WO2018204987A1 - Inhibiteurs d'anhydrase carbonique - Google Patents

Inhibiteurs d'anhydrase carbonique Download PDF

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WO2018204987A1
WO2018204987A1 PCT/AU2018/050443 AU2018050443W WO2018204987A1 WO 2018204987 A1 WO2018204987 A1 WO 2018204987A1 AU 2018050443 W AU2018050443 W AU 2018050443W WO 2018204987 A1 WO2018204987 A1 WO 2018204987A1
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optionally substituted
compound
group
alkyl
tmz
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PCT/AU2018/050443
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Chiara RIGANTI
Iris Chiara SALAROGLIO
Prashant MUJUMDAR
Sally-Ann POULSEN
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Griffith University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/45—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups at least one of the singly-bound nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom, e.g. N-acylaminosulfonamides
    • C07C311/46—Y being a hydrogen or a carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/30—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D263/34—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D263/44—Two oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
    • C07D285/01—Five-membered rings
    • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
    • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
    • C07D285/12—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles
    • C07D285/125—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles with oxygen, sulfur or nitrogen atoms, directly attached to ring carbon atoms, the nitrogen atoms not forming part of a nitro radical
    • C07D285/135—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01001—Carbonate dehydratase (4.2.1.1), i.e. carbonic anhydrase

Definitions

  • the invention relates to the field of medical treatment. More particularly, this invention relates to carbonic anhydrase inhibitors and their use as chemosensitizing agents in the treatment of cancers.
  • CAIX and CAXII are zinc metalloenzymes that contribute to pH regulation through catalysis of the reversible hydration of carbon dioxide to bicarbonate and a proton: CO 2 + H 2 O 3 ⁇ 4 HCO 3 " + H + .
  • CAIX and CAXII are highly expressed in the hypoxic core of solid tumors, where they drive tumor growth and metastasis.
  • CAXII also induces chemoresistance by generating and maintaining pH conditions optimal for the catalytic cycle of P-glycoprotein (Pgp), an efflux transporter with a broad- spectrum of substrates including many anti-cancer agents currently in clinical use.
  • Pgp P-glycoprotein
  • CAIX and CAXII are overexpressed in many solid and hypoxic tumors, and given the expression prevalence of CAXII in transformed cells, high levels of tissue-associated and circulating CAXII have been proposed as predictive markers of thyroid and squamous lung cancers, respectively.
  • CAXII overexpression has also been associated with poor prognosis in human gliomas, oral squamous cancer and esophageal squamous cell cancer.
  • CAXII is overexpressed in chemoresistant cancer cells expressing Pgp and since Pgp recognizes multiple substrates, including a broad range of chemotherapeutics, Pgp expression in cancer cells contributes to multidrug resistance.
  • CAXII physically interacts with Pgp and therefore potentiates the contribution of Pgp to multidrug resistance.
  • each incidence of R a is independently selected from the group consisting of hydrogen, hydroxyl, halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi, optionally substituted aryl, optionally substituted heterocyclic, cyano, amino, carboxyl, optionally substituted O-alkyl, optionally substituted O-aryl, oxetane, homospiro-morpholine, OCF 3 , CF 3 , S- alkyl and SO 2 NHR 2 wherein R 2 is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi and optionally substituted aryl;
  • R b is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi and optionally substituted aryl;
  • Ri is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyi, optionally substituted heterocyclic and optionally substituted aryl;
  • W is selected from -(CH 2 )m- wherein m is from 1 to 6;
  • Y is selected from optionally substituted C1 to C12 alkyl, optionally substituted C1 to C12 alkenyl, optionally substituted C1 to C12 alkylether, optionally substituted C1 to C12 acyl, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted cycloalkyi, optionally substituted heteroaryl, and PEG;
  • Z is a zinc-binding group; and wherein, when R a is a 3-bromo-4-hydroxy substitution, W is -CH 2 -, R b and Ri are hydrogen, and Y is -CH 2 CH 2 -, then Z is not -S(O) 2 NH 2 .
  • a pharmaceutical composition comprising an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • a third aspect of the invention resides in a method of reducing the chemoresistance of a cancer in a patient including the step of administering an effective amount of a compound of formula (III), or a pharmaceutically effective salt thereof, to the patient:
  • each incidence of R a is independently selected from the group consisting of hydrogen, hydroxyl, halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi, optionally substituted aryl, optionally substituted heterocyclic, cyano, amino, carboxyl, optionally substituted O-alkyl, optionally substituted O-aryl, oxetane, homospiro- morpholine, OCF 3 , CF 3 , S-alkyl and SO 2 NHR 2 wherein R 2 is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi and optionally substituted aryl;
  • R b is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi and optionally substituted aryl;
  • R-i is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyi, optionally substituted heterocyclic and optionally substituted aryl;
  • W is selected from -(CH 2 )m- wherein m is from 1 to 6;
  • Y is selected from optionally substituted C1 to C12 alkyl, optionally substituted C1 to C12 alkenyl, optionally substituted C1 to C12 alkylether, optionally substituted C1 to C12 acyl, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, and PEG; and
  • Z is a zinc-binding group.
  • a fourth aspect of the invention provides for a compound of formula (III), or a pharmaceutically effective salt thereof, for use in reducing the chemoresistance of a cancer in a patient.
  • a fifth aspect of the invention provides for use of a compound of formula (III), or a pharmaceutically effective salt thereof, in the manufacture of a medicament for reducing the chemoresistance of a cancer in a patient.
  • a sixth aspect of the invention resides in a method of modulating the activity of a carbonic anhydrase XII enzyme including the step of contacting the enzyme with a compound of formula (III).
  • a seventh aspect of the invention resides in a method of treating a cancer in a patient including the step of administering an effective amount of a compound of formula (III), or a pharmaceutically effective salt thereof, and an anti-cancer agent to the patient.
  • An eighth aspect of the invention provides for a compound of formula (III), or a pharmaceutically effective salt thereof, and an anti-cancer agent for use in the treatment of a cancer in a patient.
  • a ninth aspect of the invention provides for use of a compound of formula (III), or a pharmaceutically effective salt thereof, and an anti-cancer agent in the manufacture of a medicament for the treatment of a cancer.
  • a method of diagnosing a cancer in a mammal including the step of administering a labelled compound of formula (III), or a pharmaceutically effective salt, thereof, to the mammal or to a biological sample obtained from the mammal to facilitate diagnosis of the cancer in the mammal.
  • An eleventh aspect of the invention resides in a complex of a compound of formula (III) and a carbonic anhydrase enzyme.
  • FIG 1 A CAXII and Pgp are co-expressed and associated in glioblastoma-derived neurospheres.
  • Primary GB cells derived from three patients (unknown patient numbers, UPN1 -3) were cultured as adherent cells (AC) or as neurospheres (NS).
  • Plasma-membrane extracts were immunoprecipitated (IP) with anti-CAXII or anti-CAIX antibodies, then immunoblotted (IB) with an anti-Pgp antibody, no Ab: UPN2 NS sample immunoprecipitated without antibody.
  • D Proximity ligation assay between CAXII and Pgp in UPN2 AC and NS.
  • Bl cells incubated without primary antibodies;
  • Ab cells incubated with primary antibodies.
  • Blue nuclear staining (DAPI); green: Pgp/CAXII interaction.
  • the image is representative of one out of three experiments. A minimum of five fields/experiment were examined. Bar: 10 pm (10* ocular lens; 63* objective lens).
  • FIG 1 B Expression levels of Pgp and CAXII in AC and NS in different culture conditions.
  • Adherent cells were grown for five passages (P1 -P5) in medium of neurospheres (NS); NS were grown for five passages (P1 -P5) in medium of AC.
  • P0 AC and NS grown in their own medium, used as baseline control.
  • the expression of CAXII and Pgp was measured by immunoblotting. The figure is representative of one out of three experiments with similar results.
  • ⁇ -Tubulin was used as control of equal protein loading.
  • UPN unknown patient number.
  • FIG 2 CAXII inhibition reduces Pgp activity and increases cytotoxicity of the Pgp substrate doxorubicin in glioblastoma-derived neurospheres.
  • A Chemical structures of CAXII inhibitors used.
  • Lower circle, uppermost circle and middle circle represent the mean ⁇ SD of technical replicates of UPN1 , UPN2 and UPN3.
  • B Spectrophotometric measure of Pgp ATPase activity, detected in triplicates in NS, grown for 24 h in fresh medium (-) or in medium containing 10 nM compounds 1-5.
  • * p ⁇ 0.02 compound 4 vs.
  • FIG 3 CAXII pharmacological inhibition with 741 restores temozolomide cytotoxicity in glioblastoma-derived neurospheres.
  • NS were grown for 48 h (panels A-E) or 72 h (panel F) in fresh medium (-) or in medium containing 50 ⁇ temozolomide (T) or 10 nM compound 1 (741), alone or in association.
  • A. UPN2 NS were lysed and immunoblotted for Pgp and CAXII.
  • B Spectrophotometric measure of Pgp ATPase, detected in triplicates in NS. * p ⁇ 0.01 : T-treated vs. untreated (-) cells; ** p ⁇ 0.01 : 1 -treated vs. untreated (-) cells; *** p ⁇ 0.001 : T+1 -treated vs. untreated (-) cells; # p ⁇ 0.05: T+1 -treated vs. T-treated cells (two-way ANOVA).
  • C Intracellular content of temozolomide (TMZ), measured in duplicates after cell radiolabelling.
  • NS clones knocked out for Pgp (KO#1 , KO#2) and AC were included as control of cells with undetectable expression of Pgp.
  • *** p ⁇ 0.001 all experimental conditions vs. untreated (-) NS (two-way ANOVA).
  • D LDH release, measured spectrophotometrically in duplicates.
  • *** p ⁇ 0.001 all experimental conditions vs. untreated (-) AC/NS; ### p ⁇ 0.001 : T+1 -treated, T+KO1 /KO2 cells vs. T-treated cells; ⁇ p ⁇ 0.001 : T+KO1 /KO2 cells vs. KO1 /KO2 cells (two-way ANOVA).
  • UPN2 NS incubated as reported in A or knocked out for Pgp, were lysed and immunoblotted for procaspase and cleaved caspase 3.
  • the figure is representative of one out of three experiments.
  • F Cell viability measured by a chemiluminescent-based assay in quadruplicates. .
  • *** p ⁇ 0.001 all experimental conditions vs. untreated (-) AC/NS; ## p ⁇ 0.005: T+1 -treated vs. T-treated cells; ### p ⁇ 0.001 : T+KO1 /KO2 cells vs. T-treated cells; ⁇ p ⁇ 0.001 : T+KO1 /KO2 cells vs.
