WO2011030108A1

WO2011030108A1 - Jmjd2 demethylase inhibitors

Info

Publication number: WO2011030108A1
Application number: PCT/GB2010/001713
Authority: WO
Inventors: Christopher Joseph Schofield; Nathan Rose; Rok Sekirnik
Original assignee: Isis Innovation Limited
Priority date: 2009-09-11
Filing date: 2010-09-10
Publication date: 2011-03-17
Also published as: GB0916010D0

Abstract

The present invention relates to inhibitors of JMJD2 subfamily histone lysyl demethylase enzymes. In particular, the invention relates to compounds that are capable of inhibiting a JMJD2 subfamily histone lysyl demethylase without also inhibiting a 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase, and to assays for the identification of inhibitors of JMJD2 subfamily histone lysyl demethylase enzymes.

Description

JMJ 2 DEMETHYLASE INHIBITORS

Field of the Invention

The present invention relates to inhibitors of JMJD2 subfamily histone lysyl demethylase enzymes. In particular, the invention relates to compounds that are capable of inhibiting a JMJD2 subfamily histone lysyl demethylase without also inhibiting a 2- oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase, and to assays for the- identification of inhibitors of JMJD2 subfamily histone lysyl

demethylase enzymes.

Background to the Invention

The JmjC domain containing proteins (jmjC's), a subfamily of Fe(II) and 2- oxoglutarate (2-OG) dependent oxygenases, have been shown to be histone demethylases which catalyse the demethylation of methylated lysines of histone proteins. Methyl marks on histones have long been known to be associated with transcriptional control which is accomplished through modification of chromatin organisation. Blocking or modifying histone demethylation is rapidly gaining interest as a method of treating cancer. Loss of

trimethylation at H4 K20 and K16 is a common hallmark of human cancer and loss of the trimethylation status of H3 K27 is a predictor of poor outcome in breast, ovarian, and pancreatic cancer patients.

Among the family of human jmjC domain histone demethylases is the subfamily JMJD2. Members of this family have been shown to interact with retinoblastoma-binding protein and class I histone deacetylases and implied to have an important role in cell proliferation and oncogenesis. The JMJD2 subfamily has been shown to demethylate histone H3 9 tri- and di-methyl-lysine and H3 K36 tri- and di- methyl-lysine. JMJD2A has been implicated in prostate cancer. Esophageal squamous carcinoma cell lines were shown to overexpress histone demethylase JMJD2C. Inhibition of JMJD2C expression suppresses cell proliferation in concordance with JMJD2C's involvement in tumor growth. Members of the JMJD2 subfamily were shown to interact with and modify gene expression activity of androgen receptor. Targeting stem cells in cancer treatment has been suggested as good way to stop the cancer at its earliest stages. Knockdown of histone demethylases in embryonic fibroblasts have been shown to lock the cells in senescence. Summary of the Invention

The present invention relates to inhibitors of JMJ 2 subfamily histone lysyl demethylase enzymes. In particular, the invention provides a compound that is capable of inhibiting a JMJD2 subfamily histone lysyl demethylase without also inhibiting a 2- oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase.

The invention also relates to methods for the identification of such inhibitors. In particular, the invention provides a method of identifying an inhibitor of a JMJD2 subfamily histone lysyl demethylase, said method comprising the steps of:

(a) contacting a JMJD2 subfamily histone lysyl demethylase with a test compound; and

(b) monitoring the release of Zn(II) ions from the JMJD2 subfamily histone lysyl demethylase, wherein the release of Zn(II) ions indicates that the compound is an inhibitor of a JMJD2 subfamily histone lysyl demethylase.

Also provided are:

A compound which promotes the release of Zn(II) ions from a JMJD2 subfamily histone lysyl demethylase for use in the inhibition of a JMJD2 subfamily histone lysyl demethylase, wherein the compound does not inhibit a 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase.

- A compound which promotes the release of Zn(II) ions from a JMJD2 subfamily histone lysyl demethylase for use in the treatment of cancer, wherein the compound does not inhibit a 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase.

A method of identifying a compound that promotes the release of Zn(II) ions from a JMJD2 subfamily histone lysyl demethylase, said method comprising the steps of:

(a) contacting a subfamily JMJD2 histone lysyl demethylase with a test

compound; and

(b) monitoring the release of Zn(II) ions from the JMJD2 subfamily histone lysyl demethylase.

- A pharmaceutical composition comprising

(a) an inhibitor of a subfamily JMJD2 histone lysyl demethylase identified by a method of the invention; or

(b) a compound according to the invention

and a pharmaceutically acceptable carrier or diluent. A pharmaceutical composition according to the invention for use in the treatment of cancer.

A method of inhibiting a JMJD2 subfamily histone lysyl demethylase without also inhibiting a 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase, said method comprising:

(a) identifying an inhibitor by a method according to the invention,

(b) contacting a sample comprising at least one J JD2 subfamily histone lysyl demethylase and at least one 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase with the inhibitor of step (a) or with a compound of the invention.

A compound which is: a disulfide of formula (I) as defined herein or a

pharmaceutically acceptable salt thereof; an organoselenium compound of formula (II) as defined herein or a pharmaceutically acceptable salt thereof; a seleninic acid derivative of formula (III) as defined herein or a pharmaceutically acceptable salt thereof; a cyclic selenium compound of formula (IV) as defined herein or a pharmaceutically acceptable salt thereof; or a selenite salt; provided that the compound is other than: l-(diethylthiocarbamoyldisulfanyl)- N,N-diethyl-methanethioamide (disulfiram); bis(pyrrolidine-thiocarbamoyl) disulfide;

bis(dibenzylthiocarbamoyl) disulfide; glutathione (disulfide form); cystamine; Na₂Se0₃; PhSeOOH; PhSeSePh; PhSeCl; PhSeH; and

Brief Description of the Figures

Fig. 1 (a) is a representation of a crystal structure of the JMJD2 subfamily member, JMJD2A showing distinct catalytic Fe(II) and structural Zn(II) binding sites.

Fig. 1 (b) sets out the reaction catalyzed by JMJD2A.

Fig. 2 (a) shows kinetic time course data for Zn-ejection from JMJD2A by test compound lb at doses of (from top) 32μ , 16μΜ, 8μΜ, 4μΜ, 2μΜ, ΙμΜ and 0.5μΜ. Fig. 2 (b) shows equivalent data for test compound 16. Linear baseline correction has been applied to compensate for temperature related signal drift. Fig. 2 (c) shows dose-response curves for test compound lb (dark line/squares) and test compound 16 (lighter line/triangles). Fig. 3. Non-deconvoluted ESI-MS spectra of JMJD2A incubated with disulfiram and 2,4-PDCA. Fig. 3 (I) shows the mass spectrum of JMJD2A alone. Fig. 3 (II) is the mass spectrum when incubated with test compound la, showing covalent modification of JMJD2A with half of one disulfiram molecule, concomitant with loss of Zn(II). Fig. 3 (III) is the mass spectrum of JMJD2A when incubated with 2,4-PDCA, showing strong binding of 2,4-PDCA to JMJD2A under mild ionisation conditions (80 V sample cone voltage). Fig. 3 (IV) is the mass spectrum of JMJD2A when incubated with test compound la and 2,4-PDCA and shows binding of both 2,4-PDCA and half of test compound la under mild ionisation conditions (80V sample cone voltage), together with loss of Zn(II); by contrast, Fig. 3 (V) shows the mass spectrum of JMJD2A when incubated with test compound la and 2,4-PDCA at a higher sample cone voltage (200V). Only the binding of test compound la was retained, indicating covalent modification.

