merg- ing (right). In the right picture, the localization of TN82 in the endoplasmic reticulum and diffusely at an intracellular level is visible.
  • HeLa cervix adenocarcinoma cells
  • RFP red fluorescent protein
  • the object of the present invention is therefore an isothiocyanate and/or isoselenocyanate com- pound of general formula (I) wherein
  • Z is selected from: S, Se;
  • Y is selected from: O, NH or S;
  • R, R’ which may be identical to or different from each other, are selected from the group compris- ing:
  • alkyl with 1 to 10 carbon atoms preferably alkyl selected from the group com- prising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ; cycloalkyl with 3 to 6 carbon atoms, preferably cycloalkyl selected from the group comprising: heterocycloalkyl with 4 to 5 carbon atoms, comprising 1 or 2 heteroatoms selected from N and O, preferably heterocycloalkyl selected from the group comprising: aryl with 6, 12 or 18 carbon atoms, preferably aryl selected from the group comprising: heteroaryl with 4 to 5 carbon atoms, comprising 1 heteroatom selected from N and O, preferably heteroaryl selected from the group comprising:
  • R is selected from the group comprising: -H, -F, -Cl, -Br, -I; linear or branched alkyl with 1 to 10 carbon atoms, preferably alkyl selected from the group com- prising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ;
  • X is selected from the group comprising:
  • n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • V is selected from the group comprising: -(CO)- or -(CH 2 ) n - wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10: and Ft''' is selected from the group comprising:
  • cycloalkyl selected from the group comprising: aryl selected from the group comprising:
  • a further object of the present invention is the compound of general formula (I) as or for use as:
  • tumours solid tumours, liquid tumours, leu- kaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • tumours solid tumours, liq- uid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (I) to a subject, a patient such as a human being or an animal, needing the same;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • a further object of the present invention is an isothiocyanate and/or isoseleno- cyanate compound of general formula (II): wherein
  • Z is selected from: S, Se;
  • R and R’ which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH 2 ; alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ; cycloalkyl selected from the group comprising: heterocycloalkyl selected from the group comprising: aryl selected from the group comprising: heteroaryl selected from the group comprising:
  • R is selected from the group comprising:
  • alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ;
  • X is selected from the group comprising:
  • n is: 1 , 2, 3, 4 or 5 or 6; wherein n is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; -CH 2 CH 2 O-CH 2 CH 2 -; wherein n is: 1 , 2, 3 or 4;
  • V is selected from the group comprising: -(CO)- or -(CH 2 ) n - wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
  • cycloalkyl selected from the group comprising: aryl selected from the group comprising: or isomers or pharmaceutically acceptable salts thereof.
  • a further object of the present invention is the compound of general formula (II) as or for use as:
  • a further object of the present invention is the isothiocyanate and/or isoselenocyanate compound of general formula (II) for use in preventing and/or diagnosing and/or treating: tumours, solid tu- mours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adeno- carcinoma.
  • a further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (II) to a subject, a pa- tient such as a human being or an animal, needing the same.
  • a further object of the present invention are all the isothiocyanate and/or isoselenocyanate com- pounds, or isocyanate and/or isoselenocyanate compound, of general formula (II) for use as chem- opreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tu- mours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adeno- carcinoma.
  • a further object of the present invention as preferred forms of embodiment of the isothiocyanate and/or isoselenocyanate compound of general formula (I) or (II), are the compounds with formula selected from the group comprising:
  • a further object of the present invention are compounds with formula:
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
  • a further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the compound selected from the group comprising:
  • a further object of the present invention is an isothiocyanate and/or isoselenocyanate compound of general formula (III): wherein
  • Z is selected from: S, Se;
  • R and R’ which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH 2 ; alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ; cycloalkyl selected from the group comprising: heterocycloalkyl selected from the group comprising: aryl selected from the group comprising: heteroaryl selected from the group comprising:
  • R is selected from the group comprising:
  • alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ;
  • X is selected from the group comprising:
  • n is: 1 , 2, 3, 4 or 5 or 6; wherein n is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
  • n 1 , 2, 3 or 4;
  • V is selected from the group comprising: -(CO)- or -(CH 2 ) n - wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
  • cycloalkyl selected from the group comprising: aryl selected from the group comprising: or isomers or pharmaceutically acceptable salts thereof.
  • a further object of the present invention is the isothiocyanate and/or isoselenocyanate compound of general formula (III), as described above, as or for use as:
  • a further object of the present invention is the isothiocyanate and/or isoselenocyanate compound of general formula (III) for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uter- ine cervix adenocarcinoma;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
  • a further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (III) to a subject, a patient such as a human being or an animal, needing the same.
  • a further object of the present invention as preferred forms of embodiment of the compound of general formula (I) or (III), are the compounds of formula:
  • a further object of the present invention are compounds with formula:
  • antitumour agent effective against colon adenocarcinoma ⁇ antitumour agent effective against uterine cervix adenocarcinoma
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
  • a further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the compound selected from the group comprising:
  • a further object of the present invention is a compound of general formula (IV): wherein
  • Z is selected from: S, Se;
  • R and R’ which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH 2 ; alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ; cycloalkyl selected from the group comprising: heterocycloalkyl selected from the group comprising: aryl selected from the group comprising: heteroaryl selected from the group comprising:
  • R is selected from the group comprising:
  • alkyl selected from the group comprising: -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 ;
  • X is selected from the group comprising:
  • n is: 1 , 2, 3, 4 or 5 or 6; wherein n is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; -CH 2 CH 2 O-CH 2 CH 2 -; wherein n is: 1 , 2, 3 or 4;
  • V is selected from the group comprising: -(CO)- or -(CH 2 ) n - wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
  • cycloalkyl selected from the group comprising: heteroaryl selected from the group comprising: or isomers or pharmaceutically acceptable salts thereof.
  • a further object of the present invention is the isothiocyanate and/or isoselenocyanate compound of general formula (IV), as or for use as:
  • a further object of the present invention is the isothiocyanate and/or isoselenocyanate compound of general formula (IV) for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • isothiocyanate and/or isoselenocyanate compounds of general formula (IV) for use as chemopreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
  • a further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (IV) to a subject, a patient such as a human being or an animal, needing the same.
  • a further object of the present invention as preferred forms of embodiment of the compound of general formula (I) or (IV), are the compounds of formula: or isomers or pharmaceutically acceptable salts thereof.
  • a further object of the present invention are compounds with formula:
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
  • tumours solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
  • a further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the compound selected from the group comprising: or isomers or pharmaceutically acceptable salts thereof, to a subject, a patient such as a human being or an animal, needing the same.
  • aminoalcohols 3 available on the market or synthesized according to the proce- dures described in literature, were firstly protected towards the primary amino group and thereafter activated with tosylchloride to obtain the compounds of genera! formula 4.
  • duly substituted rhodol nucleus 5 was accomplished according to the proce- dure described in literature (Mottram L et al Org. Lett., 2007, 9(19), pages 3741-3744).
  • the corre- spending aminoalcohols 4 were reacted with the proper substituted nucleus 5 in a basic environ- ment in DMF with reflux, fo obtain the adducts which were submitted to add hydrolysis to obtain the corresponding primary amines 6.
  • the compounds with the isothiocyanate group of general formula 1 were obtained by reacting 6 with dicarbonyl dipyridone, whereas the compounds with the isoselenocyanate group 2 were ob- tained by treating the derivatives 6 initially with sodium hydroxide in the presence of Aliquat 336 and thereafter with selenium.
  • the corresponding monoprotected diamines 12 were reacted with foe duly substituted rhodol nu- cleus 11 according to foe procedure described in literature (Meinig J et al. Angew.ChemJntEd. 2015, 54, 9696-9699) to obtain foe corresponding amines 13 which are submitted to acid hydroly- sis to obtain the corresponding primary amines 14.
  • the compounds with the isothiocyanate group of general formula 7, related to general structures (I) and (III), were obtained by reacting the primary amines 14 with dicarbonyl dipyridone, whereas the compounds with the isoseienocyanate group or general rormuia u, related to general structures (i) and (III), were obtained by treating the primary amines 14 initially with sodium hydroxide In the presence of Aliquat 336 and thereafter with selenium (Zakrzewski J. et al. Synthesis 2016, 48, 85-
  • the compounds of general formula 16 were synthesized starting from the tosylamines 15 treated initially with thiourea and thereafter with NaOH, according to the procedure described in literature (Snow A.W. etal. Synthesis 2003, No. 4, 509-512).
  • the compounds with the isothiocyanate group of general formula 9, related to general structures (I) and (IV), were obtained by reacting the primary amines 18 with thiocarbonyl dipyridone.
  • the compounds with the isoselenocyanate group 10, related to general structures (! and (IV), were obtained by treating the primary amines 18 initially with sodium hydroxide in the presence of Ali- quat 336 and thereafter with selenium (Zakrzewski J. et al. Synthesis 2016, 48, 85-96).
  • the apoptotic response is rapid and both the intrinsic and the extrinsic pathway are involved, hence doubling chances of success in treating apoptosis-resistant tumour cells.
  • Apoptosis may be triggered by a variety of events.
  • DNA single-strand breaks are probably a crucial stimulus: the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the gen- eral formula (II), according to any one of the forms of embodiment of the present invention, DNA single-strand breaks are probably a crucial stimulus: the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate
  • Another consequence of the combination of the large accumulation of breaks and of the hard repa- rability thereof is the mutagenic effect observed for the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II).
  • the most promising isothiocyanate, sulforaphane can damage DNA through an indi- rect mechanism, depending on the generation of radical species centred on oxygen (ROS), pro- prised at a mitochondrial level and thereafter diffused to the nucleus.
  • ROS radical species centred on oxygen
  • the latter are mediated by ROS generation, whereas the DNA injuries caused by the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothio- cyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocya- nate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocya- nate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocya- nate and/or isoselenocyanate compound(s) described herein related to the general formula (II), ac- cording to any one of the forms of embodiment of the present invention, are not, but rather depend on an event
  • the DNA damage caused by sulforaphane allows the cytotoxic response thereof, but does not de- termine it; in the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), pref- erably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the gen- eral formula (II), according to any one of the forms of embodiment of the present invention, a good correlation was remarked
  • cytotoxic potential is ascribable to the induction of apoptosis, measured by quantifying caspase 3 and 8 activity and mitochondrial transmembrane potential and observed by means of a transmission electron microscope.
  • N-acetylcysteine only promotes a slight and insignif- icant protection against the toxicity of the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described here- in related to the general formula (II), according to any one of the forms of embodiment of the pre- sent invention.
  • a clinically relevant piece of data is the cytotoxic potential of the isothiocyanate and/or isoseleno- cyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, observed in the ex vivo model, consisting of blasts from leu- kaemia patients.
  • the ex vivo model of leukaemia represents in this context a highly relevant approach in predicting the therapeutic potential of the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of gen- eral formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more prefera- b
  • tumour cells The knowledge of the selectivity features for tumour cells allows safety operating windows to be outlined, inside which treatment can be adjusted; all this will have a significant relevance in plan- ning any clinical trials.
  • the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I) preferably the isothiocyanate and/or isoselenocyanate compound(s) of gen- eral formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more prefera- bly the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, based on findings relating to cytotoxicity and other effects (for example those on DNA), experimental feedbacks show that: at doses/
  • Threshold value in the pharmaco-toxicological field designates a dosage level separating non- effect from effect.
  • Tosylchloride (0.877 g, 0.0046 mol) is added to a solution of ferf-butyl(6-hydroxyhexyl)carbamate (1 g, 0.0046 mol), triethylamine (0.64 ml, 0.0046 mol) and DMAP (cat.) in dichloromethane and the resulting solution is stirred at room temperature for 12 h.
  • 6-((tert-butoxycarbonyl)amino)hexyl 4-methylbenzenesulfonate (0.286 g, 0.77 mmol) and potassi- um carbonate (0.131 g, 0.95 mmol) are added to a solution of 6-hydroxy-9-(o-toluoyl)-3H-xanthen- 3-one (0.354 g, 0.95 mmol) in DMF and the resulting mixture is stirred at reflux for 6 h. The solution is diluted with ethyl acetate and washed with water.
  • the organic phase is dried over sodium sulphate and evaporated.
  • the obtained residue is purified through flash chromatography using as mobile phase a mixture of dichloromethane / methanol in a 9.5/0.5 ratio.
  • HCI 6NA About 5 ml of HCI 6NA is added dropwise at 0 °C to a solution of tert-butyl (6-((3-oxo-9-(o-toluoyl)- 3H-xanthen-6-yl)oxy)hexyl)carbamate (0.2 g, 0.5 mmol) in methanol and the resulting solution is stirred at room temperature for 12h. Potassium bicarbonate is added up to pH 7 and the solvent is thereafter evaporated. The residue is treated with dichloromethane and washed with water, dried over sodium sulphate.
  • the solvent is evaporated and the residue is purified using as mobile phase a mixture of petroleum ether / ethyl acetate in a 4:6 ratio.
  • Tosylchloride (3.43 g, 0.026 mol) is added to a solution of tert-butyl (2-(2- hydroxyethoxy)ethyl)carbamate (2.63 g, 0.012 mol), triethylamine (2.51 ml, 0.026 mol) and DMAP (cat.) and the resulting solution is stirred at room temperature for 12 h.
  • the obtained residue is purified through flash chromatography using as mobile phase a mixture of petroleum ether / ethyl acetate in a 1 :1 ratio.
  • Potassium bicarbonate is added up to pH 7 and the solvent is thereafter evaporated.
  • the solvent is evaporated and the residue is purified using as mobile phase a mixture of petroleum ether / ethyl acetate in a 4:6 ratio.
  • 6-((tert-butoxycarbonyl)amino)hexyl 4-methylbenzenesulfonate (0.500 g, 1.3 mmol) and potassium carbonate (0.186 g, 1.3 mmol) are added to a solution of 2,7-difluoro-6-hydroxy-9-(o-toluoyl)-3H- xanthen-3-one(0.380 g, 1 .1 mmol) in DMF and the resulting mixture is stirred at reflux for 6 h.
  • the solution is diluted with ethyl acetate and washed with water.
  • the obtained residue is purified through flash chromatography using as mobile phase a mixture of dichloromethane / methanol in a 9.5/0.5 ratio.
  • Potassium bicarbonate is added up to pH 7 and the solvent is thereafter evaporated.
  • the solvent is evaporated and the residue is purified using as mobile phase a mixture of petroleum ether / ethyl acetate in a 4:6 ratio.
  • IC50 a concentration inhibiting cell viabil- ity by 50% compared to control cultures
  • IC50 a concentration inhibiting cell viabil- ity by 50% compared to control cultures
  • TK6 human lymphoblastoid cells
  • CT26 which are cells of colon carcinoma.
  • TN82 and TN46 in- prised a dose-dependent and significant decrease of cell viability also on these two cell lines (Fig- ure 1).
  • the small insert of Figure 1A also shows the cytotoxicity of the parental compound, sulforaphane, which, under the same experimental conditions set forth above, has an IC50 of 29.7 ⁇ M.
  • the Jurkat cells were thereafter analysed to delve into the mechanism of cell death through the use of different techniques.
  • Figure 3A the cells treated with TN82 (1 h of treatment followed by 5 h of incubation in a medium without the compounds under study according to the present invention) show clear signs of apoptosis. It is actually possible to remark the con- densation of chromatin, cup-shaped masses and micronuclei; occasionally, secondary necrosis is observed.
  • Cell death by apoptosis was biochemically confirmed by the rapid dose-dependent in- crease of the activity of caspase 3 and caspase 8 and by the increase of the percentage of cells with reduced mitochondrial activity (Figure 3B-D). The combination of these biochemical responses indicates the involvement of both intrinsic and extrinsic apoptosis.
  • TN82 and TN46 cause direct DNA and RNA damage and are mutagenic
  • the next step was the analysis of the genotoxic activity of the two TN compounds. Their effect on nuclear DNA was studied through the fast alkaline halo test. Through this assay, the DNA frag- ments resulting from DNA breakage diffuse from the nucleus in inverse proportion to their size, thus producing a concentric halo whose radius reflects the extent of DNA damage: at the micro- scope, the smaller the fragments look (highly damaged DNA), the larger the generated halo. Also, it should be noted that DNA single-strand fragments diffusing from the nucleus may derive either from a direct DNA breakage or from the presence of apurinic sites converted into DNA single- strand breaks at alkaline pH.
  • the halo formation can be properly monitored in single cells through fluorescence microscopy, as can be seen in the representative graph shown in Figure 4C.
  • Exposition for 1 h to both TN compounds induced a concentration-dependent and statistically sig- nificant increase of the formation of DNA single-strand breaks (SSBs) (Figure 4A) in Jurkat cells.
  • Micromolar concentration of TN 82 or 46 (2-8 ⁇ M) caused an extent of DNA single-strand breakage comparable to that provoked by 30 minutes of treatment with H 2 O 2 10-50 ⁇ M, a well-established and powerful agent inducing DNA damage, and far higher than that induced by sulforaphane (SFR) ( Figure 4 D). Accordingly, both TNs proved to be able to damage the DNA rapidly (within 1 h), ef- fectively and in a dose-dependent manner.
  • DNA-SSBs DNA single-strand breaks caused by the two theranostic TN compounds according to the present invention were repaired more slowly than those produced by H2O250 ⁇ M. Indeed, only 26.4% ⁇ 3.55 of the initial SSBs caused by the oxidizer were observed after 30 minutes and no residual damage was observed after 3 h of culture in a medium without the oxidiz- ing agent.
  • TK6 human lym- phoblastoid cells
  • TK6 cells proved to be sensitive to the DNA injuring activ- ity of TN82 and TN46 ( Figure 4B); as observed in Jurkat cells, TN46 proved to be more active than TN82 in TK6 cells as well.
  • TN82 and TN46 were defined through Kd, which measures the affinity of the compounds under study (according to the present invention) with regard to the DNA and is given by the ratio of Kon (association con- stant) to Koff (dissociation constant). The behaviour of the two compounds is very different.
  • TN46 actually appears to have a much higher affinity for the DNA than TN82, as shown by the Kd value in the nanomolar range for TN46 (0.009 ⁇ 0.001 ⁇ M) and in the submicromolar range for TN82 (0.097 ⁇ 0.017 ⁇ M).
  • Figure 6 shows the graphs obtained by comparing the extent of DNA single-strand breaks (DNA- SSBs) and the corresponding values of cell proliferation inhibition caused by TN82 and TN46. A good correlation was recorded between these two parameters for both compounds. In actual fact, the analysis of linear regression provided r 2 values of 0.8279 for TN82 and 0.7618 for TN46.
  • the two TNs can be classified as mutagen- ic.
  • SFR reference compound sulforaphane
  • previous studies proved the non- mutagenicity thereof [C. Fimognari, F. Berti, R. lori, G. Cantelli-Forti, P. Hrelia, Micronucleus for- mation and induction of apoptosis by different isothiocyanates and a mixture of isothiocyanates in human lymphocyte cultures, Mutat. Res. 582 (2005)1-10].