  • FIG 4 CAXII pharmacological inhibition with 729 restores temozolomide cytotoxicity in glioblastoma-derived stem cells.
  • UPN2 NS were grown for 48 h (panels A-E) or 72 h (panel F) in fresh medium (-) or in medium containing 50 ⁇ temozolomide (T) or 10 nM compound 729, alone or in association.
  • B A.
  • FIG 5 CAXII knock out rescues glioblastoma-derived neurosphere sensitivity to temozolomide.
  • NS were growth in fresh medium (- ), transduced with a non-targeting vector (scrambled vector; scr) or with two CRISPR pCas CAX/ argeting vectors (KO#1 , KO#2).
  • a non-targeting vector scr
  • CRISPR pCas CAX/ argeting vectors KO#1 , KO#2
  • 50 ⁇ temozolomide (T) was added for 48 h (panels B-E) or 72 h (panel F).
  • AC were included as control of cells with undetectable CAXII levels.
  • UPN2 NS were lysed and immunoblotted with the indicated antibodies. The figure is representative of one out of three experiments.
  • B Spectrophotometric measure of Pgp ATPase, detected in triplicates in NS. * p ⁇ 0.05: T-treated vs. scrambled-treated (-) cells; *** p ⁇ 0.001 : KO1 /KO2 or T+KO1 /KO2 cells vs. scrambled-treated (-) cells; ### p ⁇ 0-001 : T+KO1 /KO2 cells vs.
  • T-treated cells ⁇ p ⁇ 0.01 : T+KO1 /KO2 cells vs. KO1 /KO2 cells (two-way ANOVA).
  • C Intracellular content of temozolomide (TMZ), measured in duplicates after cell radiolabelling. *** p ⁇ 0.001 : all experimental conditions vs. untreated (-) NS (two-way ANOVA).
  • D LDH release, measured spectrophotometrically in duplicates. *** p ⁇ 0.001 : all experimental conditions vs. untreated (-) AC/NS; ### p ⁇ 0.001 : T+1 -treated, T+KO1 /KO2 cells vs.
  • T-treated cells ⁇ p ⁇ 0.001 : T+KO1 /KO2 cells vs. KO1 /KO2 cells (two-way ANOVA).
  • E UPN2 NS were lysed and immunoblotted for procaspase and cleaved caspase 3. The figure is representative of one out of three experiments.
  • F Cell viability measured by a chemiluminescent-based assay in quadruplicates. *** p ⁇ 0.001 : all experimental conditions vs. untreated (- ) AC/NS; ### p ⁇ 0.001 : T+1 -treated, T+KO1 /KO2 cells vs. T-treated cells; ⁇ p ⁇ 0.001 : T+KO1 /KO2 cells vs. KO1 /KO2 cells (two-way ANOVA).
  • FIG 6 Effects of the combination of CAXII inhibitor 741 , temozolomide and Pgp substrates in glioblastoma-derived neurospheres.
  • UPN2 NS were grown for 48 h (panels A-B) or 72 h (panel C) in fresh medium (-) or in medium containing 10 nM compound 1 (741 ), 50 ⁇ temozolomide (T), 5 ⁇ doxorubicin (dox), 10 ⁇ etoposide (eto), 10 ⁇ topotecan (top), 10 ⁇ irinotecan (iri), in different combinations.
  • dox/eto/top/iri- treated cells ## p ⁇ 0.01 , ### p ⁇ 0.001 : vs. cells treated with drug+compound 1 ; ⁇ p ⁇ 0.01 , ⁇ p ⁇ 0.001 : vs. cells treated with drug+T (two-way ANOVA).
  • FIG 7 Effects of the combination of CAXII inhibitor 729, temozolomide and Pgp substrates in glioblastoma-derived stem cells.
  • UPN2 NS were grown for 48 h (panels A-B) or 72 h (panel C) in fresh medium (-) or in medium containing 10 nM compound 729, 50 ⁇ temozolomide (T), 5 ⁇ doxorubicin (dox), 10 ⁇ etoposide (eto), 10 ⁇ topotecan (top), 10 ⁇ irinotecan (iri), in different combinations.
  • T fresh medium
  • T 50 ⁇ temozolomide
  • dox doxorubicin
  • eto etoposide
  • top 10 ⁇ topotecan
  • irinotecan 10 ⁇ irinotecan
  • FIG 8 Compound 741 (1) improves temozolomide efficacy against orthotopically implanted glioblastoma neurosphere-derived tumors.
  • A Representative in vivo bioluminescence imaging of orthotopically implanted UPN2 NS, in animals treated with vehicle (Ctrl), compound 1 and temozolomide (TMZ), as follows: 1 ) control group, treated with 0.2 ml saline solution i.v.; 2) 1 group, treated with 3800 ng/kg compound 1 i.v.; 3) TMZ group, treated with 50 mg/kg TMZ p.o.; 4) TMZ+1 group, treated with 50 mg/kg TMZ p.o.
  • UPN2 p ⁇ 0.002: TMZ+1-group vs. all the other groups of treatment.
  • UPN3 p ⁇ 0.001 : TMZ+1 -group vs. Ctrl and 1-group; p ⁇ 0.05: TMZ+1 group vs. TMZ-group; p ⁇ 0.01 : TMZ-group vs. Ctrl and 1-group (log rank test; not reported in the figure).
  • FIG 9 Compound 729 improves temozolomide efficacy against orthotopically implanted glioblastoma stem cells-derived tumors. 1 ⁇ 10 6
  • NS cells from UPN2, stably expressing luciferase were stereotactically injected into the right caudatus nucleus into 6-8 week olds female BALB/c nulnu mice.
  • animals (6 mice/groups) were randomized and treated with 2 cycles of 5 consecutive days (days: 7-1 1 ; 17-21 after randomization) as it follows: 1 ) control (Ctrl) group, treated with 0.2 imL saline solution i.v.; 2) 729 group, treated with 3413 ng/kg compound 729 (in 0.2 imL saline solution; final concentration: 1 ⁇ ) i.v.; 3) temozolomide (TMZ) group, treated with 50 mg/kg TMZ p.o.
  • TMZ temozolomide
  • TMZ + 729 group treated with 50 mg/kg TMZ p.o. and 3413 ng/kg compound 729 i.v. Animals were euthanized at day 30.
  • B. Tumor volume was measured by caliper on excised GB for each group of treatment. Data are presented as means + SD (n 6): 269.17 + 59.17 (Ctrl group); 259.67 + 54.60 (729 group); 227.00 + 74.57 (TMZ group); 125.83 + 36.43 (TMZ+729 group). * p ⁇ 0.02: vs.
  • C Representative intratumor staining with hematoxylin and eosin (HE), immunostaining for Ki67, an index of cell proliferation, or for cleaved (Asp175)caspase 3, an index of apoptosis. The photographs are representative of sections from 4 tumors/group of treatment (bar: 10 ⁇ ).
  • D Quantification of immunohistochemical images, performed on sections on 6 animals/group (1 1 1 -94 nuclei/field).
  • the percentage of proliferating cells was determined by the ratio Ki67-positive nuclei/total number (hematoxylin-positive) of nuclei using ImageJ software (http://imaqei.nih.gov/ii/). The Ctrl group percentage was considered 100%.
  • the percentage of caspase 3-positive cells was determined by Photoshop program. Data are presented as means + SD. * p ⁇ 0.001 : vs. untreated (-) cells; ° p ⁇ 0.001 vs TMZ-treated cells (for Ki67-positive cells); * p ⁇ 0.001 : vs. untreated (-) cells; ° p ⁇ 0.01 vs TMZ-treated cells (for cleaved caspase 3-positive cells).
  • FIG 10 Effects of CAXII inhibitors on etoposide cytotoxicity in glioblastoma cells.
  • Cells were grown for 24 h (panel a) or 72 h (panel b) in fresh medium (-) or in medium containing 10 nM of compounds 1-5, in the absence or presence of 10 ⁇ etoposide (eto).
  • Lower circle, uppermost circle and middle circle represent the mean+SD of technical replicates of UPN1 , UPN2 and UPN3.
  • a Release of LDH, measured spectrophotometrically in duplicates. *** p ⁇ 0.001 : treated AC/NS vs.
  • FIG 1 1 Effects of CAXII inhibitors on topotecan cytotoxicity in glioblastoma cells.
  • FIG 12 Effects of CAXII inhibitors on irinotecan cytotoxicity in glioblastoma cells.
  • Cells were grown for 24 h (panel a) or 72 h (panel b) in fresh medium (-) or in medium containing 10 nM of compounds 1-5, in the absence or presence of 10 ⁇ irinotecan (iri).
  • Lower circle, uppermost circle and middle circle represent the mean+SD of technical replicates of UPN1 , UPN2 and UPN3.
  • a Release of LDH, measured spectrophotometrically in duplicates.
  • FIG 13 Pgp knocking out in glioblastoma-derived stem cells.
  • UPN2 NS were grown in fresh medium (-), transduced with a non-targeting vector (scrambled vector; scr) or with a CRISPR pCas ABCCI/Pgp-targeting vector (KOPgp). AC were included as control of Pgp-lowly expressing cells. Cells were lysed and immunoblotted for Pgp and CAXII. ⁇ -tubulin level was used as control of equal protein loading. The figure is representative of one out of three experiments with similar results.
  • FIG 14 in vivo antitumor activity of different combination of temozolomide and compound 741.
  • A. Six week-old female BALB/c nu/nu mice were inoculated s.c. with 1 x10 6 AC from UPN2.
  • mice were randomized into the following groups (10 animals/group) and treated as it follows: 1 ) control (Ctrl) group, treated with 0.2 ml saline solution i.v., for 2 cycles of 5 consecutive days (days: 1 -5; 1 1 -15 after randomization); 2) TMZ 3 group, treated with 50 mg/kg TMZ p.o., for 3 consecutive days (days: 1 -3 after randomization); 3) TMZ 3(x2) group, treated with 50 mg/kg TMZ p.o., for 2 cycles of 3 consecutive days (days: 1 -3; 7-9 after randomization); 4) TMZ 5 group, treated with 50 mg/kg TMZ p.o., for 5 consecutive days (days: 1 -5 after randomization); 5) TMZ 5(x2), treated with 50 mg/kg TMZ p.o., 2 cycles of 5 consecutive days (days: 1 -5; 1 1 -15 after randomization).