Fig. 4 (A)(i) shows kinetic time course data for Zn-ejection from JMJD2A by test compounds comprising sulphur at 2μΜ, namely (from top): test compounds lb, la, and along the baseline test compounds 2, 4, 9, compound 17 and a blank (control). Fig. 4 (A)(ii) shows equivalent data for selenium containing compounds at 2μΜ, namely (from top): test compounds 12, 16, 13, 11, 14, 15 and along the baseline a blank (control). Fig. 4 (B)(i) shows the same data for compounds comprising sulphur at 50μΜ, with again (from top) test compounds la and lb, with all remaining compounds clustered close to the baseline. Fig. 4 (B)(ii) shows the same data for compounds comprising selenium at 50μΜ, (from top) test compounds 12, 16, 13, 14, 15, 11 and blank along the baseline.

Fig. 5(A) and (B) show the effect on JMJD2A demethylase activity of preincubation of 2μΜ JMJD2A with, respectively, test compounds lb and 12 (both at 37μΜ) for different time intervals (0-1800s) before initiation of the demethylation reaction was initiated by addition of the peptide substrate (10 μΜ). The reaction was quenched with MeOH after 30 minutes and demethylation analyzed by MALDI-TOF-MS. Experiments were conducted in triplicate. The minimum and the maximum JMJD2A activities observed were normalized to 0 and 1, respectively, and the plot shows (1 - fraction activity) (y-axis) against time (x-axis) indicating the dependence of JMJD2A inhibition on the duration of preincubation. Fig. 5(C) and (D) show kinetic time course data for Zn-ejection from JMJD2A by, respectively, test compounds lb and 12 at (from top), 11 ΙμΜ, 37 μΜ, 12.3 μΜ, 4.1 μΜ, 1.37 μΜ, 0.46 μΜ and 0 (blank control).

Fig. 6 shows a proposed mechanism of Zn-ejection. After the initial nucleophilic attack on a sulfur electrophile by a cysteine residue, an internal disulfide bond formation can take place with retention of covalent modification and a loss of Zn²⁺(1); internal displacement of the covalent modification leading to internal disulfide bond formation (2); after initial covalent modification of one Cys residue, another modification can take place, leading to a doubly modified Zn-binding site (3).

Detailed Description of the Invention

The present invention relates to inhibitors of JMJD2 subfamily histone lysyl demethylase enzymes. In particular, the invention provides a compound that is capable of inhibiting a JMJD2 subfamily histone lysyl demethylase without also inhibiting a 2- oxoglutarate (2-OG) dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase.

The JMJD2 subfamily enzymes are 2-OG dependent oxygenases which are capable of demethylating both the H3 9me3/me2 and H3K36me3/me 2 marks. Most inhibitors developed for 2-OG dependent oxygenases chelate the active site Fe(II) and also compete with 2-OG. Because the studied 2-OG dependent oxygenases all have a requirement for Fe(II) and have conserved Fe(II) and 2-OG binding residues, achieving selectivity between the 2- OG dependent oxygenase subfamilies with an inhibitor is challenging. Furthermore, use of an Fe(II)-chelating inhibitor in any form of therapy is difficult because of potential side-effects associated with the chelation of iron.

However, unlike related 2-OG dependent oxygenases, the JMJD2 subfamily enzymes have a Zn(II) binding site which is considered to contribute to structural stability. For example, JMJD2A has a Cys₃-His Zn(II) binding site (see Figure 1). None of the other 2-OG dependent oxygenase subfamilies have a comparable Zn(II) binding site. Thus, a compound or inhibitor of the invention, or a compound or inhibitor identified according to a method of the invention inhibits a JMJD2 subfamily histone lysyl demethylase without also inhibiting a 2-oxoglutarate (2-OG) dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase. For example, a compound or inhibitor of the invention, or a compound or inhibitor identified according to a method of the invention inhibits JMJD2A and/or does not inhibit PHD2 (prolyl hydroxylase domain 2).

The invention provides a method of identifying an inhibitor of a JMJD2 subfamily histone lysyl demethylase, said method comprising the steps of:

(a) contacting a JMJD2 subfamily histone lysyl demethylase with a test compound; and (b) monitoring the release of Zn(II) ions from the JMJD2 subfamily histone lysyl demethylase, wherein the release of Zn(II) ions indicates that the compound is an inhibitor of a JMJD2 subfamily histone lysyl demethylase.

Step (a) requires an enzyme from the JMJD2 subfamily. Suitable enzymes include J JD2A, JMJD2B, JMJD2C, JMJD2D, JMJD2E and JMJD2F. The enzyme is preferably JMJD2A.

The JMJD2 polypeptide may comprise the sequence shown in SEQ ID NO: 1 , or may be a fragment or variant of SEQ ID NO: 1 having lysyl demethylase activity. Fragments of JMJD2 are described in more detail below. The JMJD2 polypeptide may have an amino acid sequence having at least about 60% sequence identity, for example at least about 70% sequence identity, with SEQ ID NO: 1 over its entire length or over an active fragment thereof, typically greater than about 80% or 90%, such as about 95% or about 99% sequence identity.

Sequence identity may be calculated using any suitable algorithm. For example, the UWGCG Package provides the BESTFIT program can be used to infer homology (for example used on its default settings) (Devereux et al. (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to infer homology or line up sequences (typically on their default settings), for example as described in Latched (1993) J. Mol. Evol 36:290-300; Latched et al. (1990) J. Mol. Biol. 215:403-10.

The JMJD2 polypeptide may be a polypeptide encoded by any naturally occurring

JMJD2 gene. The naturally occurring JMJD2 gene may comprise the sequence shown in SEQ ID NO: 1 or may be a variant of SEQ ID NO: 1. Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the polypeptide retains lysyl methylase activity.

Amino acid substitutions of SEQ ID NO: 1 , or a fragment thereof, may be made, for example from about 1 , 2 or 3 to about 10, 20 or 30 substitutions. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. ALIPHATIC Non-polar G A P

I L V

Polar-uncharged C S T M

N Q

Polar-charged D E

K R

AROMATIC H F W Y

Variant polypeptides within the scope of the invention may be generated by any suitable method, for example by gene shuffling techniques.