  • RNA integrity number (RIN), which is calculated by means of the areas underlying the peaks of rRNA 18S and 28S. Elecropherograms representing untreated Jurkat cells and Jurkat cells treated with TN82 or TN46 6 -12 - 18 ⁇ M for 24h are shown in Figure 8A. The peaks relating to the subunits of rRNA 18S and 28S appeared to be progressively smaller, in a concentration-dependent manner, in cells treated with TN82 (RIN value at 18 ⁇ M: 6.55 ⁇ 0.21) compared to untreated cells (RIN values: 9.38 ⁇ 0.22) (Figure 8B). Conversely, TN46 did not induce a dose-dependent RNA degradation, since at a concentration of 18 ⁇ M the RIN value rose back to 8.73 ⁇ 0.42 ( Figure 8B).
  • TN82 and TN46 are cytotoxic and genotoxic also under metabolically limiting conditions
  • the cytotoxic and genotoxic effects of the two theranostic TN compounds were also studied at metabolically limiting temperatures, namely by treating cells at a temperature of 4°C.
  • cytotoxic and genotoxic activity of TN82 and TN46 is not mediated by oxidative kinds of mech anisms
  • ROS reactive oxygen species
  • GSH glutathione
  • GSH deple- tion was rapid and substantial: after 1 h of treatment, TN82 and TN46 induced a statistically signifi- cant GSH reduction in the neighbourhood of 50-60% (TN82 and TN462 ⁇ M induced a reduction of 49.86 ⁇ 9.84% and 63.87 ⁇ 11.16%, respectively). However, GSH depletion did not appear to be dose-dependent. During the following exposition hours, GSH levels were restored and, after 24 h of treatment, the GSH content increased in cells treated with both TNs, achieving higher values than those detected in control cells for most of the tested concentrations.
  • the Jurkat cells were pre-treated with NAC (N-acetylcysteine) 5 mM or with BSO (buthionine sul- foximine) 0.2 mM for 24 h.
  • NAC N-acetylcysteine
  • BSO buthionine sul- foximine
  • TN concentrations close to Glso levels (6 ⁇ M for TN82 and 4 ⁇ M for TN46) were selected in order to ascertain the ability to alter cell proliferation in relation with the variation of GSH intracellular content.
  • the increase of GSH levels only partially improved the cell proliferation rate after 1 h of exposition to TN compounds ( Figures 11 C and 11 D).
  • the GSH depletion rate induced by TN derivatives was not influenced by pre-treatment with NAC.
  • a reduced GSH content worsened cell proliferation ability, although in a statistically signif- icant manner only for TN46 ( Figures 11 E and 11 F).
  • the ex vivo model is an excellent surrogate for the determination of the patient’s cellular response to treatment and for the prediction of clinical response thereto (Andrew G. Bosanquet and Philip B. Bell. Ex vivo therapeutic index by drug sensitivity assay using fresh human normal and tu- mour cells. 2004. 4(2): 145-154).
  • FLT3-negative namely they did not show tyrosine kinase domain mutations of FMS-like tyrosine-kinase 3 (FLT3), whereas others were FLT3-positive.
  • FLT3 mutations are observed in about 1/3 of patients with acute myeloid leukaemia (Leick, M.B.; Levis, M.J. The Future of Targeting FLT3 Activation in AML. Curr Hematol Malig Rep 2017, 12, 153-167) and are often associated to a high incidence of relapses and a short survival time after antitumour chemotherapy or transplantation (Larrosa-Garcia, M.; Baer, M.R.
  • peripheral blood lymphocytes obtained from healthy donors were treated with the compounds under study (according to the present invention) within the concentration range 4- 32 ⁇ M for 4 or 24 h.
  • the peripheral blood lymphocytes from healthy donors are the untransformed counterpart of Jurkat cells.
  • TN82 and TN46 did not prove to be cytotoxic on normal lymphocytes ( Figure 14).
  • apoptosis is a cell death mechanism triggered by several stimuli, among which DNA injuries are particularly important (40, 41).
  • DNA single-strand breaks are a crucial stimulus.
  • both compounds are able to damage the DNA extensively and irrespective of the cell type.
  • the concentrations and the times which can damage the DNA are the same which can cause an outstanding cytotoxic response.
  • DNA breaks are already observed after 1 h of treatment, hence their formation precedes the appearance of cell apoptotic modifications.
  • Sulforaphane is able to damage the DNA by means of an indirect mechanism, dependent on ROS, involving the intramitochondrial production of H202 and the following diffusion thereof in the nucle- us (Sestili P, Fimognari C. Cytotoxic and Antitumor Activity of Sulforaphane: The Role of Reactive Oxygen Species. Biomed Res Int 2015;2015:402386. doi: 10.1155/2015/402386).
  • DNA injuries caused by TN82 and by TN46 have a different nature compared to those caused by the parental compound sulforaphane. Indeed, the activity of the latter is mediated by ROS gener- ation secondary to mitochondrial respiratory chain inhibition, whereas DNA injuries caused by the TNs are not, but rather depend on a metabolically correlated event, such as direct reactions with nuclear DNA.
  • the deep, structural and mechanistic difference of the DNA injuries caused by the TNs from those by sulforaphane is also substantiated by the circumstance that, unlike sul- foraphane, the TNs entail micronuclei formation.
  • DNA injuries caused by the TN theranostic agents, TNs compounds according to the present invention are causally correlated to the cytotoxic response observed in intoxicated cells.
  • DNA injuries caused by sulforaphane contribute to, but do not determine, the cytotoxic response thereof (Ses- tili, P., Paolillo, M., Lenzi, M., Colombo, E., Vallorani, L, Casadei, L, Martinelli, C., and Fi- mognari, C.
  • Sulforaphane induces DNA single strand breaks in cultured human cells. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2010.
  • condensation with rhodol significantly modifies the cellular pharmacokinetics of the residual ITC and, in particular, eases the targeting of the residual ITC on the nucleus through energy- independent mechanisms (namely passive diffusion in nuclei).
  • TN82 and TN46 are characterized by quantitatively and qual- itatively different toxic profiles compared to their parental compounds.
  • the cytotoxicity of the TNs seems to mainly depend on their DNA-injuring activity.
  • the identification of these new properties suggests the pharmacological exploitation of these TN theranostic agents according to the present invention as potential antitumour chemotherapeutic agents.
  • the ability to massively damage the DNA is an important mechanism of action of a broad number of widely used antitumour drugs, such as anthracyclines, cisplatin, mafosfamide and other alkylating agents (Ho- soya, N. and Miyagawa, K., Targeting DNA damage response in cancer therapy. Cancer Sci, 2014. 105(4): pages 370-88).
  • TN82 is localized at an intracellular level
  • HeLa cells human cervix adenocarcinoma cells
  • HeLa cells were transfected with CellLight® ER-RFP (red fluorescent protein)-calreticulin and thereafter treated treated with TN82 1 ⁇ M for 6 h, fixed with 4% paraformaldehyde for 10 minutes, washed and observed through the Olympus FV1000 fluorescence microscope. The analysis was accomplished by using the Image J software.
  • the left picture of Figure 15 shows the green fluorescence of TN82
  • the central picture shows the red fluo- rescence of RFP
  • the right picture shows the merged (MERGING) green fluorescence of TN82 and the red fluorescence of RFP, allowing TN82 to be localized in the endoplasmic reticulum.
  • the green fluorescence of TN82 is diffused inside the cell (for example, also in mitochon- dria), suggesting a non specific localization of the ITC derivative under study (the compound TN82 according to the present invention).
  • the high reactivity of the ITC molecule might account for the molecule diffusion.
  • the amine or thiol groups of the proteins and molecules found inside the cell might attract the ITC electrophilic carbon and hinder the exclusive localization of TN82 inside the endoplasmic reticulum.
  • TN such as for example TN82 and TN46
  • parental compounds considered on an individual basis, sulforaphane on one side and the fluorophore compounds RF27 and RF42, respectively, on the other side.
  • the cytotoxicity increase shown by the TN compounds according to the present invention is always at least 10-30 times higher.
  • the ability to damage the DNA is very high in TN82 and TN46, whereas said ability is 20-30 times lower in sulforaphane and lacking in the two corresponding fluorophore compounds RF27 and RF42.
  • DNA injuries caused by the compounds according to the present invention, such as for example TN82 and TN46 have a different nature compared to those caused by sulforaphane.
  • the deep, structural and mechanic difference of the DNA injuries caused by the TN compounds accord- ing to the present invention from those by sulforaphane is also substantiated by the circumstance that, unlike sulforaphane, the TN compounds according to the present invention, such as TN82 and TN46, entail micronuclei formation.
  • the DNA injuries caused by the TN compounds ac- cording to the present invention are causally correlated to the cytotoxic response observed in intoxicated cells.
  • the TN compounds according to the present invention such as TN82 and TN46
  • a good correlation between the extent of DNA damage and that of the cytotoxic response was in- variably observed, regardless of significantly different experimental conditions (namely metabolical- ly permissive versus metabolically limiting temperatures).
  • TN82 and TN46 also dam- age the other nucleic acid, RNA.
  • sulforaphane not only is not toxic for the RNA, but it even performs a protective action with regard to RNA-injuring agents.
  • the compounds according to the present invention are characterized by technical effects which were surprising and fully unexpected for those skilled in the art, hence being non- obvious with regard to available prior art.
  • Patent EP 1 961 418 A1 suggests the use of some natural and synthetic isothiocyanates including, but not limited thereto, benzyl isothiocyanate (BITC) (1), phenethyl isothiocyanate (PEITC) (2), allyl isothiocyanate (AITC) (3) and 4-sulfophenylisothiocyanate (4), as described in line 20, on page 3, for treating benign prostatic hyperplasia, prostatitis and skin tumour, their use for formulating prep- arations useful in the above pathologies, lastly suggesting some methods (with surfactants and solubilizing agents) for preparing the above formulations.
  • benzyl isothiocyanate benzyl isothiocyanate
  • PEITC phenethyl isothiocyanate
  • AITC allyl isothiocyanate
  • 4-sulfophenylisothiocyanate (4) as described in line 20, on page 3, for treating benign prostatic hyperplasia,
  • the compounds described therein induce several effects: induction of phase 2 enzyme expression, repression of androgenic receptor and PSA expression, reduction of prostate weight, reduction of inflammation, inhibition of the prolifera- tion of a prostate tumour line and of a melanoma line, reduction of tumour incidence in a mouse xenograft model of prostate tumour and melanoma.
  • the compounds described in EP 1 961 418 A1 are chemically and structurally different from the compounds according to the present invention: the compounds described in EP 1 961 418 A1 do not show fluorescent portions, whereas the com- pounds according to the present invention comprise fluorophore portions such as rhodol.
  • IC50 values relating to the cytotoxic and cytostatic effect of the most powerful compounds (BITC and PEITC) described in EP 1 961 418 A1 range from 0.8 to 1.5 ⁇ M in the used prostate tumour line, but said values are obtained only after a continuous exposition for 3 or 7 days (corre- sponding to 72 or 168 hours).
  • the compounds according to the present invention show similar IC50 values, but said values are obtained after an exposition of only 1 h, followed by post- incubation up to 72 h in a drug-free medium.
  • time factor (which is fundamental in the CxT equation applied to antitumour agent effectiveness) must increase by 72 or 168 times for the compounds BITC and PEITC described in EP 1 961 418 A1 to produce cytotoxic levels comparable to those of the compounds according to the present invention.
  • Patent WO 03/059149 describes the use of fluorophore glucose or deoxyglucose conjugates as a strategy for detecting cancerous or pre-cancerous cells in patients.
  • the emission wavelength of fluorophores ranges from 400 to 1200 nm.
  • the conjugates are used for endoscopic or visual (for example in the case of melanomas) surveys, based on the ability of tumour cells to avidly take up glucose.
  • the compounds described in WO 03/059149 are used by tumour cells as energy sub- strates to allow surgeons to understand the localization of the tumour in a patient.
  • the fluorophore can be fluorescein isothiocyanate, used as a flu- orescent probe, not provided with antitumour activity.
  • the compounds according to the present in- vention do not comprise fluorophore glucose or deoxyglucose conjugates.
  • Patent US 2013/0079401 A1 proposes the use of a natural isothiocyanate contained in Wasabia japonica, 6 methyl-sulfinylhexyl isothiocyanate, and of a derivative thereof, 6 methyl-sulfonylhexyl isothiocyanate (compounds identified with numbers I7457 and I7557, respectively), as possible an- titumour agents, alone or in combination with other chemotherapeutic agents or ionizing radiations, even for chemoresistant tumours.
  • the invention described in US 2013/0079401 A1 is based on the finding of the cell replication inhib- iting activity of the two compounds, which effect was proved in several tumour cell lines.
  • the cyto- toxic/cytostatic effect is preserved also with regard to cells (K572) which have become resistant to the conventional antitumour drug Imatinib.
  • the compounds seem to interact with proteins which are highly involved in the cell cycle dynamic, allegedly the preferential target of their effect.
  • the mole- cules associated to the two isothiocyanates are not fluorophore structures.
  • cytotoxic and cyto- static effect of the two compounds described in US 2013/0079401 A1 is much lower that that of the compounds according to the present invention: the IC50 values are always higher than 10 ⁇ M in the different tested cell lines, but said values are obtained only after continuous exposition for 48 or 72h. Although tested on different cell lines, which are however comparable to the former, the compounds according to the present invention showed IC50 values of less than 10 ⁇ M. How- ever, said values were obtained after an exposition of only 1 h, followed by post-incubation up to 72 h in a drug-free medium.
  • time factor (which is fundamental in the CxT equation applied to antitumour agent effectiveness) must increase by 48-72 times for the compounds I7457 and I7557 described in US 2013/0079401 A1 to produce cytotoxic levels barely comparable to those of the compounds according to the present invention.
  • the action of the compounds I7457 and I7557 is typically dependent on cell cycle, a function needing an active metabolism be performed; the compounds according to the present invention conversely act with an actually different mechanism, namely by damaging the DNA in a manner which operates also at a metabolically non permissive temperature (4°C).
  • Patent US 4,304,720 concerns ester and ether derivatives of fluorescein, the synthesis thereof and the possibility to incorporate some derivatives, such as for example dioleyl fluorescein, into low- density lipoproteins (LDL), thus allowing cells to be visualized.
  • LDL low- density lipoproteins
  • isothiocyanate or isoselenocyanate groups are totally absent.
  • the purpose of the use of the compounds described in US 4,304,720 is “only” the visualization of cells, since no kind of bio- logical activity is described.

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Abstract

The present invention relates to a new family of isothiocyanates and isoselenocyanates compounds suitable to be used as theranostic agents.

Description

Title:
“Isothiocyanate and isoselenocyanate compounds”
DESCRIPTION
Technical Field of the Invention
The present invention relates to a new family of isothiocyanate and isoselenocyanate compounds suitable to be used as antitumour drugs, theranostic agents with combined properties of tumour di- agnosis and treatment, and as fluorescent probes having also chemopreventive, diagnostic and an- titumour properties.
Prior Art
Recent estimates show that cancer is the second cause of death, bound to to become the first with elderly people. Due to demographic changes alone, by 2030 the number of new cancer cases might increase by 70% worldwide (World Health Organization, 2018).
The lack of effective diagnostic tools for early detection of several tumours, the scarce availability of pharmacological options to treat patients at an advanced stage of the disease and the develop- ment of drug resistance involve adverse prognosis and high mortality rate. The moderate increase of survival, the comparatively low improvement of the toxicity profile of antitumour drugs and the high costs of antitumour therapies show that a limit has been achieved in terms of clinical benefits and patient tolerance of therapy.
Said situation demands the identification and development of new drugs and innovative antitumour strategies to oppose the morbidity and mortality associated with cancer. This is intended to over- come the limits of traditional antitumour therapy, which is affected by problems such as the devel- opment of chemoresistance and the low therapeutic index characterizing most chemotherapeutic drugs.
Furthermore, the growing cost of health care and the new opportunities offered by the most ad- vanced therapeutic strategies increase pressures on healthcare budget. Over the last few years, the diagnostic industry in particular has been the object of a series of cost reductions by govern- ments and institutions, aimed at restricting the use of diagnostic tests (Gilham, 2002). In its turn, this put a significant pressure on the diagnostic industry to maintain the standards of innovation and product improvement despite the limited growth of the market. Similar pressures were exerted on the pharmaceutical field, with increasingly strong demands for evidence of the effectiveness of therapies and costs, along with evidence of safety by regulatory authorities. In this context, theranostics is an emerging strategy with enormous potentialities, since the technologies and de- velopment abilities of the diagnostic field are increasingly applied to improve the efficiency and cost-effectiveness of the finding, of the development and of the marketing of drugs. The term theranostics designates the development of diagnostic tests directly connected with the application of specific therapies. Theranostics represents a combinatory diagnosis and a therapeutic approach to cancer, aimed at removing multi-step procedures, hence reducing treatment delays and improv- ing the patient’s treatment. It offers several benefits, including better diagnosis, specific administra- tion of drugs, reduction of toxic effects on normal tissues and the like (Palekar-Shanbhag et al. , 2013). The development of multifunctional theranostic agents is an intriguing undertaking for cus- tomized oncology, because it can integrate tumour diagnosis and therapy and provide a potential earning for pharmaceutical and diagnostic companies. In parallel, for prevention and diagnosis purposes in the tumoural field, particular interest is given to the development of new reactive fluorescent probes involving a minimum perturbation of the biolog- ical system. This is essential in order to understand the structure and function of cellular processes. Fluorescent probes must be adjusted to the constraints imposed by the complex intracellular envi- ronment. Some key problems must be addressed when designing a probe suitable for use in living cells. For example, the probe should be able to penetrate the outer lipid/phospholipid membrane at a relatively high speed while preserving integrity and performance at a cellular level, should feature an intracellular localization profile which may be observed with a microscope and should target a specific organelle while preserving cell viability and proliferation as well as membrane permeability. The available fluorescent dyes which are able to label/target specific organelles, such as LysoTracker™, ER-Tracker™ and MitoTracker™, allow specific functions of the corresponding or- ganelles to be monitored and can be used at low concentrations for any experimental approach (Perry et al. , 2011). For example, the chloromethyl derivatives of fluorescent probes based on ros- amine (MitoTracker™), which are currently used as mitochondrial probes, are lipophilic and cation- ic compounds which can electrophoretically accumulate in mitochondria in reply to changes of the mitochondrial membrane potential (Scorrano et al., 1999). Basically, the reactive chloromethyl groups may form covalent bonds with SH groups of mitochondrial proteins. This prevents them from being released even if mitochondria depolarize (Scorrano et al., 1999). Other types of mole- cules which are localized in mitochondria are based on europium (III) and terbium (III) complexes of heptadentate ligands bearing azaxanthone or azathioxanthone (Kielar et al., 2008; Law et al., 2009; Murray et al., 2008). Co-colouring experiments with these complexes showed that mitochon- dria fusion with lysosomes occurred only after significantly long incubation times (> 4 hours of in- cubation) (Manning et al., 2006).