  • 1 control (Ctrl) group, treated
  • mice were randomized into the following groups (10 animals/group) and treated with 2 cycles (days: 1 -5; 1 1 -15 after randomization) as it follows: 1 ) control (Ctrl) group, treated with 0.2 ml saline solution i.v.; 2) 741 low dose (LD) group, treated with 38 ng/kg compound 741 (in 0.2 ml saline solution; final concentration: 10 nM) i.v.; 3) 741 high dose (HD) group, treated with 3800 ng/kg compound 741 (in 0.2 ml saline solution; final concentration: 1 ⁇ ) i.v.; 4) temozolomide (TMZ) group, treated with 50 mg/kg TMZ p.o.; 5) TMZ + 741 LD group, treated with 50 mg/kg TMZ p.o.
  • TMZ temozolomide
  • TMZ + 741 HD group treated with 50 mg/kg TMZ p.o. and 3800 ng/kg compound 741 i.v..
  • Tumor growth was monitored by caliper measure.
  • Data are presented as means ⁇ SD. * p ⁇ 0.05: TMZ/TMZ+741 (LD)/TMZ+741 (HD) groups vs. Ctrl group (day 30); ° p ⁇ 0.001 : TMZ+741 (LD)/TMZ+741 (HD) groups vs. TMZ group (day 30).
  • C Photographs of representative tumors from each treatment group after mice sacrifice.
  • FIG 15 Pgp knocking out in glioblastoma-derived neurospheres.
  • UPN1 and UPN3 NS were grown 48 h in in fresh medium (-) or in medium containing 50 ⁇ temozolomide (T) or 10 nM compound 1 , alone or in association. Cells were lysed and immunoblotted for Pgp and CAXII. The figure is representative of one out of three experiments on UPN1 and UPN3 NS.
  • b UPN1 and UPN3 NS were grown 48 h in in fresh medium (-) or in medium containing 50 ⁇ temozolomide (T) or 10 nM compound 1 , alone or in association. Cells were lysed and immunoblotted for Pgp and CAXII. The figure is representative of one out of three experiments on UPN1 and UPN3 NS.
  • T ⁇ temozolomide
  • UPN1 , UPN2 and UPN3 NS were grown in fresh medium (-), transduced with a non-targeting vector (scrambled vector; scr) or with two CRISPR pCas /4SCC7/Pc3 ⁇ 4D-targeting vectors (KO #1 and #2). AC were included as control of Pgp-lowly expressing cells. Cells were lysed and immunoblotted with the indicated antibodies. The figure is representative of one out of three experiments, c. UPN1 and UPN3 NS, incubated as reported in a or knocked out for Pgp, were lysed and immunoblotted for procaspase and cleaved caspase 3. The figure is representative of one out of three experiments.
  • FIG 16 CAXII knocking out in glioblastoma-derived neurospheres.
  • UPN1 and UPN3 NS were growth in fresh medium (-), transduced with a non-targeting vector (scrambled vector; scr) or with two CRISPR pCas CAX7/-targeting vectors (KO#1 , KO#2), then lysed and immunoblotted with the indicated antibodies.
  • AC were included as control of cells with undetectable CAXII levels.
  • the figure is representative of one out of three experiments.
  • UPN1 and UPN3 NS, treated as reported in a were lysed and immunoblotted for procaspase and cleaved caspase 3. The figure is representative of one out of three experiments.
  • FIG 17 Quantification of UPN1 -3 NS-derived bioluminescence imaging of orthotopically implanted UPN2 NS, in animals treated with vehicle (Ctrl), compound 729 and temozolomide (TMZ), as follows: 1 ) control group, treated with 0.2 ml saline solution i.v.; 2) 729 group, treated with 3800 ng/kg compound 729 i.v.; 3) TMZ group, treated with 50 mg/kg TMZ p.o.; 4) TMZ+729 group, treated with 50 mg/kg TMZ p. o. +3800 ng/kg compound 729 i.v. (6 animals/group), taken as index of tumor growth.
  • the present invention is predicated, at least in part, on the finding that certain sulfonamide compounds display useful efficacy in the inhibition of carbonic anhydrase XII (CAXII).
  • CAXII carbonic anhydrase XII
  • these compounds, and analogs thereof may be used as effective chemosensitizers.
  • the compounds of the invention may be useful components in the treatment of glioblastoma.
  • such an approach can be used to increase the intracellular retention of existing anti-cancer agents, such as temozolomide (TMZ), and restore the cytotoxicity of such front-line therapies.
  • temozolomide TMZ
  • this approach should mean that other chemotherapy drugs (i.e. second-line treatments) that are Pgp-substrates may become useful treatment options if dosage is accompanied with a CAXII inhibitor, as described herein.
  • administering or “administration”, and the like, describe the introduction of the compound or composition to a mammal such as by a particular route or vehicle.
  • Routes of administration may include topical, parenteral and enteral which include oral, buccal, sub-lingual, nasal, anal, gastrointestinal, subcutaneous, intramuscular and intradermal routes of administration, although without limitation thereto.
  • treat means administration of the compound or composition to a subject to at least ameliorate, reduce or suppress existing signs or symptoms of the disease, disorder or condition experienced by the subject.
  • prevent prophylactically administering the formulation to a subject who does not exhibit signs or symptoms of a disease disorder or condition, but who is expected or anticipated to likely exhibit such signs or symptoms in the absence of prevention.
  • Preventative treatment may at least lessen or partly ameliorate expected symptoms or signs.
  • effective amount refers to the administration of an amount of the relevant compound or composition sufficient to prevent the occurrence of symptoms of the condition being treated, or to bring about a halt in the worsening of symptoms or to treat and alleviate or at least reduce the severity of the symptoms.
  • the effective amount will vary in a manner which would be understood by a person of skill in the art with patient age, sex, weight etc. An appropriate dosage or dosage regime can be ascertained through routine trial.
  • the terms "subject” or “individual” or “patient” may refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy is desired.
  • Suitable vertebrate animals include, but are not restricted to, primates, avians, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes).
  • a preferred subject is a human in need of treatment for a cancer as described herein. However, it will be understood that the aforementioned terms do not imply that symptoms are necessarily present.
  • pharmaceutically acceptable salt refers to salts which are toxicologically safe for systemic or localised administration such as salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids.
  • the pharmaceutically acceptable salts may be selected from the group including alkali and alkali earth, ammonium, aluminium, iron, amine, glucosamine, chloride, sulphate, sulphonate, bisulphate, nitrate, citrate, tartrate, bitarate, phosphate, carbonate, bicarbonate, malate, maleate, napsylate, fumarate, succinate, acetate, benzoate, terephthalate, palmoate, piperazine, pectinate and S-methyl methionine salts and the like.
  • the pharmaceutically acceptable salts include acid addition salts, base addition salts, salts of pharmaceutically acceptable esters and the salts of quaternary amines and pyridiniums.
  • the acid addition salts are formed from a compound of the first aspect and a pharmaceutically acceptable inorganic or organic acid including but not limited to hydrochloric, hydrobromic, sulphuric, phosphoric, methanesulfonic, toluenesulphonic, benzenesulphonic, acetic, propionic, ascorbic, citric, malonic, fumaric, maleic, lactic, salicyclic, sulfamic, or tartartic acids.
  • the counter ion of quaternary amines and pyridiniums include chloride, bromide, iodide, sulfate, phosphate, methansulfonate, citrate, acetate, malonate, fumarate, sulfamate, and tartate.
  • the base addition salts include but are not limited to salts such as sodium, potassium, calcium, lithium, magnesium, ammonium and alkylammonium.
  • basic nitrogen-containing groups may be quaternised with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • the salts may be made in a known manner, for example by treating the compound with an appropriate acid or base in the presence of a suitable solvent.
  • alkyl refers to a straight-chain or branched alkyl substituent containing from, for example, 1 to about 12 carbon atoms, preferably 1 to about 9 carbon atoms, more preferably 1 to about 6 carbon atoms, even more preferably from 1 to about 4 carbon atoms, still yet more preferably from 1 to 2 carbon atoms.
  • substituents may be selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, te/t-butyl, pentyl, isoamyl, 2-methylbutyl, 3-methylbutyl, hexyl, heptyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-ethylbutyl, 3- ethylbutyl, octyl, nonyl, decyl, undecyl, dodecyl and the like.
  • the number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.
  • Substituted alkyl includes alkyl substituted with one or more moieties selected from the group consisting of halo ⁇ e.g., CI, F, Br, and I); halogenated alkyl ⁇ e.g., CF 3 , 2-Br-ethyl, CH 2 F, CH 2 CI, CH 2 CF 3 , or CF 2 CF 3 ); hydroxyl; amino; carboxylate; carboxamido; alkylamino; arylamino; alkoxy; aryloxy; nitro; azido; cyano; thio; sulfonic acid; sulfate; phosphonic acid; phosphate; and phosphonate as well as those described under the definition of Optionally substituted'.
  • alkenyl refers to optionally substituted unsaturated linear or branched hydrocarbon groups, having 2 to 12 carbon atoms, preferably 2 to 9 carbon atoms, more preferably 2 to 6 carbon atoms and having at least one carbon-carbon double bond.
  • the alkenyl group may have a specified number of carbon atoms, for example, C 2 -C 6 alkenyl which includes alkenyl groups having 2, 3, 4, 5 or 6 carbon atoms in linear or branched arrangements.
  • the number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents.
  • substituents may be selected from the group consisting of ethenyl, propenyl, isopropenyl, butenyl, s- and t-butenyl, pentenyl, hexenyl, hept-l,3-diene, hex-l,3-diene, non-l,3,5-triene and the like.
  • Substituted alkenyl includes alkenyl substituted with one or more moieties selected from the group consisting of halo ⁇ e.g., CI, F, Br, and I); halogenated alkyl ⁇ e.g., CF 3 , 2-Br-ethyl, CH 2 F, CH 2 CI, CH 2 CF 3 , or CF 2 CF 3 ); hydroxyl; amino; carboxylate; carboxamido; alkylamino; arylamino; alkoxy; aryloxy; nitro; azido; cyano; thio; sulfonic acid; sulfate; phosphonic acid; phosphate; and phosphonate as well as those described under the definition of Optionally substituted'.
  • halo ⁇ e.g., CI, F, Br, and I
  • halogenated alkyl ⁇ e.g., CF 3 , 2-Br-ethyl, CH 2 F, CH 2
  • halo or halogen as used herein means selected from fluorine, chlorine, bromine, and iodine.
  • cycloalkyl refers to optionally substituted saturated monocyclic, bicyclic or tricyclic carbon groups.
  • the cycloalkyl group may have a specified number of carbon atoms, for example, C 3 -C 6 cycloalkyl is a carbocyclic group having 3, 4, 5 or 6 carbon atoms.
  • Non-limiting examples may include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl and the like.
