WO2018204987A1

WO2018204987A1 - Carbonic anhydrase inhibitors

Info

Publication number: WO2018204987A1
Application number: PCT/AU2018/050443
Authority: WO
Inventors: Chiara RIGANTI; Iris Chiara SALAROGLIO; Prashant MUJUMDAR; Sally-Ann POULSEN
Original assignee: Griffith University
Priority date: 2017-05-12
Filing date: 2018-05-11
Publication date: 2018-11-15
Also published as: AU2017203184A1

Abstract

Compounds are provided which are inhibitors of the CAXII enzyme. Due to the interaction between CAXII and Pgp, such compounds may be useful in lowering the chemoresistance of a cancer allowing for co-administration with existing anti-cancer agents.

Description

CARBONIC ANHYDRASE INHIBITORS

FIELD OF THE INVENTION

[0001 ] 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.

BACKGROUND TO THE INVENTION

[0002] Any reference to background art herein is not to be construed as an admission that such art constitutes common general knowledge in Australia or elsewhere.

[0003] Carbonic anhydrases (CA) are zinc metalloenzymes that contribute to pH regulation through catalysis of the reversible hydration of carbon dioxide to bicarbonate and a proton: CO₂ + H₂O ¾ HCO₃ ^" + H⁺. In contrast to other CA members, CAIX and CAXII are highly expressed in the hypoxic core of solid tumors, where they drive tumor growth and metastasis. Importantly, 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.

[0004] Since 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.

[0005] It has been demonstrated that 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.

[0006] Most compounds that are known to directly inhibit Pgp have failed in preclinical and clinical settings as a consequence of their low specificity and high toxicity. It is therefore desirable to provide new inhibitors of CAXII to maintain or improve the efficacy of anti-cancer agents and to assist with the problem of chemoresistance based on Pgp clearance of current anti -cancer agents.

SUMMARY OF INVENTION

[0007] According to a first aspect of the invention, there is provided a compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof:

Formula (I) wherein, 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, OCF₃, CF₃, S- alkyl and SO₂NHR₂ wherein R₂ 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 -(CH₂)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 -CH₂-, R^b and Ri are hydrogen, and Y is -CH₂CH₂-, then Z is not -S(O)₂NH₂.

[0008] According to a second aspect of the invention there is provided 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.

[0009] 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:

Formula (III) wherein, 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, OCF₃, CF₃, S-alkyl and SO₂NHR₂ wherein R₂ is selected from 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 -(CH₂)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.

[0010] 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.

[001 1 ] 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.

[0012] 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).

[0013] 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.

[0014] 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.

[0015] 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.

[0016] In a tenth aspect of the invention there is provided 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. [0017] An eleventh aspect of the invention resides in a complex of a compound of formula (III) and a carbonic anhydrase enzyme.

[0018] The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, mutatis mutandis. Consequently features specified in one section may be combined with features specified in other sections as appropriate.

[0019] Further features and advantages of the present invention will become apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order that the invention may be readily understood and put into practical effect, preferred embodiments will now be described by way of example with reference to the accompanying figures wherein :

[0021 ] 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). A. Cells were lysed and immunoblotted with the indicated antibodies. The figure is representative of one out of three experiments. B. Cell surface expression of CAXII and Pgp was detected by flow cytometry in replicate. The histograms are representative of one out of three experiments. C. 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. The figure is representative of one out of three experiments. 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). [0022] FIG 1 B: Expression levels of Pgp and CAXII in AC and NS in different culture conditions. Adherent cells (AC) 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. At each passage, the expression of CAXII and Pgp was measured by immunoblotting. The figure is representative of one out of three experiments with similar results. β-Tubulin was used as control of equal protein loading. UPN: unknown patient number.

[0023] 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. For panels B- D: pooled data for UPN1 -3 are presented as means±SD (n=3). 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. untreated (-) cells: ^***p<0.001 : compounds 1 and 3 vs. untreated (-) cells (two- way ANOVA). C. Fluorimetric detection of doxorubicin (dox) accumulation, measured in duplicates in cells treated 24 h with 5 μΜ dox, alone (-) or in the presence of 10 nM compounds 1-5. ^*p<0.05: NS treated with compound 4 vs corresponding AV ^***p<0.001 : untreated NS or treated with compounds 2 and 5 vs corresponding AC; ^##p<0.002: NS treated with compound 1 and 3 versus untreated (-) NS (two-way ANOVA). D. Release of LDH, measured spectrophotometrically in duplicates, in cells grown for 24 h in fresh medium (-) or in media containing 10 nM compounds 1-5, in the absence or presence of 5 μΜ dox. ^*p<0.05: treated AC/NS vs. corresponding "- dox" cells; ^***p<0.001 : treated AC/NS vs. corresponding "- dox" cells; ^##p<0.002: NS treated with compound 1 and 3 versus "+dox" NS (two-way ANOVA). E. Viability of cells, measured by a chemiluminescent-based assay in quadruplicates, after 72 h in fresh medium (-) or in media containing 10 nM compounds 1-5, in the absence or presence of 5 μΜ dox. ^***p<0.001 : t treated AC/NS vs. corresponding "- dox" cells; ^###p<0.001 : NS treated with compound 1 , 3 and 4 versus "+dox" NS (two- way ANOVA).

[0024] 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 μΜ temozolomide (T) or 10 nM compound 1 (741), alone or in association. Panels B-C-D-F: pooled data for UPN1 -3 are presented as means+SD (n=4). Lower circle, uppermost circle and middle circle represent the mean±SD of technical replicates of UPN1 , UPN2 and UPN3. A. UPN2 NS were lysed and immunoblotted for Pgp and CAXII. The figure is representative of one out of three experiments. 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). E. 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. KO1 /KO2 cells (two-way ANOVA). [0025] 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 μΜ temozolomide (T) or 10 nM compound 729, alone or in association. A. Cells were lysed and immunoblotted for Pgp and CAXII. β- tubulin level was used as control of equal protein loading. The figure is representative of one out of three experiments with similar results. B. The Pgp ATPase activity was measured spectrophotometrically on Pgp-rich vesicles extracted from membrane fractions. Data are presented as means + SD (n = 3). ^* p <0.01 : vs. untreated (-) cells; ° p < 0.005: vs. temozolomide-treated cells. C. The intracellular content of temozolomide (TMZ) was measured radiometrically. NS knocked out for Pgp (KOPgp) and AC were included as control of cells with undetectable or very low expression of Pgp. Data are presented as means + SD (n = 4). ^* p < 0.001 : vs. untreated (-) cells; ° p < 0.001 : vs. temozolomide- treated cells. D. The release of LDH in the extracellular medium was measured spectrophotometrically. Data are presented as means + SD (n = 4). ^* p < 0.001 : vs. untreated (-) NS or AC; ° p < 0.001 : vs. temozolomide-treated cells. E. Cells were lysed and immunoblotted for procaspase and cleaved caspase 3. β- tubulin level was used as control of equal protein loading. The figure is representative of one out of three experiments with similar results. F. The absorbance of viable cells was measured spectrophotometrically. Data are presented as means + SD (n = 4). ^* p < 0.01 : vs. untreated (-) NS or AC; ° p < 0.001 : vs. temozolomide-treated cells.

[0026] 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). When indicated, 50 μΜ 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. Panels B-C-D-F: pooled data for UPN1 -3 are presented as means+SD (n=4). Lower circle, uppermost circle and middle circle represent the mean+SD of technical replicates of UPN1 , UPN2 and UPN3. A. 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).

[0027] 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 μΜ temozolomide (T), 5 μΜ doxorubicin (dox), 10 μΜ etoposide (eto), 10 μΜ topotecan (top), 10 μΜ irinotecan (iri), in different combinations. Panels A-B: data are presented as means+SD (n=4). A. LDH release, measured spectrophotometrically in duplicates. ^***p<0.001 : all experimental conditions vs. untreated (-) cells; ^ooop<0.001 : vs. dox/eto/top/iri-treated cells; ^###p<0.001 : vs. cells treated with drug+compound 1 ; ^§§§p<0.001 : vs. cells treated with drug+T (two-way ANOVA). B. Cells were lysed and immunoblotted for procaspase and cleaved caspase 3. The figure is representative of one out of three experiments. C. Cell viability, measured by a chemiluminescent-based assay in quadruplicates. ^***p<0.001 : all experimental conditions vs. untreated (-) cells; ^ooop<0.001 : vs. 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).

[0028] 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 μΜ temozolomide (T), 5 μΜ doxorubicin (dox), 10 μΜ etoposide (eto), 10 μΜ topotecan (top), 10 μΜ irinotecan (iri), in different combinations. A. The release of LDH in the extracellular medium was measured spectrophotometrically. Data are presented as means + SD (n = 4). ^* p < 0.001 : vs. untreated (-) cells; ° p < 0.001 : vs. dox/eto/top/iri-treated cells; ^# p < 0.001 : vs. cells treated with compound 729 or temozolomide alone. B. Cells were lysed and immunoblotted for procaspase and cleaved caspase 3. β-tubulin level was used as control of equal protein loading. The figure is representative of one out of three experiments with similar results. C. The absorbance of viable cells was measured spectrophotometrically. Data are presented as means + SD (n = 4). ^* p < 0.001 : vs. untreated (-) cells; ° p < 0.002: vs. dox/eto/top/iri-treated cells; ^# p < 0.02: vs. cells treated with compound 729 or temozolomide alone.

[0029] 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. +3800 ng/kg compound 1 i.v. (6 animals/group). B. Quantification of UPN1 - 3 NS-derived bioluminescence, taken as index of tumor growth. Data are presented as means+SD (6 animals/group). At day 24: ^**p<0.005, ^***p < 0.001 : TMZ+1 group vs. all the other groups of treatment;^oop<0.005, ⁰⁰⁰p <0.01 TMZ+1 group vs TMZ-group (two-way ANOVA). C. Overall survival probability was calculated using the Kaplan-Meier method. UPN1 : p < 0.02: TMZ+1-group vs. all the other groups of treatment. 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). D. Representative intratumor staining with hematoxylin and eosin (HE) or the indicated antibodies. The photographs are representative of sections from 5 tumors/group of treatment. Bar=10 μιη (10χ ocular lens, 20χ objective). E. Quantification of immunohistochemical images, performed on sections with 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 CAXII, Pgp and caspase 3-positive cells was determined by Photoshop program. Data are presented as means+SD. ^***p < 0.001 : TMZ+1 group vs. all the other groups of treatment; ^coop <0.01 TMZ+1 group vs TMZ-group (two-way ANOVA).

[0030] FIG 9: Compound 729 improves temozolomide efficacy against orthotopically implanted glioblastoma stem cells-derived tumors. 1 χ 10⁶

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. At day 7 after implantation, 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 μΜ) i.v.; 3) temozolomide (TMZ) group, treated with 50 mg/kg TMZ p.o. ; 4) 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. A. Representative in vivo bioluminescence imaging, performed on days 6, 14, and 24 after implantation. 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. untreated (-) cells; ° p < 0.02 vs TMZ-treated cells. 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 μιη). 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).

[0031 ] 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 μΜ etoposide (eto). Pooled data for UPN1 -3 are presented as means+SD (n=4). 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. corresponding "- eto" cells; ^###p<0.001 : NS treated with compound 1 , 3 and 4 versus "+ eto" NS (two-way ANOVA). b. Viability of cells, measured by a chemilunescent-based assay in quadruplicates. ^***p<0.001 : treated AC/NS vs. corresponding "- eto" cells; ^###p<0.001 : NS treated with compound 1 , 3 and 4 versus "+eto" NS (two-way ANOVA). [0032] FIG 1 1 : Effects of CAXII inhibitors on topotecan 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 μΜ topotecan (top). Pooled data for UPN1 -3 are presented as means+SD (n=4). 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. corresponding "- top" cells; ^###p<0.001 : NS treated with compound 1 , 3 and 4 versus "+ top" NS (two-way ANOVA). b. Viability of cells, measured by a chemilunescent-based assay in quadruplicates. ^***p<0.001 : treated AC/NS vs. corresponding "- top" cells; ^###p<0.001 : NS treated with compound 1 , 3 and 4 versus "+ top" NS (two-way ANOVA).

[0033] 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 μΜ irinotecan (iri). Pooled data for UPN1 -3 are presented as means+SD (n=4). 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<0.01 , ^***p<0.001 : treated AC/NS vs. corresponding "- iri" cells; ^##p<0.01 , ^###p<0.001 : NS treated with compound 1 , 3 and 4 versus "+ iri" NS (two-way ANOVA). b. Viability of cells, measured by a chemilunescent-based assay in quadruplicates. ^***p<0.001 : treated AC/NS vs. corresponding "- iri" cells; ^###p<0.001 : NS treated with compound 1 , 3 and 4 versus "+ iri" NS (two-way ANOVA).

[0034] 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. β-tubulin level was used as control of equal protein loading. The figure is representative of one out of three experiments with similar results.

[0035] 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 x10⁶ AC from UPN2. When the tumor reached the volume of 50 mm³, the 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). Tumor growth was monitored by caliper measure. Data are presented as means ± SD. ^* p < 0.005: TMZ 5/TMZ 5(x2) groups vs. Ctrl group (days 21 -30). B. Six week-old female BALB/c nu/nu mice were inoculated s.c. with 1 x10⁶ UPN2 AC or NS. When the tumor reached the volume of 50 mm³, the 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 μΜ) 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. and 38 ng/kg compound 741 i.v.); 6) 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.

[0036] FIG 15: Pgp knocking out in glioblastoma-derived neurospheres. a. UPN1 and UPN3 NS were grown 48 h in in fresh medium (-) or in medium containing 50 μΜ 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 , UPN2 and UPN3 NS were grown in fresh medium (-), transduced with a non-targeting vector (scrambled vector; scr) or with two CRISPR pCas /4SCC7/Pc¾D-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.

[0037] FIG 16: CAXII knocking out in glioblastoma-derived neurospheres. a. 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, b. 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.