More recently, most of the developed probes have been based on emissive metal complexes (Gill et al., 2009), recombinant proteins (Festy et al., 2007), semiconductor nanoparticles (Michalet et al., 2005) (often designated as quantum dots) and low molecular weight organic fluorophores, such as rhodamine, fluorescein, cyanine dyes and dipyrroylmethane (Domaille et al., 2008; Lavis et al., 2008; New et al., 2010; Weissleder et al., 2007). However, the application of these probes is limited due to several properties, such as: low solubility in water and in culture media, pH range, toxicity and cell penetration (Wolfbeis, 2005).
Based on the foregoing, the need was therefore strongly felt to implement a new family of com- pounds having such properties as to be usable concurrently as:
- innovative drugs with an outstanding profile of therapeutic effectiveness, so as to improve the ap- proach to the tumoural pathology compared to current drugs,
- theranostic agents to simultaneously diagnose and treat the oncological pathology;
- fluorescent probes for direct observation of specific cellular organelles in biomedical applications.
Summary of the Invention
While proceeding with research in the present technical field, the Applicants surprisingly and unex- pectedly implemented, as objects of the present invention:
- a new family of isothiocyanate and/or isoselenocyanate compounds of general formula (I):
Figure imgf000005_0001
wherein
Z is selected from: S, Se;
Y is selected from: O, NH or S;
R, R’, which may be identical to or different from each other, are selected from the group compris- ing:
-H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; linear or branched alkyl with 1 to 10 carbon atoms, preferably alkyl selected from the group com- prising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl with 3 to 6 carbon atoms, preferably cycloalkyl selected from the group comprising:
Figure imgf000005_0002
heterocycloalkyl with 4 to 5 carbon atoms, comprising 1 or 2 heteroatoms selected from N and O, preferably heterocycloalkyl selected from the group comprising:
Figure imgf000005_0003
aryl with 6, 12 or 18 carbon atoms, preferably aryl selected from the group comprising:
Figure imgf000005_0004
heteroaryl with 4 to 5 carbon atoms, comprising 1 heteroatom selected from N and O, preferably heteroaryl selected from the group comprising:
Figure imgf000005_0005
alkylenylcycloalkyl with 4 to 10 carbon atoms, wherein the cycloalkyl is a ring selected from 3, 4, 5 or 6 carbon atoms and the remaining alkylenyl moiety is linear or branched; cycloalkenylalkyl with 4 to 10 carbon atoms, wherein the cycloalkenyl is a ring selected from 3, 4, 5 or 6 carbon atoms and the remaining alkyl moiety is linear or branched; alkylenylheterocycloalkyl with 5 to 10 carbon atoms, wherein the heterocycloalkyl is a ring selected from 4 or 5 carbon atoms comprising 1 or 2 heteroatoms selected from N and O and the remaining alkylenyl moiety is linear or branched; heterocycloalkenylalkyl with 5 to 10 carbon atoms, wherein the heterocycloalkenyl is a ring select- ed from 4 or 5 carbon atoms comprising 1 or 2 heteroatoms selected from N and O and the remain- ing alkyl moiety is linear or branched; alkylenylaryl with 7 to 20 carbon atoms, wherein the aryl consists of 6, 12 or 18 carbon atoms and the remaining alkylenyl moiety is linear or branched; arylalkyl with 7 to 20 carbon atoms, wherein the aryl consists of 6, 12 or 18 carbon atoms and the remaining alkyl moiety is linear or branched; heteroarylalkyl with 5 to 10 carbon atoms, wherein the heteroaryl is a ring with 4 to 5 carbon atoms comprising 1 heteroatom selected from N and O and the remaining alkyl moiety is linear or branched; alkylenylheteroaryl with 5 to 10 carbon atoms, wherein the heteroaryl is a ring with 4 to 5 carbon atoms comprising 1 heteroatom selected from N and O and the remaining alkylenyl moiety is linear or branched;
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; linear or branched alkyl with 1 to 10 carbon atoms, preferably alkyl selected from the group com- prising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000006_0001
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; -CH2CH2O-CH2CH2-;
Figure imgf000007_0001
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
-CH2CH2NHCH2CH2-; -CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; -CH2CONH(CH2)nNHCOCH2- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000007_0002
aryl selected from the group comprising:
Figure imgf000007_0003
heteroaryl selected from the group comprising:
Figure imgf000007_0004
or isomers or pharmaceutically acceptable salts thereof;
- preferably isothiocyanate and/or isoselenocyanate compounds of general formula (I) wherein R and R’ are identical to each other, more preferably isothiocyanate and isoselenocyanate com- pounds of general formula (I) wherein R and R’ are identical to each other and Y is S, even more preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein R and R’ are identical to each other and Y is NH, most preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein R and R’ are identical to each other and Y is O;
- preferably isothiocyanate and/or isoselenocyanate compounds of general formula (I) wherein Z is S and R and R’ are identical to each other, more preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein Z is S and R and R’ are identical to each other and Y is S, even more preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein Z is S and R and R’ are identical to each other and Y is NH, most preferably isothiocya- nate and isoselenocyanate compounds of general formula (I) wherein Z is S and R and R’ are iden- tical to each other and Y is O, particularly all these isothiocyanate and isoselenocyanate com- pounds of general formula (I) wherein preferably R and R’ are both H or halogen, more preferably H or F, R” is -CH3 and X is selected from: -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5 or 6 or -CH2CH2-[O-CH2CH2]n- wherein n is: 1 , 2, 3 or 4;
- preferably isothiocyanate and/or isoselenocyanate compounds of general formula (I) wherein Z is Se and R and R’ are identical to each other, more preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein Z is Se and R and R’ are identical to each other and Y is S, even more preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein Z is Se and R and R’ are identical to each other and Y is NH, most preferably isothiocya- nate and isoselenocyanate compounds of general formula (I) wherein Z is Se and R and R’ are identical to each other and Y is O, particularly all these isothiocyanate and isoselenocyanate com- pounds of general formula (I) wherein preferably R and R’ are both FI or halogen, more preferably H or F, R” is -CH3 and X is selected from: -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5 or 6 or -CH2CH2-[O-CH2CH2]n- wherein n is: 1 , 2, 3 or 4;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (I) as described above as or for use as:
■ medicine/medicament,
■ antitumour agent,
■ antitumour agent effective against solid tumours,
■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma,
■ antitumour agent effective against uterine cervix adenocarcinoma,
■ theranostic agent,
■ biomedical probe,
■ fluorescent probe;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (I) for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leu- kaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- as well as a method for diagnosing and/or preventing and/or treating: tumours, solid tumours, liq- uid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (I) to a subject, a patient such as a human being or an animal, needing the same;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (I) for use as chemopreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- a new sub-family of isothiocyanate and/or isoselenocyanate compounds as preferred form of em- bodiment of the family of isothiocyanate and/or isoselenocyanate compounds of general formula (I), said sub-family being of general formula (II):
Figure imgf000009_0001
wherein
Z is selected from: S, Se;
R and R’, which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000009_0002
heterocycloalkyl selected from the group comprising:
Figure imgf000009_0003
aryl selected from the group comprising:
Figure imgf000009_0004
heteroaryl selected from the group comprising:
Figure imgf000009_0005
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4 or 5 or 6;
Figure imgf000010_0001
wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000010_0002
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000010_0003
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
-CH2CH2O-CH2CH2-;
Figure imgf000010_0004
wherein n is: 1 , 2, 3 or 4;
-CH2CH2NHCH2CH2-; -CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 2, 3, 4 or 5; -CH2CONH(CH2)nNHCOCH2- wherein n is: 2, 3, 4 or 5;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000010_0005
aryl selected from the group comprising:
Figure imgf000010_0006
heteroaryl selected from the group comprising:
Figure imgf000011_0001
or isomers or pharmaceutically acceptable salts thereof;
- all said isothiocyanate and/or isoselenocyanate compounds of general formula (II) as described above as or for use as:
■ medicine/medicament,
■ antitumour agent,
■ antitumour agent effective against solid tumours,
■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma,
■ antitumour agent effective against uterine cervix adenocarcinoma,
■ theranostic agent,
■ biomedical probe,
■ fluorescent probe;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (II) for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- as well as a method for diagnosing and/or preventing and/or treating: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (II) to a subject, a patient such as a human being or an animal, needing the same;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (II) for use as chemopreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- a new sub-family of isothiocyanate and/or isoselenocyanate compounds as further preferred form of embodiment of the family of isothiocyanate and/or isoselenocyanate compounds of general for- mula (I), said sub-family being of general formula (III):
Figure imgf000012_0001
wherein
Z is selected from: S, Se;
R and R’, which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000012_0002
heterocycloalkyl selected from the group comprising:
Figure imgf000012_0003
aryl selected from the group comprising:
Figure imgf000012_0004
heteroaryl selected from the group comprising:
Figure imgf000012_0005
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4 or 5 or 6;
Figure imgf000013_0001
wherein n is: 1 , 2, 3 or 4;
-CH2CH2NHCH2CH2-; -CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 2, 3, 4 or 5; -CH2CONH(CH2)nNHCOCH2- wherein n is: 2, 3, 4 or 5;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 40 5; and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000013_0002
aryl selected from the group comprising:
Figure imgf000013_0003
heteroaryl selected from the group comprising:
Figure imgf000014_0001
or isomers or pharmaceutically acceptable salts thereof;
- all said isothiocyanate and/or isoselenocyanate compounds of general formula (III) as described above as or for use as:
■ medicine/medicament,
■ antitumour agent,
■ antitumour agent effective against solid tumours,
■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma,
■ antitumour agent effective against uterine cervix adenocarcinoma,
■ theranostic agent,
■ biomedical probe,
■ fluorescent probe;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (III) for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- as well as a method for diagnosing and/or preventing and/or treating: tumours, solid tumours, liq- uid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (III) to a subject, a patient such as a human being or an animal, needing the same;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (III) for use as chemopreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- a new sub-family of isothiocyanate and/or isoselenocyanate compounds as further preferred form of embodiment of the family of isothiocyanate and/or isoselenocyanate compounds of general for- mula (I), said sub-family being of general formula (IV):
Figure imgf000015_0001
wherein
Z is selected from: S, Se;
R and R’, which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000015_0002
heterocycloalkyl selected from the group comprising:
Figure imgf000015_0003
aryl selected from the group comprising:
Figure imgf000015_0004
heteroaryl selected from the group comprising:
Figure imgf000015_0005
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4 or 5 or 6;
Figure imgf000016_0001
wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000016_0002
Figure imgf000016_0003
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000016_0004
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000016_0005
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
-CH2CH2O-CH2CH2-;
Figure imgf000016_0006
wherein n is: 1 , 2, 3 or 4;
-CH2CH2NHCH2CH2-; -CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 2, 3, 4 or 5; -CH2CONH(CH2)nNHCOCH2- wherein n is: 2, 3, 4 or 5;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000016_0007
aryl selected from the group comprising:
Figure imgf000016_0008
heteroaryl selected from the group comprising:
Figure imgf000017_0001
or isomers or pharmaceutically acceptable salts thereof;
- all said isothiocyanate and/or isoselenocyanate compounds of general formula (IV) as described above as or for use as:
■ medicine/medicament, antitumour agent,
■ antitumour agent effective against solid tumours,
■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma,
■ antitumour agent effective against uterine cervix adenocarcinoma,
■ theranostic agent,
■ biomedical probe,
■ fluorescent probe;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (IV) for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leu- kaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- as well as a method for diagnosing and/or preventing and/or treating: tumours, solid tumours, liq- uid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (IV) to a subject, a patient such as a human being or an animal, needing the same;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (IV) for use as chemopreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
As is known to those skilled in the art, in all the structural formulas: (I), (II), (III) or (IV) of the com- pounds according to the present invention, the symbols:
Figure imgf000017_0002
where present, are to be construed as the attachment point on the corresponding cycloalkyl, aryl or heterocyclic substituent group, whereby said group establishes a covalent bond with the remaining part of the relevant structural formulas (I), (II), (III) or (IV) of the compounds according to the pre- sent invention.
Brief Description of the Figures
Figure 1 shows through graphs the cytotoxicity of the theranostic (TN) compounds according to the present invention, identified by code TN82 {6-((6-isothiocyanatohexyl)oxy)-9-(o-toluoyl)-3H- xanthen-3-one} and by code TN46 (6-(2-(2-isothiocyanatoethoxy)ethoxy)-9-(o-toluoyl)-3H-xanthen- 3-one}, respectively, and identified inside the description of the present invention; in actual fact, cells of human t-cell acute lymphoblastic leukaemia (Jurkat) were treated with TN82 (circles), with TN46 (squares) (panel A) or with sulforaphane (SFR, insert) for 1 h and then cultured for an addi- tional 72 h in a medium without the compounds under study (according to the present invention); human lymphoblastoyd cells (TK6) were treated for 4 h with the compounds under study (according to the present invention) and cultured for an additional 24 h in a medium without the compounds under study (according to the present invention) (panel B); cells of colon carcinoma (CT26) were treated for 24 h with TN82 (0-40 μM) (C) and with TN46 (0-40 μM) (D). Cytotoxicity was determined by means of (Trypan Blue) dye exclusion tests or by means of analyses of intracellular alkaline es- terase (MUH assay). * p<0.05; ** p<0.01 ; **** p<0.0001 versus control cultures (untreated cells). Figure 2 shows through graph the cytotoxicity of the reference compounds, whose synthesis is re- ported, identified with code RF27 (said reference compound: 6-((6-aminoexyl)oxy)-9-(o-toluoyl)-3FI- xanthen-3-one, being the prodromal compound of the theranostic compound TN82) and with code RF42 (said reference compound: 6-(2-(2-aminoethoxy)ethoxy)-9-(o-toluoyl)-3FI-xanthen-3-one, be- ing the prodromal compound of the theranostic compound TN46), respectively, and identified inside the description of the present invention and on Jurkat cells (A) treated for 24 h with the compounds under study (according to the present invention). ** p<0.01 ; **** p<0.0001 versus control cultures (untreated cells).
Figure 3 shows through graphs the data confirming the apoptosis of Jurkat cells due to the com- pounds according to the present invention, such as TN82 and TN46, which induce apoptotic cell death. Apoptosis in Jurkat cells treated with TN82 or TN46. Panel A: TEM (transmission electron microscope) picture of cells which were treated for 1 h with TN82 and cultured for an additional 5 h in a medium without the compounds under study (according to the present invention). The pres- ence of apoptotic cells (ap) and secondary necrotic cells (n) is remarked. Caspase 3 (panel B) and caspase 8 (panel D) activity and the mitochondrial transmembrane potential (panel C) in cells treated for 1 h with TN82 or with TN46 and cultured for an additional 3 h or for 5 minutes with the mitochondrial uncoupler Carbonyl Cyanide m-Chlorophenylhydrazine (CCCP) used as positive control. The dashed lines in B-D refer to the values obtained in control cells (untreated cells). * p<0.05; ** p<0.01 ; *** p<0.001 ; **** p<0.0001 versus control cultures (untreated cells).
Figure 4 shows through graphs and picture the data confirming that the compounds according to the present invention, such as TN82 and TN46, induce DNA single-strand breakage in Jurkat and TK6 cells. The cells were treated with TN82 (open circles) or with TN46 (squares) for 1 h and im- mediately submitted to analysis to assess the presence of DNA strand breaks through fast halo as- say. Panels A and B show the extent of DNA damage in Jurkat cells (A) and in TK6 cells (B). The extent of DNA single-strand breakage is expressed as nuclear diffusion factor (NDF). (C) Picture representing Jurkat cells treated with 6 μM of TN46 and analysed through fast halo assay. (D) Ex- tent of DNA damage caused by 30 minutes of treatment with H2O2 (solid circles) or for 3 h with sul- foraphane (SFR, triangles) in Jurkat cells. ** p<0.01 ; **** p<0.0001 versus control cells (untreated cells).
Figure 5 shows through graph the data confirming that the reference compounds RF27 and RF42 do not induce DNA single-strand breakage in Jurkat cells. The cells were treated with RF27 (cir- cles) or RF42 (squares) for 1 h and immediately submitted to analysis to assess the presence of DNA strand breaks through fast halo assay. The extent of DNA single-strand breakage is ex- pressed as nuclear diffusion factor (NDF).
Figure 6 shows through graphs the data confirming the correlation between DNA damage and cell proliferation inhibition. The Jurkat cells were treated with TN82 (A) or TN46 (B) at concentrations of 0 - 2 - 4 - 6 μM for 1 h and immediately checked for DNA damage or incubated for 72 h in a culture medium without the compounds under study (according to the present invention) and counted in order to determine their proliferation.
Figure 7 shows through graph the data relating to the formation of micronuclei (MN) induced by treatment with TN82 or TN46. The TK6 cells were treated with increasing concentrations of TN82 (white bar graphs) or TN46 (black bar graphs) for 4 h, left to proliferate for an additional 20 h in a culture medium without the compounds under study (according to the present invention) and ana- lysed to measure the MN number. The dashed line refers to the baseline of MN measured in con- trol cells (untreated cells). The graph also shows the number of MN induced by mitomycin C (0.8 μg/ml) and vinblastine (2 μg/ml) used as positive controls.* p<0.05; ** p<0.01 ; *** p<0.001 ; **** p<0.0001 versus control cells (untreated cells).
Figure 8 shows through graph the data confirming the RNA damage induced by TN82 or by TN46 in Jurkat cells. The cells were treated with TN82 or with TN46 at concentrations of 0 - 6 - 12 - 18 μM for 24 h. (A) Elecropherograms representing the RNA distribution depending on size. (B) RIN (RNA Integrity Number) values. * p<0.05; *** p<0.001 versus control cells (untreated cells).
Figure 9 shows through graphs the data confirming that the compounds according to the present invention, such as TN82 and TN46, are cytotoxic and genotoxic under metabolically limiting condi- tions (4°C). Jurkat cells were treated with TN82 (circles) or TN46 (squares) for 1 h at 4°C and left to proliferate for an additional 72 h in a medium without the compounds under study (according to the present invention) (A) or immediately checked for DNA damage through fast halo assay (B). The panels C and D show the correlation between DNA damage and the corresponding cytotoxic re- sponses. ** p<0.01 ; **** p<0.0001 versus control cells (untreated cells).
Figure 10 shows through graphs the data relating to the effects of ortho-phenanthroline (o-phen) and rotenone (rot) on the cytotoxic and genotoxic activity of TN82 and TN46. The cells were treat- ed with TN82 (6 μM) or with TN46 (4 μM) for 1 h, or with sulforaphane (SFR, 30 μM for 3 h), in the presence or in the absence of ortho-phenanthroline (3μM) or rotenone (1 μM); the cells were im- mediately checked for DNA damage through fast halo assay (A) or for cytotoxic activity after 48 h of growth in a medium without the compounds under study (according to the present invention) (B). *p < 0.01 compared to SFR alone.