  • aryl refers to an unsubstituted or substituted aromatic carbocyclic substituent, as commonly understood in the art. It is understood that the term aryl applies to cyclic substituents that are planar and comprise 4n+2 ⁇ electrons, according to Huckel's Rule.
  • Aryl includes biaryl, such as naphthyl, and so may include C5 to C12 aryl (these numbers referring only to the ring carbons). C-6 aryl is preferred.
  • heteroaryl refers to an aryl group containing from one or more (particularly one to four) non-carbon atom(s) (particularly N, O or S) or a combination thereof, which heteroaryl group is optionally substituted at one or more carbon or nitrogen atom(s). Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.
  • Heteroaryl includes, but is not limited to, 5-membered heteroaryls having one hetero atom (e.g., thiophenes, pyrroles, furans); 5 membered heteroaryls having two heteroatoms in 1 ,2 or 1 ,3 positions (e.g., oxazoles, pyrazoles, imidazoles, thiazoles, purines); 5-membered heteroaryls having three heteroatoms (e.g., triazoles, thiadiazoles); 5-membered heteroaryls having four heteroatoms (e.g., tetrazoles); 6-membered heteroaryls with one heteroatom (e.g., pyridine, quinoline, isoquinoline, phenanthrine, 5,6-cycloheptenopyridine); 6-membered heteroaryls with two heteroatoms (e.g., pyridazines, cinnolines, phthalazines, pyrazines,
  • Heterocyclic refers to a non-aromatic ring having 4 to 8 atoms in the ring and of those atoms 1 to 4 are heteroatoms. Heterocyclic rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings. Heterocyclic includes partially and fully saturated heterocyclic groups. Heterocyclic systems may be attached to another moiety via any number of carbon atoms or heteroatoms of the radical and may be both saturated and unsaturated.
  • heterocyclic examples include pyrrolidinyl, pyrrolinyl, pyranyl, piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolinyl, dithiolyl, oxathiolyl, oxetane, dioxanyl, dioxinyl, oxazinyl, azepinyl, diazepinyl, thiazepinyl, oxepinyl and thiapinyl, imidazolinyl, thiomorpholinyl, thiophene, thiadiazole, dithiazole, dithiolane, and the like.
  • Optionally substituted in each incidence of its use herein, and in the absence of an explicit listing for any particular moiety, refers to substituent groups optionally substituted with one or more moieties, for example, those selected from the group consisting of optionally substituted C1 -10 alkyl (e.g., optionally substituted C1 -6 alkyl); optionally substituted C3-6 cycloalkyl (e.g., optionally substituted cyclopropyl); optionally substituted hydroxyalkyi; optionally substituted C1 -10 alkoxy (e.g., optionally substituted C1 -6 alkoxy); optionally substituted C2-10 alkenyl; optionally substituted C2-10 alkynyl; optionally substituted C6-C12 aryl; aryloxy; optionally substituted heteroaryl; optionally substituted heterocyclic; halo (e.g., CI, F, Br, and I); hydroxyl; halogenated alkyl (e.g., CF
  • each incidence of R a is independently selected from the group consisting of hydrogen, hydroxyl, halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic, cyano, amino, carboxyl, optionally substituted O-alkyl, optionally substituted O-aryl, oxetane, homospiro-morpholine, OCF 3 , CF 3 , S- alkyl and SO 2 NHR 2 wherein R 2 is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi and optionally substituted aryl;
  • R b is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi and optionally substituted aryl;
  • Ri is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyi, optionally substituted heterocyclic and optionally substituted aryl;
  • W is selected from -(CH 2 )m- wherein m is from 1 to 6;
  • Y is selected from optionally substituted C1 to C12 alkyl, optionally substituted C1 to C12 alkenyl, optionally substituted C1 to C12 alkylether, optionally substituted C1 to C12 acyl, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted cycloalkyi, optionally substituted heteroaryl, and PEG;
  • Z is a zinc binding group; and wherein, when R a is a 3-bromo-4-hydroxy substitution, W is -CH 2 -, R b and Ri are hydrogen, and Y is -CH 2 CH 2 -, then Z is not -S(O) 2 NH 2 .
  • each incidence of R a is independently selected from the group consisting of hydrogen, hydroxyl, halo, optionally substituted C1 to C6 alkyl, optionally substituted C1 to C6 alkenyl, optionally substituted C1 to C6 cycloalkyi, optionally substituted C5 or C6 aryl, optionally substituted C5 or C6 heterocyclic, cyano, amino, carboxyl, optionally substituted C1 to C6 O-alkyl, optionally substituted C5 or C6 O-aryl.
  • each incidence of R a is independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro, chloro, iodo, optionally substituted C1 to C6 alkyl, optionally substituted C1 to C6 alkenyl, cyano, amino and carboxyl.
  • each incidence of R a is independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro, chloro, cyano, amino and carboxyl.
  • each incidence of R a is independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro and chloro.
  • R b is selected from the group consisting of hydrogen, optionally substituted C1 to C6 alkyl, optionally substituted C1 to C6 alkenyl, optionally substituted C5 or C6 cycloalkyi and optionally substituted C5 or C6 aryl.
  • R b is selected from the group consisting of hydrogen, optionally substituted C1 to C6 alkyl and optionally substituted C5 or C6 cycloalkyi.
  • Ri is selected from the group consisting of hydrogen, optionally substituted C1 to C6 alkyl, optionally substituted C5 or C6 heterocyclic and optionally substituted phenyl.
  • Ri is selected from hydrogen and a protecting group.
  • protecting groups include benzyl (Bn) and tetrahydropyranyl (THP).
  • R- is a protecting group then that group may be cleaved prior to the compound binding to CAXII.
  • W is selected from the group consisting of -CH 2 -, - CH2CH2-, -CH2CH2CH2-, and -CH2CH2CH2CH2-.
  • W is -CH 2 -.
  • Y is selected from optionally substituted C1 to C6 alkyl, optionally substituted C1 to C6 alkenyl, optionally substituted C5 or C6 heterocyclic, optionally substituted C5 or C6 aryl, C2 to C16 PEG and optionally substituted C5 or C6 heteroaryl.
  • Y is selected from optionally substituted C1 to C4 alkyl, optionally substituted C5 or C6 heterocyclic, optionally substituted phenyl and optionally substituted C5 or C6 heteroaryl.
  • Y is heterocyclic or heteroaryl it may be selected from C5 or C6 nitrogen and/or sulphur containing heterocyclic or heteroaryl.
  • Y when Y is heterocyclic or heteroaryl then it may be selected from pyrazole, furan, tetrahydrofuran, tetrahydropyran, pyran, pyrrolidine, pyrrole, triazole, tetrazole, imidazole, pyridine, morpholine, piperazine, piperidine, pyrazine, pyrimidine, thiophene, thiadiazole, dithiazole and dithiolane, all of which may be optionally substituted as appropriate.
  • Y may be selected from 1 ,2,3-thiadiazole, 1 ,2,4-thiadiazole, 1 ,2,5-thiadiazole and 1 ,3,4-thiadiazole.
  • Zinc-binding groups are known in the art and reference to various such groups can be found in, for example, Kawai et al, Eu. J Med. Chem. 51 , 2012, pp 271 -276.
  • Z is selected from -S(O) 2 NR c R d , -OS(O) 2 NR c R d and optionally substituted C5 or C6 heterocyclic, wherein R c and R d are independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl and optionally substituted aryl or at least one of R c and R d may be a component of a mono- or bicyclic ring system with the nitrogen to which they are attached and the sulfur of the sulfamate or sulfonamide group.
  • R c and R d are independently selected from hydrogen, optionally substituted C1 to C6 alkyl, optionally substituted C1 to C6 alkenyl, optionally substituted C5 or C6 cycloalkyl and optionally substituted C5 or C6 aryl.
  • R c and R d may be a component of a mono- or bicyclic ring system with the nitrogen to which they are attached and the sulfur of the sulfamate or sulfonamide group then they may form an optionally substituted thiazole or benzothiazole ring system.
  • the thiazole or benzothiazole ring system may be an optionally substituted thiazole-trione or benzothiazole-trione.
  • Z is optionally substituted C5 or C6 heterocyclic then it may be a 5 or 6-membered nitrogen and/or oxygen-containing heterocycle.
  • Z when Z is optionally substituted C5 or C6 heterocyclic then it may be an oxazolidinedione.
  • Z is a 2,4- oxazolidinedione.
  • R c and R d are preferably hydrogen. That is, it may be preferable that Z is a primary sulfonamide or sulfamate.
  • reference to optionally substituted C5 or C6 aryl includes optionally substituted phenyl.
  • the compound of formula (I) is a compound of formula (II):
  • R e and R' are hydrogen.
  • R f , R 9 and R h are independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro, chloro, cyano, amino and carboxyl.
  • R f , R 9 and R h are independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro and chloro.
  • R c and R d are hydrogen.
  • the compound of formula (I) or formula (II) is selected from the group consisting of:
  • prodrugs are compounds which, when administered to a mammal, are converted in whole or in part to a compound of the invention.
  • the prodrugs are pharmacologically inert chemical derivatives that can be converted in vivo to the active drug molecules to exert a therapeutic effect. Any of the compounds described herein can be administered as a prodrug to increase the activity, bioavailability, or stability of the compound or to otherwise alter the properties of the compound.
  • Typical examples of prodrugs include compounds that have biologically labile protecting groups on a functional moiety of the active compound.
  • Prodrugs include, but are not limited to, compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, and/or dephosphorylated to produce the active compound.
  • prodrug ligands are known.
  • alkylation, acylation, or other lipophilic modification of one or more heteroatoms of the compound, such as a free amine or carboxylic acid residue may reduce polarity and allow for the compound's passage into cells.
  • substituent groups that can replace one or more hydrogen atoms on a free amine and/or carboxylic acid moiety include, but are not limited to, the following: aryl; steroids; carbohydrates (including sugars); 1 ,2-diacylglycerol; alcohols; acyl (including lower acyl); alkyl (including lower alkyl); sulfonate ester (including alkyl or arylalkyl sulfonyl, such as methanesulfonyl and benzyl, wherein the phenyl group is optionally substituted with one or more substituents as provided in the definition of an aryl given herein); optionally substituted arylsulfonyl; lipids (including phospholipids); phosphotidylcholine; phosphocholine; amino acid residues or derivatives; amino acid acyl residues or derivatives; peptides; cholesterols; or other pharmaceutically acceptable leaving groups which, when administered in vivo, provide the free amine
  • Esters of the active agent compounds according to the present invention may be prepared through functionalization of hydroxyl and/or carboxyl groups that may be present within the molecular structure of the compound.