[0038] 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. Data are presented as means+SD (6 animals/group). At day 24: UPN2 ^*p < 0.001 : TMZ+729 group vs. all the other groups of treatment; °p <0.01 TMZ+729 group vs TMZ-group (two- way ANOVA); UPN1 : ^*p < 0.005: TMZ+729 group vs. all the other groups of treatment; not significant TMZ+729 group vs TMZ-group (two-way ANOVA); UPN3: ^*p < 0.005: TMZ+729 group vs. all the other groups of treatment; °p <0.05 TMZ+729 group vs TMZ-group (two-way ANOVA). Overall survival probability was calculated using the Kaplan-Meier method. UPN2: p < 0.001 : TMZ+729-group vs. all the other groups of treatment. UPN2: p < 0.001 : TMZ+729-group vs. all the other groups of treatment. UPN1 : p < 0.05: TMZ+729-group vs. all the other groups of treatment. UPN3: p < 0.001 : TMZ+729-group vs. Ctrl and 729-group; not significant: TMZ-group vs. Ctrl and 729-group (log rank test; not reported in the figure).

DETAILED DESCRIPTION

[0039] 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). In cancers which express P-glycoprotein (Pgp) and CAXII, these compounds, and analogs thereof, may be used as effective chemosensitizers. Particularly, the compounds of the invention may be useful components in the treatment of glioblastoma.

[0040] As shown herein, 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. Further, 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.

Definitions [0041 ] In this patent specification, the terms 'comprises', 'comprising', 'includes', 'including', or similar terms are intended to mean a non-exclusive inclusion, such that a method or composition that comprises a list of elements does not include those elements solely, but may well include other elements not listed.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as would be commonly understood by those of ordinary skill in the art to which this invention belongs.

[0043] As generally used herein, the terms "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.

[0044] By "treat", "treatment" or "treating" is meant 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.

[0045] By "prevent", "preventing" or "preventative" is meant 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.

[0046] As used herein, "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.

[0047] As used herein, 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.

[0048] The term "pharmaceutically acceptable salt", as used herein, 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.

[0049] More particularly, 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. Also, 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.

[0050] The term "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. Examples of such 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., CF₃, 2-Br-ethyl, CH₂F, CH₂CI, CH₂CF₃, or CF₂CF₃); 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'. [0051 ] The term "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. Where appropriate, the alkenyl group may have a specified number of carbon atoms, for example, C₂-C₆ 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. Examples of such 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., CF₃, 2-Br-ethyl, CH₂F, CH₂CI, CH₂CF₃, or CF₂CF₃); 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'.

[0052] The term "halo" or "halogen" as used herein means selected from fluorine, chlorine, bromine, and iodine.

[0053] The term "cycloalkyl" refers to optionally substituted saturated monocyclic, bicyclic or tricyclic carbon groups. Where appropriate, the cycloalkyl group may have a specified number of carbon atoms, for example, C₃-C₆ 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.

[0054] The term "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 π 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.

[0055] The term "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, pyrimidines, quinazolines); 6-membered heretoaryls with three heteroatoms (e.g., 1 ,3,5- triazine); and 6-membered heteroaryls with four heteroatoms. "Substituted heteroaryl" means a heteroaryl having one or more non-interfering groups as substituents and including those defined under Optionally substituted'.

[0056] "Heterocyclic" as used herein 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. Non-limiting examples of heterocyclic 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.

[0057] 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., CF₃, 2-Br-ethyl, CH₂F, CH₂CF₃, and CF₂CF₃); amino (e.g., NH₂, NR₁₂H, and NR ₂R ₃); alkylamino; arylamino; acyl; amido; CN; NO₂; N₃; CH₂OH; CONH₂; CONR₂₄R₂₅; CO₂R₂₄; CH₂OR₂₄; NHCOR₂₄; NHCO₂R₂₄; C1 -3 alkylthio; sulfate; sulfonic acid; sulfonate esters such as alkyl or aralkyl sulfonyl, including methanesulfonyl; phosphonic acid; phosphate; phosphonate; mono-, di-, or triphosphate esters; trityl or monomethoxytrityl; R₂₄SO; R₂₄SO₂; CF₃S; and CF₃SO₂; trialkylsilyl such as dimethyl-t-butylsilyl or diphenylmethylsilyl; and R₂₄ and R₂₅ are each independently selected from H or optionally substituted C1 -10 alkyl, C1 -6 alkyl or C1 -4 alkyl.

[0058] Whenever a range of the number of atoms in a structure is indicated (e.g., a C Ci ₂, C1 -C10, C1 -C9, CrC₆, CrC₄, alkyl, etc.), it is specifically contemplated that any sub-range or individual number of carbon atoms falling within the indicated range also can be used. Thus, for instance, the recitation of a range of 1 -12 carbon atoms (e.g., C-|-C ₂), 1 -9 carbon atoms (e.g., CrC₉), 1 -6 carbon atoms (e.g., CrC₆) , 1 -4 carbon atoms (e.g., CrC₄), 1 -3 carbon atoms (e.g., C C₃), or 2-8 carbon atoms (e.g., C₂-C₈) as used with respect to any chemical group (e.g., alkyl, etc.) referenced herein encompasses and specifically describes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , and/or 12 carbon atoms, as appropriate, as well as any sub-range thereof (e.g., 1 -2 carbon atoms, 1 -3 carbon atoms, 1 -4 carbon atoms, 1 -5 carbon atoms, 1 -6 carbon atoms, 1 -7 carbon atoms, 1 -8 carbon atoms, 1 -9 carbon atoms, 1 -10 carbon atoms, 1 -1 1 carbon atoms, 1 -12 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms, 2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 2-9 carbon atoms, 2-10 carbon atoms, 2-1 1 carbon atoms, 2-12 carbon atoms, 3-4 carbon atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms, 3-9 carbon atoms, 3-10 carbon atoms, 3-1 1 carbon atoms, 3-12 carbon atoms, 4-5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8 carbon atoms, 4-9 carbon atoms, 4-10 carbon atoms, 4-1 1 carbon atoms, and/or 4-12 carbon atoms, etc., as appropriate).

[0059] According to a first aspect of the invention, there is provided a compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof:

Formula (I) wherein, 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, OCF₃, CF₃, S- alkyl and SO₂NHR₂ wherein R₂ is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi and optionally substituted aryl;

W is selected from -(CH₂)m- wherein m is from 1 to 6;

Z is a zinc binding group; and wherein, when R^a is a 3-bromo-4-hydroxy substitution, W is -CH₂-, R^b and Ri are hydrogen, and Y is -CH₂CH₂-, then Z is not -S(O)₂NH₂.

[0060] In embodiments, 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.

[0061 ] In certain embodiments, 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.

[0062] In further embodiments, each incidence of R^a is independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro, chloro, cyano, amino and carboxyl.

[0063] Suitably, each incidence of R^a is independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro and chloro.

[0064] In embodiments, 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.

[0065] In certain embodiments, R^b is selected from the group consisting of hydrogen, optionally substituted C1 to C6 alkyl and optionally substituted C5 or C6 cycloalkyi.

[0066] In embodiments, 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.

[0067] In certain embodiments, Ri is selected from hydrogen and a protecting group. Non-limiting examples of such protecting groups include benzyl (Bn) and tetrahydropyranyl (THP).

[0068] It will be appreciated that when R-, is a protecting group then that group may be cleaved prior to the compound binding to CAXII.

[0069] Preferably, W is selected from the group consisting of -CH₂-, - CH2CH2-, -CH2CH2CH2-, and -CH2CH2CH2CH2-. Suitably, W is -CH₂-.

[0070] In embodiments, 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. [0071 ] In embodiments, 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.

[0072] When Y is heterocyclic or heteroaryl it may be selected from C5 or C6 nitrogen and/or sulphur containing heterocyclic or heteroaryl.

[0073] Particularly, 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.

[0074] In certain embodiments, Y may be selected from 1 ,2,3-thiadiazole, 1 ,2,4-thiadiazole, 1 ,2,5-thiadiazole and 1 ,3,4-thiadiazole.

[0075] 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.

[0076] In embodiments, Z is selected from -S(O)₂NR_cR_d, -OS(O)₂NR_cR_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.

[0077] In embodiments, 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.

[0078] When 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 then they may form an optionally substituted thiazole or benzothiazole ring system. In one non-limiting example, the thiazole or benzothiazole ring system may be an optionally substituted thiazole-trione or benzothiazole-trione.

[0079] When Z is optionally substituted C5 or C6 heterocyclic then it may be a 5 or 6-membered nitrogen and/or oxygen-containing heterocycle.

[0080] Preferably, when Z is optionally substituted C5 or C6 heterocyclic then it may be an oxazolidinedione. In one non-limiting example, Z is a 2,4- oxazolidinedione.

[0081 ] In certain embodiments where Z is selected from -S(O)₂NR_cR_d and - OS(O)₂NR_cRd then R^c and R^d are preferably hydrogen. That is, it may be preferable that Z is a primary sulfonamide or sulfamate.

[0082] In any of the embodiments described herein, reference to optionally substituted C5 or C6 aryl includes optionally substituted phenyl.

[0083] In one embodiment of the first aspect, the compound of formula (I) is a compound of formula (II):

Formula II wherein, W, R^b, R_{1 ;} Y, R^c and R^d are as previously described for any embodiment of formula (I); each incidence of R^e, R^f, R⁹, R^h and R¹ is independently selected from those groups as set out above for any embodiment of R^a; and wherein, when R^a is a 3-bromo-4-hydroxy substitution, W is -CH₂-, R^b, R_{1 ;} R^c and R^d are all hydrogen, then Y is not -CH₂CH₂-.

[0084] In embodiments, R^e and R' are hydrogen.

[0085] In certain embodiments, R^f, R⁹ and R^h are independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro, chloro, cyano, amino and carboxyl.

[0086] Suitably, R^f, R⁹ and R^h are independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro and chloro.

[0087] In embodiments of formula (I) and (II) it is preferred that at least one position on the phenyl ring is not hydrogen. That is, in formula (II), it is preferred that at least one of R^e, R^f, R⁹, R^h and R' is not hydrogen.

[0088] Certain embodiments of formula (I) and (II) are 3-halo substituted on the phenyl ring. That is, in formula (II), it is preferred that R^h is halo.

[0089] In certain embodiments, R^c and R^d are hydrogen.

[0090] In one embodiment, the compound of formula (I) or formula (II) is selected from the group consisting of:

[0092] In some embodiments of the present invention, therapeutically inactive prodrugs of the compounds of the first aspect are provided. Prodrugs are compounds which, when administered to a mammal, are converted in whole or in part to a compound of the invention. In most embodiments, 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.

[0093] A number of prodrug ligands are known. In general, 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. Examples of 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. Any of these moieties can be used in combination with the disclosed active agents to achieve a desired effect. [0094] 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. Moreover, 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, Ν,Ν-dimethylformamide) at a temperature of 0 ^QC to 60 ^QC. 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.

[0095] In some embodiments, 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.

[0096] 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. Examples of 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.

[0097] Various methods are known in the art for preparing optically active forms and determining activity. Such methods include standard tests described herein and other similar tests which are well known in the art. Examples of methods that can be used to obtain 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;

ii) simultaneous crystallization whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state;

iii) enzymatic resolutions whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme;

iv) 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;

v) chemical asymmetric synthesis whereby the desired enantiomer is synthesized from an achiral precursor under conditions that produce asymmetry {i.e., chirality) in the product, which may be achieved using chiral catalysts or chiral auxiliaries;

vi) diastereomer separations whereby a racemic compound is reacted with an enantiomerically pure reagent (the chiral auxiliary) that converts the individual enantiomers to diastereomers. The resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer;

vii) first- and second-order asymmetric transformations whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer. The desired enantiomer is then released from the diastereomers;

viii) 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;

ix) enantiospecific synthesis from non-racemic precursors whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis;

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;

xi) chiral gas chromatography whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase;

xii) extraction with chiral solvents whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; and

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.

[0098] 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%.

[0099] The terms (R), (S), (R,R), (S,S), (R,S) and (S,R) as used herein mean that the composition contains a greater proportion of the named isomer of the compound in relation to other isomers. In a preferred embodiment, 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. In some embodiments, 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.

Synthesis

[00100] Scheme 1 , below, 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.

Compound 1 Compound 2 Compound 3 Compound 4 Compound 5

Ri H, R₂ = H R^ ^— H, R2 " ^■■ H 6, 63% Ri H, R₂ = H 10, 58% R, H, R₂ = H, R₃ = Bn 14, 79% Ri Br, R₂ = H R, = Br, R₂ = H 7, 73% Ri Br, R₂ = H 11, 34% R, Br, R₂ = H, R₃ = THP 15, 58% Ri H, R₂ = OH R^ ⁼ H, R2 "^■■ OAc 8, 64% Ri H, R₂ = OH 12, 90% R, H, R₂ = OH, R₃ = THP 16, 44% Ri Br, R₂ = OH R, = Br, R₂ = OAc 9, 85% Ri Br, R₂ = OH 13, 65% R, Br, R₂ = OH, R₃ = THP 17, 54%

RT = H, R₂ = H, R₃ = Bn, R4 = S0₂NH₂ 18, 20% Ri ^: H, R₂ = H, R₃ = S0₂NH₂ 2, 30%

RT = Br, R₂ = H, R₃ = THP, R4 = SO_zNH₂ 19, 51% Ri : Br, R₂ = H, R₃ = S0₂NH₂ 3, 77%

RT = H, R₂ = OH, R₃ = THP, R4 = S0₂NH₂ 20, 37% Ri ^: H, R₂ = OH, R₃ = S0₂NH₂ 4, 55%

RT = Br, R₂ = OH, R₃ = THP, R4 = S0₂NH₂ 21, 46% Ri : Br, R₂ = OH, R₃ = S0₂NH₂ 1, 80%

R₁ = Br, R₂ = OH, R₃ = THP, R4 = CONH₂ 22, 43% Ri : Br, R₂ = OH, R₃ = CONH₂ 5, 75%

Scheme 1 : Synthesis of sulfonamides 1 to 5. ^aReagents and conditions: (i) N- acetyl glycine, NaOAc, Ac₂O, 120 °C, 3 h; (ii) Aq. HCI (10%), reflux, 15 h; (iii) HCI.H₂NOBn or H₂NOTHP, dry pyridine, rt, 15 h; (iv) (a) EDC.HCI, HOSu, dry 1 ,4-dioxane, 2-3 h, rt; (b) β-aminoethanesulfonamide hydrochloride or 3- aminopropanamide, dry NEt₃, dry 1 ,4-dioxane, dry MeOH, rt, 12 h; (v) Pd/C, dry NEt₃, HCOOH, abs. EtOH 60 °C, 3-4 h or (vi) 4M HCI in 1 ,4-dioxane, 0 °C, 8-10 h. Note that R-, to R₃ in this scheme are used entirely separately to those identifiers in the claims and formula (I) to (III) herein. [00101 ] Scheme 2, below, shows the use of this general synthetic route to access a further compound of the invention, compound 729. Further detail on this synthesis is provided in the experimental section.