Figure 11 shows through graphs the data relating to the determination of GSFI levels in Jurkat cells treated with TN82 (A) or with TN46 (B) 0 - 2 - 4 - 6 μM for 1 - 3 - 6 - 24h. Influence of higher (C, D) or lower (E, F) intracellular GSH levels on cell proliferation. Jurkat cells were pre-treated with NAC 5 mM or with BSO 0,2 mM for 24 h, treated with TN82 6 μM or with TN46 4 μM for 1 h and left to proliferate for 48-72 h in a medium without the compounds under study (according to the present invention). * p<0.05; ** p<0.01 ; *** p<0.001 .
Figure 12 shows through graphs the date relating to the determination of intracellular GSH levels and the formation of DNA single-strand breakage in Jurkat cells which were pre-treated or were not pre-treated with NAC 5mM or with BSO 0.2 mM for 24 h and which were thereafter treated with TN82 or with TN46 (B) 0 - 2- 4 - 6 μM for 1 h: determination of intracellular GSH levels (A) and for- mation of DNA single-strand breakage (B and C).
Figure 13 shows through graphs the data confirming that the compounds according to the present invention, such as TN82 and TN46, are cytotoxic on blasts isolated from leukaemia patients. The blasts taken from peripheral blood (A) or bone marrow (B) were treated with the TNs for 24 h.
Figure 14 shows through graphs the data confirming that the compounds according to the present invention, such as TN82 and TN46, are not cytotoxic on human lymphocytes of peripheral blood at any of the tested concentrations after 4 h of treatment; after 24 h of treatment, TN82 is not cytotox- ic at any of the tested concentrations, whereas TN46 is cytotoxic at the highest tested concentra- tion (32 μM). Lymphocytes were treated at different concentrations of TN82 or TN46 (4-32 μM) for 4 h (A) or for 24 h (B).
Figure 15 shows a series of pictures confirming the use of the compounds according to the present invention, such as TN82, as biomedical probe, particularly as fluorescent probe. Intracellular locali- zation of TN82. Microscopic analysis of cervix adenocarcinoma cells (HeLa) transfected with red fluorescent protein (RFP) and treated with subtoxic concentrations (1 μM) of TN82. Two pictures representing the co-localization of TN82 (left) and RFP (centre) were selected to perform the merg- ing (right). In the right picture, the localization of TN82 in the endoplasmic reticulum and diffusely at an intracellular level is visible.
Detailed Description and Forms of Embodiment of the Invention
The object of the present invention is therefore an isothiocyanate and/or isoselenocyanate com- pound of general formula (I)
Figure imgf000020_0001
wherein
Z is selected from: S, Se;
Y is selected from: O, NH or S;
R, R’, which may be identical to or different from each other, are selected from the group compris- ing:
-H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; linear or branched alkyl with 1 to 10 carbon atoms, preferably alkyl selected from the group com- prising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl with 3 to 6 carbon atoms, preferably cycloalkyl selected from the group comprising:
Figure imgf000021_0001
heterocycloalkyl with 4 to 5 carbon atoms, comprising 1 or 2 heteroatoms selected from N and O, preferably heterocycloalkyl selected from the group comprising:
Figure imgf000021_0002
aryl with 6, 12 or 18 carbon atoms, preferably aryl selected from the group comprising:
Figure imgf000021_0003
heteroaryl with 4 to 5 carbon atoms, comprising 1 heteroatom selected from N and O, preferably heteroaryl selected from the group comprising:
Figure imgf000021_0004
alkylenylcycloalkyl with 4 to 10 carbon atoms, wherein the cycloalkyl is a ring selected from 3, 4, 5 or 6 carbon atoms and the remaining alkylenyl moiety is linear or branched; cycloalkenylalkyl with 4 to 10 carbon atoms, wherein the cycloalkenyl is a ring selected from 3, 4, 5 or 6 carbon atoms and the remaining alkyl moiety is linear or branched; alkylenylheterocycloalkyl with 5 to 10 carbon atoms, wherein the heterocycloalkyl is a ring selected from 4 or 5 carbon atoms comprising 1 or 2 heteroatoms selected from N and O and the remaining alkylenyl moiety is linear or branched; heterocycloalkenylalkyl with 5 to 10 carbon atoms, wherein the heterocycloalkenyl is a ring select- ed from 4 or 5 carbon atoms comprising 1 or 2 heteroatoms selected from N and O and the remain- ing alkyl moiety is linear or branched; alkylenylaryl with 7 to 20 carbon atoms, wherein the aryl consists of 6, 12 or 18 carbon atoms and the remaining alkylenyl moiety is linear or branched; arylalkyl with 7 to 20 carbon atoms, wherein the aryl consists of 6, 12 or 18 carbon atoms and the remaining alkyl moiety is linear or branched; heteroarylalkyl with 5 to 10 carbon atoms, wherein the heteroaryl is a ring with 4 to 5 carbon atoms comprising 1 heteroatom selected from N and O and the remaining alkyl moiety is linear or branched; alkylenylheteroaryl with 5 to 10 carbon atoms, wherein the heteroaryl is a ring with 4 to 5 carbon atoms comprising 1 heteroatom selected from N and O and the remaining alkylenyl moiety is linear or branched;
R” is selected from the group comprising: -H, -F, -Cl, -Br, -I; linear or branched alkyl with 1 to 10 carbon atoms, preferably alkyl selected from the group com- prising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000022_0001
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000022_0002
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000022_0003
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000022_0004
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000022_0005
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
-CH2CH2O-CH2CH2-;
Figure imgf000022_0006
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
-CH2CH2NHCH2CH2-; -CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; -CH2CONH(CH2)nNHCOCH2- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10: and Ft''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000022_0007
aryl selected from the group comprising:
Figure imgf000023_0001
Figure imgf000023_0002
or isomers or pharmaceutically acceptable salts thereof;
- preferably isothiocyanate and/or isoselenocyanate compounds of general formula (I) wherein R and R’ are identical to each other, more preferably isothiocyanate and isoselenocyanate com- pounds of general formula (I) wherein R and R’ are identical to each other and Y is S, even more preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein R and R’ are identical to each other and Y is NH, most preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein R and R’ are identical to each other and Y is O;
- preferably isothiocyanate and/or isoselenocyanate compounds of general formula (I) wherein Z is S and R and R’ are identical to each other, more preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein Z is S and R and R’ are identical to each other and Y is S, even more preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein Z is S and R and R’ are identical to each other and Y is NH, most preferably isothiocya- nate and isoselenocyanate compounds of general formula (I) wherein Z is S and R and R’ are iden- tical to each other and Y is O, particularly all these isothiocyanate and isoselenocyanate com- pounds of general formula (I) wherein preferably R and R’ are both H or halogen, more preferably H or F, R” is -CH3 and X is selected from: -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5 or 6 or -CH2CH2-[O- CH2CH2]n- wherein n is: 1 , 2, 3 or 4;
- preferably isothiocyanate and/or isoselenocyanate compounds of general formula (I) wherein Z is Se and R and R’ are identical to each other, more preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein Z is Se and R and R’ are identical to each other and Y is S, even more preferably isothiocyanate and isoselenocyanate compounds of general formula (I) wherein Z is Se and R and R’ are identical to each other and Y is NH, most preferably isothiocya- nate and isoselenocyanate compounds of general formula (I) wherein Z is Se and R and R’ are identical to each other and Y is O, particularly all these isothiocyanate and isoselenocyanate com- pounds of general formula (I) wherein preferably R and R’ are both H or halogen, more preferably H or F, R” is -CH3 and X is selected from: -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5 or 6 or -CH2CH2-[O- CH2CH2]n- wherein n is: 1 , 2, 3 or 4.
A further object of the present invention is the compound of general formula (I) as or for use as:
■ medicine/medicament,
■ antitumour agent,
■ antitumour agent effective against solid tumours, ■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma,
■ antitumour agent effective against uterine cervix adenocarcinoma,
■ theranostic agent,
■ biomedical probe,
■ fluorescent probe;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (I) for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leu- kaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- as well as a method for diagnosing and/or preventing and/or treating: tumours, solid tumours, liq- uid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (I) to a subject, a patient such as a human being or an animal, needing the same;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (I) for use as chemopreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
A further object of the present invention, as preferred form of embodiment of the isothiocyanate and/or isoselenocyanate compound of general formula (I), is an isothiocyanate and/or isoseleno- cyanate compound of general formula (II):
Figure imgf000024_0001
wherein
Z is selected from: S, Se;
R and R’, which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000025_0001
heterocycloalkyl selected from the group comprising:
Figure imgf000025_0002
aryl selected from the group comprising:
Figure imgf000025_0003
heteroaryl selected from the group comprising:
Figure imgf000025_0004
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4 or 5 or 6;
Figure imgf000025_0005
wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000025_0006
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000025_0007
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000025_0008
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000026_0001
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; -CH2CH2O-CH2CH2-;
Figure imgf000026_0002
wherein n is: 1 , 2, 3 or 4;
-CH2CH2NHCH2CH2-; -CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 2, 3, 4 or 5; -CH2CONH(CH2)nNHCOCH2- wherein n is: 2, 3, 4 or 5;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000026_0003
aryl selected from the group comprising:
Figure imgf000026_0004
Figure imgf000026_0005
or isomers or pharmaceutically acceptable salts thereof.
A further object of the present invention is the compound of general formula (II) as or for use as:
■ medicine/medicament,
■ antitumour agent,
■ antitumour agent effective against solid tumours,
■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma,
■ antitumour agent effective against uterine cervix adenocarcinoma,
■ theranostic agent, ■ biomedical probe,
■ fluorescent probe.
A further object of the present invention is the isothiocyanate and/or isoselenocyanate compound of general formula (II) for use in preventing and/or diagnosing and/or treating: tumours, solid tu- mours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adeno- carcinoma.
A further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (II) to a subject, a pa- tient such as a human being or an animal, needing the same.
A further object of the present invention are all the isothiocyanate and/or isoselenocyanate com- pounds, or isocyanate and/or isoselenocyanate compound, of general formula (II) for use as chem- opreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tu- mours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adeno- carcinoma.
A further object of the present invention, as preferred forms of embodiment of the isothiocyanate and/or isoselenocyanate compound of general formula (I) or (II), are the compounds with formula selected from the group comprising:
Figure imgf000027_0001
A further object of the present invention are compounds with formula:
Figure imgf000028_0001
or isomers or pharmaceutically acceptable salts thereof, as or for use as:
■ medicine/medicament, antitumour agent,
■ antitumour agent effective against solid tumours,
■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma,
■ antitumour agent effective against uterine cervix adenocarcinoma,
■ theranostic agent,
■ biomedical probe,
■ fluorescent probe. as well as all said isothiocyanate and/or isoselenocyanate compounds for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- as well as all said isothiocyanate and/or isoselenocyanate compounds for use as chemopreven- tive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
A further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the compound selected from the group comprising:
Figure imgf000029_0001
or isomers or pharmaceutically acceptable salts thereof, to a subject, a patient such as a human being or an animal, needing the same.
A further object of the present invention, as preferred form of embodiment of the compound of gen- eral formula (I), is an isothiocyanate and/or isoselenocyanate compound of general formula (III):
Figure imgf000029_0002
wherein
Z is selected from: S, Se;
R and R’, which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000030_0001
heterocycloalkyl selected from the group comprising:
Figure imgf000030_0002
aryl selected from the group comprising:
Figure imgf000030_0003
heteroaryl selected from the group comprising:
Figure imgf000030_0004
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4 or 5 or 6;
Figure imgf000030_0005
wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000030_0006
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000030_0007
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000030_0008
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000031_0001
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
-CH2CH2O-CH2CH2-;
Figure imgf000031_0002
wherein n is: 1 , 2, 3 or 4;
-CH2CH2NHCH2CH2-; -CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 2, 3, 4 or 5; -CH2CONH(CH2)nNHCOCH2- wherein n is: 2, 3, 4 or 5;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000031_0003
aryl selected from the group comprising:
Figure imgf000031_0004
Figure imgf000031_0005
or isomers or pharmaceutically acceptable salts thereof.
A further object of the present invention is the isothiocyanate and/or isoselenocyanate compound of general formula (III), as described above, as or for use as:
■ medicine/medicament,
■ antitumour agent,
■ antitumour agent effective against solid tumours,
■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma,
■ antitumour agent effective against uterine cervix adenocarcinoma, ■ theranostic agent,
■ biomedical probe,
■ fluorescent probe.
A further object of the present invention is the isothiocyanate and/or isoselenocyanate compound of general formula (III) for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uter- ine cervix adenocarcinoma;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (III) for use as chemopreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
A further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (III) to a subject, a patient such as a human being or an animal, needing the same.
A further object of the present invention, as preferred forms of embodiment of the compound of general formula (I) or (III), are the compounds of formula:
Figure imgf000032_0001
A further object of the present invention are compounds with formula:
Figure imgf000033_0001
or isomers or pharmaceutically acceptable salts thereof, as or for use as:
■ medicine/medicament, antitumour agent, ■ antitumour agent effective against solid tumours,
■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma, ■ antitumour agent effective against uterine cervix adenocarcinoma,
■ theranostic agent,
■ biomedical probe,
■ fluorescent probe. as well as all said isothiocyanate and/or isoselenocyanate compounds for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- as well as all said isothiocyanate and/or isoselenocyanate compounds for use as chemopreven- tive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma. A further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the compound selected from the group comprising:
Figure imgf000034_0001
or isomers or pharmaceutically acceptable salts thereof, to a subject, a patient such as a human being or an animal, needing the same.
A further object of the present invention, as preferred form of embodiment of the compound of gen- eral formula (I), is a compound of general formula (IV):
Figure imgf000034_0002
wherein
Z is selected from: S, Se;
R and R’, which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000035_0001
heterocycloalkyl selected from the group comprising:
Figure imgf000035_0002
aryl selected from the group comprising:
Figure imgf000035_0003
heteroaryl selected from the group comprising:
Figure imgf000035_0004
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4 or 5 or 6;
Figure imgf000035_0005
wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000035_0006
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000035_0008
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000035_0007
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000036_0001
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; -CH2CH2O-CH2CH2-;
Figure imgf000036_0002
wherein n is: 1 , 2, 3 or 4;
-CH2CH2NHCH2CH2-; -CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 2, 3, 4 or 5; -CH2CONH(CH2)nNHCOCH2- wherein n is: 2, 3, 4 or 5;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000036_0003
Figure imgf000036_0004
heteroaryl selected from the group comprising:
Figure imgf000036_0005
or isomers or pharmaceutically acceptable salts thereof.
A further object of the present invention is the isothiocyanate and/or isoselenocyanate compound of general formula (IV), as or for use as:
■ medicine/medicament,
■ antitumour agent,
■ antitumour agent effective against solid tumours,
■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma,
■ antitumour agent effective against uterine cervix adenocarcinoma, ■ theranostic agent,
■ biomedical probe,
■ fluorescent probe.
A further object of the present invention is the isothiocyanate and/or isoselenocyanate compound of general formula (IV) for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- as well as all said isothiocyanate and/or isoselenocyanate compounds of general formula (IV) for use as chemopreventive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
A further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the isothiocyanate and/or isoselenocyanate compound of general formula (IV) to a subject, a patient such as a human being or an animal, needing the same.
A further object of the present invention, as preferred forms of embodiment of the compound of general formula (I) or (IV), are the compounds of formula:
Figure imgf000037_0001
or isomers or pharmaceutically acceptable salts thereof.
A further object of the present invention are compounds with formula:
Figure imgf000038_0001
or isomers or pharmaceutically acceptable salts thereof, as or for use as:
■ medicine/medicament,
■ antitumour agent,
■ antitumour agent effective against solid tumours,
■ antitumour agent effective against liquid tumours,
■ antileukemic agent,
■ antitumour agent effective against acute leukaemia,
■ antitumour agent effective against colon adenocarcinoma,
■ antitumour agent effective against uterine cervix adenocarcinoma,
■ theranostic agent,
■ biomedical probe,
■ fluorescent probe. as well as all said isothiocyanate and/or isoselenocyanate compounds for use in preventing and/or diagnosing and/or treating: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma;
- as well as all said isothiocyanate and/or isoselenocyanate compounds for use as chemopreven- tive and/or chemodiagnostic and/or chemotherapeutic agent against: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma.
A further object of the present invention is a method for diagnosing and/or preventing and/or treat- ing: tumours, solid tumours, liquid tumours, leukaemia, acute leukaemia, colon adenocarcinoma, uterine cervix adenocarcinoma, said method comprising the administration of an effective amount of the compound selected from the group comprising:
Figure imgf000039_0001
or isomers or pharmaceutically acceptable salts thereof, to a subject, a patient such as a human being or an animal, needing the same. Some Synthesis Methods
General Synthesis Scheme I
Figure imgf000040_0001
The process of the General Synthesis Scheme I describes the general synthesis of compounds of general formula (II), wherein R, R1, R” and X are defined as described according to the present in- vention.
Particularly, the aminoalcohols 3, available on the market or synthesized according to the proce- dures described in literature, were firstly protected towards the primary amino group and thereafter activated with tosylchloride to obtain the compounds of genera! formula 4.
The synthesis of the duly substituted rhodol nucleus 5 was accomplished according to the proce- dure described in literature (Mottram L et al Org. Lett., 2007, 9(19), pages 3741-3744). The corre- spending aminoalcohols 4 were reacted with the proper substituted nucleus 5 in a basic environ- ment in DMF with reflux, fo obtain the adducts which were submitted to add hydrolysis to obtain the corresponding primary amines 6.
The compounds with the isothiocyanate group of general formula 1 were obtained by reacting 6 with dicarbonyl dipyridone, whereas the compounds with the isoselenocyanate group 2 were ob- tained by treating the derivatives 6 initially with sodium hydroxide in the presence of Aliquat 336 and thereafter with selenium.
The following compounds listed below were prepared according to the General Synthesis Scheme I, modified by using the appropriate chemical reagents to obtain the desired compounds (foe struc- ture of foe compounds listed below was confirmed by 1H-NMR spectroscopy):
Figure imgf000041_0001
General Synthesis Scheme il
The process of the General Synthesis Scheme II describes foe general synthesis of compounds of general formula (III), wherein R, R’, R" and X are defined as described according to foe present in- vention.
The synthesis of foe duly substituted rhodol nucleus 11 was accomplished according to the proce- dure described in literature (Meinig J et al. Angew.Chem.lntEd. 2015, 54, 9696-9699).
The corresponding monoprotected diamines 12 were reacted with foe duly substituted rhodol nu- cleus 11 according to foe procedure described in literature (Meinig J et al. Angew.ChemJntEd. 2015, 54, 9696-9699) to obtain foe corresponding amines 13 which are submitted to acid hydroly- sis to obtain the corresponding primary amines 14.