  • Amides and prodrugs may also be prepared using techniques known to those skilled in the art. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine.
  • esters and amides of compounds of the invention can be made by reaction with a carbonylating agent ⁇ e.g., ethyl formate, acetic anhydride, methoxyacetyl chloride, benzoyl chloride, methyl isocyanate, ethyl chloroformate, methanesulfonyl chloride) and a suitable base ⁇ e.g., 4-dimethylaminopyridine, pyridine, triethylamine, potassium carbonate) in a suitable organic solvent ⁇ e.g., tetrahydrofuran, acetone, methanol, pyridine, ⁇ , ⁇ -dimethylformamide) at a temperature of 0 Q C to 60 Q C.
  • a carbonylating agent e.g., ethyl formate, acetic anhydride, methoxyacetyl chloride, benzoyl chloride, methyl isocyanate, ethyl chloroformate, methan
  • Prodrugs are typically prepared by covalent attachment of a moiety, which results in a compound that is therapeutically inactive until modified by an individual's metabolic system.
  • Examples of pharmaceutically acceptable solvates include, but are not limited to, compounds according to the invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.
  • compounds with one or more chiral centers are provided. While racemic mixtures of compounds of the invention may be active, selective, and bioavailable, isolated isomers may be of interest as well.
  • the compounds of the first aspect may, in some instances, contain chiral centers, which may be either of the (R) or (S) configuration, or which may comprise a mixture thereof. Accordingly, the present invention also includes stereoisomers of the compounds described herein, where applicable, either individually or admixed in any proportions. Stereoisomers may include, but are not limited to, enantiomers, diastereomers, racemic mixtures, and combinations thereof. Such stereoisomers can be prepared and separated using conventional techniques, either by reacting enantiomeric starting materials, or by separating isomers of compounds and prodrugs of the present invention. Isomers may include geometric isomers.
  • geometric isomers include, but are not limited to, cis isomers or trans isomers across a double bond.
  • Other isomers are contemplated among the compounds of the present invention.
  • the isomers may be used either in pure form or in admixture with other isomers of the compounds described herein.
  • optical isomers of the compounds according to the present invention include the following: i) physical separation of crystals whereby macroscopic crystals of the individual enantiomers are manually separated. This technique may particularly be used when crystals of the separate enantiomers exist ⁇ i.e., the material is a conglomerate), and the crystals are visually distinct;
  • enzymatic asymmetric synthesis a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer;
  • kinetic resolutions comprising partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions;
  • x) chiral liquid chromatography whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase.
  • the stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions;
  • xiii) transport across chiral membranes whereby a racemate is placed in contact with a thin membrane barrier.
  • the barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane which allows only one enantiomer of the racemate to pass through.
  • the compound optionally may be provided in a composition that is enantiomerically enriched, such as a mixture of enantiomers in which one enantiomer is present in excess, in particular, to the extent of 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, including 100%.
  • a composition that is enantiomerically enriched such as a mixture of enantiomers in which one enantiomer is present in excess, in particular, to the extent of 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, including 100%.
  • compositions contain a greater proportion of the named isomer of the compound in relation to other isomers.
  • these terms indicate that the composition contains at least 90% by weight of the named isomer and 10% by weight or less of the one or more other isomers; or more preferably about 95% by weight of the named isomer and 5% or less of the one or more other isomers.
  • the composition may contain at least 99% by weight of the named isomer and 1 % or less by weight of the one or more other isomers, or may contain 100% by weight of the named isomer and 0% by weight of the one of more other isomers. These percentages are based on the total amount of the compound of the present invention present in the composition.
  • Scheme 1 shows one pathway by which compounds 1 to 4, of the invention (referred to occasionally in the biological results section and figures as compounds 741 , 737, 739, and 744 respectively), and the control, 5 (referred to as compound 787 in the biological results section), can be synthesised. Further detail on this approach is provided in the experimental section.
  • Scheme 1 Synthesis of sulfonamides 1 to 5. a Reagents and conditions: (i) N- acetyl glycine, NaOAc, Ac 2 O, 120 °C, 3 h; (ii) Aq.
  • HCI.H 2 NOBn or H 2 NOTHP dry pyridine, rt, 15 h;
  • EDC.HCI HOSu, dry 1 ,4-dioxane, 2-3 h, rt;
  • ⁇ -aminoethanesulfonamide hydrochloride or 3- aminopropanamide dry NEt 3 , dry 1 ,4-dioxane, dry MeOH, rt, 12 h;
  • Pd/C dry NEt 3 , HCOOH, abs.
  • Scheme 2 Synthetic scheme for compound 729. a Reagents and conditions: (i) AAacetyl glycine, NaOAc, Ac 2 0, 120 °C, 3 h, 67%; (ii) Aq. HCI (10%), reflux, 15 h, 62%; (iii) HCI.H 2 NOBn or H 2 NOTHP, dry pyridine, rt, 15 h, 41 %; (iv) EDC.HCI, HOBT.H 2 0, 5-amino-1 ,3,4-thiadiazole-2-sulfonamide, dry DMF, 24 h, 54%; (v) 4M HCI in 1 ,4-dioxane, 0 °C, 8-10 h, 70%.
  • a pharmaceutical composition comprising an effective amount of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may include more than one compound of formula (I) or (II). When the composition includes more than one compound then the compounds may be in any ratio.
  • the composition may further comprise known co-actives, delivery vehicles or adjuvants.
  • the compound of formula (I) or (II), is present in the pharmaceutical composition in an amount sufficient to chemosensitize the cancer which is the subject of treatment. Suitable dosage forms and rates of the compounds and the pharmaceutical compositions containing such may be readily determined by those skilled in the art.
  • Diluents may include one or more of microcrystalline cellulose, lactose, mannitol, calcium phosphate, calcium sulfate, kaolin, dry starch, powdered sugar, and the like.
  • Binders may include one or more of povidone, starch, stearic acid, gums, hydroxypropylmethyl cellulose and the like.
  • Disintegrants may include one or more of starch, croscarme!iose sodium, crospovidone, sodium starch glycolate and the like.
  • Solvents may include one or more of ethanol, methanol, isopropanol, chloroform, acetone, methylethyl ketone, methylene chloride, water and the like.
  • Lubricants may include one or more of magnesium stearate, zinc stearate, calcium stearate, stearic acid, sodium stearyl fumarate, hydrogenated vegetable oil, glyceryl behenate and the like.
  • a glidant may be one or more of colloidal silicon dioxide, talc or cornstarch and the like.
  • Buffers may include phosphate buffers, borate buffers and carbonate buffers, although without limitation thereto.
  • Fillers may include one or more gels inclusive of gelatin, starch and synthetic polymer gels, although without limitation thereto.
  • Coatings may comprise one or more of film formers, solvents, plasticizers and the like.
  • Suitable film formers may be one or more of hydroxypropyl methyl cellulose, methyl hydroxyethyl cellulose, ethyl cellulose, hydroxypropyl cellulose, povidone, sodium carboxymethyl cellulose, polyethylene glycol, acrylates and the like.
  • Suitable solvents may be one or more of water, ethanol, methanol, isopropanol, chloroform, acetone, methylethyl ketone, methylene chloride and the like.
  • Plasticizers may be one or more of propylene glycol, castor oil, glycerin, polyethylene glycol, polysorbates, and the like.
  • composition may be in the form of a tablet, capsule, caplet, powder, an injectable liquid, a suppository, a slow release formulation, an osmotic pump formulation or any other form that is effective and safe for administration.
  • Compounds of the first aspect may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
  • Compounds of general formula (I) may also be administered in combination with cyclodextrins for enhanced aqueous solubility.
  • Dosage levels of the compound of first aspect may be of the order of about 1 ,000 ng to about 10,000 ng per kilogram body weight, with a preferred dosage range between about 2,000 ng to about 6,000 ng per kilogram body weight per day (including from about 3,000 ng to about 5,000 ng per kilogram body weight per day).
  • the amount of active ingredient which may be combined with the carrier materials to produce a single dosage will vary, depending upon the host to be treated and the particular mode of administration.
  • the compounds of the first aspect may additionally be combined with other compounds, in the composition of the second aspect, to provide an operative combination. It is intended to include any chemically compatible combination of pharmaceutically-active agents, as long as the combination does not eliminate the activity of the compound of the first aspect. In an embodiment, they are used in combination with therapeutic agents, for example anti-cancer agents to improve the efficacy of said anti-cancer agent.
  • therapeutic agents for example anti-cancer agents to improve the efficacy of said anti-cancer agent.
  • the invention thus provides in a further aspect a combination comprising a compound of the first aspect or a pharmaceutically acceptable salt or derivative thereof together with another therapeutically active agent, in particular an anti-cancer agent.
  • the pharmaceutical composition is for the treatment or prophylaxis of a disease, disorder or condition responsive to carbonic anhydrase XII inhibition.
  • the pharmaceutical composition is for the treatment or prevention of a disease, disorder or condition in a mammal.
  • a third aspect of the invention resides in a method of reducing the chemoresistance of a cancer in a patient including the step of administering an effective amount of a compound of formula (III), or a pharmaceutically effective salt thereof, to the patient:
  • each incidence of R a is independently selected from the group consisting of hydrogen, hydroxyl, halo, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi, optionally substituted aryl, optionally substituted heterocyclic, cyano, amino, carboxyl, optionally substituted O-alkyl, optionally substituted O-aryl, oxetane, homospiro- morpholine, OCF 3 , CF 3 , S-alkyl and SO 2 NHR 2 wherein R 2 is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi and optionally substituted aryl;
  • R b is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi and optionally substituted aryl;
  • R-i is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyi, optionally substituted heterocyclic and optionally substituted aryl;
  • W is selected from -(CH 2 )m- wherein m is from 1 to 6;
  • Y is selected from optionally substituted C1 to C12 alkyl, optionally substituted C1 to C12 alkenyl, optionally substituted C1 to C12 alkylether, optionally substituted C1 to C12 acyl, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted cycloalkyi, optionally substituted heteroaryl, and PEG; and
  • Z is a zinc binding group
  • R a , R-i , W, R b , Y, and Z may be as described for any embodiment of formula (I) or (II).
  • R c and R d may be as described for any embodiment of formula (I).
  • the compound of formula (III) may also have a structure as shown in formula (II) with R e , R f , R 9 , R h and R' being as previously described. [00120]
  • the compounds of formula (III) therefore include all of the compounds encompassed by formula (I) and formula (II), but additionally includes Psammaplin C.