Scheme 2: Synthetic scheme for compound 729. ^aReagents and conditions: (i) AAacetyl glycine, NaOAc, Ac₂0, 120 °C, 3 h, 67%; (ii) Aq. HCI (10%), reflux, 15 h, 62%; (iii) HCI.H₂NOBn or H₂NOTHP, dry pyridine, rt, 15 h, 41 %; (iv) EDC.HCI, HOBT.H₂0, 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%.

[00102] According to a second aspect of the invention there is provided 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.

[00103] 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.

[00104] 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.

[00105] 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.

[00108] Reference is made to the Handbook of Excipients 8^th Edition, Eds. Rowe, Sheskey & Quinn (Pharmaceutical Press), which provides non-limiting examples of excipients which may be useful according to the invention.

[00107] It will be appreciated that the choice of pharmaceutically acceptable carriers, diluents and/or excipients will, at least in part, be dependent upon the mode of administration of the formulation. By way of example only, the 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.

[00108] 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.

[00109] 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.

[001 10] It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[001 1 1 ] 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. [001 12] 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.

[001 13] The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus such formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier therefore comprise a further aspect of the invention.

[001 14] Suitably, the pharmaceutical composition is for the treatment or prophylaxis of a disease, disorder or condition responsive to carbonic anhydrase XII inhibition.

[001 15] Suitably, the pharmaceutical composition is for the treatment or prevention of a disease, disorder or condition in a mammal.

[001 16] 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:

W is selected from -(CH₂)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.

[001 17] R^a, R-i , W, R^b, Y, and Z may be as described for any embodiment of formula (I) or (II).

[001 18] When Z is selected from -S(O)₂NR_cR_d and -OS(O)₂NR_cR_d then R^c and R^d may be as described for any embodiment of formula (I).

[001 19] The compound of formula (III) may also have a structure as shown in formula (II) with R^e, R^f, R⁹, 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.

[00121 ] Such an approach of reducing chemoresistance through CAXII inhibition differs from direct Pgp inhibition, due to the targeting of a Pgp- adjuvant protein (CAXII) that is specifically expressed in tumor cells and is usually poorly expressed in most healthy cells. The use of 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.

[00122] 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.

[00123] 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.

[00124] 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).

[00125] 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.

[00126] 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.

[00127] 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.

[00128] 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.

[00129] In a tenth aspect of the invention there is provided 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.

[00130] 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.

[00131 ] The use of the compounds of formula (III) in diagnosing cancers, such as those listed herein, may be achieved by near infrared fluorescent imaging and ex vivo characterisation of by degree of inhibition of CAXII levels. In vivo diagnostics using positron emission tomography (PET) may be appropriate. PET is a molecular imaging technique that requires specific probes radiolabeled with short-lived positron emitting radionuclides. Typical isotopes include ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁴Cu, ⁶²Cu, ¹²⁴l, ⁷⁶Br, ⁸²Rb and ⁶⁸Ga, with ¹⁸F being the most clinically utilized. In particular it is possible to produce in a simple manner a labelled halogen on the aromatic ring of formula (I), (II) and (III), or 1 1 C or 13N one or more of the compounds of formula (III). This enables rapid preparation of a diagnostic probe for radioimaging, PET and the like whereby the intensity, location and temporal accretion of the diagnostic probe is able to identify the degree and/or the location of cancer cells over expressing CAXII.

[00132] An eleventh aspect of the invention resides in a complex of a compound of formula (III) and a carbonic anhydrase enzyme.

[00133] Suitably, the carbonic anhydrase enzyme is a carbonic anhydrase IX or XII. Most preferably, a carbonic anhydrase XII enzyme, including a human carbonic anhydrase XII enzyme.

[00134] In any embodiment of the aforementioned aspects, the cancer is one which is responsive to inhibition of carbonic anhydrase XII enzyme. Suitably, 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.

[00135] In certain embodiments, 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.

[00136] In embodiments of the aforementioned aspects employing an anticancer agent, the anti-cancer agent may be selected from any known clinically useful anti-cancer agent. Particularly, 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.

[00137] In certain embodiments, the anti-cancer agent may be a second-line chemotherapeutic agent. [00138] In certain embodiments, 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.

[00139] In embodiments, 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.

[00140] In certain embodiments, the anti-cancer agent may be two or more anti-cancer agents.

[00141 ] In one embodiment, in any of the aspects described, the compound of formula (I), (II) or (III) is not Psammaplin C as shown below:

[00142] In certain embodiments, the compound below may be excluded from formula (I), (II) or (III):

[00143] The following experimental section describes in more detail the characterisation of certain of the compounds of the invention and their antiviral activity. The intention is to illustrate certain specific embodiments of the compounds of the invention and their efficacy without limiting the invention in any way.

EXPERIMENTAL

[00144] In the results and discussion which follows, and in the related figures, the compounds may be identified by the below numerals where, for example, for compound 1 , the identifiers 1 and 741 are interchangeable.

Compound 1 Compound 2 Compound 3 Compound 4

(#741 in figure data) (#737) (#739) (#744)

Compound 5 Compound 729

(#787, control)

Chemistry

General

[00145] All reactions were carried out in dry solvents under anhydrous conditions, unless otherwise mentioned. All chemicals were purchased commercially and used without further purification. All reactions were monitored by TLC using silica plates with visualization of product bands by UV fluorescence (λ = 254 nm) and charring with Vanillin (6 g vanillin in 100 mL of EtOH containing 1 % (v/v) concentrated sulfuric acid) stain. Silica gel flash chromatography was performed using silica gel 60 A (230-400 mesh). NMR (¹H, ¹³C, COSY, HSQC and HMBC) spectra were recorded on the 500 MHz spectrometer at 25 °C. Chemical shifts for ¹H and ¹³C NMR obtained in DMSO- d₆ are reported in ppm relative to residual solvent proton (δ = 2.50 ppm) and carbon (δ = 39.5 ppm) signals, respectively. Multiplicity is indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet), bs (broad signal). Coupling constants are reported in hertz (Hz). High- and low- resolution mass spectra were acquired using electrospray as the ionization technique in positive-ion and/or negative-ion modes as stated. All MS analysis samples were prepared as solutions in methanol. Purity of all compounds was >95% as determined by HPLC (Agilent HPLC 1 100 series) with UV detection. The melting points are uncorrected.

General Procedure 1 : Synthesis of benzylidene oxazolones 6-9

[00146] To a mixture of anhydrous NaOAc (1 .0 equiv) and AAacetyl glycine (1 .0 equiv) suspended in Ac₂O (10.0 equiv) at room temperature under inert atmosphere, was added the relevant benzaldehyde (1 .0 equiv). The reaction mixture was stirred at 120 °C for 4 h and then cooled to room temperature. The crude product precipitated and was collected by suction filtration then added to ice cold water (25-30 mL) and vigorously stirred for 15-20 min. The solution was filtered and the residual crude oxazolone solid dried under vacuum and used for the next step without further purification. A sample of compound 6-9 was purified for characterization purposes as described. The characterization of compound 9 has been reported previously [Mujumdar, 2016].

General Procedure 2: Synthesis of O-Bn or O-THP protected oximino acids 14-17 from benzylidene oxazolones 6-9.

[00147] A mixture of benzylidine oxazolone 6-9 (1 equiv) in 10% aqueous HCI (10 imL/mmol) was stirred at 100 °C overnight (12-14 h) then allowed to cool to room temperature (~3 h). The product precipitated and was filtered, washed with ice-cold water (10-15 mL) and dried under vacuum. The filtrate was washed with EtOAc (3 ^χ 20 mL), then the combined organic fractions washed with water (1 ^χ 20 mL) and saturated aqueous NaCI (1 ^χ 20 mL), dried over MgSO₄ and concentrated in vacuo. The oily residue was triturated with a minimal amount of n-hexane. The n-hexane portion was decanted and the crude solid was dried under vacuum. The crude aryl pyruvic acid fractions 10- 13 (one collected by filtration and the other by extraction-trituration) were combined and this material used without further purification.

[00148] To a mixture of 10-13 (1 equiv) and either benzyloxyamine hydrochloride (NH₂OBn.HCI, 1 .5 equiv) for 14 or 0-(tetrahydro-2H-pyran-2-yl) hydroxylamine (NH₂OTHP, 1 .5 equiv) for 15-17 was added anhydrous pyridine (2 mL/mmol). The reaction mixture was stirred at room temperature under an argon atmosphere overnight (12-14 h). Pyridine was removed in vacuo. 1 .0 M HCI (5.5 mL/mmol) was added to the remaining residue and extracted in EtOAc (3 x 25 mL). The combined organic fractions were washed with saturated aqueous NaCI (1 ^χ 20 mL/mmol), dried over MgSO₄ and concentrated in vacuo. The target O-Bn or O-THP protected oximino acids 14-17 were purified as described below. The characterisation of compound 17 has been reported previously [Mujumdar, 2016].

General Procedure 3: Synthesis of O-Bn and O-THP protected psammaplin C derivatives 18-22

[00149] To a mixture of O-Bn or O-THP protected oximino acid 14-17 (1 .0 equiv), EDC.HCI (1 .7 equiv) and HOSu (1 .9 equiv) under an argon atmosphere was added anhydrous 1 ,4-dioxane (10 mL/mmol) and the resulting solution stirred at room temperature for 3 h. The solvent was evaporated under vacuum and the residue dissolved in EtOAc (30-35 mL), washed with saturated aqueous NaHCO₃ (2 x 15 mL/mmol), 1 .0 M aqueous HCI (2 ^χ 15 mL/mmol) and saturated aqueous NaCI (1 ^χ 20 mL/mmol). The organic fraction was dried over MgSO₄ and concentrated in vacuo. The succinate ester intermediate was dissolved in anhydrous 1 ,4-dioxane (4 mL/mmol) under argon and a solution of β-aminoethanesulfonamide hydrochloride (1 .2 equiv) or 3-aminopropanamide (1 .2 equiv) and NEt₃ (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].

General Procedure 4: Deprotection of O-THP protected compounds 19-22

[00150] To the oxime THP ether (1 .0 mmol) 19-22 was added 4.0 M HCI in 1 ,4-dioxane solution (15 imL/mmol) at 0 °C, the reaction mixture was stirred at the same temperature. After completion of reaction (TLC monitoring) the solvent evaporated in vacuo and the desired compounds 1 and 3-5 were purified as described. The synthesis of compound 1 has been reported previously [Mujumdar, 2016].

(4iE)-2-Methyl-4-(phenylmethylidene)-4,5-dihydro-1 ,3-oxazol-5-one (6)

[00151 ] Compound 6 was synthesised from benzaldehyde (4.0 g, 37.7 mmol) according to general procedure 1 as yellow solid (4.5 g, 63%, crude). A small amount of crude solid was purified by flash chromatography (Gradient: 5-8% acetone in n-hexane) for characterisation purposes. R_f= 0.46 (20% acetone in n-hexane). Mp = 148-150 °C. ¹H NMR (500 MHz, DMSO-tf₆) δ_Η = 8.18-8.17 (m, 2H, H_Ar), 7.51 -7.47 (m, 3H, H_Ar), 7.22 (s, 1 H, Ph-CH=C), 2.39 (s, 3H, CH₃), general assignments were confirmed by ¹ H-¹ H gCOSY. ¹³C NMR (125 MHz, DMSO-of₆) 5c = 167.3 (C_quat), 166.7 (C_quat), 133.0 (C_quat), 132.6 (C_quat), 131 .9 (2 x CH_Ar), 131 .0 (1 x CH_Ar), 129.7 (Ph-CH=C), 128.8 (2 ^χ CH_Ar), 15.3 (CH₃), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z= 188 [M + H]⁺.

(4iE)-4-[(3-Bromophenyl)methylidene]-2-methyl-4,5-dihydro-1 ,3-oxazol-5- one (7)

[00152] Compound 7 was synthesised from 3-bromobenzaldehyde (5.0 g, 27.0 mmol) according to general procedure 1 as yellow solid (5.25 g, 73%, crude). A small amount of crude solid was purified by flash chromatography (Gradient: 5-8% acetone in n-hexane) for characterisation purposes. R_f= 0.45 (20% acetone in n-hexane). Mp = 120-122 °C. ¹H NMR (500 MHz, DMSO-tf₆) δ_Η = 8.42 (s, 1 H, H_Ar), 8.14 (d, J = 8.1 Hz, 1 H, H_Ar), 7.67 (d, J = 7.05 Hz, 1 H, H_Ar), 7.45 (t, J = 7.9 Hz, 1 H, H_Ar), 7.20 (s, 1 H, Ph-CH=C), 2.41 (s, 3H, CH₃), general assignments were confirmed by ¹ H-¹ H gCOSY. ¹³C NMR (125 MHz, DMSO-ak) 5c = 167.3 (C_quat), 166.6 (C_quat), 135.0 (C_quat), 133.4 (C_quat), 133.3 (CH_Ar), 133.0 (CH_Ar), 130.5 (CH_Ar), 130.4 (CH_Ar), 127.3 (Ph-CH=C), 121 .7 (Cquat) , 15.1 (CH₃), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z = 266 [M + H, ⁷⁹Br]⁺, 268 [M+ H, ⁸¹ Br]⁺.