The compounds with the isothiocyanate group of general formula 7, related to general structures (I) and (III), were obtained by reacting the primary amines 14 with dicarbonyl dipyridone, whereas the compounds with the isoseienocyanate group or general rormuia u, related to general structures (i) and (III), were obtained by treating the primary amines 14 initially with sodium hydroxide In the presence of Aliquat 336 and thereafter with selenium (Zakrzewski J. et al. Synthesis 2016, 48, 85-
96).
Figure imgf000042_0001
The following compounds listed below were prepared according to the General Synthesis Scheme II, modified by using the appropriate chemical reagents to obtain the desired compounds (the struc- ture of the compounds listed below was confirmed by 1H-NMR spectroscopy):
Figure imgf000043_0001
General Synthesis Scheme III
The process of the General Synthesis Scheme III describes the general synthesis of compounds of general formula (IV), wherein R, R’, R” and X are defined as described according to the present in- vention.
The compounds of general formula 16 were synthesized starting from the tosylamines 15 treated initially with thiourea and thereafter with NaOH, according to the procedure described in literature (Snow A.W. etal. Synthesis 2003, No. 4, 509-512).
The compounds of general formula 16 were reacted with the duly substituted rhodol nucleus 11 us- ing Pd2(dba) as catalyst, to obtain the corresponding compounds 17.
Said compounds 17, submitted to acid hydrolysis, allow the corresponding primary amines 18 to be synthesized.
The compounds with the isothiocyanate group of general formula 9, related to general structures (I) and (IV), were obtained by reacting the primary amines 18 with thiocarbonyl dipyridone. The compounds with the isoselenocyanate group 10, related to general structures (!) and (IV), were obtained by treating the primary amines 18 initially with sodium hydroxide in the presence of Ali- quat 336 and thereafter with selenium (Zakrzewski J. et al. Synthesis 2016, 48, 85-96).
Figure imgf000044_0001
The following compounds listed below were prepared according to the General Synthesis Scheme III, modified by using the appropriate chemical reagents to obtain the desired compounds (the structure of the compounds listed below was confirmed by 1H-NMR spectroscopy):
Figure imgf000045_0001
The isothiocyanate and/or isoselenocyanate compounds according to the present invention, related to general formulas (I) or (II) or (III) or (IV), are listed below with the relevant structural formula and chemical name:
Figure imgf000045_0002
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
ADVANTAGES
Medicine/medicament/Antitumour Agent
The isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specif- ic isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, are new compounds which can address the complexity of cancer through multiple and complex mecha- nisms of action: they induce the death of tumour cells, block the proliferation of tumour cells, can damage the DNA and RNA of tumours cells and are localized in mitochondria and in the endo- plasmic reticulum. A clinically relevant observation is that their cytotoxic activity was detected also on primary cells from patients affected by acute myeloblastic leukaemia.
The isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specif- ic isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, show a con- siderable cytotoxic activity, unrelated to the cell type.
The kind of cellular death caused by the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described here- in related to the general formula (II), according to any one of the forms of embodiment of the pre- sent invention, is apoptotic, as biochemically and microscopically ascertained.
The apoptotic response is rapid and both the intrinsic and the extrinsic pathway are involved, hence doubling chances of success in treating apoptosis-resistant tumour cells.
Apoptosis may be triggered by a variety of events.
In the case of the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the gen- eral formula (II), according to any one of the forms of embodiment of the present invention, DNA single-strand breaks are probably a crucial stimulus: the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, can extensively damage the cell DNA and a causal relation- ship exists between DNA damage and cytotoxic response.
The cytotoxic impact of the DNA injuries induced by the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, is further confirmed by the remark that these are not rapidly repaired. Accordingly, their persistence represents a significant and causal toxic event.
Another consequence of the combination of the large accumulation of breaks and of the hard repa- rability thereof is the mutagenic effect observed for the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II).
It should be noted that the activity of the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described here- in related to the general formula (II), according to any one of the forms of embodiment of the pre- sent invention, is completely different from that of isothiocyanates or ITCs or isothiocyanate moie- ties.
For example, the most promising isothiocyanate, sulforaphane, can damage DNA through an indi- rect mechanism, depending on the generation of radical species centred on oxygen (ROS), pro- duced at a mitochondrial level and thereafter diffused to the nucleus.
Unlike sulforaphane, the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocya- nate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocya- nate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more prefera- bly, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, damage the nuclear DNA through a mechanism which is independent from ROS.
Overall, the results outlined so far have two important implications.
Firstly: the DNA injuries caused by the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described here- in related to the general formula (II), according to any one of the forms of embodiment of the pre- sent invention, have a different nature compared to those caused by sulforaphane.
In actual fact, the latter are mediated by ROS generation, whereas the DNA injuries caused by the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothio- cyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocya- nate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocya- nate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocya- nate and/or isoselenocyanate compound(s) described herein related to the general formula (II), ac- cording to any one of the forms of embodiment of the present invention, are not, but rather depend on an event which is unrelated to metabolism, such as the direct reaction with the nuclear DNA.
The fact that the DNA injuries caused by the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of gen- eral formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more prefera- bly the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, and those caused by sulforaphane are deeply, structurally and mechanically dif- ferent is also substantiated by the fact the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described here- in related to the general formula (II), according to any one of the forms of embodiment of the pre- sent invention, involve the formation of micronuclei, whereas sulforaphane does not.
Secondly: the DNA injuries caused by the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described here- in related to the general formula (II), according to any one of the forms of embodiment of the pre- sent invention, unlike those caused by sulforaphane, are causally related to the cytotoxic response observed in the treated cells.
The DNA damage caused by sulforaphane allows the cytotoxic response thereof, but does not de- termine it; in the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), pref- erably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the gen- eral formula (II), according to any one of the forms of embodiment of the present invention, a good correlation was remarked between the extent of DNA damage and the extent of the cytotoxic re- sponse, regardless of radically different experimental conditions.
Overall, these results show the pleiotropic activity of the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, on tumour cells.
The multiple mechanisms related to the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described here- in related to the general formula (II), according to any one of the forms of embodiment of the pre- sent invention, such as the cytotoxic effect, the induction of apoptosis independently from ROS, the genotoxic and mutagenic potential, significantly improve the effectiveness of the antitumour treat- ment and represent an interesting offer in the oncological field.
The isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specif- ic isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, perform a powerful cytostatic and cytotoxic activity, act on both solid and blood tumours, bind to the DNA and accumulate at the level of the mitochondria and endoplasmic reticulum of tumour cells, fostering the eradication of tumour cells.
Theranostic Agent
The cytotoxic potential of the isothiocyanate and/or isoselenocyanate compound(s) of general for- mula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothio- cyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothio- cyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more pref- erably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present inven- tion, on tumour cells, their fluorescence and their ability to be localized in mitochondria and in the endoplasmic reticulum suggest their use as theranostic drugs in cancer models.
Furthermore, the cytotoxicity they show at higher doses, along with their fluorescence and their ability to be localized in mitochondria and in the endoplasmic reticulum, suggest their use as theranostic drugs in the oncological field.
Moreover, the fluorescence characterizing the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, enables them to perform simultaneously a diagnostic and a therapeutic action, based on the identification of tumour cells by means of imaging techniques and following addressing of cytotoxicity to tumour cells, accordingly their use as new theranostic drugs. Biological Probe/Fluorescent Probe
The ability of the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), pref- erably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the gen- eral formula (II), according to any one of the forms of embodiment of the present invention, not to alter, at certain concentrations, cell viability, their fluorescence, their rapid intracellular accumula- tion and their ability to be localized in mitochondria and in the endoplasmic reticulum suggest their use as probes for biomedical applications.
The isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specif- ic isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, are fluores- cent, are localized in specific organelles and, at low concentrations, preserve cell viability. In addi- tion, the fact that the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the gen- eral formula (II), according to any one of the forms of embodiment of the present invention, are lo- calized in both mitochondria and the endoplasmic reticulum in just few hours (1-3 hours of incuba- tion) makes them ideal candidates as specific cellular probes, on account of the close bond be- tween endoplasmic reticulum and mitochondria. These observations show that they might be suita- ble as probes to be used in living cells.
Also,
1) the ability of the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the gen- eral formula (II), according to any one of the forms of embodiment of the present invention, to penetrate the cell membrane without requiring particular procedures to increase cell permeability and without requiring derivatization,
2) their time-dependent intracellular localization,
3) their preservation of cell viability at certain concentrations, and
4) their fluorescence provide proof of concept of their use as markers in biomedical studies. EXPERIMENTAL FINDINGS
In vitro experiments were performed on different tumour and untransformed cell lines (Jurkat, TK6, HeLa, Ct26) in order to study the cytotoxic potential of the compound(s) according to any one of the forms of embodiment of the present invention.
The isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specif- ic isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, were able to significantly reduce cell viability in all cell lines with IC50 values (concentration inhibiting cell viabil- ity by 50% compared to control cultures) ranging from 3 to 5 μM (Jurkat cells).
The mechanism underlying their cytotoxic potential is ascribable to the induction of apoptosis, measured by quantifying caspase 3 and 8 activity and mitochondrial transmembrane potential and observed by means of a transmission electron microscope.
The isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specif- ic isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, show a genotoxic and mutagenic profile, as proved by fast halo assay as well as by micronucleus test, re- spectively, also under metabolically limiting conditions.
In order to specifically probe into the role of glutathione depletion in the cytotoxic and genotoxic ac- tivity of the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocya- nate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specif- ic isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, the cells were pre-treated with N-acetylcysteine, a condition which significantly increases the base level of intracellular glutathione.
Cells enriched with glutathione were exposed to the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, at a concentration about corresponding to GI50 (concentra- tion inhibiting cell viability by 50% compared to control cultures) for 1 hour and were thereafter cul- tured for an additional 72 hours to compare their proliferation with that of cells treated with the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothio- cyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocya- nate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocya- nate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocya- nate and/or isoselenocyanate compound(s) described herein related to the general formula (II), ac- cording to any one of the forms of embodiment of the present invention, but not with N- acetylcysteine.
Despite the significant increase of glutathione, N-acetylcysteine only promotes a slight and insignif- icant protection against the toxicity of the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described here- in related to the general formula (II), according to any one of the forms of embodiment of the pre- sent invention.
Likewise, the extent of DNA damage induced by the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, was hardly and insignificantly influenced by pretreatment with N-acetylcysteine.
A clinically relevant piece of data is the cytotoxic potential of the isothiocyanate and/or isoseleno- cyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, observed in the ex vivo model, consisting of blasts from leu- kaemia patients.
Since a cell line may strongly differ from the leukemic population taken from the patient in terms of growth kinetics and pharmacological determinants, ex vivo studies are in the oncological field an excellent surrogate for the determination of the patient’s cellular response to treatment and a highly relevant model to predict clinical response thereto (Bosanquet e Bell, J Exp Ther Oncol 4,145,2004); the ex vivo model of leukaemia represents in this context a highly relevant approach in predicting the therapeutic potential of the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of gen- eral formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more prefera- bly the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, and provides a unique contribution to relating the activity of the agents under study with the cytogenetic, molecular and immunological features of patients.
Lastly, the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocya- nate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specif- ic isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, seem to fea- ture a selective cytotoxicity towards tumour cells, namely the ability to interact only with tumour cells and not with untransformed cells.
This piece of data is extremely important, since it might be the foundation to assume their favoura- ble toxicological profile.
The knowledge of the selectivity features for tumour cells allows safety operating windows to be outlined, inside which treatment can be adjusted; all this will have a significant relevance in plan- ning any clinical trials.
Other in vitro experiments were performed in order to determine the cellular localization of the compound(s) according to any one of the forms of embodiment of the present invention.
The isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specif- ic isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, are local- ized in both mitochondria and the endoplasmic reticulum after 1-3 hours of cell incubation. Their fluorescence spectrum was also registered.
Their emissions cover a wide range of wavelengths, with maximum emission being achieved at 518 nm.
It must be noted that the cytotoxic effects of the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate com- pound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, are dose-dependent.
This means that concentrations without cytotoxic effects of the isothiocyanate and/or isoselenocya- nate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more preferably the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate com- pound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, can be identified.
Their fluorescence spectrum, similar to that of most fluorescent probes used in flow cytometry and in microscopy studies, as well as their biological features, proves their use as promising probes for biomedical applications (at not cytotoxic doses) and as drugs (at cytotoxic doses).
More generally, it is found that, regarding the isothiocyanate and/or isoselenocyanate compound(s) of general formula (I), preferably the isothiocyanate and/or isoselenocyanate compound(s) of gen- eral formula (II) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (III) or the isothiocyanate and/or isoselenocyanate compound(s) of general formula (IV), more prefera- bly the isothiocyanate and/or isoselenocyanate compound(s) of general formula (II), as well as, even more preferably, the specific isothiocyanate and/or isoselenocyanate compound(s) described herein related to the general formula (II), according to any one of the forms of embodiment of the present invention, based on findings relating to cytotoxicity and other effects (for example those on DNA), experimental feedbacks show that: at doses/concentrations <2 microM (microMolar or μM) they can be used as probe (whereby the accumulate at intracellular level, but do not perturb cell homeostasis); at doses equal to or higher than 2 microMolar (μM) the compounds according to the invention are used as/are theranostic agents (they are localized at intracellular level AND induce cytotoxic and genotoxic effects).
Threshold value in the pharmaco-toxicological field designates a dosage level separating non- effect from effect.
At concentrations equal to or higher than 2 microM the cytotoxic effect starts, increasing as dose increases. Examples
SYNTHESIS EXAMPLES tert-butyl (6-hydroxyhexyl)carbamate
Figure imgf000058_0001
Ditertbutyldicarbonate (7.4 g, 0.034 mol), solubilized in 20 ml of dichloromethane, is added at zero degrees to a solution of 6-aminoethanol (2 g, 0.017 mol) and triethylamine (7.09 ml, 0.051 mol) in 20 ml of dichloromethane and the resulting mixture is stirred at room temperature for 12 hours. The mixture is washed with distilled water, dried over sodium sulphate and thereafter evaporated with rotavapor to obtain the compound at issue, which is used in the following step without further purifi- cation. Yield: 95 %. 1H-NMR (400 MHz, CDCI3) 1 .25-1 .77 (m, 17H), 3.15 (t, 2H), 3.72 (t, 2H). 6-((tert-butoxycarbonyl)amino)hexyl 4-methylbenzenesulfonate
Figure imgf000058_0002
Tosylchloride (0.877 g, 0.0046 mol) is added to a solution of ferf-butyl(6-hydroxyhexyl)carbamate (1 g, 0.0046 mol), triethylamine (0.64 ml, 0.0046 mol) and DMAP (cat.) in dichloromethane and the resulting solution is stirred at room temperature for 12 h.
The solution is washed with water, brine, dried over sodium sulphate and thereafter evaporated to obtain the compound at issue, which is used in the following step without further purification. Yield: 67 %. 1H-NMR (400 MHz, CDCI3) 1.19-1.81 (m, 17H), 2.47 (s, 3H), 3.11 (t, 2H), 4.16 (t, 2H), 7.32 (d, 2H), 7.79 (d, 2H). tert-butyl (6-((3-oxo-9-(o-toluoyl)-3H-xanthen-6-yl)oxy)hexyl)carbamate
Figure imgf000058_0003
6-((tert-butoxycarbonyl)amino)hexyl 4-methylbenzenesulfonate (0.286 g, 0.77 mmol) and potassi- um carbonate (0.131 g, 0.95 mmol) are added to a solution of 6-hydroxy-9-(o-toluoyl)-3H-xanthen- 3-one (0.354 g, 0.95 mmol) in DMF and the resulting mixture is stirred at reflux for 6 h. The solution is diluted with ethyl acetate and washed with water.
The organic phase is dried over sodium sulphate and evaporated. The obtained residue is purified through flash chromatography using as mobile phase a mixture of dichloromethane / methanol in a 9.5/0.5 ratio.
Yield: 53%. 1H-NMR (400 MHz, CDCI3)1.22 (s, 9H), 1.29-1.57 (m, 4H), 1.69-1.92 (m, 4H), 2.03 (s, 3H), 3.57 (t, 2H), 4.11 (t, 2H), 6.38 (d, 1 H), 6.41-6.49 (m, 1 H), 6.62-6.73 (m, 1 H), 6.81-6.98 (m, 3H), 7.05 (d, 1 H), 7.20-7.36 (m, 2H). 6-((6-aminohexyl)oxy)-9-(o-toluoyl)-3H-xanthen-3-one (RF27)
Figure imgf000059_0001
About 5 ml of HCI 6NA is added dropwise at 0 °C to a solution of tert-butyl (6-((3-oxo-9-(o-toluoyl)- 3H-xanthen-6-yl)oxy)hexyl)carbamate (0.2 g, 0.5 mmol) in methanol and the resulting solution is stirred at room temperature for 12h. Potassium bicarbonate is added up to pH 7 and the solvent is thereafter evaporated. The residue is treated with dichloromethane and washed with water, dried over sodium sulphate.
The solvent is thereafter evaporated and the residue is purified through flash chromatography us- ing as mobile phase a mixture of dichloromethane / methanol /aqueous ammonia (33%) in a 9:1 :0.1 ratio to obtain the compound at issue. Yield: quantitative. 1H-NMR (400 MHz, CDCI3) 1.33- 1 .47 (m, 6H), 1 .74-1 .83 (m, 4H), 1 .99 (brs, 1 H), 3.99 (t, 2H), 6.37 (d, 1 H), 6.47-6.50 (m, 1 H), 6.67- 6.69 (m, 1 H), 6.85-6.89 (m, 3H), 7.07 (d, 1 H), 7.29-7.40 (m, 3H). 6-((6-isothiocyanatohexyl)oxy)-9-(o-toluoyl)-3H-xanthen-3-one (TN82)
Figure imgf000059_0002
A solution of 1 ,1 '-Thiocarbonyldi-2(1 H)-pirydone (0.078 g, 0.34 mmol) is added dropwise to a solu- tion of 6-((6-aminohexyl)oxy)-9-(o-toluoyl)-3H-xanthen-3-one (0.135 g, 0.34 mmol) in dichloro- methane and the mixture is stirred at room temperature for 12h.
The solvent is evaporated and the residue is purified using as mobile phase a mixture of petroleum ether / ethyl acetate in a 4:6 ratio.
Yield: 67%. 1H-NMR (400 MHz, CDCI3) 1.52-1.58 (m, 4H), 1.72-1.78 (m, 2H), 1.85-2.06 (m, 2H), 3.54 (t, 2H), 4.09 (t, 2H), 6.51 (s, 1 H), 6.58-6.61 (dd, 1 H), 6.67-6.79 (dd, 1 H), 6.95-6.99 (m, 3H), 7.15 (d, 1 H), 7.35-7.40 (m, 2H), 7.44-7.47 (m, 1 H); 13C-NMR (400 MHz, CDCI3) 19.5, 25.2, 26.2,
28.6, 29.6, 44.9, 68.6, 100.7, 105.6, 114.0, 114.3, 118.0, 126.0, 128.9, 129.4, 129.5, 129.7, 130.5,
130.6, 132.4, 136.0, 150.1 , 154.7, 158.9, 163.9, 185.2. ESI-MS (+) 443 (M+H)+. tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate
Figure imgf000059_0003
Ditertbutyldicarbonate (7.4 g, 0.034 mol), solubilized in methanol, is added at zero degrees to a so- lution of 2-(2-aminoethoxy)ethanol (1.90 ml, 0.019 mol) and triethylamine (7.14 ml, 0.051 mol) in methanol and the resulting mixture is stirred at room temperature for 12 hours.