  • CAXII inhibitors to indirectly reduce Pgp activity, may provide a selective and more effective cancer-targeting approach when administered with standard chemotherapeutic drugs to patients. Additionally, the separate administration within a defined time frame or co-administration of CAXII inhibitors may reduce the chemotherapy dosing required to achieve a reduction in tumour size.
  • a fourth aspect of the invention provides for a compound of formula (III), or a pharmaceutically effective salt thereof, for use in reducing the chemoresistance of a cancer in a patient.
  • a fifth aspect of the invention provides for use of a compound of formula (III), or a pharmaceutically effective salt thereof, in the manufacture of a medicament for reducing the chemoresistance of a cancer in a patient.
  • a sixth aspect of the invention resides in a method of modulating the activity of a carbonic anhydrase XII enzyme including the step of contacting the enzyme with a compound of formula (III).
  • a seventh aspect of the invention resides in a method of treating a cancer in a patient including the step of administering an effective amount of a compound of formula (III), or a pharmaceutically effective salt thereof, and an anti-cancer agent to the patient.
  • the compound of formula (III), or the pharmaceutically effective salt thereof, and the anti-cancer agent may be administered separately or may be co-administered. In any event, the two will be administered within a defined time frame in which the compound of formula (III) is operating to reduce the chemoresistance of the cancer.
  • An eighth aspect of the invention provides for a compound of formula (III), or a pharmaceutically effective salt thereof, and an anti-cancer agent for use in the treatment of a cancer in a patient.
  • a ninth aspect of the invention provides for use of a compound of formula (III), or a pharmaceutically effective salt thereof, and an anti-cancer agent in the manufacture of a medicament for the treatment of a cancer.
  • a method of diagnosing a cancer in a mammal including the step of administering a labelled compound of formula (III), or a pharmaceutically effective salt, thereof, to the mammal or to a biological sample obtained from the mammal to facilitate diagnosis of the cancer in the mammal.
  • the compounds of formula (III) are potent and direct inhibitors of CAXII. Accordingly, a chemical probe specific for CAXII, which is present in cancer cells has potential utility in diagnosing those cancers.
  • a CAXII activation probe comprising a compound of formula (III) could act as an effective surrogate biomarker of cancer for ex vivo (blood) or in vivo (MRI, PET etc.) diagnostics.
  • PET positron emission tomography
  • Typical isotopes include 11 C, 13 N, 15 O, 18 F, 64 Cu, 62 Cu, 124 l, 76 Br, 82 Rb and 68 Ga, with 18 F being the most clinically utilized.
  • An eleventh aspect of the invention resides in a complex of a compound of formula (III) and a carbonic anhydrase enzyme.
  • the carbonic anhydrase enzyme is a carbonic anhydrase IX or XII.
  • a carbonic anhydrase XII enzyme including a human carbonic anhydrase XII enzyme.
  • the cancer is one which is responsive to inhibition of carbonic anhydrase XII enzyme.
  • the cancer is one in which CAXII and Pgp are overexpressed. Any cancer cells/cancer stem cells where CAXII and Pgp proteins co-immunoprecipitate, as described in the immunoblotting component of the experimental section, may be an appropriate cancer for co-treatment using the compounds of the invention.
  • the cancer is selected from glioblastoma (GB), glioblastoma-derived stem cells (GB-SC), thyroid cancers, squamous lung cancers, gliomas, oral squamous cancer and esophageal squamous cell cancer, human colon cancer, lung cancer, breast cancer, osteosarcoma, and prostate, ovarian and pancreatic solid tumors.
  • GB glioblastoma
  • GB-SC glioblastoma-derived stem cells
  • thyroid cancers squamous lung cancers
  • gliomas gliomas
  • oral squamous cancer and esophageal squamous cell cancer human colon cancer
  • lung cancer breast cancer
  • osteosarcoma osteosarcoma
  • prostate ovarian and pancreatic solid tumors.
  • the anti-cancer agent may be selected from any known clinically useful anti-cancer agent.
  • the anti-cancer agent may be one useful in the treatment of cancers in which Pgp is overexpressed but which has its efficacy reduced due to chemoresistance.
  • the anti-cancer agent may be a second-line chemotherapeutic agent.
  • the anti-cancer agent may be selected from an alkylating agent, anti-tumour antibiotics, topoisomerase inhibitors, mitotic inhibitors, and any chemotherapeutic agent that is a substrate of Pgp and antimetabolites.
  • the anti-cancer agent may be selected from Altretamine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cyclophosphamide, dacarbazine, Doxorubicin, Daunorubicin, Epirubicin, Lomustine, Melphalan, Temozolomide, Thiotepa, Etoposide, Topotecan, Irinotecan, Vinblastine, and Vincristine.
  • the anti-cancer agent may be two or more anti-cancer agents.
  • the compound of formula (I), (II) or (III) is not Psammaplin C as shown below:
  • the compound below may be excluded from formula (I), (II) or (III):
  • the compounds may be identified by the below numerals where, for example, for compound 1 , the identifiers 1 and 741 are interchangeable.
  • the succinate ester intermediate was dissolved in anhydrous 1 ,4-dioxane (4 mL/mmol) under argon and a solution of ⁇ -aminoethanesulfonamide hydrochloride (1 .2 equiv) or 3-aminopropanamide (1 .2 equiv) and NEt 3 (1 .1 equiv) in anhydrous MeOH (5 mL/mmol) added.
  • the reaction mixture was stirred at room temperature overnight (12-14 h).
  • the solvent was evaporated in vacuo and the target compounds 18-22 were purified as described below.
  • the characterisation of compound 21 has been reported previously [Mujumdar, 2016].
  • U87-MG cells a commercially available line of glioma cells, were purchased from from ATCC (Manassas, VA) and authenticated by microsatellite analysis, using the PowerPlex kit (Promega Corporation, Madison, Wl; last authentication: December 2016).
  • Primary human GBM cells (CV17, 010627, No3) were obtained from surgical samples of three patients, operated on at the Department of Neuroscience, Neurosurgical Unit, Universities of Torino and Novara, Italy, or DIBIT San Raffaele, Milan, Italy. The samples are designated as "unknown patient number" (UPN) UPN 1 , UPN 2 and UPN 3.
  • UPN unknown patient number
  • AC adherent cells
  • NS stem-cell like cells
  • FBS penicillin-streptomycin
  • FBS fetal bovine serum
  • NS DMEM-F12 medium was supplemented with 1 M Hepes, 0.3 img/mL glucose, 75 pg/mL NaHCO 3 , 2 img/mL heparin, 2 img/mL bovine serum albumin, 2 imM progesterone, 20 ng/mL EGF, 10 ng/mL bFGF.
  • AC were obtained from dissociated NS cells, centrifuged at 1 ,200 ⁇ g for 5 min and seeded in AC medium. Morphological analysis and phenotypic characterization of differentiation and stemness markers, in vitro clonogenicity and self-renewal, in vivo tumorigenicity properties are detailed in [Caldera, 201 1 ; Riganti, 2013].
  • anti-CD133 Miltenyi Biotec, Bergisch Gladbach, Germany
  • anti-nestin Millipore, Billerica, MA
  • anti-Musashi-1 Millipore
  • Gal-C anti-galactocerebroside
  • FITC Fluorescein- isothiocyanate
  • TRITC rabbit anti-mouse tetramethyl rhodamine iso-thiocyanate
  • DAPI 4',6-diamidino-2-phenylindole
  • the observations were made by immunofluorescence on a Zeiss Axioskop microscope equipped with an AxioCam5MRSc and coupled to an imaging system (AxixoVision Release 4.5, Zeiss; 63 x oil immersion objective ; 10 x ocular lens). For each experimental point, a minimum of 5 microscopic fields were examined.
  • the percentage of cells positive for general stemness markers was further quantified by flow cytometry.
  • Cells were washed with PBS, detached with Cell Dissociation Solution (Sigma Chemical Co.), re-suspended in culture medium containing 5 ⁇ /100 ml FBS, incubated with antibodies recognizing Nanog (Cell Signaling Technology, Danvers, MA), Oct4 (Cell Signaling Technology), SOX2 (Biolegend, San Diego, CA) and ABCG2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by the secondary fluorescein isothiocyanate (FITC)-conjugated antibody (30 min at 4 °C) and fixation in 25 pg/ml paraformaldehyde.
  • FITC fluorescein isothiocyanate
  • Aldehyde dehydrogenase-Based Cell Detection Kit (Stemcell Technologies, Vancouver, Canada) was used to calculate the percentage of Aldehyde dehydrogenase (ALDH) br ' 9ht cells, 5 x 1 0 5 cells were analyzed by the Guava® easyCyte flow cytometer, using the InCyte software (Millipore). Control experiments included incubation of cells with nonimmune isotypic antibody, followed by secondary antibody.
  • the cells were rinsed with ice-cold lysis buffer (50 mM, Tris, 1 0 imM EDTA, 1 % v/v Triton-X1 00), supplemented with the protease inhibitor cocktail set III (80 ⁇ aprotinin, 5 mM bestatin, 1 .5 mM leupeptin, 1 mM pepstatin; Calbiochem, San Diego, CA), 2 mM phenylmethylsulfonyl fluoride and 1 mM Na 3 VO 4 , sonicated and centrifuged at 1 3,000 x g for 1 0 min at 4 °C.
  • protease inhibitor cocktail set III 80 ⁇ aprotinin, 5 mM bestatin, 1 .5 mM leupeptin, 1 mM pepstatin; Calbiochem, San Diego, CA
  • 2 mM phenylmethylsulfonyl fluoride and 1 mM Na 3 VO
  • the membranes were washed with Tris-buffered saline-Tween 0.1 % v/v solution, and the proteins were detected by enhanced chemiluminescence (Bio-Rad Laboratories). Plasma membrane-associated proteins were evaluated in biotinylation assays, using the Cell Surface Protein Isolation kit (Thermo Fisher Scientific Inc., Waltham, MA), as previously reported [De Boo, 2009]. An anti- pancadherin antibody (H-300; Santa Cruz Biotechnology Inc.) was used to confirm equal protein loading.
  • cells were washed once in PBS, twice with 10 imM Hepes/Hank's balanced salt solution, fixed with 4% v/v PFA in PBS for 5 min. After a washing step in Hepes, cells were permeabilized in 0.1 % w/v saponin/Hepes and incubated with an anti-ABCB1 /Pgp (MRK16; Kamiya, Seattle, WA) antibody 45 min at 4 °C, washed in 0.1 % w/v saponin/Hepes, incubated with a secondary anti-mouse FITC-conjugated antibody for 30 min at 4 °C, washed twice in 0.1 % w/v saponin/Hepes and once in Hepes.