The present invention also includes use of active portions, fragments, derivatives and functional mimetic of the polypeptides of the invention. An "active portion" of a polypeptide means a peptide which is less than said full-length polypeptide, but which retains lysyl demethylase activity. An active fragment of JMJD2 may typically be identified by

monitoring for 2-OG dependent oxygenase activity. Such an active fragment may be included as part of a fusion protein.

The fragment may have up to about 60, 70, 80, 100, 150, 200, 300, 400, 500, 600, 700,

800, 900 or 1000 amino acids.

The fragment may comprise any region from about amino acid 1 to about 1064 of the amino acid sequence shown in SEQ ID NO: 1, such as from amino acid 2, 3, 4, 5 or about 10 to about amino acid 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060. Other suitable fragments may readily be identified, for example by comparing the JMJD2 amino acid sequence to the amino acid sequence of one or more known 2-OG dependent oxygenase and identifying which regions are homologous to regions having catalytic activity. The regions having catalytic activity are typically included in the active fragments. Such fragments can be used to construct chimeric molecules.

Fragments of any JMJD2 polypeptide having at least about 60%, such as at least about

70%, 80%, 90%, 95% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, which fragments have lysyl demethylase activity may also be used in an assay of the invention and are encompassed within the term "JMJD2 polypeptide" used herein. Step (b) requires a means for monitoring the release of Zn(II) ions. Any suitable means may be used. The means is typically a Zn(II) binding or Zn(II) -chelating agent. The means typically produces a detectable signal when bound to Zn(II) ions, the signal thereby produced being proportional to the amount of Zn(II) ions present. The signal is typically a fluorescent signal. In this case, the signal will typically be measured as Relative Fluorescence Units (RFU), which can be converted to a quantitative measure as is normal in the art by plotting a standard curve for a given Zn(II)-binding or chelating agent. The Zn(II) binding or Zn(II) - chelating agent is typically any Zn-specific chelator that fluoresces when bound to Zn(II) in solution. Suitable Zn(II)-chelating agents are commercially available, for example under the name FluoZin.

The method of the invention may further comprise the step of:

(c) determining whether or not the test compound changes the melting temperature (T_m) of the JMJD2 subfamily histone lysyl demethylase, wherein a reduction in T_m indicates that the compound is an inhibitor of a JMJD2 subfamily histone lysyl demethylase; and/or (d) comparing the amount of Zn(II) released in the presence and in the absence of said test compound, wherein a greater amount of Zn(II) released in the presence of the compound compared with the amount of Zn(II) released in the absence of the compound indicates that the compound is an inhibitor of a JMJD2 subfamily histone lysyl demethylase.

The change in T_m may be measured by any suitable method. Typically, T_m may be determined using differential scanning fluorimetry using methods as previously described in, for example, Niesen et al, Nat. Protocols 2007 p2212-2221). An inhibitor of the invention or an inhibitor identified by a method of the invention will typically reduce the T_m of a JMJD2 subfamily enzyme relative to the T_m in the absence of the inhibitor. An inhibitor of the invention or an inhibitor identified by a method of the invention will not increase the T_m of a JMJD2 subfamily enzyme relative to the T_m in the absence of the inhibitor.

The reduction in T_m produced by an inhibitor of the invention may be at least 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 20°C, 25°C, 30^°C or 35°C. The reduction in T_m is preferably at least 10^°C

The amount of Zn(II) released may be monitored as described above. An inhibitor of the invention or an inhibitor identified by a method of the invention will typically increase the quantity of Zn(II) ions released relative to the amount of Zn(II) ions released in the absence of the inhibitor. The amount of Zn(II) ions released will typically be at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold or 100 fold greater than the amount of Zn(II) ions released in the absence of the inhibitor. The amount of Zn(II) ions released in the presence of an inhibitor of the invention or an inhibitor identified by a method of the invention will typically be at least 2μΜ when the inhibitor is present at a concentration of 5μΜ.

The IC₅₀ of an inhibitor of the invention or an inhibitor identified by a method of the invention will typically be no greater than 200μΜ, preferably no greater than 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2 or ΙμΜ. Most preferably, the IC50 of an inhibitor of the invention or an inhibitor identified by a method of the invention will be no greater than 30μΜ, no greater than 50 μΜ or no greater than 100 μΜ. ICso may be determined by any appropriate method. For example, IC50 may be determined by a MALDI-TOF mass spectrometry (MS) turnover assay, formaldehyde dehydrogenase (FDH) coupled inhibition assay, or assays monitoring the release of labelled (typically radioactively labelled) C0₂..

In a typical MALDI-TOF MS turnover assay a JMJD2 subfamily enzyme is incubated with 2-OG and Fe(II) in the presence and absence of a potential inhibitor at various concentrations. A suitable substrate for the JMJD2 subfamily enzyme is included. Suitable substrates may include the native sequence of a histone protein or a fragment thereof incorporating a H3 K9 tri or di methyl-lysine and H3 K36 tri or di methyl-lysine site upon which the JMJD2 subfamily enzyme will act. Suitable fragments incorporating such sites are typically 7, 8, 9, 10, 1 1 or 12 residues in length. An example of a suitable fragment is the 8- residue histone H3 fragment (ARKme₃STGGK), which comprises a H3K9me₃ site.

The sample is then analysed by MALDI-TOF MS. The relative intensities of different methylation states of the JMJD2 subfamily enzyme substrate are observed in the mass spectra and are used to calculate percentage demethylation. IC50 is calculated from the variation in percentage demethylation at different inhibitor concentrations.

In a typical FDH coupled assay, a JMJD2 subfamily enzyme and FDH are incubated with 2-OG and Fe(II) in the presence and absence of a potential inhibitor at various concentrations. A suitable substrate for the JMJD2 subfamily enzyme (as described above for the MALDI-TOF assay) is then included, together with NAD+ for the FDH reaction. The coupled reaction proceeds and yields formaldehyde from the JMJD2 subfamily enzyme reaction, which provides substrate for the FDH, and accordingly NADH is produced and may be monitored, typically by spectrophotometry. The relative amounts of NADH produced are used to calculate JMJD2 subfamily enzyme activity. IC₅₀ is calculated from the variation in activity at different inhibitor concentrations. Test compounds suitable for use in the method of the invention include any compound which may be reasonably expected to promote the release of Zn(II) ions from a protein Zn(II) binding site. Such compounds typically comprise selenium and/or sulphur. Examples of suitable compounds include derivatives of l-(diethylthiocarbamoyldisulfanyl)-N,N-diethyl- methanethioamide (disulfiram) or 2-phenyl-l, 2-benzisoselenazol-3(2H)-one (ebselen). For instance, the test compound may be a disulfide of formula (I) as defined herein or a pharmaceutically acceptable salt thereof; an organoselenium compound of formula (II) as defined herein or a pharmaceutically acceptable salt thereof; a seleninic acid derivative of formula (III) as defined herein or a pharmaceutically acceptable salt thereof; a cyclic selenium compound of formula (IV) as defined herein or a pharmaceutically acceptable salt thereof; or a selenite salt.