The solvent is evaporated and the residue is treated with dichloromethane, washed with water and dried over sodium sulphate to obtain the product at issue, which is used in the following step with- out further purification.
Yield: 79%. 1H-NMR (400 MHz, CDCI3) 1.39 (s, 9H), 3.25-3.31 (m, 2H), 3.49-3.61 (m, 4H), 3.89 (t, 2H).
2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl 4-methylbenzenesulfonate
Figure imgf000060_0001
Tosylchloride (3.43 g, 0.026 mol) is added to a solution of tert-butyl (2-(2- hydroxyethoxy)ethyl)carbamate (2.63 g, 0.012 mol), triethylamine (2.51 ml, 0.026 mol) and DMAP (cat.) and the resulting solution is stirred at room temperature for 12 h.
Water is added to the solution, the organic portion is extracted, washed with brine, dried over sodi- um sulphate and the solvent is evaporated.
The obtained residue is purified through flash chromatography using as mobile phase a mixture of petroleum ether / ethyl acetate in a 1 :1 ratio.
Yield: 81%. 1H-NMR (400 MHz, CDCI3) 1.41 (s, 9H), 2.42 (s, 3H), 3.21-3.26 (m, 2H), 3.48 (t, 2H), 3.71 (t, 2H), 4.22 (t, 2H), 7.29 (d, 2H), 7.469 (d, 2H). tert-butyl (2-(2-((3-oxo-9-(o-toluoyl)-3H-xanthen-6-yl)oxy)ethoxy)ethyl)carbamate
Figure imgf000060_0002
2-(2-(tert-butoxycarbonyl)amino)ethoxy)ethyl 4-methylbenzenesulfonate (0.413 g, 1.1 mmol) and potassium carbonate (0.160 g, 1.1 mmol) are added to a solution of 6-hydroxy-9-(o-toluoyl)-3H- xanthen-3-one (0.348 g, 1.1 mmol) in DMF and the resulting mixture is stirred at reflux for 12 h. The solution is diluted with ethyl acetate and washed with water.
The organic phase is dried over sodium sulphate and evaporated. The obtained residue is purified through flash chromatography using as eluent a mixture of ethyl acetate / petroleum ether in a 8:2 ratio.
Yield: 61%. 1H-NMR (400 MHz, CDCI3) 1.35 (s, 9H), 1.98 (s, 3H), 3.24-3.28 (m, 2H), 3.53-3.56 (m, 2H), 3.78 (t, 2H), 4.17 (t, 2H), 6.40 (d, 1 H), 6.49-6.51 (dd, 1 H), 6.74-6.77 (dd, 1 H), 6.87-6.93 (m, 3H), 7.07-7.09 (dd, 1 H), 7.26-7.40 (m, 3H), 7.92 (brs, 1 H). 6-(2-(2-aminoethoxy)ethoxy)-9-(o-toluoyl)-3H-xanthen-3-one (RF42)
Figure imgf000061_0001
About 5 ml of HCI 6N is added dropwise at 0 °C to a solution of tert-butyl (2-(2-((3-oxo-9-(o-toluoyl)- 3H-xanthen-6-yl)oxy)ethoxy)ethyl)carbamate (0.250 g, 0.64 mmol) in methanol and the resulting solution is stirred at room temperature for 12h.
Potassium bicarbonate is added up to pH 7 and the solvent is thereafter evaporated.
The residue is treated with dichloromethane and washed with water, dried over sodium sulphate. The solvent is thereafter evaporated and the residue is purified through flash chromatography us- ing as mobile phase a mixture of dichloromethane / methanol /aqueous ammonia (33%) in a 9:1 :0.1 ratio to obtain the compound at issue.
Yield: quantitative.
1H-NMR (400 MHz, CDCI3) 2.0 (s, 3H), 2.89-2.94 (m, 2H), 3.61 (t, 2H), 3.86 (t, 2H), 4.24 (t, 2H), 6.44 (d, 1 H), 6.53-6.56 (dd, 1 H), 6.77-6.79 (dd, 1 H), 6.90-6.97 (m, 3H), 7.11 -7.14 (dd, 1 H), 7.32- 7.46 (m, 3H).
6-(2-(2-isothiocyanatoethoxy)ethoxy)-9-(o-toluoyl)-3H-xanthen-3-one (TN46)
Figure imgf000061_0002
A solution of 1 ,1 '-Thiocarbonyldi-2(1 H)-pirydone (0.051 g, 0.22 mmol) is added dropwise to a solu- tion of 6-(2-(2-aminoethoxy)ethoxy)-9-(o-toluoyl)-3H-xanthen-3-one (0.100 g, 0.23 mmol) in di- chloromethane and the mixture is stirred at room temperature for 12h.
The solvent is evaporated and the residue is purified using as mobile phase a mixture of petroleum ether / ethyl acetate in a 4:6 ratio.
Yield: 67%.
1H-NMR (400 MHz, CDCI3) 1 .99 (s, 3H), 3.64 (t, 2H), 3.69 (t, 2H), 3.86 (t, 2H), 4.19 (t, 2H), 6.38 (d, 1 H), 6.47-6.51 (dd, 1 H), 6.74-6.77 (dd, 1 H), 6.86-6.93 (m, 3H), 7.07-7.09 (dd, 1 H), 7.29-7.40 (m, 3H);
13C-NMR (400 MHz, CDCI3) 19.6, 45.3, 68.2, 69.4, 69.5, 101.1 , 105.7, 113.9, 114.5, 118.2, 126.1 , 129.0, 129.5, 129.9, 130.6, 132.4, 136.1 , 149.7, 154.5, 158.9, 163.4, 185.7; ESI-MS (+) 431 (M+H)+. tert-butyl (6-((2,7-difluoro-3-oxo-9-(o-toluoyl)-3H-xanthen-6-yl)oxy)hexyl)carbamate
Figure imgf000062_0001
6-((tert-butoxycarbonyl)amino)hexyl 4-methylbenzenesulfonate (0.500 g, 1.3 mmol) and potassium carbonate (0.186 g, 1.3 mmol) are added to a solution of 2,7-difluoro-6-hydroxy-9-(o-toluoyl)-3H- xanthen-3-one(0.380 g, 1 .1 mmol) in DMF and the resulting mixture is stirred at reflux for 6 h.
The solution is diluted with ethyl acetate and washed with water.
The organic phase is dried over sodium sulphate and evaporated.
The obtained residue is purified through flash chromatography using as mobile phase a mixture of dichloromethane / methanol in a 9.5/0.5 ratio.
Yield: 61%.
1H-NMR (400 MHz, CDCI3) 1.27 (s, 9H), 1.35-1.57 (m, 4H), 1.72-1.95 (m, 4H), 2.11 (s, 3H), 3.62 (t, 2H), 4.25 (t, 2H), 6.61 (s, 1 H), 6.67 (d, 1 H), 6.81 (d, 1 H), 6.99-7.10 (m, 2H), 7.28-7.37 (m, 2H), 7.45-7.52 (m, 1 H).
6-((6-aminohexyl)oxy)-2,7-difluoro-9-(o-toluoyl)-3H-xanthen-3-one
Figure imgf000062_0002
About 2.5 ml of HCI 6N is added dropwise at 0 °C to a solution of tert-butyl (6-((2,7-difluoro-3-oxo- 9-(o-toluoyl)-3H-xanthen-6-yl)oxy))hexyl)carbamate (0.100 g, 0.23 mmol) in methanol and the re- sulting solution is stirred at room temperature for 12h.
Potassium bicarbonate is added up to pH 7 and the solvent is thereafter evaporated.
The residue is treated with dichloromethane and washed with water, dried over sodium sulphate. The solvent is thereafter evaporated and the residue is purified through flash chromatography us- ing as mobile phase a mixture of dichloromethane / methanol /aqueous ammonia (33%) in a 9:1 :0.1 ratio to obtain the compound at issue.
Yield: quantitative.
1H-NMR (400 MHz, CDCI3) 1 .29-1 .49 (m, 4H), 1 .70-1 .91 (m, 4H), 2.13 (s, 3H), 2.69 (t, 2H), 4.29 (t, 2H), 6.60 (s, 1 H), 6.64 (d, 1 H), 6.84 (d, 1 H), 7.01 -7.12 (m, 2H), 7.30-7.39 (m, 2H), 7.42-7.50 (m, 1 H). 2,7-difluoro-6-((6-isothiocyanatohexyl)amino)-9-(o-toluoyl)-3H-xanthen-3-one
Figure imgf000063_0001
A solution of 1 ,1 '-Thiocarbonyldi-2(1 H)-pirydone (0.022 g, 0.11 mmol) is added dropwise to a solu- tion of 6-((6-aminohexyl)amino)-2,7-difluoro-9-(o-toluoyl)-3H-xanthen-3-one (0.050 g, 0.11 mmol) in dichloromethane and the mixture is stirred at room temperature for 12h.
The solvent is evaporated and the residue is purified using as mobile phase a mixture of petroleum ether / ethyl acetate in a 4:6 ratio.
Yield: 59%. 1H-NMR (400 MHz, CDCI3) 1.39-1.49 (m, 4H), 1.65-1.70 (m, 2H), 1.84-1.89 (m, 2H), 1.99 (s, 3H), 3.48 (t, 2H), 4.12 (t, 2H), 6.57 (s, 1 H), 6.60 (d, 1 h), 6.71 (d, 1 H), 7.02-7.09 (m, 2H), 7.31 -7.37 (m, 2H), 7.41 -7.45 (m, 1 H); ESI-MS (+) 437 (M+H)+.
6-((6-isoselenocyanatohexyl)amino)-9-(o-toluoyl)-3H-xanthen-3-one
Figure imgf000063_0002
6-((6-aminohexyl)amino)-9-(o-toluoyl)-3H-xanthen-3-one, chloroform, Aliquat 336 and NaOH 59% aq in dichloromethane are vigorously stirred until the starting amine disappears. Elementary sele- nium is now added and the solution is stirred at room temperature. Water and dichloromethane are added and the solid portion is filtered. The organic phase is separated from the aqueous phase, dried over sodium sulphate and evaporated to obtain a residue which is purified through flash chromatography using as mobile phase a mixture of petroleum ether / ethyl acetate in a 9:1 ratio. Yield: 41%. ESI-MS (+) 490 (M+H)+.
BIOLOGICAL EXAMPLES OF EXPERIMENTAL PROCEDURES
Ascertaining the cytotoxic properties of the theranostic compounds according to the invention iden- tified with codes TN82 and TN46
The cytotoxic effect of the reference compounds, as described above and identified with codes RF27 {6-((6-aminohexyl)oxy)-9-(o-toluoyl)-3H-xanthen-3-one} and RF42 (6-(2-(2- aminoethoxy)ethoxy)-9-(o-toluoyl)-3H-xanthen-3-one}, respectively, compared to that of the corre- sponding theranostic compounds according to the present invention, as described above and iden- tified with codes TN82 {6-((6-isothiocyanatohexyl)oxy)-9-(o-toluoyl)-3H-xanthen-3-one} and TN46 { 6-(2-(2-isothiocyanatoethoxy)ethoxy)-9-(o-toluoyl)-3H-xanthen-3-one}, respectively, was studied on cells of human T-cell acute lymphoblastic leukaemia: 1 h of treatment at micromolar concentrations of TN82 and TN46 followed by 3 days of treatment in a medium without the compounds caused a dose-dependent decrease of cell viability. The values of IC50 (a concentration inhibiting cell viabil- ity by 50% compared to control cultures) are 5.96 μM for TN82 and 3.65 μM for TN46 (Figure 1). The cytotoxic effect was also tested on two other cell lines: TK6, which are human lymphoblastoid cells, and CT26, which are cells of colon carcinoma. As shown in Figure 1 , TN82 and TN46 in- duced a dose-dependent and significant decrease of cell viability also on these two cell lines (Fig- ure 1). For comparative purposes, the small insert of Figure 1A also shows the cytotoxicity of the parental compound, sulforaphane, which, under the same experimental conditions set forth above, has an IC50 of 29.7 μM.
The two other reference compounds (RF27 e RF42), under the same experimental conditions, did not cause cytotoxicity up to the concentrations of 32 μM on Jurkat cells (Figure 2).
The Jurkat cells were thereafter analysed to delve into the mechanism of cell death through the use of different techniques. At a microscopic level (Figure 3A), the cells treated with TN82 (1 h of treatment followed by 5 h of incubation in a medium without the compounds under study according to the present invention) show clear signs of apoptosis. It is actually possible to remark the con- densation of chromatin, cup-shaped masses and micronuclei; occasionally, secondary necrosis is observed. Cell death by apoptosis was biochemically confirmed by the rapid dose-dependent in- crease of the activity of caspase 3 and caspase 8 and by the increase of the percentage of cells with reduced mitochondrial activity (Figure 3B-D). The combination of these biochemical responses indicates the involvement of both intrinsic and extrinsic apoptosis.
It should be noted that the apoptotic responses described so far were triggered by the same con- centrations for both TN derivatives, which induce the very high cytotoxic effects shown in Figure 1 . TN82 and TN46 cause direct DNA and RNA damage and are mutagenic
The next step was the analysis of the genotoxic activity of the two TN compounds. Their effect on nuclear DNA was studied through the fast alkaline halo test. Through this assay, the DNA frag- ments resulting from DNA breakage diffuse from the nucleus in inverse proportion to their size, thus producing a concentric halo whose radius reflects the extent of DNA damage: at the micro- scope, the smaller the fragments look (highly damaged DNA), the larger the generated halo. Also, it should be noted that DNA single-strand fragments diffusing from the nucleus may derive either from a direct DNA breakage or from the presence of apurinic sites converted into DNA single- strand breaks at alkaline pH. The halo formation can be properly monitored in single cells through fluorescence microscopy, as can be seen in the representative graph shown in Figure 4C. Exposition for 1 h to both TN compounds induced a concentration-dependent and statistically sig- nificant increase of the formation of DNA single-strand breaks (SSBs) (Figure 4A) in Jurkat cells. Micromolar concentration of TN 82 or 46 (2-8 μM) caused an extent of DNA single-strand breakage comparable to that provoked by 30 minutes of treatment with H2O2 10-50 μM, a well-established and powerful agent inducing DNA damage, and far higher than that induced by sulforaphane (SFR) (Figure 4 D). Accordingly, both TNs proved to be able to damage the DNA rapidly (within 1 h), ef- fectively and in a dose-dependent manner.
As is the case with cytotoxic activity, TN46’s ability to damage the DNA proved to be higher than TN82’s. The DNA single-strand breaks (DNA-SSBs) caused by the two theranostic TN compounds according to the present invention were repaired more slowly than those produced by H2O250 μM. Indeed, only 26.4% ± 3.55 of the initial SSBs caused by the oxidizer were observed after 30 minutes and no residual damage was observed after 3 h of culture in a medium without the oxidiz- ing agent. In the case of TN 82 and 46 (8 μM), a significant level (59.2% ± 6.1 and 48.7% ± 6.55, respectively) of the initial SSBs did not appear to have been repaired after 3 h of culture in a medi- um without the compounds under study (according to the present invention).
In conclusion, DNA damage was probed into also in a different cell line, consisting of human lym- phoblastoid cells (TK6). Like Jurkat cells, TK6 cells proved to be sensitive to the DNA injuring activ- ity of TN82 and TN46 (Figure 4B); as observed in Jurkat cells, TN46 proved to be more active than TN82 in TK6 cells as well.
It should be noted that the reference compounds RF27 and RF42 (containing only the rhodol group) under the same experimental conditions do not cause DNA damage up to the concentration of 32 μM (Figure 5). This rules out the possibility that the cytotoxic and genotoxic effects of the theranostic TN compounds according to the present invention under study are the expression of rhodol intrinsic properties.
The interaction of TN82 and TN46 with double-stranded DNA, determined by means of fluorimetry, was defined through Kd, which measures the affinity of the compounds under study (according to the present invention) with regard to the DNA and is given by the ratio of Kon (association con- stant) to Koff (dissociation constant). The behaviour of the two compounds is very different. TN46 actually appears to have a much higher affinity for the DNA than TN82, as shown by the Kd value in the nanomolar range for TN46 (0.009 ± 0.001 μM) and in the submicromolar range for TN82 (0.097 ± 0.017 μM).
Figure 6 shows the graphs obtained by comparing the extent of DNA single-strand breaks (DNA- SSBs) and the corresponding values of cell proliferation inhibition caused by TN82 and TN46. A good correlation was recorded between these two parameters for both compounds. In actual fact, the analysis of linear regression provided r2 values of 0.8279 for TN82 and 0.7618 for TN46.
The following experiments were performed in order to determine whether the DNA single-strand breaks caused by TN82 and TN46 entailed the formation of micronuclei, which are secondary gen- otoxic events, more complex and denoting chromosome instability. The two compounds showed the trend to promote micronuclei formation in a concentration-dependent manner. The increase proved to be statistically significant at concentrations ≥4 μM, whereas only for TN46 already at a concentration of 2 μM: TN82 and TN46 4 μM induced 13.07 ± 0.07 and 19.59 ± 2.4 MN/1000 binu- cleated cells, respectively (Figure 7). The effect of both the TNs is comparable to that of mitomycin C and vinblastine, used as positive controls. Therefore, the two TNs can be classified as mutagen- ic. As regards the reference compound sulforaphane (SFR), previous studies proved the non- mutagenicity thereof [C. Fimognari, F. Berti, R. lori, G. Cantelli-Forti, P. Hrelia, Micronucleus for- mation and induction of apoptosis by different isothiocyanates and a mixture of isothiocyanates in human lymphocyte cultures, Mutat. Res. 582 (2005)1-10].
The ability to induce RNA damage by the TN compounds was determined by assessing the RNA integrity number (RIN), which is calculated by means of the areas underlying the peaks of rRNA 18S and 28S. Elecropherograms representing untreated Jurkat cells and Jurkat cells treated with TN82 or TN46 6 -12 - 18 μM for 24h are shown in Figure 8A. The peaks relating to the subunits of rRNA 18S and 28S appeared to be progressively smaller, in a concentration-dependent manner, in cells treated with TN82 (RIN value at 18 μM: 6.55 ± 0.21) compared to untreated cells (RIN values: 9.38 ± 0.22) (Figure 8B). Conversely, TN46 did not induce a dose-dependent RNA degradation, since at a concentration of 18 μM the RIN value rose back to 8.73 ± 0.42 (Figure 8B).