  • an anti-ABCB1 /Pgp MRK16; Kamiya, Seattle, WA
  • PKA Proximity ligation assay
  • CAXII-Pgp interaction was measured with the DuoLink In Situ kit (Sigma Chemicals Co.), using a mouse anti-human Pgp (F4; Sigma Chemical Co.) and a rabbit anti-human CAXII (2310047E01 Rik; NovoPro, Shangai, China) antibody, respectively, as per the manufacturer's instructions. Nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Cells were examined using a Leica TCS SP2 AOP confocal laser-scanning microscope (10 ⁇ ocular lens; 63 ⁇ objective lens; Leica Microsystem, Wetzlar, Germany). For each experimental condition, a minimum of five fields were examined.
  • Doxorubicin content was measured fluorimetrically as detailed elsewhere [Riganti, 2005]. The results were expressed as nmol doxorubicin/mg cell proteins, according to a titration curve previously set. Temozolomide content was measured by incubating cells with 10 ⁇ [ 3 H]-temozolomide (0.7 Ci/ml; Moravek Biochemical Inc., Brea, CA). The amount of [ 3 H]-temozolomide in cell lysate was measured by liquid scintillation counting. The results were expressed as nmol [ 3 H]-temozolomide/mg cell proteins, according to a titration curve previously set.
  • LDH activity was measured in the extracellular medium and in the cell lysate: 50 ⁇ of supernatant from extracellular medium or 5 ⁇ of cell lysate were incubated at 37°C with 5 imM NADH. The reaction was started by adding 20 imM pyruvic acid and was followed for 6 min, measuring absorbance at 340 nm with a Packard EL340 microplate reader (Bio-Tek Instruments, Winooski, VT). The reaction kinetics was linear throughout the time of measurement. Both intracellular and extracellular enzyme activities were expressed as ⁇ NADH oxidized/min/dish, then extracellular LDH activity was calculated as percentage of the total LDH activity in the dish.
  • RNA vector CRISPR pCas guide vector, Origene, Rockville, MD
  • 1 g non-targeting vector mixed with 1 g donor DNA vector (Origene), following the manufacturer's instructions.
  • Stable KO cells were selected in medium containing 1 g/mL puromycin for six weeks. The efficacy of Pgp and CAXII KO was evaluated by immunoblotting, as reported above. in vivo tumor growth
  • mice were randomized into the following groups (10 animals/group) and treated with 2 cycles of 5 consecutive days (days: 1 -5; 1 1 -15 after randomization) as follows: 1 ) control group, treated with 0.2 imL saline solution intravenously (i.v.); 2) 741 low dose (LD) group, treated with 38 ng/kg compound 741 (in 0.2 imL saline solution; final concentration: 10 nM) i.v.; 3) 741 high dose (HD) group, treated with 3800 ng/kg compound 741 (in 0.2 imL saline solution; final concentration: 1 ⁇ ) i.v.; 4) TMZ group, treated with 50 mg/kg TMZ per os (p.o.); 5) TMZ + 741 LD group, treated with 50 mg/kg TMZ p.o.
  • TMZ + 741 HD group with 50 mg/kg TMZ p.o. and 3800 ng/kg compound 741 i.v.
  • the dose of TMZ i.e. the dose that significantly reduced the growth of AC-derived tumors but was significantly less effective in NS-derived tumors
  • the dose of TMZ was chosen after trying different experimental protocols (detailed in the legend of Figure 14, panel A). Tumor volumes were monitored daily by caliper and animals were euthanized by injecting zolazepam (0.2 ml/kg) and xylazine (16 mg/kg) intramuscle (i.m.) at day 30. Tumors were excised and photographed immediately after mice sacrifice.
  • Tumor growth was monitored by in vivo bioluminescence (Xenogen IVIS Spectrum, PerkinElmer, Waltham, MA) at day 6, 14 and 24 post-implantation.
  • animals were randomized into the following groups (6 animals/group) and treated with 2 cycles of 5 consecutive days (days: 7-1 1 ; 17-21 after randomization) as it follows: 1 ) control group, treated with 0.2 imL saline solution i.v.; 2) 741 group, treated with 3800 ng/kg compound 741 (in 0.2 imL saline solution; final concentration: 1 ⁇ ) i.v.; 3) TMZ group, treated with 50 mg/kg TMZ p.o.; 4) TMZ + 741 group, treated with 50 mg/kg TMZ p.o.
  • mice were randomized into the following groups (6 animals/group) and treated with 2 cycles of 5 consecutive days (days: 7-1 1 ; 17-21 after randomization) as it follows: 1 ) control group, treated with 0.2 mL saline solution i.v.; 2) 729 group, treated with compound 729 (in 0.2 mL saline solution; final concentration: 1 ⁇ ) i.v.; 3) TMZ group, treated with 50 mg/kg TMZ p.o. ; 4) TMZ + 729 group, treated with 50 mg/kg TMZ p.o. and 1 ⁇ of compound 729 i.v., respectively. Animals were euthanized at day 30, as reported.
  • the hemocromocytometric analysis was performed with a UniCel DxH 800 Coulter Cellular Analysis System (Beckman Coulter, Miami, FL) on 0.5 ml of blood collected immediately after mice sacrifice.
  • the hematochemical parameters LDH, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), creatinine, CPK were measured on the same blood samples, using the respective kits from Beckman Coulter Inc. Animal care and experimental procedures were approved by the Bio-Ethical Committee of the Italian Ministry of Health (#122/2015-PR). in Vitro Plasma Stability
  • Mouse plasma (Animal Resource Centre, Perth, Australia, pooled from multiple mice) was stored frozen at -80 °C. On the day of the experiment, frozen plasma was thawed in a water bath maintained at 37 °C. Compound 741 was spiked into plasma to a nominal concentration of 1000 ng/mL (final DMSO and acetonitrile concentrations were 0.2 and 0.4% v/v, respectively), vortex mixed and then aliquoted (50 ⁇ ). Spiked plasma aliquots were incubated at 37 °C for 4 h, and at various time points, triplicate plasma samples were taken and immediately snap-frozen in dry ice.
  • the metabolic stability assay was performed in mouse liver microsomes (Xenotech, Tokyo, Japan, lot#1510043). Compounds 741 and 729 (1 ⁇ ) were incubated with liver microsomes at a final protein concentration of 0.4 img/mL at 37 °C. The metabolic reaction was initiated by the addition of a NADPH-regenerating system, and subsequently quenched with acetonitrile (containing diazepam as internal standard) at 2, 30 and 60 min. Compounds were also incubated in the absence of NADPH cofactor to monitor the non- cytochrome P450-mediated metabolism in the microsomal matrix.
  • a species scaling factor [Ring, 201 1 ] was used to convert the in vitro clearance (CL int ) ⁇ L/min/mg) to an in vivo CL mX (mL/min/kg).
  • Hepatic blood clearance and the corresponding hepatic extraction ratio (E H ) were calculated using the well- stirred model of hepatic extraction according to the "in vitro T 1/2 " approach described in [Obach, 1999].
  • the E H was then used to classify compounds as low ( ⁇ 0.3), intermediate (0.3-0.7), high (0.7-0.95) or very high (> 0.95) extraction compounds and are shown in table 7. Studies were performed by Centre for Drug Candidate Optimisation, Monash University, Melbourne, Australia.
  • Compound 1 (0.25 to 20 ⁇ ) was incubated with CYP substrate in human liver microsomes (batch #1410230; XenoTech LLC, Lenexa, KS, USA) at 37 °C. The total organic solvent concentration was 0.47% v/v. The specific incubation conditions for each CYP isoform/substrate are summarized in Table 1 . The reactions were initiated by the addition of a NADPH-regenerating system and quenched by the addition of ice cold acetonitrile containing analytical internal standard (0.15 pg/mL diazepam).
  • Metabolite concentrations in quenched samples were determined by UPLC-MS (Waters/Micromass Xevo TQD triple-quadrupole) relative to calibration standards prepared in quenched microsomal matrix.
  • the inhibitory effect of compound 1 was assessed based on the reduction in the formation of the specific CYP-mediated metabolite relative to a control for maximal CYP enzyme activity.
  • Glioblastoma-derived stem cells co-express CAXII and Pgp
  • Inhibitors of CAXII reduce Pgp activity in glioblastoma-derived stem cells, increasing retention and cytotoxicity of Pgp substrates
  • CAXII inhibition enhances temozolomide cytotoxicity in neurospheres by reducing Pgp activity
  • TMZ is a substrate of Pgp.
  • compound 1 was the most effective in restoring the effects of Pgp substrates in resistant GB NS, this compound was selected for further characterization in TMZ-treated cells.
  • TMZ downregulated Pgp expression (Figure 3a; Figure 15a) and activity (Figure 3b) in NS, consistent with previous observations.
  • Compound 1 did not change Pgp expression in NS, either when used alone or when used in combination with TMZ ( Figure 3a; Figure 15a).
  • Compound 1 reduced Pgp-ATPase activity in NS, while the combination of compound 1 with TMZ showed a further reduction on Pgp-ATPase activity (Figure 3b).
  • CAXII inhibition with compound 729 enhances temozolomide cytotoxicity in glioblastoma-derived stem cells by reducing Pgp activity
  • TMZ did not increase the release of LDH ( Figure 4D), nor activate caspase 3 (Figure 4E) or reduce viability in NS ( Figure 4F).
  • the co-incubation of TMZ with compound 729 did however increase TMZ accumulation in NS ( Figure 4C) and restored its cytotoxic effects to the extent that it was as effective as TMZ cytotoxicity in AC ( Figure 4D-F).
  • a UPN2 clone knocked-out for Pgp but with unaltered CAXII levels Figure 13 was produced.
  • KO NS (Pgp-negative/CAXII- positive) had the same response to TMZ than wild-type (Pgp-positive/CAXII- positive) NS treated with compound 729 and TMZ or AC (Pgp-negative/CAXII- negative; Figure 4C-F).
  • CAXII knocking out restores sensitivity to temozolomide in neurospheres
  • CAXII inhibition enhances the cytotoxicity of temozolomide in combination with Pgp substrates in neuropsheres
  • CAXII inhibition with compound 729 enhances the cytotoxic effect of temozolomide and Pgp substrates in glioblastoma-derived neuropsheres subjected to combination treatments
  • CAXII inhibition restores the efficacy of temozolomide in tumors derived from resistant glioblastoma neurospheres in vivo
  • compound 1 was administered at two dosages, 38 ng/kg and 3800 ng/kg, in mice bearing patient 2-derived NS: the lower concentration was chosen according to the CAXII j; the latter concentration was chosen to increase the amount of compound 1 that reached the tumor, balancing the haematic and lymphatic clearance.