The invention also provides compounds identified as inhibitors according to the method of the invention.

The invention also provides a method of identifying compounds which promote the release of Zn(II) ions from a JMJD2 subfamily enzyme comprising the steps of:

(a) contacting a subfamily JMJD2 histone lysyl demethylase with a test compound; and

Release of Zn(II) ions may be monitored according to any suitable method as described above. Any suitable JMJD2 subfamily enzyme may be used as set out above.

The invention also provides compounds which promote the release of Zn(II) ions from a JMJD2 subfamily enzyme for use in the inhibition of a JMJD2 subfamily enzyme, wherein the compound does not also inhibit a 2-OG dependent oxygenase which is not a JMJD2 subfamily enzyme. Such a use may typically be as part of an in vitro assay.

The invention also provides compounds which promote the release of Zn(II) ions from a JMJD2 subfamily enzyme for use in the treatment of cancer, wherein the compound does not also inhibit a 2-OG dependent oxygenase which is not a JMJD2 subfamily enzyme.

The JMJD2 subfamily enzymes are believed to have an important role in cell proliferation and oncogenesis. More specifically, the JMJD2 family has been implicated in esophogael squamous cell carcinoma and prostate cancer. Accordingly, the cancer to be treated by the compound of the invention may typically be esophogael cancer, prostate cancer and/or other forms of squamous cell carcinoma. The JMJD2 family has also been shown to interact with and modify gene expression activity of androgen receptors. The compound of the invention may therefore be used to treat any disease associated with androgen receptor malfunction. Prostate cancer is linked with abnormal androgen receptor activity.

The compound of the invention may typically be any compound which may be reasonably expected to promote the release of Zn(II) ions from a protein Zn(II) binding site. Such compounds typically comprise selenium and/or sulphur. Examples of suitable compounds include disulfiram, ebselen, and derivatives thereof.

In one embodiment the compound of the invention is a compound which comprises sulphur or selenium. In one embodiment, the compound is a compound which comprises sulfur. In another embodiment, the compound is a compound which comprises selenium. In another embodiment, the compound comprises both sulfur and selenium.

Typically, the compound of the invention is:

- a disulfide of formula (I)

wherein R¹ and R², which are the same or different, are independently selected from unsubstituted or substituted C_1-20 alkyl, unsubstituted or substituted C_2-2o alkenyl,

unsubstituted or substituted C2_-20 alkynyl, unsubstituted or substituted C_3-2o carbocyclyl, unsubstituted or substituted C_3-2o heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, cyano, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, and a group of formula (lb)

wherein Y is O or S, and wherein R¹² and R¹³ are the same or different and are independently selected from hydrogen, unsubstituted or substituted C_1-10 alkyl, unsubstituted or substituted C₂-₁₀ alkenyl, unsubstituted or substituted C_2-io alkynyl, unsubstituted or substituted C_3-20 carbocyclyl, unsubstituted or substituted C3_-2o heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, carboxy, unsubstituted or substituted ester, and unsubstituted or substituted acyl, provided that R¹² and R¹³ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-20 heterocyclyl group or an unsubstituted or substituted heteroaryl group, wherein said C_1-2o alkyl, C_2-2o alkenyl, C_2-20 alkynyl, C_1-10 alkyl, C_2-10 alkenyl and C_2-10 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C ₄ alkyl;

or a pharmaceutically acceptable salt thereof; or

- an organoselenium compound of formula II)

wherein

R is unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C₃.₂o carbocyclyl, unsubstituted or substituted C3_-20 heterocyclyl, unsubstituted or substituted ^.₂₀ alkyl, unsubstituted or substituted C_2-20 alkenyl,

unsubstituted or substituted C₂,₂₀ alkynyl, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester, or unsubstituted or substituted acyl, wherein said Ci_-20 alkyl, C₂.₂o alkenyl and C_2-20 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C1 -4 alkyl; and R²¹ is hydrogen, halo, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C_3-20 carbocyclyl, unsubstituted or substituted C_3-20 heterocyclyl, unsubstituted or substituted Ci_-2o alkyl, unsubstituted or substituted C2_-20 alkenyl, unsubstituted or substituted C_2-20 alkynyl, cyano, amino, hydroxyl, thiol,

unsubstituted or substituted Ci_-10 alkylamino, unsubstituted or substituted di(Ci_-10)alkylarnino, unsubstituted or substituted arylamino, unsubstituted or substituted diarylamino, unsubstituted or substituted arylalkylamino, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, unsubstituted or substituted acyloxy, unsubstituted or substituted Ci-io alkoxy, unsubstituted or substituted aryloxy, unsubstituted or substituted C_1-10 alkylthio, unsubstituted or substituted arylthio, or -Se-R²², wherein R²² is as defined above for R²⁰, wherein said C_1-2o alkyl, C_2-2o alkenyl and C_2-20 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C_t-₄ alkyl;

or a pharmaceutically acceptable salt thereof; or

- a seleninic acid derivative of formula (III)

wherein R is unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted Cj.₂₀ alkyl, unsubstituted or substituted C_2-20 alkenyl,

unsubstituted or substituted C_2-2o alkynyl, unsubstituted or substituted C₃.₂o carbocyclyl, unsubstituted or substituted C₃.₂o heterocyclyl, wherein said C_1-20 alkyl, C2-₂₀ alkenyl and C2-20 alkynyl groups are optionally interrupted by 0, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C ₁-₄ alkyl; and

R²⁵ is hydrogen, unsubstituted or substituted C]_-20 alkyl, unsubstituted or substituted C_2-2o alkenyl, unsubstituted or substituted C_2-20 alkynyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C_3-20 carbocyclyl, unsubstituted or substituted C₃.₂₀ heterocyclyl, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, unsubstituted or substituted amido, unsubstituted or substituted thioamido or -Se(0)R²⁶, wherein R²⁶ is as defined above for R²⁴;

or a pharmaceutically acceptable salt thereof; or

- a cyclic selenium compound of formula (IV)

(IV)

wherein

Ar is an unsubstituted or substituted aryl or heteroaryl ring;

L¹ is -C(O)- or unsubstituted or substituted C_1-4 alkylene; and

X is N(R²⁷), O, S, C(O) or C(R²⁷)(R²⁸)-, wherein R²⁷ and R²⁸, which are the same or different when both are present, are independently selected from hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted d-₁₀ alkyl, unsubstituted or substituted C₂-i₀ alkenyl, unsubstituted or substituted C_2-10 alkynyl, unsubstituted or substituted C_3-20 carbocyclyl, unsubstituted or substituted C_3-2o heterocyclyl, cyano, amino, halo, nitro, hydroxyl, thiol, unsubstituted or substituted C_1-10 alkylamino, unsubstituted or substituted di(Ci.₁₀)alkylamino, unsubstituted or substituted arylamino, unsubstituted or substituted diarylamino, unsubstituted or substituted arylalkylamino, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, unsubstituted or substituted acyloxy, unsubstituted or substituted C_1-10 alkoxy, unsubstituted or substituted aryloxy, unsubstituted or substituted C_M0 alkylthio, and unsubstituted or substituted arylthio, wherein said Ci_-10 alkyl, C₂-i₀ alkenyl and C_2-10 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C_r4 alkyl; or a pharmaceutically acceptable salt thereof; or

- a selenite salt.