4-{[(4iE)-2-methyl-5-oxo-4,5-dihydro-1 ,3-oxazol-4-ylidene]methyl}phenyl acetate (8)

[00153] Compound 8 was synthesised from 4-hydroxybenzaldehyde (5.0 g, 40.9 mmol) according to general procedure 1 as yellow solid (6.5 g, 64%, crude). A small amount of crude solid was purified by flash chromatography (Gradient: 8-10% acetone in n-hexane) for characterisation purposes. R_f= 0.29 (20% acetone in n-hexane). Mp = 130-132 °C. ¹H NMR (500 MHz, DMSO-tf₆) δ_Η = 8.22 (d, J = 8.7 Hz, 2H, H_Ar), 7.27 (d, J = 8.65 Hz, 2H, H_Ar), 7.23 (s, 1 H, Ph-CH=C), 2.39 (s, 3H, CH₃), 2.29 (s, 3H, CH₃), general assignments were confirmed by ¹H-¹H gCOSY. ¹³C NMR (125 MHz, DMSO-tf₆) 5_C = 168.8 (C_quat), 167.3 (Cquat) , 166.8 (C_quat), 152.3 (C_quat), 133.2 (2 χ CH_Ar), 132.4 (C_quat), 130.7 (Cquat) , 128.8 (Ph-CH=C), 122.4 (2 χ CH_Ar), 20.8 (CH₃), 15.3 (CH₃), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z= 246 [M + H]⁺.

(2ir)-2-[(Benzyloxy)imino]-3-phenylpropanoic acid (14)

[00154] Benzylidine oxazolone 6 was converted to intermediate aryl pyruvic acid 10 and then compound 14 was synthesised from 10 (0.5 g, 3.0 mmol) according to general procedure 2. Compound 14 was purified by flash chromatography (Gradient: 0.5-1 % MeOH in DCM) as a white solid (0.65 g, 79%). R_f = 0.37 (10% MeOH in DCM). Mp = 75-77 °C. ¹H NMR (500 MHz, DMSO-de) δ_Η = 13.16 (bs, 1 H, COOH), 7.36-7.16 (m, 10H, H_Ar), 5.27 (s, 2H, Ph- CH₂-O), 3.85 (s, 2H, Ph-CH₂-C), general assignments were confirmed by ¹H-¹H gCOSY. ¹³C NMR (125 MHz, DMSO-tf₆) 5_C = 164.3 (C_quat) , 151 .2 (C_quat), 136.9 (Cquat), 135.9 (Cquat), 128.5 (2 ^χ CH_Ar), 128.4 (2 x CH_Ar), 128.3 (2 ^χ CH_Ar), 128.0 (CHAr), 127.9 (2 x CHAr), 126.4 (CH_Ar), 76.5 (Ph-CH₂-O), 30.8 (Ph-CH₂-C), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z= 270 [M + H]⁺, 292 [M + Na]⁺. HRMS (ESI): calcd for [Ci₆H₁₅NO₃ - H]⁺ 268.0979, found 268.0978.

(2ir)-3-(3-Bromophenyl)-2-[(oxan-2-yloxy)imino]propanoic acid (15)

[00155] Benzylidine oxazolone 7 was converted to intermediate aryl pyruvic acid 11 and then compound 15 was synthesised from 11 (1 .0 g, 4.1 mmol) according to general procedure 2. Compound 15 was purified by flash chromatography (Gradient: 1 -2% MeOH in DCM) as a white solid (0.82 g, 58%). R_f = 0.28 (10% MeOH in DCM). Mp = 64-66 °C. ¹H NMR (500 MHz, DMSO-de) δ_Η = 13.32 (bs, 1 H, COOH), 7.45 (s, 1 H, H_Ar), 7.42 (d, J = 7.65 Hz, 1 H, H_Ar), 7.27 (t, J = 7.65 Hz, 1 H, H_Ar), 7.24-7.22 (m, 1 H, H_Ar), 5.37 (s, 1 H, CHTHP), 3.89 (d, J = 13.7 Hz, 1 H, Ph-CHH-C), 3.84 (d, J = 13.75 Hz, 1 H, Ph- CHH-C), 3.48-3.41 (m, 2H, H_THp), 1 .75-1 .67 (m, 3H, H_THp), 1 .60-1 .52 (m, 2H, HTHP), 1 -46-1 .44 (m, 1 H, H_THP), general assignments were confirmed by ¹ H-¹ H gCOSY. ¹³C NMR (125 MHz, DMSO-tf₆) 5_C = 164.4 (C_quat), 151 .2 (C_quat), 138.9

(Cquat), 131 .6 (CHAr), 130.6 (CHAr), 129.3 (CHAr), 127.6 (CHAr), 121 .5 (Cquat),

100.8 (CHTHP), 61 .2 (CH₂-THP), 30.7 (Ph-CH₂-C), 28.0 (CH₂-THP), 24.5 (CH₂- THP), 18.3 (CH₂-THP), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z = 364, 366 [M + Na, ⁷⁹Br, ⁸¹Br]⁺. HRMS (ESI): calcd for [C₁₄H₁₆BrNO₄ - H, ⁷⁹Br]⁺ 340.0189, found 340.0186, calcd for [C₁₄H₁₆BrNO₄ - H, ⁸¹Br]⁺ 342.0169, found 342.0166.

(2ir)-3-(4-Hydroxyphenyl)-2-[(oxan-2-yloxy)imino]propanoic acid (16)

[00156] Benzylidine oxazolone 8 was converted to intermediate aryl pyruvic acid 12 and then compound 16 was synthesised from 12 (1 .9 g, 10.6 mmol) according to general procedure 2. Compound 16 was purified by flash chromatography (Gradient: 3-4% MeOH in DCM) as a white solid (1.30 g, 44%). R_f= 0.17 (10% MeOH in DCM). ¹ H NMR (500 MHz, DMSO-tf₆) δ_Η = 9.25 (s, 1 H, Ph-OH), 7.04 (d, J = 8.6 Hz, 2H, H_Ar), 6.67 (d, J = Hz, 2H, H_Ar), 5.34- 5.33 (m, 1 H, CH_THp), 3.76 (d, J= 13.4 Hz, 1 H, Ph-CHH-C), 3.70 (d, J= 13.4 Hz, 1 H, Ph-CHH-C), 3.56-3.47 (m, 2H, H_THp), 1 .76-1 .69 (m, 3H, H_THp), 1 .60-1 .53 (m, 2H, HTHP), 1 .48-1 .44 (m, 1 H, H_THP), COOH proton in exchange, general assignments were confirmed by ¹ H-¹ H gCOSY. ¹³C NMR (125 MHz, DMSO-ak) 5c = 164.6 (Cquat), 155.9 (C_quat), 152.4 (C_quat), 129.7 (2 ^χ CH_Ar), 126.0 (C_quat),

1 15.2 (2 x CH_Ar), 100.7 (CH_THp), 61 .4 (CH₂-THP), 30.0 (Ph-CH₂-C), 28.2 (CH₂- THP), 24.6 (CH₂-THP), 18.6 (CH₂-THP), general assignments were confirmed by ¹H-¹³C HSQC. LRMS (ESI): m/z = 302 [M + Na]⁺. HRMS (ESI): calcd for [C₁₄H₁₇NO₅ - H]⁺ 278.1034, found 278.1032.

(2£)-2-[(Benzyloxy)imino]-3-phenyl-A-(2-sulfamoylethyl)propanamide (18)

[00157] Compound 18 was synthesised from compound 14 (0.4 g, 1 .5 mmol) according to general procedure 3. The crude solid was purified by flash chromatography (Gradient: 0-1 .5% MeOH in DCM) to afford the title compound as white solid (0.1 15 g, 20%). R_f= 0.32 (5% MeOH in DCM). Mp = 105-107 °C. ¹ H NMR (500 MHz, DMSO-tf₆) δ_Η = 8.24 (t, J= 5.75 Hz, 1 H, N-H), 7.38-7.31 (m, 5H, H_Ar), 7.26-7.23 (m, 2H, H_Ar), 7.19-7.16 (m, 3H, H_Ar), 6.92 (s, 2H, SO₂NH₂), 5.23 (s, 2H, Ph-CH₂-O), 3.84 (s, 2H, Ph-CH₂-C), 3.55 (q, J = 6.25 Hz, 2H, HN- Chb-CHs), 3.15 (t, J = 6.85 Hz, 2H, HN-CH₂-CH₂), general assignments were confirmed by ¹H-¹H gCOSY. ¹³C NMR (125 MHz, DMSO-tf₆) 5_C = 162.2 (C_quat),

152.3 (Cquat), 136.8 (C_quat), 136.0 (C_quat), 128.7 (2 ^χ CH_Ar), 128.32 (2 ^χ CH_Ar), 128.29 (2 x CH_Ar), 128.00 (2 ^χ CH_Ar), 127.97 (CH_Ar), 126.2 (CH_Ar), 76.4 (Ph- CH₂-O), 53.3 (HN-CH₂-CH₂), 34.3 (HN-CH₂-CH₂), 29.8 (Ph-CH₂-C), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z= 376 [M + H]⁺, 398 [M + Na]⁺, HRMS (ESI): calcd for [C₁₈H₂₁N₃O₄S + H]⁺ 376.1325, found 376.1328.

(2£)-3-(3-bromophenyl)-2-[(oxan-2-yloxy)imino]-A/-(2- sulfamoylethyl)propanamide (19) [00158] Compound 19 was synthesised from compound 15 (0.195 g, 0.6 mmol) according to general procedure 3. The crude solid was purified by flash chromatography (Gradient: 3-5% MeOH in DCM) to afford the title compound as white solid (0.133 g, 51 %). fl, = 0.29 (10% MeOH in DCM). Mp = 135-137 °C. ¹ H NMR (500 MHz, DMSO-tf₆) δ_Η = 8.35 (t, J = 5.9 Hz, 1 H, N-H), 7.48 (s, 1 H, H_Ar), 7.40 (dt, J = 6.65, 1 .85 Hz, 1 H, H_Ar), 7.28-7.23 (m, 2H, H_Ar), 6.93 (m, 2H, SO₂NH₂), 5.35 (s, 1 H, CH_THp), 3.89 (d, J= 13.35 Hz, 1 H, Ph-CHH-C), 3.82 (d, J = 13.35 Hz, 1 H, Ph-CHH-C), 3.62-3.50 (m, 2H, HN-CH₂-CH₂), 3.46-3.42 (m, 1 H, HTHP), 3.37 (td, J = 10.55, 3.0 Hz, 1 H, H_THp), 3.17 (t, J = 7.25 Hz, 2H, HN-CH₂-CH₂), 1 .75-1 .69 (m, 3H, H_THp), 1 -60-1 .51 (m, 2H, H_THp), 1 -45-1 .43 (m, 1 H, HTHP), general assignments were confirmed by ¹H-¹H gCOSY. ¹³C NMR (125 MHz, DMSO-ak) 5_C = 162.4 (C_quat), 152.5 (C_quat), 139.1 (C_quat), 131 .8 (CH_Ar), 130.6 (CH_Ar), 129.2 (CH_Ar), 127.9 (CH_Ar), 121 .5 (C_quat), 100.6 (CH_THp),

61 .0 (CH₂-THP), 53.3 (HN-CH2-CH2), 34.4 (HN-CH₂-CH₂), 29.8 (Ph-CH₂-C),

28.1 (CH₂-THP), 24.6 (CH₂-THP), 18.2 (CH₂-THP), general assignments were confirmed by ¹H-¹³C HSQC. LRMS (ESI): 470, 472 [M + Na, ⁷⁹Br, ⁸¹ Br]⁺, HRMS (ESI): calcd for

- H, ⁷⁹Br]⁺ 446.0390, found 446.0383, calcd for [Ci₆H₂₂BrN₃O₅S - H, ⁸¹ Br]⁺ 448.0370, found 448.0362.

(2£)-3-(4-Hydroxyphenyl)-2-[(oxan-2-yloxy)imino]-/V-(2- sulfamoylethyl)propanamide (20)

[00159] Compound 20 was synthesised from compound 16 (0.25 g, 0.8957 mmol) according to general procedure 3. The crude solid was purified by flash chromatography (Gradient: 3-5% MeOH in DCM) to afford the title compound as off-white oil (0.13 g, 37%). R, = 0.30 (10% MeOH in DCM). ¹ H NMR (500 MHz, DMSO-tf₆) δ_Η = 9.22 (s, 1 H, Ph-OH), 8.25 (t, J = 5.85 Hz, 1 H, N-H), 7.03 (d, J = 8.55 Hz, 2H, H_Ar), 6.93 (s, 2H, SO₂NH₂), 6.65 (d, J = 8.55 Hz, 2H, H_Ar), 5.33 (s, 1 H, CHTHP), 3.77 (d, J = 13.05 Hz, 1 H, Ph-CHH-C), 3.70 (d, J = 13.0 Hz, 1 H, Ph-CHH-C), 3.58-3.50 (m, 3H, HN-CH₂-CH₂ and H_THp), 3.48-3.45 (m, 1 H, HTHP), 3.14 (t, J = 7.25 Hz, 2H, HN-CH₂-CH₂), 1 .76-1 .72 (m, 3H, H_THP), 1 .60-1 .52 (m, 2H, H_THP), 1 .47-1 .45 (m, 1 H, H_THP), general assignments were confirmed by ¹H-¹H gCOSY. ¹³C NMR (125 MHz, DMSO-tf₆) 5_C = 162.6 (C_quat), 155.8 (Cquat), 153.6 (C_quat), 129.9 (2 x CH_Ar), 126.1 (C_quat), 1 15.1 (2 x CH_Ar), 100.5 (CHTHP), 61 .3 (CH₂-THP), 53.4 (HN-CH₂-CH₂), 34.3 (HN-CH₂-CH₂), 29.1 (Ph-CH₂-C), 28.3 (CH₂-THP), 24.7 (CH₂-THP), 18.5 (CH₂-THP), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z= 386 [M + H]⁺, 408 [M + Na]⁺, HRMS (ESI): calcd for [C₁₆H₂₃N₃O₆S - H]⁺ 384.1234, found 384.1229.