  • Compound 1 alone did not reduce NS-derived tumor growth, but when combined with TMZ it significantly enhanced the anti-tumor efficacy of TMZ against NS-derived tumors, in particular - as expected - at the higher dose ( Figure 14b-c).
  • compound 1 caused no hematopoiesis, liver, kidney or muscle toxicity.
  • TMZ slightly reduced the number of platelets, however the co-administration of TMZ and compound 1 did not further decrease this parameter (Table 6).
  • compound 729 was not toxic for haematopoiesis, liver, kidney and muscles according to the haematochemical parameters of the animals at the time of sacrifice, and did not worsen the TMZ-induced decrease in platelets.
  • the experiments were repeated for all 3 patient samples (UPN1 , 2 and 3) and the results shown in Figure 17.
  • CAXII and Pgp co-expression are a hallmark of chemoresistance in GB SC.
  • the results presented above show that CAXII expression is increased in GB SC derived from primary tumors.
  • the trend of CAXII inhibition by the compounds discussed above was 729 ⁇ 741 > 739 > 744 > 737 while 787 is not an inhibitor of CAXII.
  • Compound 787 is not expected to exhibit any CA inhibition properties as it lacks the primary sulfonamide functional group for interaction with the CA active site Zn and was employed as a control.
  • Compounds 741 , 729 and 739 were also the strongest inhibitors of Pgp ATPase activity; accordingly, they rescued the cytotoxic efficacy of the Pgp substrates doxorubicin, etoposide, topotecan and irinotecan.
  • These topoisomerase II and I inhibitors are not included in first-line treatment for GB, however they are under evaluation in preclinical models and/or in phase l/ll clinical trials as second-line treatments.
  • CAXII inhibitors, including those disclosed herein, may represent useful enhancers of these agents, with the added value of being particularly effective against the GB NS. This is of great importance as an improvement in therapy against the SC-component of GB is an area of high need.
  • compound 741 and compound 729 have improved potency for CAXII and compound 741 has greater selectivity for CAXII over all other CA isozymes compared to acetazolamide (Table 4).
  • This improved potency and selectivity provides an enhanced therapeutic window and also has beneficial implications to the effective dosage of the chemotherapeutic agent used.
  • an added value of the compounds reported here is their high efficacy against CAXII-positive GB SC, the most difficult GB component to be eradicated.
  • CAXII has minimal expression in healthy cells (https://www.proteinatlas.orq/ENSG00000074410-CA12/tissue)
  • the present approach differs from direct Pgp inhibition as the compounds disclosed target a Pgp-adjuvant protein - i.e. CAXII - that is specifically expressed in tumor cells and poorly expressed in most healthy cells.
  • CAXII inhibitors may thus provide a selective, safer, and more effective GB-targeting approach when administered with either TMZ or with second-line chemotherapeutic drugs (provided the drug is a substrate of Pgp).
  • the exemplified combination treatments of GB second-line chemotherapeutic drugs (Tonder 2014), (Reynes 2016), topoisomerase I and II inhibitors, plus compound 1 demonstrate that potent and selective CAXII inhibition may rescue the efficacy of these drugs against NS.
  • the co-administration of CAXII inhibitors, TMZ and the second-line chemotherapeutic drugs was even more effective against GB NS. If translated into a clinical setting the use of CAXII inhibitors may have the potential to reduce the chemotherapy dose of TMZ and/or Pgp substrates required to achieve a significant GB reduction.
  • TMZ improved efficacy of TMZ can be explained by the co-expression in NS-derived tumors of CAXII and Pgp: as occurred in NS cultures, it is also hypothesized that, in vivo, compound 1 indirectly reduced Pgp activity by inhibiting CAXII. This mechanism may increase intratumor retention of TMZ and improve the drugs anti-tumor efficacy, independent of other resistance factors, such as MGMT status. Indeed, the combination treatment of compound 1 and TMZ reduced Pgp-expression and tumor cell proliferation, and increased intratumor apoptosis, producing in vivo the same cell death mechanisms observed in primary NS cultures in vitro.
  • a further notable result is that the combination of compound 741 , TMZ and inhibitors of topoisomerase I (topotecan, irinotecan) or II (etoposide, doxorubicin) were highly effective in inducing cell death and reducing cell viability of GB SC. It is postulated that compound 741 increased the retention of TMZ and topoisomerase inhibitors owing to its indirect inhibition exerted on Pgp activity, thus producing synergistic cytotoxic effects between TMZ and topoisomerase l/ll inhibitors. This same effect/trend occurred with the combination of compound 729, TMZ and inhibitors of topoisomerase I (topotecan, irinotecan) or II (etoposide, doxorubicin).
  • Compound 741 is shown to be effective in rescuing the antitumor activity of TMZ in xenografts derived from primary TMZ-refractory NS. Either a very low dosage (i.e. 10 nM, ⁇ 12x the CAXII i) or a 100-fold higher dosage (i.e. 1 ⁇ ) systemically administered significantly reduced NS-derived tumor growth. As expected, the higher dosage was more effective and reduced the growth of NS-derived tumors as TMZ-alone did in sensitive AC-derived tumors. Moreover, at both dosages compound 741 did not elicit liver, kidney or muscle toxicity, and did not worsen the platelet reduction elicited by TMZ.
  • CAXII inhibition indirectly reduces P-glycoprotein activity in neurospheres to restore sensitivity to temozolomide.
  • the overall survival in orthotopic patient-derived xenografts of temozolomide-resistant neurospheres, co-dosed with Psammaplin C and temozolomide, is significantly increased over temozolomide only, without detectable signs of systemic toxicity.
  • the compounds hereby claimed, as CAXII inhibitors, in combination with anti-cancer agents, such as temozolomide for example may provide a new and effective approach to reverse chemoresistance and improve outcomes of certain cancers, including glioblastoma.
  • CYP cytochrome P450
  • Table 1 In vitro cytochrome P450 (CYP) isoforms (CYPs 1 A2, 2C9, 2C19, 2D6 and 3A4) metabolism data for compound 1 in human liver microsomes. a Where less than 50% inhibition was observed at 20 ⁇ (i.e. the highest concentration tested), the IC 50 value is deemed to be >20 ⁇ . n.m.i. No measurable inhibition of CYP activity at the highest concentration of the test compound used.
  • CYP cytochrome P450
  • Radiotherapy 60 Gy (30 fractions).
  • Chemotherapy 75 mg/m 2 temozolomide (TMZ), per os, daily, concurrently to radiotherapy, followed by 200 mg/m 2 TMZ, per os, days 1 -5, every 28 days, 6 cycles.
  • Post-recurrence therapy radiotherapy: 60 Gy (30 fractions); chemotherapy: 80 mg/m 2 carmustine (BCNU), days 1 -3, every 8 weeks, 3 cycles.
  • Time to recurrence interval between the surgery and the appearance of tumor relapse at magnetic resonance imaging (MRI).
  • Overall survival interval between diagnosis and patient death.
  • UPN unknown patient number.
  • MGMT O 6 -methylguanine-DNA methyltransferase. Fully methylated: promoter methylation of both alleles; partially methylated: promoter methylation of one allele.
  • EGFR epithelial growth factor receptor. Amplified: > 2 copies of EGFR genes; not amplified: ⁇ 2 copies of EGFR gene. IDH: isocitrate dehydrogenase. Markers UPNl UPNl UPN2 UPN2 UPN3 UPN3 AC NS AC NS AC NS AC NS
  • Adherent cells (AC) and neurospheres (NS) were analyzed by immunofluorescence microscopy for the neural sternness markers CD133, nestin, Musashi-1 , and for the neural differentiation markers glial fibrillary acidic protein (GFAP) and galactocerebroside (Gal-C), Positivity of staining were graded as it follows: -: undetectable; +/-: low expression ( ⁇ 25% cells positive); +: moderate expression (26%-74% cells positive); ++: high expression (> 75% cells positive).
  • the percentage of Nanog-, Oct4- SOX2-,ABCG2- and aldehyde dehydrogenase (ALDH) b ⁇ w -positive cells was determined by flow cytometry.
  • mice treated with saline solution mice treated with 38 ng/kg compound 1 i.v.
  • 1 HD mice treated with 3800 ng/kg compound 1 i.v.
  • TMZ mice treated with 50 mg/kg temozolomide (TMZ) p.o. *p ⁇ 0.05: vs. Ctrl group.
  • Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/p-catenin pathway. Neuro Oncol. 2013 Nov;15(1 1 ):1502-17.

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Abstract

L'invention concerne des composés qui sont des inhibiteurs de l'enzyme CAXII. Selon la présente invention, et à cause de l'interaction entre CAXII et Pgp, de tels composés peuvent être utiles dans la réduction de la chimiorésistance d'un cancer permettant une co-administration avec des agents anticancéreux existants.
PCT/AU2018/050443 2017-05-12 2018-05-11 Inhibiteurs d'anhydrase carbonique WO2018204987A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115925675A (zh) * 2022-11-10 2023-04-07 中国海洋大学 一种Psammaplin A类衍生物及其制备方法和应用

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DE19860543A1 (de) * 1998-12-23 2000-06-29 Knoell Hans Forschung Ev Verwendung substituierter Tyrosinderivate als Protease-Inhibitoren

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DATABASE CAPLUS retrieved from STN Database accession no. 2014:46891 *
KOPECKA, J. ET AL.: "Carbonic anhydrase XII is a new therapeutic target to overcome chemoresistance in cancer cells", ONCOTARGET, vol. 6, no. 9, pages 6776 - 6793, XP055555604 *
KOTTAKOTA, S. K. ET AL.: "Versatile Routes to Marine Sponge Metabolites through Benzylidene Rhodanines", ORGANIC LETTERS, vol. 14, no. 24, 2012, pages 6310 - 6313, XP055555588 *
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MUJUMDAR, P. ET AL.: "An Unusual Natural Product Primary Sulfonamide: Synthesis, Carbonic Anhydrase Inhibition, and Protein X-ray Structures of Psammaplin C", JOURNAL OF MEDICINAL CHEMISTRY, vol. 59, no. 11, 2016, pages 5462 - 5470, XP055555572 *
NICHOLAS, G. M. ET AL.: "Inhibition and Kinetics of Mycobacterium Tuberculosis and Mycobacterium Smegmatis Mycothiol-S-conjugate Amidase by Natural Product Inhibitors", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 11, no. 4, 2003, pages 601 - 608, XP055555565 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115925675A (zh) * 2022-11-10 2023-04-07 中国海洋大学 一种Psammaplin A类衍生物及其制备方法和应用

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