In one embodiment, the compound of the invention is a disulfide of formula (I). Typically, in this embodiment, R¹ and R², which are the same or different, are independently selected from unsubstituted or substituted C_1- o alkyl, unsubstituted or substituted C₂-₂o alkenyl, unsubstituted or substituted C_2-2o alkynyl, unsubstituted or substituted C3-20 carbocyclyl, unsubstituted or substituted C3_-20 heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, cyano, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, and a group of formula (lb) as defined above. It will be understood that substituted C_1-2o alkyl groups include alkaryl groups, as defined herein. R¹ and/or R² may for instance be a group of formula -Z-Ar, wherein Z is

alkylene and Ar is aryl or heteroaryl.

Typically, the disulfide of formula (I) is other than cystamine (compound 9). Usually, the disulfide of formula (I) is other than the disulfide form of glutathione (compound 5). Typically, the disulfide of formula (I) is other than bis(dibenzyltbiocarbamoyl) disulfide (compound lc).

1 2

In one embodiment, at least one of R and R is a group of formula (lb) as defined above. Typically, both R¹ and R², which are the same or different, are groups of formula (lb) as defined above.

Typically, Y, in the group of formula (lb) is S.

Typically, R¹² and R¹³ in the group of formula (lb) are independently selected from hydrogen, unsubstituted or substituted CMO alkyl and unsubstituted or substituted aryl, provided that R¹² and R¹³ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C3_-20 heterocyclyl group or an unsubstituted or substituted heteroaryl group.

The disulfide of formula I) may be a thiuram disulfide of formula (la)

wherein

R³, R⁴, R⁵ and R⁶ are the same or different and are independently selected from hydrogen, unsubstituted or substituted C_{1 -10} alkyl, unsubstituted or substituted C₂.₁₀ alkenyl, unsubstituted or substituted C_2-i₀ alkynyl, unsubstituted or substituted C₃.₂₀ carbocyclyl, unsubstituted or substituted C3_-20 heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, carboxy, unsubstituted or substituted ester, and unsubstituted or substituted acyl, wherein said C_1-10 alkyl, C₂.₁₀ alkenyl and C_2-io alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or Ci-₄ alkyl, provided that R³ and R⁴ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-2o heterocyclyl group or an unsubstituted or substituted heteroaryl group, and

provided that R⁵ and R⁶ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-20 heterocyclyl group or an- unsubstituted or substituted heteroaryl group.

Typically, the thiuram disulfide of formula (la) is other than

bis(dibenzylthiocarbamoyl) disulfide (compound lc).

Typically, R³ and R⁴ are the same or different and are independently selected from unsubstituted or substituted CMO alkyl and unsubstituted or substituted aryl, provided that R and R⁴ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-2₀ heterocyclyl group or an unsubstituted or substituted heteroaryl group, and provided that R⁵ and R⁶ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C₃.₂o heterocyclyl group or an unsubstituted or substituted heteroaryl group.

The substituted C_1-10 alkyl group may be an aryl-substituted Ci_-10 alkyl group.

In one embodiment, R³ and R⁴ are the same and are selected from unsubstituted or substituted C_1-10 alkyl and unsubstituted or substituted aryl.

Typically, R⁵ and R⁶ are the same or different and are independently selected from unsubstituted or substituted C_1-10 alkyl and unsubstituted or substituted aryl, provided that R and R⁴ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-20 heterocyclyl group or an unsubstituted or substituted heteroaryl group, and provided that R⁵ and R⁶ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-2o heterocyclyl group or an unsubstituted or substituted heteroaryl group.

The substituted C_1-10 alkyl group may be an aryl-substituted C_1-10 alkyl group.

In one embodiment, R⁵ and R⁶ are the same and are selected from unsubstituted or substituted C_1-10 alkyl and unsubstituted or substituted aryl.

In one embodiment, R³, R⁴, R⁵ and R⁶ are the same and are unsubstituted or substituted C_1-10 alkyl or unsubstituted or substituted aryl. In one embodiment, R³ and R⁴ together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-20 heterocyclyl group or an unsubstituted or substituted heteroaryl group.

Typically, in this embodiment, R³ and R⁴ together form a bidendate C_1-10 alkylene group, which is substituted or unsubstituted and optionally interrupted by O, S, or N(R"), wherein R" is H, aryl or C1-4 alkyl. Usually, R³ and R⁴ together form a bidendate C o alkylene group which is unsubstituted, more typically an unsubstituted C_3-6 alkylene group, for instance a -CH2CH2CH2CH2- (butylene) group.

Typically, R⁵ and R⁶ together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C₃.₂o heterocyclyl group or an unsubstituted or substituted heteroaryl group.

Typically, in this embodiment, R⁵ and R⁶ together form a bidendate C_1-10 alkylene group, which is substituted or unsubstituted and optionally interrupted by O, S, or N(R"), wherein R" is H, aryl or Cr₄ alkyl. Usually, R³ and R⁴ together form a bidendate Cj.io alkylene group which is unsubstituted, more typically an unsubstituted C _-6 alkylene group, for instance a -CH₂CH₂CH2CH₂- (butylene) group.

In one embodiment, the thiuram disulfide of formula (la) is

l-(diemyltWocarbamoyldismfanyl)-N,N-diemyl-memanemioarnide (compound la;

disulfiram) or bis(pyrrolidine-thiocarbarnoyl) disulfide (compound lb).

Disulfide compounds of formula (I) may be prepared by routine chemical synthetic methods using commercially available starting materials, for instance by methods which are known in the art or by routine modifications thereof. The syntheses of 1- (diemyltWocarbamoyldisulfanyl)-N,N-diethyl-methanetWoamide (compound la; disulfiram), bis(pyrrolidine-thiocarbamoyl) disulfide (compound lb) and bis(diben2ylthiocarbamoyl) disulfide (compound lc) are for instance described in the Example herein.

In one embodiment, the compound of the invention is an organoselenium compound of formula (II).

Typically, in this embodiment, R is unsubstituted or substituted aryl or heteroaryl; and R²¹ is hydrogen, halo, unsubstituted or substituted Ci.2₀ alkyl, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester,

9 9 9Π

unsubstituted or substituted acyl, or -Se-R , wherein R is as defined for R wherein said Ci-20 alkyl is optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C\-4 alkyl. Usually, R²⁰ is unsubstituted or substituted aryl, for instance unsubstituted or substituted phenyl. R²⁰ may for instance be unsubstituted phenyl.