(2£)-3-(3-Bromo-4-hydroxyphenyl)-yV-(2-carbamoylethyl)-2-[(oxan-2- yloxy)imino]propanamide (22)

[00160] Compound 22 was synthesised from compound 17 (0.1 g, 0.3 mmol) according to general procedure 3. The crude material was purified by flash chromatography (Gradient: 2.5-5% MeOH in DCM) to afford the title compound as colorless oil (0.053 g, 43%). R_f = 0.29 (10% MeOH in DCM). ¹ H NMR (500 MHz, DMSO-de) δ_Η = 10.08 (s, 1 H, Ph-OH), 8.08 (t, J = 5.9 Hz, 1 H, N-H), 7.38 (d, J = 2.05 Hz, 1 H, H_Ar), 7.34 (bs, 1 H, O=C-NHH), 7.05 (dd, J = 8.5, 2.05 Hz, 1 H, H_Ar), 6.86 (d, J = 8.3 Hz, 1 H, H_Ar), 6.84 (bs, 1 H, O=C-NHH), 5.35 (s, 1 H, CHTHP), 3.77 (d, J = 13.1 Hz, 1 H, Ph-CHH-C), 3.70 (d, J = 13.1 Hz, 1 H, Ph- CHH-C), 3.48-3.46 (m, 2H, H_THp), 3.36-3.34 (m, 2H, HN-CH₂-CH₂), 2.29 (t, J = 7.4 Hz, 2H, HN-CH₂-CH₂), 1 .77-1 .73 (m, 3H, H_THp), 1 .64-1 .55 (m, 2H, H_THp), 1 .49-1 .46 (m, 1 H, H_THP), general assignments were confirmed by ¹H-¹H gCOSY. ¹³C NMR (125 MHz, DMSO-ak) 5_C = 172.6 (C_quat), 162.2 (C_quat), 153.3 (C_quat), 152.5 (Cquat), 133.3 (CH_Ar), 129.1 (CH_Ar), 128.2 (C_quat), 1 16.2 (CH_Ar), 108.9 (Cquat), 100.5 (CHTHP), 61 .1 (CH₂-THP), 35.5 (HN-CH₂-CH₂), 34.5 (HN-CH₂- CH₂), 28.8 (Ph-CH₂-C), 28.2 (CH₂-THP), 24.7 (CH₂-THP), 18.4 (CH₂-THP), general assignments were confirmed by ¹H-¹³C HSQC. LRMS (ESI): m/z= 450, 452 [M + Na, ⁷⁹Br, ⁸¹Br]⁺, HRMS (ESI): calcd for [Ci ₇H₂₂BrN₃O₅ + Na, ⁷⁹Br]⁺ 450.0635, found 450.0626, calcd for [C₁₇H₂₂BrN₃O5 + Na, ⁸¹Br]⁺ 452.0615, found 452.0605.

(2iE)-2-(A^Hydroxyimino)-3-phenyl-yV-(2-sulfamoylethyl)propanamide (2) [00161 ] To the solution of compound 18 (0.1 g, 0.2666 mmol) in abs. EtOH (5 ml_) was added Pd/C (0.025 g, 25% by weight of 18), dry NEt₃ (1 1 μΙ_, 0.3 equiv) and formic acid (50 μΙ_, 5 equiv) at room temperature under Ar atmosphere. The resulting reaction mixture was heated to 60 °C and stirred for 3-4 hours. After completion of reaction (TLC), the hot reaction mixture was filtered through Celite bed, washed with acetone (3 x 1 0 imL) and the filtrate was evaporated to dryness to afford the crude solid. The crude solid was purified by flash chromatography (Gradient: 3-5% MeOH in DCM) to afford the title compound as white solid (0.023 g, 30%). R_f = 0.22 (10% MeOH in DCM). Mp = 1 36- 1 38 °C. ¹H NMR (500 MHz, DMSO-tf₆) δ_Η = 1 1 .9 (s, 1 H, N-OH), 8.06 (t, J= 5.85 Hz, 1 H, N-H), 7.27-7.15 (m, 5H, H_Ar), 6.91 (s, 2H, SO₂NH₂), 3.82 (s, 2H, Ph-CH₂-C), 3.55 (q, J = 6.4 Hz, 2H, HN-CH₂-CH₂), 3.14 (t, J = 6.7 Hz, 2H, HN-CH₂-CH₂), general assignments were confirmed by ¹H-¹H gCOSY. ¹³C NMR (125 MHz, DMSO-ak) 5_C = 163.2 (C_quat), 151 .5 (C_quat), 136.7 (C_quat), 128.7 (2 χ CH_Ar), 128.2 (2 x CH_Ar), 126.0 (CH_Ar), 53.5 (HN-CH₂-CH₂), 34.1 (HN-CH₂-CH₂), 28.9 (Ph-CH₂-C), general assignments were confirmed by ¹H-¹³C HSQC. LRMS (ESI): m/z= 286 [M + H]⁺, 308 [M + Na]⁺, HRMS (ESI): calcd for [CnH₁₅N₃O₄S - H]⁺ 284.0710, found 284.0707.

(2£)-3-(3-Bromophenyl)-2-(yV-hydroxyimino)-yV-(2- sulfamoylethyl)propanamide (3)

[00162] Compound 3 was synthesised from compound 19 (0.105g, 0.2348 mmol) according to general procedure 4. The crude solid was purified by flash chromatography (Gradient: 5-6% MeOH in DCM) to afford the title compound as white solid (0.066 g, 77%). R_f = 0.28 (10% MeOH in DCM). Mp = 1 78- 1 80 °C. ¹ H NMR (500 MHz, DMSO-tf₆) δ_Η = 12.05 (s, 1 H, N-OH), 8.12 (t, J= 5.9 Hz, 1 H, N-H), 7.39-7.37 (m, 2H, H_Ar), 7.25-7.20 (m, 2H, H_Ar), 6.93 (s, 2H, SO₂NH₂), 3.81 (s, 2H, Ph-CH₂-C), 3.56 (q, J= 7.25 Hz, 2H, HN-CH2-CH₂), 3.15 (t, J= 7.4 Hz, 2H, HN-CH^CJi), general assignments were confirmed by ¹ H-¹ H gCOSY. ¹³C NMR (1 25 MHz, DMSO-ak) 5_C = 1 63.0 (C_quat), 1 50.9 (C_quat), 1 39.6 (C_quat), 131 .4 (CH_Ar), 130.5 (CH_Ar), 129.0 (CH_Ar), 127.9 (CH_Ar), 121 .5 (C_quat), 53.5 (HN- CH₂-CH₂), 34.2 (HN-CH₂-CH₂), 28.7 (Ph-CH₂-C), general assignments were confirmed by ¹H-¹³C HSQC. LRMS (ESI): m/z = 364, 366 [M + H, ⁷⁹Br, ⁸¹Br]⁺, 386, 388 [M + Na, ⁷⁹Br, ⁸¹ Br]⁺, HRMS (ESI): calcd for [CnH₁₄BrN₃O₄S - H, ⁷⁹Br]⁺ 361 .9815, found 361 .981 1 , calcd for [Cn H₁₄BrN₃O₄S - H, ⁸¹Br]⁺ 363.9795, found 363.9790.

(2£)-2-(yV-Hydroxyimino)-3-(4-hydroxyphenyl)-yV-(2- sulfamoylethyl)propanamide (4)

[00163] Compound 4 was synthesised from compound 20 (0.1 g, 0.2596 mmol) according to general procedure 4. The crude solid was purified by flash chromatography (Gradient: 5-8% MeOH in DCM) to afford the title compound as pale yellow solid (0.043 g, 55%). R_f = 0.1 1 (10% MeOH in DCM). Mp = 170-172 °C. ¹ H NMR (500 MHz, DMSO-tf₆) δ_Η = 1 1 .82 (s, 1 H, N-OH), 9.17 (s, 1 H, Ph-OH), 8.03 (t, J = 5.9 Hz, 1 H, N-H), 7.00 (d, J = 8.55 Hz, 2H, H_Ar), 6.93 (s, 2H, SO₂NH₂), 6.63 (d, J= 8.55 Hz, 2H, H_Ar), 3.68 (s, 2H, Ph-CH₂-C), 3.54 (q, J = 7.6 Hz, 2H, HN-CH₂-CH₂), 3.13 (t, J = 7.45 Hz, 2H, HN-CH₂-CH₂), general assignments were confirmed by ¹ H-¹ H gCOSY. ¹³C NMR (125 MHz, DMSO-tf₆) 5c = 163.3 (Cquat), 155.6 (C_quat), 152.1 (C_quat), 129.8 (2 ^χ CH_Ar), 126.8 (C_quat), 1 15.0 (2 x CH_Ar), 53.6 (HN-CH₂-CH₂), 34.1 (HN-CH₂-CH₂), 28.0 (Ph-CH₂-C), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z= 302 [M + H]⁺, HRMS (ESI): calcd for [Οιι Η₁₅Ν₃Ο₅8 - H]⁺ 300.0659, found 300.0656.

(2£)-3-(3-Bromo-4-hydroxyphenyl)-yV-(2-carbamoylethyl)-2-(yV- hydroxyimino)propanamide (5)

[00164] Compound 5 was synthesised from compound 22 (0.040 g, 0.0936 mmol) according to general procedure 4. The crude material was purified by flash chromatography (Gradient: 4-8% MeOH in DCM) to afford the title compound as off-white solid (0.024 g, 75%). R_f = 0.M (10% MeOH in DCM). Mp = 160-165 °C, decomposition. ¹ H NMR (500 MHz, DMSO-tf₆) δ_Η = 1 1 .88 (bs, 1 H, N-OH), 7.88 (t, J = 5.85 Hz, 1 H, N-H), 7.35 (bs, 1 H, O=C-NHH), 7.29 (d, J = 1 .9 Hz, 1 H, H_Ar), 7.00 (dd, J = 8.3, 1 .9 Hz, 1 H, H_Ar), 6.84 (bs, 1 H, O=C- NHH), 6.82 (d, J = 8.3 Hz, 1 H, H_Ar), 3.67 (s, 2H, Ph-CH₂-C), 3.31 (q, J = 6.35 Hz, 2H, HN-CH₂-CH₂), 2.26 (t, J= 6.95 Hz, 2H, HN-CH₂-CH₂), Ph-OH proton in exchange, general assignments were confirmed by ¹H-¹H gCOSY. ¹³C NMR (125 MHz, DMSO-ak) 5_C = 172.8 (C_quat), 162.8 (C_quat), 152.5 (C_quat), 151 .7 (Cquat), 132.8 (CH_Ar), 129.1 (CH_Ar), 128.7 (C_quat), 1 16.2 (CH_Ar), 108.9 (C_quat), 35.3 (HN-CH₂-CH₂), 34.5 (HN-CH₂-CH₂), 27.6 (Ph-CH₂-C), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z = 344, 346 [M + H, ⁷⁹Br, ⁸¹Br]⁺, 366, 368 [M + Na, ⁷⁹Br, ⁸¹ Br]⁺, HRMS (ESI): calcd for [C₁₂H₁₄BrN₃O₄ + Na, ⁷⁹Br]⁺ 366.0059, found 366.0053, calcd for [Cn H₁₄BrN₃O₄ + Na, ⁸¹Br]⁺ 368.0039, found 368.0032.

Synthesis of Compound 729

[00165] The compound and intermediate numbers and letters used in the below synthetic experimental for compound 729, refer to those as set out in scheme 2.

(4iE)-2-Methyl-4-(phenylmethylidene)-4,5-dihydro-1 ,3-oxazol-5-one (A)

[00166] To a mixture of anhydrous NaOAc (2.32 g, 28.27 mmol, 1 .0 equiv) and AAacetyl glycine (3.31 g, 28.27 mmol, 1 .0 equiv) suspended in Ac₂O (29 mL, 282.7 mmol, 10.0 equiv) at room temperature under inert atmosphere, was added the benzaldehyde (3.0 g, 28.27 mmol, 1 .0 equiv). The reaction mixture was stirred at 120 °C for 4 h and then cooled to room temperature. The crude product precipitated and was collected by suction filtration then added to ice cold water (25-30 mL) and vigorously stirred for 15-20 min. The solution was filtered and the residual crude oxazolone solid dried under vacuum to obtain the title compound A as yellow solid (3.55 g, 67%, crude). A small amount of crude solid was purified by flash chromatography (Gradient: 5-8% acetone in n- hexane) for characterisation purposes. R_f = 0.46 (20% acetone in n-hexane). Mp = 148-150 °C. ¹H NMR (500 MHz, DMSO-tf₆) δ_Η = 8.18-8.17 (m, 2H, H_Ar), 7.51 -7.47 (m, 3H, H_Ar), 7.22 (s, 1 H, Ph-CH=C), 2.39 (s, 3H, CH₃), general assignments were confirmed by ¹ H-¹ H gCOSY. ¹³C NMR (125 MHz, DMSO-tf₆) 5_C = 167.3 (Cquat), 66.7 (C_quat), 133.0 (C_quat), 132.6 (C_quat), 131 .9 (2 x CH_Ar), 131 .0 (1 x CH_Ar), 129.7 (Ph-CH=C), 128.8 (2 χ CH_Ar), 15.3 (CH₃), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z= 188 [M + H]⁺.