TN82 and TN46 are cytotoxic and genotoxic also under metabolically limiting conditions The cytotoxic and genotoxic effects of the two theranostic TN compounds were also studied at metabolically limiting temperatures, namely by treating cells at a temperature of 4°C. This approach has already been used in order to determine by comparison whether temperature-dependent met- abolic events have an impact on or contribute to the real cytotoxic and genotoxic response caused by specific agents (such as X-rays or oxidizing agents) and observed at both a physiological and a metabolically permissive temperature [Cantoni, O., Cattabeni, F., Stocchi, V., Meyn, R.E., Cerutti, P., and Murray, D., Hydrogen peroxide insult in cultured mammalian cells: relationships between DNA single-strand breakage, poiy(ADP-ribose) metabolism and cell killing. Biochim Biophys Acta, 1989. 1014(1): pages 1-7; Cantoni, O., Sestili, P., Guidarelli, A., Giacomoni, P.U., and Cattabeni, F., Effects of L-histidine on hydrogen peroxide-induced DNA damage and cytotoxicity in cultured mammalian cells. Molecular pharmacology, 1992. 41(5): pages 969-974] To this end, DNA dam- age and cell proliferation inhibition were examined using the same experimental protocols as were used for the experiments illustrated in Figures 1 and 4, with the only exception that treatment for 1 h with TN82 or with TN46 was performed in ice, namely at 4°C. It is interesting to note that, under these conditions, both TN compounds caused an accumulation of DNA single-strand breaks com- parable to the one observed at a physiological temperature (Figure 9B), but promoted a significant- ly higher cytotoxic effect (Figure 9A). Indeed, IC50 values at 4°C were of 3,99 μM and 2,54 μM for TN82 and for TN46, respectively, compared to the corresponding values of 5,96 μM and 3,65 μM obtained at 37°C. Once again, a good correlation was detected between the extent of DNA dam- age and the corresponding cytotoxic responses caused by treatments in ice (Figures 9C and D).
The cytotoxic and genotoxic activity of TN82 and TN46 is not mediated by oxidative kinds of mech anisms
The production of reactive oxygen species (ROS) at a mitochondrial level plays a significant role in the cytotoxic and genotoxic activity of sulforaphane (SFR) [Xiao, D., Powolny, A. A., Antosiewicz, J., Hahm, E.R., Bommareddy, A., Zeng, Y., Desai, D., Amin, S., Herman-Antosiewicz, A., and Singh, S.V., Cellular responses to cancer chemopreventive agent D,L-sulforaphane in human prostate cancer cells are initiated by mitochondrial reactive oxygen species. Pharm Res, 2009. 26(7): pages 1729-38; Singh, S.V., Srivastava, S.K., Choi, S., Lew, K.L., Antosiewicz, J., Xiao, D., Zeng, Y., Watkins, S.C., Johnson, C.S., Trump, D.L., Lee, Y.J., Xiao, H., and Herman-Antosiewicz, A., Sul- foraphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen spe cies. J Biol Chem, 2005. 280(20): pages 19911-24; Choi, W.Y., Choi, B.T., Lee, W.H., and Choi, Y.H., Sulforaphane generates reactive oxygen species leading to mitochondrial perturbation for apoptosis in human leukemia U937 cells. Biomed Pharmacother, 2008. 62(9): pages 637-44; Sesti- li, P., Paolillo, M., Lenzi, M., Colombo, E., Vallorani, L., Casadei, L., Martinelli, C., and Fimognari, C., Sulforaphane induces DNA single strand breaks in cultured human cells. Mutation Re- search/Fundamental and Molecular Mechanisms of Mutagenesis, 2010. 689(1): pages 65-73]. To assess whether the same mechanisms are involved also in the activity of the TN compounds, the Jurkat cells were treated for 1 h with TN82 6 μM or with TN46 4 μM in the absence or in the pres- ence of ortho-phenanthroline (3 μM, which is able to chelate iron) or of the mitochondrial complex I inhibitor rotenone (1 μM). Both compounds entail, with a different mechanism, the blocking of the Fenton reaction and hence prevent ROS production at a mitochondrial level. Unlike the reference ITC sulforaphane SFR, neither the DNA damage nor the cytotoxicity caused by the TN theranostic compounds according to the present invention can be relieved by using these conditions (Figures 10A and B).
GSH levels and their relevance for the cytotoxicity of TN82 and TN46
Since it is well known that the main metabolic pathway involving the ITC group or isothiocyanate group is mediated by glutathione (GSH) (Yagishita Y, Fahey JW, Dinkova Kostova AT, Kensler TW. Broccoli or Sulforaphane: Is It the Source or Dose That Matters? Molecules 2019, 24, 3593; doi:10.3390/molecules24193593), the variation of the GSH content was analysed during an exposi- tion to TN compounds ranging from 1 h to 24 h (Figures 11 A and 11 B). The observed GSH deple- tion was rapid and substantial: after 1 h of treatment, TN82 and TN46 induced a statistically signifi- cant GSH reduction in the neighbourhood of 50-60% (TN82 and TN462 μM induced a reduction of 49.86 ± 9.84% and 63.87 ± 11.16%, respectively). However, GSH depletion did not appear to be dose-dependent. During the following exposition hours, GSH levels were restored and, after 24 h of treatment, the GSH content increased in cells treated with both TNs, achieving higher values than those detected in control cells for most of the tested concentrations.
In order to understand the importance of GSH levels on the cytotoxic activity of the TN compounds, the Jurkat cells were pre-treated with NAC (N-acetylcysteine) 5 mM or with BSO (buthionine sul- foximine) 0.2 mM for 24 h. NAC is a glutathione precursor, whereas BSO is a gamma- glutamylcysteine synthetase inhibitor. Therefore, treatment with NAC entailed a 50% increase and treatment with BSO entailed a reduction by 80% of GSH intracellular levels compared to untreated cells (data not shown). Thereafter, TN concentrations close to Glso levels (6 μM for TN82 and 4 μM for TN46) were selected in order to ascertain the ability to alter cell proliferation in relation with the variation of GSH intracellular content. The increase of GSH levels only partially improved the cell proliferation rate after 1 h of exposition to TN compounds (Figures 11 C and 11 D). Furthermore, the GSH depletion rate induced by TN derivatives was not influenced by pre-treatment with NAC. By contrast, a reduced GSH content worsened cell proliferation ability, although in a statistically signif- icant manner only for TN46 (Figures 11 E and 11 F).
Similarly to experiments concerning cell proliferation, also the genotoxicity induced by the TN com- pounds appeared to be hardly affected by GSH intracellular levels. As a matter of fact, the same extent of DNA dingle-strand breakage was observed after treatment with the TN compounds both in cells preserved in unaltered culture conditions, thus without GSH, and in cells preserved in cul- ture conditions with increased GSH levels (Figure 12).
The end stage of the study envisaged the analysis of the effects of TN82 and TN46 on an ex vivo model, consisting of blasts isolated from peripheral blood or bone marrow of leukaemia patients. The ex vivo model is an excellent surrogate for the determination of the patient’s cellular response to treatment and for the prediction of clinical response thereto (Andrew G. Bosanquet and Philip B. Bell. Ex vivo therapeutic index by drug sensitivity assay using fresh human normal and tu- mour cells. 2004. 4(2): 145-154). To this end, blasts were taken from eight patients with acute myeloid leukaemia. Some patients were FLT3-negative, namely they did not show tyrosine kinase domain mutations of FMS-like tyrosine-kinase 3 (FLT3), whereas others were FLT3-positive. FLT3 mutations are observed in about 1/3 of patients with acute myeloid leukaemia (Leick, M.B.; Levis, M.J. The Future of Targeting FLT3 Activation in AML. Curr Hematol Malig Rep 2017, 12, 153-167) and are often associated to a high incidence of relapses and a short survival time after antitumour chemotherapy or transplantation (Larrosa-Garcia, M.; Baer, M.R. FLT3 Inhibitors in Acute Myeloid Leukemia: Current Status and Future Directions. Mol Cancer Ther 2017, 16, 991-1001). The blasts were treated for 24 h with different concentrations of TN82 or TN46 (4-32 μM). Both compounds showed cytotoxic effects on the blasts of both FLT3-positive and FLT3-negative patients (Figure 13).
One of the major problems associated with antitumour chemotherapeutic treatments is the low therapeutic index characterizing most antitumour drugs. This is ascribable to their inability to dis- criminate between tumour cells and highly proliferating untransformed cells. In order to ascertain whether the activity of the compounds under study (according to the present invention) is selective for leukaemia cells, peripheral blood lymphocytes obtained from healthy donors were treated with the compounds under study (according to the present invention) within the concentration range 4- 32 μM for 4 or 24 h. The peripheral blood lymphocytes from healthy donors are the untransformed counterpart of Jurkat cells. TN82 and TN46 did not prove to be cytotoxic on normal lymphocytes (Figure 14).
In conclusion, apoptosis is a cell death mechanism triggered by several stimuli, among which DNA injuries are particularly important (40, 41). In the case of TN82 and TN46, DNA single-strand breaks are a crucial stimulus. Indeed, unlike the compounds containing only the rhodol group (RF27 and RF42), both compounds are able to damage the DNA extensively and irrespective of the cell type. In both Jurkat and TK6 cells, the concentrations and the times which can damage the DNA are the same which can cause an outstanding cytotoxic response. Moreover, DNA breaks are already observed after 1 h of treatment, hence their formation precedes the appearance of cell apoptotic modifications. Overall, such data highlight the existence of a causal relation between DNA damage and cytotoxic activity for the derivatives under study (according to the present inven- tion).
The cytotoxic relevance of the DNA injuries caused by the TNs is further confirmed by the observa- tion that these are not rapidly repaired (T½ > 3h). This means that their persistence is probably a significant and complex toxic event. In actual fact, a direct consequence of the combination of the large accumulation of DNA breaks and of the hard reparability thereof is the mutagenic effect ob- served in TK-6 cells, wherein a significant increase of micronuclei after exposition to the two TNs was detected. In particular, the lack of any mutagenic effect of sulforaphane implicitly but clearly highlights the differences existing between the nature of the injuries caused by the ITC and by the two TB theranostic agents according to the present invention.
Sulforaphane is able to damage the DNA by means of an indirect mechanism, dependent on ROS, involving the intramitochondrial production of H202 and the following diffusion thereof in the nucle- us (Sestili P, Fimognari C. Cytotoxic and Antitumor Activity of Sulforaphane: The Role of Reactive Oxygen Species. Biomed Res Int 2015;2015:402386. doi: 10.1155/2015/402386). Unlike sul- foraphane, the DNA-damaging activity of TN82 and TN46 was not blocked either by the iron chela- tor o-phenanthroline, which blocks the Haber-Weiss reaction accounting for the generation of oxy- gen radicals (Sestili, P., Piedimonte, G., Cattabeni, F., and Cantoni, O., Induction of DNA breakage and suppression of DNA synthesis by the OH radical generated in a Fenton-like reaction. Biochem- istry international, 1986. 12(3): pages 493-501 ; Koppenol, W.H. and Hider, R.H., Iron and redox cycling. Do's and don'ts. Free Radic Biol Med, 2018), or by the mitochondrial electron transport chain complex I inhibitor (Teeter, M.E., Baginsky, M.L., and Hatefi, Y., Ectopic inhibition of the complexes of the electron transport system by rotenone, piericidin A, demerol and antimycin A. Bi- ochim Biophys Acta, 1969. 172(2): pages 331-3), which prevents the superoxide anion production induced by sulforaphane/complex III interactions (Xiao, D., Powolny, A. A., Antosiewicz, J., Hahm, E.R., Bommareddy, A., Zeng, Y., Desai, D., Amin, S., Herman-Antosiewicz, A., and Singh, S.V., Cellular responses to cancer chemopreventive agent D,L-sulforaphane in human prostate cancer cells are initiated by mitochondrial reactive oxygen species. Pharm Res, 2009. 26(7): pages 1729- 38.; Sestili, P., Paolillo, M., Lenzi, M., Colombo, E., Vallorani, L, Casadei, L, Martinelli, C., and Fimognari, C., Sulforaphane induces DNA single strand breaks in cultured human cells. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2010. 689(1): pages 65-73). These observations rule out the possibility that the TNs damage nuclear DNA through a ROS- dependent mechanism.
Further mechanistic information derive from the outcome of the experiments accomplished under metabolically limiting exposition conditions, namely 4°C, an experimental approach used to outline the contribution of cell metabolism to the overall toxic response provoked by specific agents [Can- toni, O., Cattabeni, F., Stocchi, V., Meyn, R.E., Cerutti, P., and Murray, D., Hydrogen peroxide in- sult in cultured mammalian cells: relationships between DNA single-strand breakage, poly(ADP- ribose) metabolism and cell killing. Biochim Biophys Acta, 1989. 1014(1): pages 1-7; Cantoni, O., Sestili, P., and Cattabeni, F., Randomly distributed DNA single strand breaks are not lethal for mammalian cells. Xenobiotica, 1988. 18(12): pages 1481-1487; Cantoni, O., Sestili, P., Guidarelli, A., Giacomoni, P.U., and Cattabeni, F., Effects of L-histidine on hydrogen peroxide-induced DNA damage and cytotoxicity in cultured mammalian cells. Molecular pharmacology, 1992. 41 (5): pages 969-974; Sestili, P., Piedimonte, G., Cattabeni, F., and Cantoni, O., Induction of DNA breakage and suppression of DNA synthesis by the OH radical generated in a Fenton-like reaction. Biochemistry international, 1986. 12(3): pages 493-501]. Surprisingly, we remarked that TN82 and TN46 cause a slightly, although not significantly, broader damage to nuclear DNA compared to what was ob- served at 37°C. In other words, the two derivatives of sulforaphane damage the DNA regardless of cell metabolism. It is hence likely that DNA injuries induced by the two TNs derive from direct inter- actions with double-stranded DNA, as later proved by the measurements of the affinity of the com- pounds under study (according to the present invention) for the DNA and by the definition of the relevant Kd values. The slightly higher accumulation of DNA single-strand breaks observed at 4°C is probably due to the fact that, like other temperature-sensitive/dependent cellular processes, DNA repair as well is inhibited at 4°C.
Overall, our findings entail three important implications: 1) DNA injuries caused by TN82 and by TN46 have a different nature compared to those caused by the parental compound sulforaphane. Indeed, the activity of the latter is mediated by ROS gener- ation secondary to mitochondrial respiratory chain inhibition, whereas DNA injuries caused by the TNs are not, but rather depend on a metabolically correlated event, such as direct reactions with nuclear DNA. The deep, structural and mechanistic difference of the DNA injuries caused by the TNs from those by sulforaphane is also substantiated by the circumstance that, unlike sul- foraphane, the TNs entail micronuclei formation.
2) unlike those caused by sulforaphane, DNA injuries caused by the TN theranostic agents, TNs compounds according to the present invention, are causally correlated to the cytotoxic response observed in intoxicated cells. In actual fact, whereas we have previously proved that DNA injuries caused by sulforaphane contribute to, but do not determine, the cytotoxic response thereof (Ses- tili, P., Paolillo, M., Lenzi, M., Colombo, E., Vallorani, L, Casadei, L, Martinelli, C., and Fi- mognari, C., Sulforaphane induces DNA single strand breaks in cultured human cells. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2010. 689(1): pages 65-73), in the TNs a good correlation between the extent of DNA damage and that of the cytotoxic re- sponse was observed, regardless of significantly different experimental conditions (namely meta- bolically permissive versus metabolically limiting intoxication temperatures). The by far lower cy- totoxic relevance of DNA injuries by sulforaphane, compared to those caused by both TNs, is not surprising. It is actually well-known that DNA injuries induced by oxidizers, despite their abun- dance, are moderately toxic for mammalian cells (Cantoni, O., Sestili, P., and Cattabeni, F., Ran- domly distributed DNA single strand breaks are not lethal for mammalian cells. Xenobiotica, 1988. 18(12): pages 1481-1487; Coleman, J.B., Gilfor, D., and Farber, J.L., Dissociation of the accumulation of single-strand breaks in DNA from the killing of cultured hepatocytes by an oxida- tive stress. Mol Pharmacol, 1989. 36(1): pages 193-200). This might depend on the extremely high effectiveness of the nuclear systems repairing oxidative injuries, which are rapidly removed. It is interesting to remark that, in contrast with oxidative injuries, the DNA injuries caused by the TNs are characterized by a far longer persistence. This is another strong sign of their non- oxidative nature, probably accounting for their considerable toxicological impact.
3) condensation with rhodol significantly modifies the cellular pharmacokinetics of the residual ITC and, in particular, eases the targeting of the residual ITC on the nucleus through energy- independent mechanisms (namely passive diffusion in nuclei).
Overall, our findings clearly show that TN82 and TN46 are characterized by quantitatively and qual- itatively different toxic profiles compared to their parental compounds. In particular, the cytotoxicity of the TNs seems to mainly depend on their DNA-injuring activity. The identification of these new properties suggests the pharmacological exploitation of these TN theranostic agents according to the present invention as potential antitumour chemotherapeutic agents. In actual fact, the ability to massively damage the DNA is an important mechanism of action of a broad number of widely used antitumour drugs, such as anthracyclines, cisplatin, mafosfamide and other alkylating agents (Ho- soya, N. and Miyagawa, K., Targeting DNA damage response in cancer therapy. Cancer Sci, 2014. 105(4): pages 370-88).
TN82 is localized at an intracellular level
Specific studies were performed to ascertain whether the compounds under study (according to the present invention) are localized at an intracellular level. Since the thin cytoplasmic layer of Jurkat cells impaired subcellular localization, this experimental model was replaced by the use of HeLa cells (human cervix adenocarcinoma cells). HeLa cells were transfected with CellLight® ER-RFP (red fluorescent protein)-calreticulin and thereafter treated treated with TN82 1 μM for 6 h, fixed with 4% paraformaldehyde for 10 minutes, washed and observed through the Olympus FV1000 fluorescence microscope. The analysis was accomplished by using the Image J software. The left picture of Figure 15 shows the green fluorescence of TN82, the central picture shows the red fluo- rescence of RFP and the right picture shows the merged (MERGING) green fluorescence of TN82 and the red fluorescence of RFP, allowing TN82 to be localized in the endoplasmic reticulum. However, the green fluorescence of TN82 is diffused inside the cell (for example, also in mitochon- dria), suggesting a non specific localization of the ITC derivative under study (the compound TN82 according to the present invention). The high reactivity of the ITC molecule might account for the molecule diffusion. As a matter of fact, the amine or thiol groups of the proteins and molecules found inside the cell might attract the ITC electrophilic carbon and hinder the exclusive localization of TN82 inside the endoplasmic reticulum.
The table below concisely summarizes some biological data which further highlight the surprising and fully unexpected technical effects of the compounds according to the present invention, gener- ally designated as TN, such as for example TN82 and TN46, compared to parental compounds considered on an individual basis, sulforaphane on one side and the fluorophore compounds RF27 and RF42, respectively, on the other side.