Typically, R²¹ is hydrogen, halo, unsubstituted or substituted C_1-10 alkyl, or -Se-R²², wherein R is as defined herein for R . R may for instance be unsubstituted or substituted aryl, for instance unsubstituted or substituted phenyl. R²² may for instance be unsubstituted phenyl.

In one embodiment, the organoselenium compound of formula (II) is Ph-Se-Se-Ph (compound 13), PhSeCl (compound 14), or PhSeH (compound 15).

Organoselenium compounds of formula (II) may be prepared by routine chemical synthetic methods using commercially available starting materials, for instance by methods which are known in the art or by routine modifications thereof.

In one embodiment, the compound of the invention is a seleninic acid derivative of formula (III).

Typically, in this embodiment, R²⁴ is unsubstituted or substituted aryl, or unsubstituted or substituted C_1-10 alkyl. Usually, R²⁴ is unsubstituted or substituted aryl, for instance unsubstituted or substituted phenyl. R²⁴ may for instance be unsubstituted phenyl.

Usually, R²⁵ is hydrogen.

In one embodiment, the seleninic acid derivative of formula (III) is benzene seleninic acid, PhSe(0)OH (compound 12).

Seleninic acid derivatives of formula (III) may be prepared by routine chemical synthetic methods using commercially available starting materials, for instance by methods which are known in the art or by routine modifications thereof.

In one embodiment, the compound of the invention is a cyclic selenium compound of formula (IV).

Typically, in this embodiment, Ar is an unsubstituted or substituted aryl ring, for instance an unsubstituted or substituted phenyl ring. Alternatively, Ar may be an

unsubstituted aryl or heteroaryl ring. Usually, Ar is an unsubstituted aryl ring, for instance unsubstituted phenyl.

Typically, L¹ is -C(O)- or unsubstituted or substituted C_1-2 alkylene. Usually, L¹ is -C(O)- or CH₂ (methylene). More typically, L¹ is -C(O)-.

X is typically N(R²⁷), O, S, C(O) or -C(R²⁷)(R²⁸)-, wherein R²⁷ and R²⁸ are as defined above. Typically, when X is -C(R²⁷)(R²⁸)-, R²⁷ and R²⁸ are both hydrogen. Typically, when X is N(R ), R is as defined above. More typically, R is hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted Cno alkyl.

In one embodiment, X is N(R²⁷) and R²⁷ is as defined hereinbefore. More typically, however, R²⁷ is unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted Cno alkyl. In one embodiment, R²⁷ is an unsubstituted or substituted aryl ring, for instance an unsubstituted or substituted phenyl ring. Alternatively, R may be an unsubstituted aryl or heteroaryl ring. Usually, R is an unsubstituted aryl ring, for instance unsubstituted phenyl. Thus, X in one embodiment is N(Ph).

Typically, Ar is unsubstituted phenyl and the cyclic selenium compound of formula (IV) is a compound of formula (I

wherein L¹ and X are as defined hereinbefore.

Typically, L¹ is -C(O)- or unsubstituted or substituted C_1-2 alkylene. Usually, X is N(R²⁷), wherein R²⁷ is hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted C o alkyl. However, L¹ and/or X may be as further defined herein.

In one embodiment, the cyclic selenium compound of formula (IV) or (IVa) is

(compound 16; ebselen).

Cyclic selenium compounds of formula (IV) may be prepared by routine chemical synthetic methods using commercially available starting materials, for instance using synthesis methods which are known in the art or by routine modifications thereof.

In one embodiment, the compound of the invention is a selenite salt.

Typically, in this embodiment, the selenite salt is a selenite salt of formula (V)

[Xⁿ⁺]_P[Se0₃ ²1_{q (v)} wherein

X is a cation having a charge n+ wherein n is a positive integer; and

p and q are positive integers wherein n multiplied by p is equal to 2q. X may for instance be a monocation, for instance an alkali metal cation such as Na⁺. In that case n is 1, p is 2 and q is 1. Alternatively, X may be a dication, for instance an alkaline earth metal cation such as Ca²⁺, in which case n is 2, and p and q are both 1.

Alternatively, X may be a trication, for instance a trivalent lanthanide or actinide or a trivalent group 13 cation such as Al³⁺, in which case n is 3, p is 2 and q is 3. In another embodiment, X may be a tetravalent cation, for instance a tetravalent lanthanide or actinide, for instance Ce⁴⁺, in which case n is 4, p is 1 and q is 2.

In one embodiment, the selenite salt is Na₂Se0₃.

Such selenite salts are- commercially available and/or may be prepared by routine chemical synthetic methods using commercially available starting materials, for instance using synthesis methods which are known in the art or by routine modifications thereof.

In one embodiment, the compound of the invention is:

1 -(diemyltWocarbamoyldisulfanyl)-N,N-diemyl-methanethioamide (compound 1 a;

disulfiram), bis(pyrrolidine-thiocarbamoyl) disulfide (compound lb), Na₂Se0₃ (compound 11), PhSeOOH (compound 12; benzene seleninic acid), PhSeSePh (compound 13), PhSeCl

(compound 14), PhSeH (compound 15), or:

(compound 16; ebselen).

Compound numbers refer to the numbering system used in Table 1 of the Example. As used herein, a C_1-20 alkyl group is an unsubstituted or substituted, straight or branched chain saturated hydrocarbon radical. Typically it is C_1-10 alkyl, for example methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl, or C_1-6 alkyl, for example methyl, ethyl, propyl, butyl, pentyl or hexyl, or C_1-4 alkyl, for example methyl, ethyl, i-propyl, n-propyl, t-butyl, s-butyl or n-butyl. When an alkyl group is substituted it typically bears one or more (e.g. one, two, three or four) substituents selected from substituted or unsubstituted Ci-20 alkyl; substituted or unsubstituted aryl; substituted or unsubstituted aralkyl; cyano;

amino; Q-io alkylamino; di(Ci-i₀)alkylamino; arylamino; diarylamino; arylalkylamino; nitro; amido; thioamido; acylamido; hydroxyl; keto; oxo; halo; haloalkl (e.g. -CF₃); carboxy; ester; acyl; acyloxy; Ci_-10 alkoxy; aryloxy; haloalkyl; sulfhydryl (i.e. thiol, -SH); C_1-10 alkylthio; arylthio; -N=N-C_1-2o alkyl, which C^o alkyl is unsubstituted or substituted; -N=N-aryl, which aryl is unsubstituted or substituted; -S(0)R*; -S(0)₂R_<.; -S(0)₂ORe; -S(0)NR_¾R_c; -

S(0)₂N eR_e; -0(CR_e¾)NR_eR_€; -C(0)N¾¾; -C0₂(CR R_e)CONR_iR_<:; -OC(0)NReR_e; - R_€C(0)0¾; -R_eC(0)NRe¾; -CRe(N-ORe); -N₂ ⁺; -CFH₂; -CF₂H; or -CF₃; wherein Re is H, C_1-6 alkyl, C_1-6 carbocyclyl or aryl and if two or more Re groups are present these may be the same or different.