(2E)-2-[(oxan-2-yloxy)imino]-3-phenylpropanoic acid (C)

[00167] A mixture of benzylidine oxazolone A (3.0 g, 16.03 mmol, 1 equiv) in 10% aqueous HCI (160 mL, 10 mL/mmol) was stirred at 100 °C overnight (12- 14 h) then allowed to cool to room temperature (~3 h). The product precipitated and was filtered, washed with ice-cold water (10-15 mL) and dried under vacuum. The filtrate was washed with EtOAc (3 χ 20 mL), then the combined organic fractions washed with water (1 χ 20 mL) and saturated aqueous NaCI (1 x 20 mL), dried over MgSO₄ and concentrated in vacuo. The oily residue was triturated with a minimal amount of n-hexane. The n-hexane portion was decanted and the crude solid was dried under vacuum to obtain crude aryl pyruvic acid B as off-white solid (1 .63 g, 62%, crude). This crude aryl pyruvic acid B was used for the next step without further purification. To the mixture of 2 (1 .6 g, 9.76 mmol, 1 equiv) and 0-(tetrahydro-2 -/-pyran-2-yl) hydroxylamine (NH₂OTHP, 1 .71 g, 14.63 mmol, 1 .5 equiv) was added anhydrous pyridine (20 mL, 2 mL/mmol). The reaction mixture was stirred at room temperature under an argon atmosphere overnight (12-14 h). Pyridine was removed in vacuo. 1 .0 M HCI (5.5 mL/mmol) was added to the remaining residue and extracted in EtOAc (3 x 25 mL). The combined organic fractions were washed with saturated aqueous NaCI (1 χ 20 mL/mmol), dried over MgSO₄ and concentrated in vacuo to obtain the crude residue. The crude residue was purified by flash chromatography (0.5-1 % MeOH in DCM) to afford the title compound as white solid. (1 .065 g, 41 %). ^= 0.17 (10% MeOH in DCM). Mp = 57-59 °C. ¹ H NMR (500 MHz, DMSO-tf₆) δ_Η = 7.31 -7.28 (m, 2H, H_Ar), 7.23-7.19 (m, 3H, H_Ar), 5.35 (s, 1 H, CH_THp), 3.90 (d, J= 13.6 Hz, 1 H, Ph-CHH-C), 3.84 (d, J= 13.6 Hz, 1 H, Ph-CHH-C), 3.49-3.45 (m, 2H, H_THp) , 1 -75-1 .69 (m, 3H, H_THp) , 1 .59-1 .52 (m, 2H, H_THp), 1 -45-1 .43 (m, 1 H, H_THp) , COOH proton in exchange, general assignments were confirmed by ¹ H-¹ H gCOSY. ¹³C NMR (125 MHz, DMSO-ofe) 5c = 164.5 (C_quat), 151 .9 (C_quat), 136.1 (C_quat), 128.6 (2 ^χ CH_Ar), 128.4 (2 x CH_Ar), 126.4 (CH_Ar), 100.8 (CH_THp), 61 .3 (CH₂-THP), 31 .0 (Ph-CH₂-C), 28.1 (CH₂-THP), 24.6 (CH_2THp), 18.5 (CH₂-THP), general assignments were confirmed by ¹H-¹³C HSQC. LRMS (ESI): m/z = 286 [M + Na]⁺. HRMS (ESI): calcd for [Ci₄H₁₇NO₄ - H]⁺ 262.1085, found 262.1083.

(2iE)-2-[(oxan-2-yloxy)imino]-3-phenyl-N-(5-sulfamoyl-1 ,3,4-thiadiazol-2- yl)propanamide (D)

[00168] To a mixture of O-THP protected oxime acid C (0.245 g, 0.93 mmol, 1 .0 equiv), EDC.HCI (0.267 g, 1 .397 mmol, 1 .5 equiv) and HOBt.H₂O (0.213 g, 1 .397 mmol, 1 .5 equiv) under Ar atmosphere, was added anhydrous DMF (10 imL, 10 imL/mmol) and the resulting solution was stirred at room temperature for 45 min. Next, 5-amino-1 ,3,4-thiadiazole-2-sulfonamide (0.20 g, 1 .1 18 mmol, 1 .2 equiv) was added and the resulting mixture was stirred at room temperature for 24 h. DMF was then evaporated under high vacuum and H₂O (15 imL), added to the remaining residue, followed by extraction with EtOAc (3 ^χ 25 imL). The combined organic fractions were washed with saturated aqueous NaHCO₃ (2 ^χ 15 imL/mmol), saturated aqueous NaCI (1 ^χ 20 imL/mmol), dried over MgSO₄ and concentrated in vacuo to obtain the crude residual solid. The crude solid was purified by flash chromatography (Gradient: 2-2.5 % MeOH in DCM) to afford the title compound as white solid (0.217 g, 54%). R_f= 0.23 (5% MeOH in DCM). Mp = 92-94 °C. ¹H NMR (500 MHz, DMSO-tf₆) δ_Η = 13.22 (s, 1 H, N-H), 8.36 (s, 2H, SO₂NH₂), 7.32-7.25 (m, 4H, H_Ar), 7.23-7.19 (m, 1 H, H_Ar), 5.53 (t, J = 3.2 Hz, 1 H, CHTHP), 4.01 (d, J = 13.9 Hz, 1 H, Ph-CHH-C), 3.94 (d, J = 13.9 Hz, 1 H, Ph-CHH-C), 3.49-3.46 (m, 2H, H_THP), 1 .80-1 .77 (m, 2H, H_THP), 1 .75- 1 .68 (m, 1 H, HTHP), 1 -62-1 .53 (m, 2H, H_THp) , 1 -47-1 .44 (m, 1 H, H_THp) , general assignments were confirmed by ¹ H-¹ H gCOSY. ¹³C NMR (125 MHz, DMSO-cfe) 5c = 164.8 (Cquat), 162.5 (C_quat), 161 .2 (C_quat), 151 .5 (C_quat), 135.6 (C_quat), 128.7 (2 x CH_Ar), 128.6 (2 ^χ CH_Ar), 126.6 (CH_Ar), 101 .4 (CH_THp), 61 .4 (CH₂-THP), 30.4 (Ph-CH₂-C), 28.0 (CH₂-THP), 24.5 (CH₂-THP), 18.2 (CH₂-THP), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z= 426 [M + H]⁺, 448 [M + Na]⁺, HRMS (ESI): calcd for [C₁₆H₁₉N₅O₅S₂ - H]⁺ 424.0754, found 424.0752.

(2iE)-2-(N-hydroxyimino)-3-phenyl-N-(5-sulfamoyl-1 ,3,4-thiadiazol-2- yl)propanamide (729)

[00169] To the oxime THP ether D (0.125 g, 0.294 mmol) was added 4.0 M HCI in 1 ,4-dioxane solution (4.4 imL, 15 imL/mmol) at 0 °C, the reaction mixture was stirred at the same temperature. After completion of reaction (TLC monitoring) the solvent evaporated in vacuo and the crude residue. The crude residue was purified by flash chromatography (Gradient: 5-7% MeOH in DCM) to afford the title compound as white solid (0.070 g, 70.0%). ft^ = 0.28 (10% MeOH in DCM). Mp = 260-270 °C, decomposition. ¹H NMR (500 MHz, DMSO- d₆) δ_Η = 12.96 (s, 1 H, N-H), 12.73 (s, 1 H, N-OH), 8.34 (s, 2H, SO₂NH₂), 7.29- 7.26 (m, 2H, H_Ar), 7.24-7.23 (m, 2H, H_Ar), 7.21 -7.17 (m, 1 H, H_Ar), 3.94 (s, 2H, Ph-CH₂-C), general assignments were confirmed by ¹ H-¹ H gCOSY. ¹³C NMR (125 MHz, DMSO-ak) 5_C = 164.7 (C_quat), 163.2 (C_quat), 161 .1 (C_quat), 150.4 (Cquat) , 136.1 (Cquat), 128.6 (2 χ CH_Ar), 128.5 (2 χ CH_Ar), 126.4 (CH_Ar), 29.3 (Ph- CH₂-C), general assignments were confirmed by ¹ H-¹³C HSQC. LRMS (ESI): m/z= 342 [M + H]⁺, 364 [M + Na]⁺, HRMS (ESI): calcd for [CnHnNsO^ - H]⁺ 340.0179, found 340.01 80.

Biology

Cells

[00170] 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. The histological diagnosis of GB was performed according to WHO guidelines. Cells were cultured as adherent cells (AC) or stem-cell like cells (neurospheres, NS) as previously described [Reynolds, 1992], with minor modifications. For AC, DMEM supplemented with 1 % penicillin-streptomycin, 10% v/v fetal bovine serum (FBS; Lonza, Basel, Switzerland) was used. For NS, DMEM-F12 medium was supplemented with 1 M Hepes, 0.3 img/mL glucose, 75 pg/mL NaHCO₃, 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 ^χ 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].

[00171 ] For phenotypic characterization, the following antibodies were used: anti-CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-nestin (Millipore, Billerica, MA), anti-Musashi-1 (Millipore), considered markers of neural/GB stemness; anti-glial fibrillary acidic protein (GFAP; Dako, Glostrup, Denmark), anti-galactocerebroside (Gal-C; Millipore), considered typical markers of neural differentiation. Secondary goat anti-rabbit fluorescein- isothiocyanate (FITC)-conjugated IgG and rabbit anti-mouse tetramethyl rhodamine iso-thiocyanate (TRITC)-conjugated IgG antibodies were used. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). 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 μΙ/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. 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 0⁵ 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.

Immunoblotting

[001 72] For whole cell lysates, 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 μΜ aprotinin, 5 mM bestatin, 1 .5 mM leupeptin, 1 mM pepstatin; Calbiochem, San Diego, CA), 2 mM phenylmethylsulfonyl fluoride and 1 mM Na₃VO₄, sonicated and centrifuged at 1 3,000 x g for 1 0 min at 4 °C. 20 pg protein extracts were subjected to SDS- PAGE and probed with the following antibodies: anti-CAXII (2C6; Abeam, Cambridge, UK), anti-CAIX (NB1 00-41 7; Novus Biologicals, Littleton, CO), anti- Pgp (C21 9; Calbiochem), anti-caspase 3 (C33, GeneTex, Hsinhu City, Taiwan), anti-p-tubulin (D-1 0; Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by a peroxidase-conjugated secondary antibody (Bio-Rad Laboratories). 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. In co-immunoprecipitation experiments, 100 pg of plasma membrane-associated proteins were immunoprecipitated with the anti- CAXII and anti-CAIX antibodies, using the PureProteome protein A and protein G Magnetic Beads (Millipore, Billerica, MA). The immunoprecipitated proteins were separated by SDS-PAGE and probed with the anti-Pgp antibody, followed by a peroxidase-conjugated secondary antibody.

Flow cytometry

[00173] Cells were washed with PBS, detached with Cell Dissociation Solution (Sigma Chemical Co.) and re-suspended in culture medium containing 5 % v/v FBS. For CAXII surface analysis, samples were washed with 2.5 pg/mL bovine serum albumin/phosphate-buffered saline (BSA-PBS), incubated with the anti-CAXII antibody (Abeam) 45 min at 4 °C, washed twice and incubated with the secondary fluorescein isothiocyanate (FITC)-conjugated antibody for 30 min at 4 °C, then fixed with 2.5% v/v paraformaldehyde (PFA). For Pgp surface analysis, 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. 5 χ 10⁵ cells were analyzed by the Guava® easyCyte flow cytometer (Millipore), using the InCyte software (Millipore). Control experiments included incubation of cells with non-immune isotypic antibody, followed by secondary antibody.

Proximity ligation assay (PLA)

[00174] The 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 (10χ ocular lens; 63χ objective lens; Leica Microsystem, Wetzlar, Germany). For each experimental condition, a minimum of five fields were examined.

Pgp ATPase activity

[00175] The assay was performed on Pgp-enriched membrane vesicles as detailed in [Kopecka, 2014]. Verapamil (10 μιτιοΙ/L) was added to the reaction mix to achieve a maximal activation of the Pgp ATPase activity. Results were expressed as nmol hydrolyzed phosphate (Pi)/min/mg proteins, according to the titration curve previously prepared.

Doxorubicin and temozolomide accumulation

[00176] 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 μΜ [³H]-temozolomide (0.7 Ci/ml; Moravek Biochemical Inc., Brea, CA). The amount of [³H]-temozolomide in cell lysate was measured by liquid scintillation counting. The results were expressed as nmol [³H]-temozolomide/mg cell proteins, according to a titration curve previously set.

Lactate dehydrogenase (LDH) release

[00177] The release of LDH in cell supernatant, considered an index of cell damage and necrosis [Riganti, 2005], was measured as it follows: the extracellular medium was centrifuged at 12,000 ^χ g for 15 min to pellet cellular debris, whereas the cells were washed with fresh medium, detached with 0.01 % v/v trypsin/EDTA, re-suspended in 0.2 imL of 82.3 imM triethanolamine phosphate-HCI (pH 7.6) and sonicated on ice with two 10 s-bursts. LDH activity was measured in the extracellular medium and in the cell lysate: 50 μί of supernatant from extracellular medium or 5 μί 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 μιηοΙ NADH oxidized/min/dish, then extracellular LDH activity was calculated as percentage of the total LDH activity in the dish.

Cell viability

[00178] Cell viability was evaluated by measuring the percentage of cells stained with neutral red dye, as reported previously [Riganti, 2015]. The viability of untreated cells was considered 100%; the results were expressed as percentage of viable cells in each experimental condition versus untreated cells.

Generation of Pgp- and CAXII-knocked out (KO) clones

[00179] 5 x 10⁵ cells were transduced with 1 g RNA vector (CRISPR pCas guide vector, Origene, Rockville, MD) targeting ABCBI/Pgp or CAXII, respectively, or with 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

[00180] In a first experimental set, 1 x 10⁶ AC or NS cells from UPN2, mixed with 100 μΙ_ Matrigel, were injected subcutaneously in female BALB/c nu/nu mice (weight: 19.6 g + 2.4; Charles River Laboratories Italia, Calco). Animals were housed (5 per cage) under 12 h light/dark cycles in a barrier facility on HEPA-filtered racks and were fed with autoclaved diet. Tumor growth was measured daily by caliper and calculated according to the equation (L x W²)/2, where L = tumor length, W = tumor width. When the tumor reached a volume of 50 mm³, animals 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 μΜ) 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. and 38 ng/kg compound 741 i.v.; 6) 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) 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.