Figure imgf000071_0001
1 TK6 cells, 1 h exp + 72h post treatment, drug-free growth
2 Concentration resulting in an NDF value of 5 after 30 min challenge of Jurkat cells at 37°C
3 ND not determinable
First: The cytotoxicity increase shown by the TN compounds according to the present invention, such as TN82 and TN46, compared to the parental compounds considered on an individual basis, sulforaphane on one side and the fluorophore compounds RF27 and RF42, respectively, on the other side, is always at least 10-30 times higher. The ability to damage the DNA is very high in TN82 and TN46, whereas said ability is 20-30 times lower in sulforaphane and lacking in the two corresponding fluorophore compounds RF27 and RF42. DNA injuries caused by the compounds according to the present invention, such as for example TN82 and TN46, have a different nature compared to those caused by sulforaphane. Indeed, those by sulforaphane are mediated by an ox- idative mechanism secondary to mitochondrial respiratory chain inhibition, whereas those caused by the TN compounds according to the present invention do not depend on an event correlated to metabolism, but probably on direct reactions with nuclear DNA. This aspect is proved in that the DNA damage induced by sulforaphane only occurs at a metabolically permissive temperature (37°C and not 4°C), whereas that induced by the TN compounds according to the present inven- tion, such as TN82 and TN46, is independent from the exposition temperature (37°C º 4°C). The deep, structural and mechanic difference of the DNA injuries caused by the TN compounds accord- ing to the present invention from those by sulforaphane is also substantiated by the circumstance that, unlike sulforaphane, the TN compounds according to the present invention, such as TN82 and TN46, entail micronuclei formation.
Second: Unlike those caused by sulforaphane, the DNA injuries caused by the TN compounds ac- cording to the present invention, such as TN82 and TN46, are causally correlated to the cytotoxic response observed in intoxicated cells. In actual fact, whereas we have previously proved that DNA single-strand injuries caused by sulforaphane contribute to, but do not determine, the cytotoxic re- sponse thereof, in the TN compounds according to the present invention, such as TN82 and TN46, a good correlation between the extent of DNA damage and that of the cytotoxic response was in- variably observed, regardless of significantly different experimental conditions (namely metabolical- ly permissive versus metabolically limiting temperatures). The by far lower cytotoxic relevance of DNA injuries by sulforaphane, compared to those caused by the TN compounds according to the present invention, such as TN82 and TN46, is not surprising. It is actually well-known that DNA single-strand breaks due to radical damage, despite their abundance, are moderately toxic for mammalian cells. This might depend on the high effectiveness of the nuclear systems repairing ox- idative injuries, which are rapidly removed. It is interesting to remark that, in contrast with oxidative injuries, the DNA single-strand breaks caused by the TN compounds according to the present in- vention, such as TN82 and TN46, are characterized by a far longer persistence, which is another strong sign of their non-oxidative nature, probably accounting for their considerable toxicological impact.
Third: The TN compounds according to the present invention, such as TN82 and TN46, also dam- age the other nucleic acid, RNA. Conversely, sulforaphane not only is not toxic for the RNA, but it even performs a protective action with regard to RNA-injuring agents.
Fourth: Condensation with fluorophore platforms significantly modifies the cellular pharmacokinet- ics of the TN compounds according to the present invention, such as TN82 and TN46, and, in par- ticular, eases the targeting thereof in the nucleus through energy-independent mechanisms (name- ly passive diffusion in nuclei).
As stated above, the compounds according to the present invention are characterized by technical effects which were surprising and fully unexpected for those skilled in the art, hence being non- obvious with regard to available prior art.
Patent EP 1 961 418 A1 suggests the use of some natural and synthetic isothiocyanates including, but not limited thereto, benzyl isothiocyanate (BITC) (1), phenethyl isothiocyanate (PEITC) (2), allyl isothiocyanate (AITC) (3) and 4-sulfophenylisothiocyanate (4), as described in line 20, on page 3, for treating benign prostatic hyperplasia, prostatitis and skin tumour, their use for formulating prep- arations useful in the above pathologies, lastly suggesting some methods (with surfactants and solubilizing agents) for preparing the above formulations. The compounds described therein induce several effects: induction of phase 2 enzyme expression, repression of androgenic receptor and PSA expression, reduction of prostate weight, reduction of inflammation, inhibition of the prolifera- tion of a prostate tumour line and of a melanoma line, reduction of tumour incidence in a mouse xenograft model of prostate tumour and melanoma. The compounds described in EP 1 961 418 A1 are chemically and structurally different from the compounds according to the present invention: the compounds described in EP 1 961 418 A1 do not show fluorescent portions, whereas the com- pounds according to the present invention comprise fluorophore portions such as rhodol.
The IC50 values relating to the cytotoxic and cytostatic effect of the most powerful compounds (BITC and PEITC) described in EP 1 961 418 A1 range from 0.8 to 1.5 μM in the used prostate tumour line, but said values are obtained only after a continuous exposition for 3 or 7 days (corre- sponding to 72 or 168 hours). The compounds according to the present invention show similar IC50 values, but said values are obtained after an exposition of only 1 h, followed by post- incubation up to 72 h in a drug-free medium. This means that the time factor (which is fundamental in the CxT equation applied to antitumour agent effectiveness) must increase by 72 or 168 times for the compounds BITC and PEITC described in EP 1 961 418 A1 to produce cytotoxic levels comparable to those of the compounds according to the present invention.
Patent WO 03/059149 describes the use of fluorophore glucose or deoxyglucose conjugates as a strategy for detecting cancerous or pre-cancerous cells in patients. The emission wavelength of fluorophores ranges from 400 to 1200 nm. The conjugates are used for endoscopic or visual (for example in the case of melanomas) surveys, based on the ability of tumour cells to avidly take up glucose. The compounds described in WO 03/059149 are used by tumour cells as energy sub- strates to allow surgeons to understand the localization of the tumour in a patient. On page 13 of WO 03/059149, it is specified that the fluorophore can be fluorescein isothiocyanate, used as a flu- orescent probe, not provided with antitumour activity. The compounds according to the present in- vention do not comprise fluorophore glucose or deoxyglucose conjugates.
Patent US 2013/0079401 A1 proposes the use of a natural isothiocyanate contained in Wasabia japonica, 6 methyl-sulfinylhexyl isothiocyanate, and of a derivative thereof, 6 methyl-sulfonylhexyl isothiocyanate (compounds identified with numbers I7457 and I7557, respectively), as possible an- titumour agents, alone or in combination with other chemotherapeutic agents or ionizing radiations, even for chemoresistant tumours.
The invention described in US 2013/0079401 A1 is based on the finding of the cell replication inhib- iting activity of the two compounds, which effect was proved in several tumour cell lines. The cyto- toxic/cytostatic effect is preserved also with regard to cells (K572) which have become resistant to the conventional antitumour drug Imatinib. The compounds seem to interact with proteins which are highly involved in the cell cycle dynamic, allegedly the preferential target of their effect. The mole- cules associated to the two isothiocyanates are not fluorophore structures. The cytotoxic and cyto- static effect of the two compounds described in US 2013/0079401 A1 is much lower that that of the compounds according to the present invention: the IC50 values are always higher than 10 μM in the different tested cell lines, but said values are obtained only after continuous exposition for 48 or 72h. Although tested on different cell lines, which are however comparable to the former, the compounds according to the present invention showed IC50 values of less than 10 μM. How- ever, said values were obtained after an exposition of only 1 h, followed by post-incubation up to 72 h in a drug-free medium. This means that the time factor (which is fundamental in the CxT equation applied to antitumour agent effectiveness) must increase by 48-72 times for the compounds I7457 and I7557 described in US 2013/0079401 A1 to produce cytotoxic levels barely comparable to those of the compounds according to the present invention.
The action of the compounds I7457 and I7557 is typically dependent on cell cycle, a function needing an active metabolism be performed; the compounds according to the present invention conversely act with an actually different mechanism, namely by damaging the DNA in a manner which operates also at a metabolically non permissive temperature (4°C).
Patent US 4,304,720 concerns ester and ether derivatives of fluorescein, the synthesis thereof and the possibility to incorporate some derivatives, such as for example dioleyl fluorescein, into low- density lipoproteins (LDL), thus allowing cells to be visualized. In the compounds described in US 4,304,720, isothiocyanate or isoselenocyanate groups are totally absent. The purpose of the use of the compounds described in US 4,304,720 is “only” the visualization of cells, since no kind of bio- logical activity is described.
Asako Murata et. Al “Small molecule fluorescent probes for specific RNA targets” Chemical Com- munications, vol. 47, pages 4712-4714, deals with the development of fluorescent probes for RNA real-time detection.
Of the three mentioned compounds, only one features the fluorescein nucleus (compound 4), but no isothiocyanate or isoselenocyanate group is present in any of the compounds, whereas said group is always present in the compounds according to the present invention.
Yongbin Song et al. “Design, Synthesis and Anticancer Activity of N3,N11-Bis(2-hydroxyethyl)-14- Aryl-14H-Dibenzo[a,j]xanthenes-3,11 -Dicarboxamide” Chemical and Pharmaceutical Bullettin, Vol, 61 , no. 2, (2013), pages 167 - 175, concerns the development of a series of xanthen derivatives as antiproliferative agents. The compounds mentioned in this article are structurally very different from the compounds according to the present invention, since they do not feature any isothiocyanate or isoselenocyanate group.

Claims

1 . A compound of general formula (I):
Figure imgf000075_0001
wherein
Z is selected from: S, Se;
Y is selected from: O, NH or S;
R, R’, which may be identical to or different from each other, are selected from the group comprising:
-H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; linear or branched alkyl with 1 to 10 carbon atoms, preferably alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, - CH2CH(CH3)2; cycloalkyl with 3 to 6 carbon atoms, preferably cycloalkyl selected from the group comprising:
Figure imgf000075_0002
heterocycloalkyl with 4 to 5 carbon atoms, comprising 1 or 2 heteroatoms selected from N and O, preferably heterocycloalkyl selected from the group comprising:
Figure imgf000075_0003
aryl with 6, 12 or 18 carbon atoms, preferably aryl selected from the group comprising:
Figure imgf000075_0004
heteroaryl with 4 to 5 carbon atoms, comprising 1 heteroatom selected from N and O, preferably heteroaryl selected from the group comprising:
Figure imgf000075_0005
alkylenylcycloalkyl with 4 to 10 carbon atoms, wherein the cycloalkyl is a ring selected from 3, 4, 5 or 6 carbon atoms and the remaining alkylenyl moiety is linear or branched; cycloalkenylalkyl with 4 to 10 carbon atoms, wherein the cycloalkenyl is a ring selected from 3, 4, 5 or 6 carbon atoms and the remaining alkyl moiety is linear or branched; alkylenylheterocycloalkyl with 5 to 10 carbon atoms, wherein the heterocycloalkyl is a ring selected from 4 or 5 carbon atoms comprising 1 or 2 heteroatoms selected from N and O and the remaining alkylenyl moiety is linear or branched; heterocycloalkenylalkyl with 5 to 10 carbon atoms, wherein the heterocycloalkenyl is a ring selected from 4 or 5 carbon atoms comprising 1 or 2 heteroatoms selected from N and O and the remaining alkyl moiety is linear or branched; alkylenylaryl with 7 to 20 carbon atoms, wherein the aryl consists of 6, 12 or 18 carbon atoms and the remaining alkylenyl moiety is linear or branched; arylalkyl with 7 to 20 carbon atoms, wherein the aryl consists of 6, 12 or 18 carbon atoms and the remaining alkyl moiety is linear or branched; heteroarylalkyl with 5 to 10 carbon atoms, wherein the heteroaryl is a ring with 4 to 5 carbon atoms comprising 1 heteroatom selected from N and O and the remaining alkyl moiety is linear or branched; alkylenylheteroaryl with 5 to 10 carbon atoms, wherein the heteroaryl is a ring with 4 to 5 carbon atoms comprising 1 heteroatom selected from N and O and the remaining alkylenyl moiety is linear or branched;
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; linear or branched alkyl with 1 to 10 carbon atoms, preferably alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000076_0001
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000076_0002
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000076_0003
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000076_0004
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Figure imgf000077_0001
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and m is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; -CH2CH2O-CH2CH2-;
Figure imgf000077_0002
wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
-CH2CH2NHCH2CH2-; -CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10; -CH2CONH(CH2)nNHCOCH2- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000077_0003
aryl selected from the group comprising:
Figure imgf000077_0004
heteroaryl selected from the group comprising:
Figure imgf000077_0005
or isomers or pharmaceutically acceptable salts thereof.
2. A compound according to claim 1 , wherein said compound has the general formula (II):
Figure imgf000077_0006
wherein
Z is selected from: S, Se;
R and R’, which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000078_0001
heterocycloalkyl selected from the group comprising:
Figure imgf000078_0002
aryl selected from the group comprising:
Figure imgf000078_0003
heteroaryl selected from the group comprising:
Figure imgf000078_0004
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000078_0005
wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000078_0006
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000079_0001
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000079_0002
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000079_0003
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; -CH2CH2O-CH2CH2-;
Figure imgf000079_0004
wherein n is: 1 , 2, 3 or 4;
-CH2CH2NHCH2CH2-; -CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 2, 3, 4 or 5; -CH2CONH(CH2)nNHCOCH2- wherein n is: 2, 3, 4 or 5;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000079_0005
Figure imgf000079_0006
heteroaryl selected from the group comprising:
Figure imgf000079_0007
or isomers or pharmaceutically acceptable salts thereof.
3. A compound according to claim 2, wherein said compound is selected from the group comprising:
Figure imgf000080_0001
or isomers or pharmaceutically acceptable salts thereof.
4. A compound according to claim 1 , wherein said compound has the general formula (III):
Figure imgf000080_0002
wherein
Z is selected from: S, Se;
R and R’, which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000080_0003
heterocycloalkyl selected from the group comprising:
Figure imgf000081_0001
aryl selected from the group comprising:
Figure imgf000081_0002
heteroaryl selected from the group comprising:
Figure imgf000081_0003
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2;
X is selected from the group comprising:
-(CH2)n- wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000081_0004
wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000081_0005
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000081_0006
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000081_0007
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000081_0008
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; -CH2CH2O-CH2CH2-;
-CH2CH2NHCH2CH2-;
Figure imgf000082_0005
-CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 2, 3, 4 or 5;
-CH2CONH(CH2)nNHCOCH2- wherein n is: 2, 3, 4 or 5;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000082_0001
Figure imgf000082_0002
heteroaryl selected from the group comprising:
Figure imgf000082_0003
or isomers or pharmaceutically acceptable salts thereof.
5. A compound according to claim 4, wherein said compound is selected from the group comprising:
Figure imgf000082_0004
or isomers or pharmaceutically acceptable salts thereof.
6. A compound according to claim 1 , wherein said compound has the general formula (IV):
Figure imgf000083_0001
wherein
Z is selected from: S, Se;
R and R’, which are identical to each other, are selected from the group comprising: -H, -F, -Cl, -Br, -I, -OH, -CN, -COOH, -NH2; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000083_0002
heterocycloalkyl selected from the group comprising:
Figure imgf000083_0003
Figure imgf000083_0004
heteroaryl selected from the group comprising:
Figure imgf000083_0005
R” is selected from the group comprising:
-H, -F, -Cl, -Br, -I; alkyl selected from the group comprising: -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; X is selected from the group comprising: -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000084_0001
wherein n is: 1 , 2, 3, 4 or 5;
Figure imgf000084_0002
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000084_0003
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
Figure imgf000084_0004
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5;
-
Figure imgf000084_0005
wherein n is: 1 , 2, 3, 4 or 5 and m is: 1 , 2, 3, 4 or 5; -CH2CH2O-CH2CH2-;
Figure imgf000084_0006
wherein n is: 1 , 2, 3 or 4;
-CH2CH2NHCH2CH2-;
-CH2CH2NH(CH2)nNHCH2CH2- wherein n is: 2, 3, 4 or 5;
-CH2CONH(CH2)nNHCOCH2- wherein n is: 2, 3, 4 or 5;
-CH2CH2N(VR''')CH2CH2- wherein V is selected from the group comprising: -(CO)- or -(CH2)n- wherein n is: 1 , 2, 3, 4 or 5; and R''' is selected from the group comprising:
-CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2; cycloalkyl selected from the group comprising:
Figure imgf000084_0007
aryl selected from the group comprising:
Figure imgf000085_0001
Figure imgf000085_0002
or isomers or pharmaceutically acceptable salts thereof.
7. A compound according to claim 6, wherein said compound is selected from the group comprising:
Figure imgf000085_0003
or isomers or pharmaceutically acceptable salts thereof.
8. A compound according to any one of claims 1 to 7, for use as a medicament.
9. A compound according to any one of claims 1 to 7, for use as antitumour agent.
10. A compound according to any one of claims 1 to 7, for use as theranostic agent.
11 . A compound according to any one of claims 1 to 7, for use as biomedical probe.
12. A compound according to any one of claims 1 to 7, for use as fluorescent probe.
13. A compound according to any one of claims 1 to 7, for use in preventing and/or diagnosing and/or treating tumours.
14. A compound according to any one of claims 1 to 7, for use in preventing and/or diagnosing and/or treating liquid tumours.
15. A compound according to any one of claims 1 to 7, for use in preventing and/or diagnosing and/or treating leukaemia.
16. A compound according to any one of claims 1 to 7, for use in preventing and/or diagnosing and/or treating acute leukaemia.
17. A compound according to any one of claims 1 to 7, for use in preventing and/or diagnosing and/or treating solid tumours.
18. A compound according to any one of claims 1 to 7, for use in preventing and/or diagnosing and/or treating colon adenocarcinoma.
19. A compound according to any one of claims 1 to 7, for use in treating uterine cervix adenocarcinoma.
20. A compound according to any one of claims 1 to 7, for use as biomedical probe at a concentration of less than 2 micromolar.
21. A compound according to any one of claims 1 to 7, for use as fluorescent probe at a concentration of less than 2 micromolar.
22. A compound according to any one of claims 1 to 7, for use as theranostic agent at a concentration equal to or greater than 2 micromolar.
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Citations (4)

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WO2003059149A2 (en) * 2001-12-21 2003-07-24 Threshold Pharmaceuticals, Inc. Methods for cancer imaging
EP1961418A1 (en) * 2005-11-15 2008-08-27 Cheng, Jingcai The use of isothiocyanates compounds in treating prostatic diseases and skin cancer
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US4304720A (en) * 1980-04-30 1981-12-08 Merck & Co., Inc. Fluorescein esters and ethers and the preparation thereof
WO2003059149A2 (en) * 2001-12-21 2003-07-24 Threshold Pharmaceuticals, Inc. Methods for cancer imaging
EP1961418A1 (en) * 2005-11-15 2008-08-27 Cheng, Jingcai The use of isothiocyanates compounds in treating prostatic diseases and skin cancer
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