Examples of substituted atkyl groups include haloalkyl, hydroxyalkyl, aminoalkyl, alkoxyalkyl and alkaryl groups. The term alkaryl, as used herein, pertains to a C_1-20 alkyl group in which at least one hydrogen atom (e.g., 1, 2, 3) has been replaced with an aryl group. Examples of such groups include, but are not limited to, ben2yl (phenylmethyl, PhCH₂-), benzhydryl (Ph₂CH-), trityl (triphenylmethyl, Ph₃C-), phenethyl (phenylethyl, Ph-CH₂CH₂-), styryl (Ph-CH=CK-), cinnamyl (Ph-CH=CH-CH₂-).

Typically a substituted C_1-2o alkyl group carries 1, 2 or 3 substituents, for instance 1 or

2.

A C2-20 alkenyl group or moiety is a straight or branched group or moiety, which contains from 2 to 20 carbon atoms. One or more double bonds may be present in the alkenyl group or moiety, typically one double bond. A C₂-₂₀ alkenyl group or moiety is typically ethenyl or a C_3-1o alkenyl group or moiety, i.e. a C_2-i₀ alkenyl group, more typically a C_2-6 alkenyl group. A C_3-10 alkenyl group or moiety is typically a C_3-6 alkenyl group or moiety, for example allyl, propenyi, butenyl, pentenyl or hexenyl. A C_2-4 alkenyl group or moiety is ethenyl, propenyi or butenyl. An alkenyl group may be unsubstituted or substituted by one to four (e.g. one, two, three or four) substituents, the substituents, unless otherwise specified, being selected from those listed above for Ci_-2o alkyl groups. Where two or more substituents are present, these may be the same or different.

A C_2-20 alkynyl group or moiety is a straight or branched group or moiety which, unless otherwise specified, contains from 2 to 20 carbon atoms. One or more triple bonds, and optionally one or more double bonds may be present in the alkynyl group or moiety, typically one triple bond. A C_2-20 alkynyl group or moiety is typically ethynyl or a C_3-10 alkynyl group or moiety, i.e. a C_2-10 alkynyl group, more typically a C_2-6 alkynyl group. A C_3- io alkynyl group or moiety is typically a C_3-6 alkynyl group or moiety, for example propynyl, butynyl, pentynyl or hexynyl. A C_2-4 alkynyl group or moiety is ethynyl, propynyl or butynyl. An alkynyl group may be unsubstituted or substituted by one to four substituents (e.g. one, two, three or four), the substituents, unless otherwise specified, being selected from those listed above for C_1-20 alkyl groups. Where two or more substituents are present, these may be the same or different.

An aryl ring is an unsubstituted or substituted aromatic ring of covalently linked carbon atoms. Typically, the aryl ring is a 5- or 6- membered aryl ring, examples of which include cyclopentadienyl (C_p) and phenyl. An aryl ring may be unsubstituted or substituted by, typically, one to four substituents (e.g. one, two, three or four), the substituents, unless otherwise specified, being selected from those listed above for C]_-20 alkyl groups. Where two or more substituents are present, these may be the same or different.

A heteroaryl ring is an unsubstituted or substituted heteroaromatic ring of covalently linked atoms including one or more heteroatoms. The one or more heteroatoms are typically selected from nitrogen, phosphorus, silicon, oxygen and sulfur (more commonly from nitrogen, oxygen and sulfur). A heteroaryl ring is typically a 5- or 6- membered heteroaryl ring containing at least one heteroatom selected from nitrogen, phosphorus, silicon, oxygen and sulfur (more commonly selected from nitrogen, oxygen and sulfur). It may contain, for example, 1, 2 or 3 heteroatoms. Examples of heteroaryl rings include pyridine, pyrazine, pyrimidine, pyridazine, furan, thiofuran, pyrazole, pyrrole, oxazole, oxadiazole, isoxazole, thiadiazole, thiazole, isothiazole, imidazole and pyrazole. A heteroaryl ring may be unsubstituted or substituted by, typically, one to four substituents (e.g. one, two, three or four), the substituents, unless otherwise specified, being selected from those listed above for Ci-20 alkyl groups. Where two or more substituents are present, these may be the same or different.

A C₃-2o carbocyclyl group is an unsubstituted or substituted monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a carbocyclic ring of a carbocyclic compound, which moiety has from 3 to 20 carbon atoms (unless otherwise specified), including from 3 to 20 ring atoms. The carbocyclyl ring may be saturated or unsaturated. Thus, the term "carbocyclyl" includes the sub-classes cycloalkyl, cycloalkyenyl and cycloalkynyl. Preferably, each ring has from 5 to 7 ring atoms. Examples of groups of C_3-2o carbocyclyl groups include C_3-io carbocyclyl, C5-7 carbocyclyl and C_5-6 carbocyclyl. When a C_3-20 carbocyclyl group is substituted it typically bears one or more substituents

(typically one, two, three or four substituents) selected from those listed above for C_1-20 alkyl groups.

Examples of C_3-io carbocyclyl groups include, but are not limited to, those derived from saturated monocyclic hydrocarbon compounds:

cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅), cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄), dimethylcyclopropane (C₅), methylcyclobutane (C₅), dimethylcyclobutane (C₆), methylcyclopentane (C₆), dimethylcyclopentane (C₇), methylcyclohexane (C₇), dimethylcyclohexane (Cg), menthane (C₁₀);

unsaturated monocyclic hydrocarbon compounds: cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅), cyclopentadiene (C₅), cyclohexene (C6), cyclohexadiene (C₆), methylcyclopropene (C ), dimethylcyclopropene (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆), methylcyclopentene (C₆),

dimethylcyclopentene (C₇), methylcyclohexene (C₇), dimethylcyclohexene (C₈);

saturated polycyclic hydrocarbon compounds:

thujane (Cio), carane (C₁₀), pinane (C₁₀), bornane (Cio), norcarane (C₇), norpinane (C₇), norbornane (C₇), adamantane (Ci₀), decalin (decahydronaphthalene) (C₁₀); unsaturated polycyclic hydrocarbon compounds: camphene (C₁₀), limonene (C₁₀), pinene (Cio);

polycyclic hydrocarbon compounds having an aromatic ring:

indene (C₉), indane (e.g., 2,3-dihydro-lH-indene) (C₉), tetraline

(1,2,3,4-tetrahydronaphthalene) (C₁₀), acenaphthene (C₁₂), fluorene (Ci₃), phenalene (C₁₃), 5,5,8,8-tetramethyl tetraline (C₁₄), acephenanthrene (C15), aceanthrene (C₁₆),

cholanthrene (C₂₀).