[00181 ] In a second experimental set, 1 χ 10⁶ NS cells from UPN2, stably transfected with the pGL4.51 [luc2/CMV/Neo] vector encoding for luciferase (Promega Corporation), mixed with 300 μΙ_ sterile physiological solution, were stereotactically injected into the right caudatus nucleus into 6-8 week olds female BALB/c nulnu mice (weight: 20.3 g + 2.4), anesthetized with sodium phenobarbital (60 mg/kg) intraperitoneal^ (i.p.). Tumor growth was monitored by in vivo bioluminescence (Xenogen IVIS Spectrum, PerkinElmer, Waltham, MA) at day 6, 14 and 24 post-implantation. At day 7, 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 μΜ) 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. and 3800 ng/kg compound 741 i.v. Animals were euthanized at day 30. Brains were fixed in 4% v/v paraformaldehyde at 4°C overnight. Volume of excised tumors was measured as described above. Tumor sections were stained with hematoxylin and eosin or immunostained for Ki67 (Millipore) and cleaved (Asp175)caspase 3 (Cell Signaling Technology Inc., Danvers, MA), followed by a peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark). Sections were examined with a Leica DC100 microscope (Leica Microsystems GmbH, Wetzlar, Germany; 10 χ ocular lens, 20 χ objective).

[00182] In a third experimental set, 1 χ 10⁶ NS cells from UPN2, stably transfected with the pGL4.51 [luc2/CMV/Neo] vector encoding for luciferase (Promega Corporation), mixed with 300 μΙ_ sterile physiological solution, were stereotactically injected into the right caudatus nucleus into 6-8 week olds female BALB/c nulnu mice (weight: 20.3 g + 2.4), anesthetized with sodium phenobarbital (60 mg/kg) intraperitoneal^ (i.p.). Tumor growth was monitored by in vivo bioluminescence (Xenogen IVIS Spectrum, PerkinElmer, Waltham, MA) at day 6, 14 and 24 post-implantation. At day 7, 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 mL saline solution i.v.; 2) 729 group, treated with compound 729 (in 0.2 mL saline solution; final concentration: 1 μΜ) 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 μΜ of compound 729 i.v., respectively. Animals were euthanized at day 30, as reported. Brain were fixed in 4% v/v paraformaldehyde at 4°C overnight. Volume of excised tumors was measured as described above. Tumor sections were stained with hematoxylin and eosin or immunostained for Ki67 (Millipore) and cleaved (Asp175)caspase 3 (Cell Signaling Technology Inc., Danvers, MA), followed by a peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark). Sections were examined with a Leica DC100 microscope (Leica Microsystems GmbH, Wetzlar, Germany; 10 χ ocular lens, 20 x objective).

[00183] 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

[00184] 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 μί). 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. All plasma samples were stored frozen (-20 °C) until analysis by liquid chromatography-mass spectrometry (LC-MS, using a Micromass Xevo triple quadrupole mass spectrometer; Waters Co., Milford, MA) relative to calibration standards (741 and diazepam as internal standard) prepared in blank mouse plasma. At each sample time, the average concentration of test compound (based on triplicate samples) was expressed as a % compound remaining relative to the sample quenched at time = 5 min. Studies were performed by Centre for Drug Candidate Optimisation, Monash University, Melbourne, Australia. in Vitro Metabolic Stability

[00185] The metabolic stability assay was performed in mouse liver microsomes (Xenotech, Tokyo, Japan, lot#1510043). Compounds 741 and 729 (1 μΜ) 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.

In Vitro Cytochrome P450 (CYP) Stability

[00186] Compound 1 (0.25 to 20 μΜ) 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.

Statistical analysis

[00187] All data in the text and figures are provided as means + SD. The results were analyzed by a one-way analysis of variance (ANOVA) and Tukey's test, using Statistical Package for Social Science (SPSS) software (IBM SPSS Statistics v.1 9). The Kaplan-Meier method was used to calculate overall survival of mice. Log rank test was used to compare the outcome of the treatment groups, using MedCalc® software (v.17.4). p < 0.05 was considered significant.

RESULTS

Glioblastoma-derived stem cells co-express CAXII and Pgp

[00188] From three GB patient (Unknown patient number, UPN1 -3) were generated primary cultures grown as differentiated/adherent cells (AC) or NS. The clinical, genomic and phenotypic data for samples are provided in Table 2 and Table 3. Patient cells when cultured as NS were found to have comparable levels of CAIX and CAXII but when cultured as AC only CAIX was detectable, while CAXII was not (Figure 1 A part a). NS had higher levels of both CAXII and Pgp on the cell surface than AC (Figure 1 A part b). In NS, plasma membrane- associated CAXII, but not CAIX, co-immunoprecipitated with Pgp (Figure 1 A part c), and was physically associated with Pgp (Figure 1 A part d). Pgp and CAXII expression were independent of different culture conditions for AC and NS. Pgp and CAXII were undetectable in AC cultured in NS medium, but remained at detectable levels in NS cultured in AC medium (Figure 1 B).

Inhibitors of CAXII reduce Pgp activity in glioblastoma-derived stem cells, increasing retention and cytotoxicity of Pgp substrates

[00189] The effect of CAXII inhibition on Pgp ATPase activity in NS derived from the three patients was tested against a compound panel comprising Psammaplin C (1 ), its derivatives (2-4) and the non-CAXII inhibitor control compound (5) (Figure 2a). Compound 1 (CAXII Kj = 0.79 nM) and 3 (CAXII Kj = 43 nM), the most potent CAXII inhibitors of the panel (Table 4), produced the strongest reduction of Pgp-ATPase activity (Figure 2b). Compound 4 (CAXII Kj = 219 nM) had a less pronounced but similar effect to 1 and 3, while compounds 2 (Kj>50,000 nM) and 5 (inactive control) had no effect (Figure 2b), consistent with CAXII/Pgp expression and relative CAXII inhibition constants of the compounds. AC cells treated with doxorubicin accumulated the drug intracellular^ (Figure 2c) and toxicity was evident (Figure 2d-e). These traits were unchanged by compounds 1-5 (Figure 2c-e). NS did not accumulate doxorubicin and were resistant to the drug. Compounds 1 or 3, the two most potent CAXII inhibitors, restored the intracellular doxorubicin concentration to a level comparable to that found in doxorubicin-treated AC (Figure 2c). Additionally, compounds 1 and 3 partially restored the release of lactate dehydrogenase (LDH) induced by doxorubicin (Figure 2d) and reduced cell viability (Figure 2e) in NS.

[00190] The chemosensitizing effects of the test compounds were not drug- specific, and a similar effect to doxorubicin was observed also in AC and NS treated with other Pgp substrate chemotherapeutic drugs, including etoposide (Figure 10), topotecan (Figure 1 1 ) and irinotecan (Figure 12). Similar results were obtained in all patient derived and U87-MG AC and NS analyzed (data shown is for UPN2). As compounds 1 (741 ) and 729 were the most effective in restoring the anti-tumor effect of all these drugs, it was selected for further characterization in the following experiments.

CAXII inhibition enhances temozolomide cytotoxicity in neurospheres by reducing Pgp activity

[00191 ] TMZ is a substrate of Pgp. As 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). Accumulation of TMZ was lower in Pgp- expressing NS than in AC (Figure 3c). Furthermore, TMZ treatment of NS did not lead to a boost in LDH release (Figure 3d), activation of caspase 3 (Figure 3e) or reduction of viability (Figure 3f). Co-incubation of TMZ with compound 1 increased TMZ accumulation in NS (Figure 3c) and restored the cytotoxic effects of TMZ to the extent observed in Pgp-KO NS clones (wherein levels of CAXII are unaltered) or in AC (Figure 3d-f; Figure 15b-c).

CAXII inhibition with compound 729 enhances temozolomide cytotoxicity in glioblastoma-derived stem cells by reducing Pgp activity

[00192] A downregulation of Pgp expression in TMZ-treated UPN2 NS (Figure 4A-B) was also observed. Compound 729 had no effect on Pgp expression (Figure 4A), but it further reduced Pgp ATPase activity in TMZ- treated NS (Figure 4B) compared to TMZ treatment alone. It has been previously reported that TMZ is a substrate of Pgp. Our results show that TMZ accumulated less in Pgp-expressing NS than in AC (i.e. Pgp-negative and CAXII-negative cells) (Figure 4C). Consistent with this, 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). To verify that the chemosensitizing effects of 729 were mediated by a reduced Pgp activity, a UPN2 clone knocked-out for Pgp but with unaltered CAXII levels (Figure 13) was produced. The 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

[00193] CAX7/-KO NS clones (Figure 5a; Figure 16a) had lower Pgp-ATPase activity than wild-type NS (Figure 5b), even though the Pgp level was the same in both (Figure 5a). TMZ further reduced Pgp-ATPase activity in CAX7/-KO NS clones (Figure 5b) and produced the same phenotypic response as it did in AC, i.e. greater accumulation (Figure 5c), increased acute cell cytotoxicity (Figure 5d) and apoptosis (Figure 5e; Figure 16b), and reduced cell viability (Figure 5f). Together these findings suggest that CAXII activity mediated Pgp-induced resistance to TMZ in NS and that CAXII inhibition may overcome this resistance.

CAXII inhibition enhances the cytotoxicity of temozolomide in combination with Pgp substrates in neuropsheres

[00194] To obtain further evidence that the chemosensitizing effects of compound 1 were mediated through CAXII inhibition indirectly inhibiting Pgp activity, the effects of co-administration of compound 1 and/or TMZ with other known substrates of Pgp (doxorubicin, etoposide, topotecan and irinotecan) on patient 2-derived NS were analyzed. As expected, the four drugs, when used alone, showed no toxicity above untreated cells, including no increase of LDH release (Figure 6a), no activation of caspase 3 (Figure 6b) and no reduction of cell viability (Figure 6c). However, when either compound 1 or TMZ was combined with the four drugs, toxicity parameters became evident (Figure 6a- c). A triple combination of compound 1 , TMZ and other drug, even further enhanced the cytotoxic effects (Figure 6a-c).

CAXII inhibition with compound 729 enhances the cytotoxic effect of temozolomide and Pgp substrates in glioblastoma-derived neuropsheres subjected to combination treatments

[00195] The effects of the co-administration of compound 729, TMZ and chemotherapeutic drugs that are substrates of Pgp were analysed, namely doxorubicin, etoposide, topotecan and irinotecan, that are under evaluation for GB treatment and have different pharmacodynamics properties. The latter four drugs when used alone in NS did not exert a cytotoxic effect, that is, no release of LDH (Figure 7A), no activation of caspase 3 (Figure 7B) and no reduction in cell viability (Figure 7C) was observed. However, when either compound 729 or TMZ was combined with each of the four drugs the toxicity parameters were evident (Figure 7A-7C). Lastly, the triple combination of compound 729, TMZ and each of these drugs, enhanced further the cytotoxic effects of the drugs over the dual combination of 729 or TMZ with each drug (Figure 7A-7C).

CAXII inhibition restores the efficacy of temozolomide in tumors derived from resistant glioblastoma neurospheres in vivo

[00196] As variable schedules of TMZ administration per os have shown different efficacy against GB, it was decided to first identify an in vivo dosing schedule that maximally reduced tumor growth of AC (Figure 14a), i.e. 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). The same dosage had significantly lower efficacy against NS (Figure 14b-c). This is consistent with the poor response to TMZ in in vitro culture of NS (Figure 3).

[00197] In a preliminary dose-response experimental set, 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). Notably, compound 1 caused no hematopoiesis, liver, kidney or muscle toxicity. At the end of the treatment TMZ slightly reduced the number of platelets, however the co-administration of TMZ and compound 1 did not further decrease this parameter (Table 6).

[00198] The effect of compound 1 and TMZ in orthotopic patient-derived NS- xenografts was then investigated. The NS model is expected to be TMZ- resistant and indeed neither compound 1 nor TMZ, when used alone, reduced tumor growth (Figure 8a-b). The tumor growth was however significantly reduced by co-treatment with TMZ and compound 1 (Figure 8a-b). The decrease in tumor growth correlated with a significantly increased overall survival in all patient-derived xenografts (Figure 8c). Only mice with patient 3- derived NS showed a partial response to TMZ (Figure 8b-c): this could be due to the MGMT promoter methylation status and to the genomic profile of UPN3 GB cells (Table 6) that are suggestive of increased TMZ sensitivity. Even so, in UPN3, the addition of compound 1 significantly improved the magnitude of TMZ anti-tumor effects (Figure 8b-c).

[00199] The effects of compound 729 and TMZ in orthotopically implanted UPN2 NS was investigated. When used alone, neither compound 729 nor TMZ reduced the growth of tumor, as observed by in vivo bioluminescence imaging (Figure 9A) and by the analysis of tumor volume following resection (Figure 9B). Tumor growth and volume were significantly reduced by the combination of TMZ + compound 729 (Figure 9A-9C). Furthermore, in contrast to untreated animals or animals treated with compound 729 or TMZ alone, the combination treatment also strongly reduced the intratumor cell proliferation and increased the intratumor apoptosis (Figure 9C-9D). Similarly to compound 741 , 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.

[00200] Furthermore, in NS-derived tumors showing detectable levels of CAXII, the combination treatment of TMZ and compound 1 reduced Pgp expression and cell proliferation, and increased intra-tumor apoptosis, as indicated by Ki67 and cleaved caspase 3 staining, in contrast to untreated animals or animals treated with either compound 1 or TMZ alone (Figure 8d-e).

[00201 ] Compound 1 was stable in Balb/c mice plasma: at 4 h 94% of compound 1 remained. The in vitro metabolic stability indicated no measurable degradation of 1. Based on E_H compound 1 was classified to have low in vivo clearance and a plasma half-life > 240 min (Table 5). The potential for compound 1 to inhibit major drug metabolizing cytochrome P450 (CYP) isoforms (CYPs 1 A2, 2C9, 2C19, 2D6 and 3A4) was assessed in human liver microsomes. Compound 1 exhibited minimal reduction of the formation of CYP- mediated metabolites across the CYP panel (IC₅₀ > 20 μΜ; Table 1 ). These data suggest that compound 1 has low potential to inhibit CYP-mediated metabolism of co-administered drugs. The same protocol was applied to compound 729 with the results shown in Figure 17.

DISCUSSION

[00202] It has been proposed that 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 and 729 are sub-nanomolar inhibitors of the cancer associated CAXII ( i = 0.79 nM and 0.56 nM, respectively), while compound 741 also displayed very high isoform selectivity CAXII over most other CAs, including >100-fold selectivity over CAM - a ubiquitous CA (Table 4). Control compound 787 was ineffective in all assays, providing strong confirmation that CAXII is central to the effects observed.

[00203] 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.

[00204] The efficacy of TMZ in association with compounds 741 or 729 was tested, which produced a significant reduction in Pgp catalytic activity in GB NS. NS treated with TMZ and compound 741 or compound 729 had the same amount of Pgp than cells treated with TMZ alone, but the former exhibited lower Pgp activity. This result suggests that CAXII inhibition does not change Pgp gene transcription but does reduce the catalytic efficacy of Pgp. Without wishing to be bound by theory, it is likely that this reduction is a consequence of the altered pH of membrane microenvironment where Pgp works from optimum pH in the absence of active CAXII. Notably, the co-treatment of NS with CAXII inhibitor and TMZ switched the effect of TMZ such that it became as effective in NS as was TMZ alone in Pgp-negative AC or in SC knocked-out for Pgp.

[00205] Compared to acetazolamide, which strongly inhibits many different CA isoforms, 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. Moreover, due to the high activity and selectivity for CAXII, 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.

[00206] Since CAXII has minimal expression in healthy cells (https://www.proteinatlas.orq/ENSG00000074410-CA12/tissue), a major advantage of targeting CAXII to indirectly reduce Pgp efflux activity in tumors, including GB, is avoidance of the usual in vivo toxicity associated with Pgp inhibitors. 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. The use of 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.

[00207] The efficacy of 1 was validated in patient-derived NS xenografts. In line with the resistance to TMZ observed in NS cultures and the poor clinical response to TMZ of the corresponding patient, two out of three xenografts were fully refractory to TMZ. The third xenograft - generated from the patient with the most favorable genetic profile suggestive of TMZ sensitivity, the longest time to recurrence after TMZ treatment and highest overall survival - was partially sensitive to TMZ. Of note, in all patient-derived xenografts, compound 1 significantly improved the anti-tumor activity of TMZ and prolonged the overall survival. The 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.

[00208] 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).

[00209] 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 μΜ) 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. The combination of compound 741 and TMZ significantly reduced tumor growth and tumor cell proliferation, and increased the intratumor apoptosis in the orthotopic model, signifying an effective rescue of TMZ efficacy in the relevant in vivo setting for GB SC is achievable with CAXII inhibitors. Similarly to 741 and TMZ, the combination of compound 729 and TMZ significantly reduced tumor growth and tumor cell proliferation, and increased the intratumor apoptosis in the orthotopic model, signifying an effective rescue of TMZ efficacy in the relevant in vivo setting for GB SC is achievable with CAXII inhibitors.

[00210] It will therefore be appreciated from the above discussion and data that a new mechanism of glioblastoma-derived stem cells chemoresistance has been demonstrated by showing that patient-derived neurospheres co-express CAXII and P-glycoprotein, and that CAXII is necessary for optimum P- glycoprotein efflux of temozolomide and second-line chemotherapeutic drugs. Psammaplin C, a potent inhibitor of CAXII, re-sensitizes drug-resistant glioblastoma to temozolomide, independent of other known temozolomide resistance factors present in the patients. It has been shown that 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. It can therefore be seen that 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.

TABLES

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. ^aWhere less than 50% inhibition was observed at 20 μΜ (i.e. the highest concentration tested), the IC₅₀ value is deemed to be >20 μΜ. n.m.i. No measurable inhibition of CYP activity at the highest concentration of the test compound used.

co-deletion

Table 2. Patient clinical, pathological and genetic data Anagraphic, pathological, clinical and genetic data of patients of samples were used in the study. Radiotherapy: 60 Gy (30 fractions). Chemotherapy: 75 mg/m² temozolomide (TMZ), per os, daily, concurrently to radiotherapy, followed by 200 mg/m² TMZ, per os, days 1 -5, every 28 days, 6 cycles. Post-recurrence therapy: radiotherapy: 60 Gy (30 fractions); chemotherapy: 80 mg/m² 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: O⁶-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

CD133 - - - + - -/+

Nestin - + - ++ - +

Musashi-1 - + - ++ - +

GFAP + - + - + -

Gal-C + - +/- - + -

Nanog 1.3% 98.7% 2.7% 97.3% 1.8% 98.2%

Oct4 1.2% 98.8% 1.4% 98.6% 1.6% 98.4%

SOX2 2.4% 97.6% 2.2% 97.8% 1.7% 98.3%

ABCG2 3.4% 96.6% 5.1% 94.9% 1.9% 98.1%

ALDH ^bright 0.6% 99.4% 0.1% 99.9% 1.4% 98.6%

Table 3. Phenotypic characterization of primary patient-derived adherent cells and neurospheres. 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.

Table 4. Human CA inhibition and CAXII selectivity profile for compounds 1 -4 and the reference CA inhibitor acetazolamide. ^a Errors ± 10% of the reported values (n=3). hCA: human carbonic anhydrase; AZA: acetazolamide. Compound 729 also had a K, of 2.2 nm at CAI and 1 .3 nm at CAIX.

Table 5. In vitro mouse plasma stability and mouse microsome stability data for compound 1 . CL_int: in vitro clearance; E_H: hepatic extraction ratio.

Table 6. Hematochemical parameters of animals. Animals (n = 10/group) were treated as reported in Supplementary Figure 5B. Blood was collected immediately after euthanasia and analyzed for RBC, hemoglobin (Hb), WBC, platelets (PLT), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), creatinine, creatine phosphokinase (CPK). Ctrl: mice treated with saline solution; 1 LD: 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.

Table 7. In vitro mouse plasma stability and mouse microsome stability data for compounds 741 and 729; CL_int: in vitro clearance; E_H: hepatic extraction ratio.

References

-Mujumdar, P.; Teruya, K.; Tonissen, K.; Vullo, D.; Supuran, C; Peat, T.; Poulsen, S.-A. An Unusual Natural Product Primary Sulfonamide: Synthesis, Carbonic Anhydrase Inhibition, and Protein X-ray Structures of Psammaplin C. J. Med. Chem. 2016, 59, 5462-5470.

-Nicolaou, K. C; Hughes, R.; Pfefferkorn, J. A.; Barluenga, S.; Roecker, A. J. Combinatorial synthesis through disulfide exchange: Discovery of potent psammaplin A Type antibacterial agents active against methicillin-resistant staphylococcus aureus (MRSA). Chem. Eur. J. 2001 , 7, 4280-4295.

-Baud, M. G. J.; Leiser, T.; Meyer-Almes, F.-J.; Fuchter, M. J. New synthetic strategies towards psammaplin A, access to natural product analogues for biological evaluation. Org. Biomol. Chem. 2011 , 9, 659-662. -Prasad, K.; Jiang, X.; Slade, J. S.; Clemens, J.; Repic, 0.; Blacklock, T. J. New Trends in Palladium-Catalyzed Transfer Hydrogenations Using Formic Acid. Adv. Synth. Catal. 2005, 347, 1769-1773.

-Yoshida, M.; Saito, K.; Fujino, Y.; Doi, T. A concise total synthesis of biologically active frutinones via tnbutylphosphine-catalyzed tandem acyl transfer-cyclization. Tetrahedron 2014, 70, 3452-3458.

-Reynolds BA, Tetzlaff W, Weiss S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 1992;12:4565-4574.

-Caldera V, Mellai M, Annovazzi L, Piazzi A, Lanotte M, Cassoni P, Schiffer D. Antigenic and Genotypic Similarity between Primary Glioblastomas and Their Derived Neurospheres. J Oncol. 201 1 ; 201 1 :e314962.

- Riganti C, Salaroglio IC, Caldera V, Campia I, Kopecka J, Mellai M, Annovazzi L, Bosia A, Ghigo D, Schiffer D. 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.

- De Boo S, Kopecka J, Brusa D, Gazzano E, Matera L, Ghigo D, Bosia A, Riganti C. iNOS activity is necessary for the cytotoxic and immunogenic effects of doxorubicin in human colon cancer cells. Mol Cancer. 2009; 8: e108

- Kopecka J, Salzano G, Campia I, Lusa S, Ghigo D, De Rosa G, Riganti C. Insights in the chemical components of liposomes responsible for P- glycoprotein inhibition. Nanomedicine. 2014; 10: 77-87

- Riganti C, Miraglia E, Viarisio D, Costamagna C, Pescarmona G, Ghigo D, Bosia A. Nitric oxide reverts the resistance to doxorubicin in human colon cancer cells by inhibiting the drug efflux. Cancer Res. 2005; 65: 516-525

- Riganti C, Kopecka J, Panada E, Barak S, Rubinstein M. The Role of C/ΕΒΡ-β LIP in Multidrug Resistance. J Natl Cancer Inst 2015, 107(5): djv046 - Ring BJ, Chien JY, Adkison KK, Jones HM, Rowland M, Jones RD, Yates JW, Ku MS, Gibson CR, He H, Vuppugalla R, Marathe P, Fischer V, Dutta S, Sinha VK, Bjornsson T, Lave T, Poulin P. PhRMA CPCDC initiative on predictive models of human pharmacokinetics, part 3: comparative assessement of prediction methods of human clearance. J Pharm Sci. 201 1 Oct;100(10):4090- 1 10.

- Obach RS. Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes. Drug Metab Dispos. 1999 Nov;27(1 1 ):1350-9.

- Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP- dependent transporters. Nat Rev Cancer. 2002; 2: 48-58

- Tonder, M., Weller, M., Eisele, G. & Roth, P. Carboplatin and Etoposide in Heavily Pretreated Patients with Progressive High-Grade Glioma. Chemotherapy 60, 375-378 (2014).

- Reynes, G. et al. Phase II trial of irinotecan and metronomic temozolomide in patients with recurrent glioblastoma. Anticancer Drugs 27, 133-137 (2016).

Claims

A compound of formula (I), or a pharmaceutically acceptable salt prodrug thereof:

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 -(CH₂)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;

2. The compound of claim 1 wherein 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.

3. The compound of claim 1 or claim 2 wherein each incidence of R^a is independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro, chloro, cyano, amino and carboxyl.

4. The compound of any one of the preceding claims wherein 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 cycloalkyl and optionally substituted C5 or C6 aryl.

5. The compound of any one of the preceding claims wherein R-i is selected from the group consisting of hydrogen, optionally substituted C1 to C6 alkyl, optionally substituted C5 or C6 heterocyclic and optionally substituted phenyl.

6. The compound of any one of the preceding claims wherein 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.

7. The compound of any one of the preceding claims wherein when Y is heterocyclic or heteroaryl it is selected from C5 or C6 nitrogen and/or sulphur containing heterocyclic or heteroaryl.

8. The compound of claim 7 wherein, when Y is heterocyclic or heteroaryl, it is 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

9. The compound of any one of the preceding claims wherein Z is selected from -S(0)₂NR^cR^d, -OS(0)₂NR^cR^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 is 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.

10. The compound of claim 9 wherein when Z is selected from -S(O)₂NR^cR^d, -OS(O)₂NR^cR^d then R^c and R^d are hydrogen.

1 1 . The compound of any one of the preceding claims wherein the compound of formula (I) is a compound of formula (II):

Formula II wherein, W, R^b, Ri , Y, R^c and R^d are as defined in any one of claims 1 to

10; each incidence of R^e, R^f, R⁹, R^h and R' is independently selected from those groups defined for R^a in any one of claims 1 to 9; and wherein, when R^a is a 3-bromo-4-hydroxy substitution, W is -CH₂-, R^b, Ri , R^c and R^d are all hydrogen, then Y is not -CH₂CH₂-.

12. The compound of claim 1 1 wherein R^e and R' are hydrogen.

13. The compound of claim 1 1 or claim 12 wherein R^f, R⁹ and R^h are independently selected from the group consisting of hydrogen, hydroxyl, bromo, fluoro and chloro.

14. The compound of any one of the preceding claims wherein the compound is selected from the group consisting of:

and

15. A pharmaceutical composition comprising an effective amount of a compound of any one of the preceding claims, or a pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

16. 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:

Formula wherein, 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, OCF₃, CF₃, S-alkyl and SO₂NHR₂ wherein R₂ is selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyi and optionally substituted aryl;

W is selected from -(CH₂)m- wherein m is from 1 to 6;

Z is a zinc binding group.

17. A method of modulating the activity of a carbonic anhydrase XII enzyme including the step of contacting the enzyme with a compound of any one of claims 1 to 14.

18. A method of treating a cancer in a patient including the step of administering an effective amount of a compound of any one of claims 1 to 14, or a pharmaceutically effective salt or prodrug thereof, and an anti-cancer agent to the patient.

19. A method of diagnosing a cancer in a mammal including the step of administering a labelled compound of any one of claims 1 to 14, 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.

20. The method of claim 16, 18 or claim 19 wherein 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.

21 . A complex of a compound of any one of claims 1 to 14 and a carbonic anhydrase enzyme.

22. The complex of claim 21 wherein the carbonic anhydrase enzyme is a carbonic anhydrase IX or XII.