A C_3-1o cycloalkyl group or moiety is a 3- to 10- membered unsubstituted or substituted group or moiety, typically a 3 -to 6-membered group or moiety, which may be a monocyclic ring or which may consist of two or more fused rings. Examples of C_3-1o cycloalkyl groups or moieties include cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅), cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄), dimethylcyclopropane (C₅), methylcyclobutane (C₅), dimethylcyclobutane (C₆), methylc)'clopentane (C₆),

dimethylcyclopentane (C₇), methyl cyclohexane (C₇), dimethyl cyclohexane (C₈), menthane (C₁₀), thujane (C₁₀), carane (C₁₀), pinane (C]₀), bornane (Cio), norcarane (C₇), norpinane (C₇), norbornane (C₇), adamantane (Cio) and decalin (decahydronaphthalene) (C₁₀).

A C_3-20 heterocyclyl group is an unsubstituted or substituted monovalent, monocyclic, bicyclic or tricyclic moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms (unless otherwise specified), of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. When a C₃.₂₀ heterocyclyl group is substituted it typically bears one or more substituents selected from Ci- alkyl which is unsubstituted, aryl (as defined herein), cyano, amino, Cj.io alkylamino, di(Ci_-1o)alkylamino, arylamino, diarylamino, arylalkylamino, amido, thioamido, acylamido, hydroxyl, oxo, halo, haloalkyl (e.g. CF₃), carboxy, ester, acyl, acyloxy, C_1-2o alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, -SH), Ci-io alkylthio, arylthio, and sulfonyl. Typically a substituted C_3-2o heterocyclyl group carries 1, 2 or 3 substituents, for instance 1 or 2. Examples of groups of heterocyclyl groups include C₃.₂o heterocyclyl, C_5-20 heterocyclyl, C_3-15 heterocyclyl, C5.15 heterocyclyl, C_3-12 heterocyclyl, C5-12 heterocyclyl, C3.10 heterocyclyl, C_5-10 heterocyclyl, C_3-7 heterocyclyl, C_5-7 heterocyclyl, and C _-6 heterocyclyl.

Examples of monocyclic C_3-2o heterocyclyl groups include, but are not limited to, those derived from:

Ni: aziridine (C3), azetidine (C₄), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);

Oj : oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆), pyran (C₆), oxepin (C₇);

Si: thiirane (C3), thietane (C ), thiolane (tetrahydrothiophene) (C₅), thiane

(tetrahydrothiopyran) (C₆), thiepane (C₇);

0₂: dioxolane (C5), dioxane (C₆), and dioxepane (C₇);

0₃: trioxane (C₆);

N₂: imidazolidine (C₅), pyrrolidine (diazolidine) (C₅), imidazoline (C₅), pyrazoline

(dihydropyrazole) (C₅), piperazine (C₆);

N1O1 : tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), mo holine (C₆), tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine

(C₆);

NiSi: thiazoline (C₅), thiazolidine (C₅), t iomo holine (C₆);

N₂Oi: oxadiazine (C₆);

O1S1 : oxathiole (C₅) and oxathiane (thioxane) (C₆); and,

N1O1S1 : oxathiazine (C₆).

Examples of C₃.₂o heterocyclyl groups which are also aryl groups are described below as heteroaryl groups.

An aryl group is a substituted or unsubstituted, monocyclic or bicyclic aromatic group which typically contains from 6 to 14 carbon atoms, preferably from 6 to 10 carbon atoms in the ring portion. Examples include phenyl, naphthyl, indenyl and indanyl groups. An aryl group is unsubstituted or substituted. When an aryl group as defined above is substituted it typically bears one or more substiruents selected from unsubstituted or substituted d-₆ alkyl (to form an aralkyl group), aryl which is unsubstituted, cyano, amino, C_1-10 alkylamino, di(Ci- ₁o)alkylamino, arylamino, diarylamino, arylalkylamino, amido, thioamido, acylamido, hydroxyl, halo, haloalkyl (e.g. CF₃), carboxy, ester, acyl, acyloxy, C_1-2o alkoxy, aryloxy, haloalkyl, sulfhydryl (i.e. thiol, -SH), Ci-io lkylthio, arylthio, sulfonic acid and sulfonyl. Typically it carries 0, 1, 2 or 3 substituents. A substituted aryl group may be substituted in two positions with a single unsubstituted or substituted C_1-6 alkylene group, or with a bidentate group represented by the formula -X-Ci_-6 alkylene, or -X-C_1-6 alkylene-X-, wherein X is selected from O, S and NR, and wherein R is H, aryl or Ci_-6 alkyl. Thus a substituted aryl group may be an aryl group fused with a cycloalkyl group or with a heterocyclyl group.

The term aralkyl as used herein, pertains to an aryl group in which at least one hydrogen atom (e.g., 1, 2, 3) has been substituted with a C_1-6 alkyl group. Examples of such groups include, but are not limited to, tolyl (from toluene), xylyl (from xylene), mesityl (from mesitylene), and cumenyl (or cumyl, from cumene), and duryl (from durene).

The ring atoms of an aryl group may include one or more heteroatoms, as in a heteroaryl group. Such an aryl group (a heteroaryl group) is a substituted or unsubstituted mono- or bicyclic heteroaromatic group which typically contains from 6 to 10 atoms in the ring portion including one or more heteroatoms. It is generally a 5- or 6-membered ring, or two fused rings each of which is the same or different and typically independently selected from a 5-membered ring and a 6-membered ring, containing at least one heteroatom selected from O, S, N, P, Se and Si. It may contain, for example, 1, 2 or 3 heteroatoms. Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thienyl, pyrazolidinyl, pyrrolyl, oxazolyl, oxadiazolyl, isoxazolyl, thiadiazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, quinolyl and isoquinolyl. A heteroaryl group may be unsubstituted or substituted, for instance, as specified above for aryl. Typically it carries 0, 1 , 2 or 3 substituents.

A C_1-10 alkylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 1 to 10 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkylene" includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below. Typically it is C_1-6 alkylene. Typically it is C_1-4 alkylene, for example methylene, ethylene, i-propylene, n-propylene, t- butylene, s-butylene or n-butylene. It may also be pentylene, hexylene, heptylene, octylene and the various branched chain isomers thereof. An alkylene group may be unsubstituted or substituted, for instance, as specified above for alkyl. Typically a substituted alkylene group carries 1, 2 or 3 substituents, for instance 1 or 2.

In this context, the prefixes (e.g., C_1-4, C_1-7, C_1-10, C_2-7, C_3-7, etc.) denote the number of carbon atoms, or range of number of carbon atoms. For example, the term "C^alkylene," as used herein, pertains to an alkylene group having from 1 to 4 carbon atoms. Examples of groups of alkylene groups include C_1-4 alkylene ("lower alkylene"), Ci_-7 alkylene and Ci.₁₀ alkylene.

Examples of linear saturated C_1-7 alkylene groups include, but are not limited to, -(CH₂)_n- where n is an integer from 1 to 7, for example, -CH₂- (methylene), -CH₂CH₂- (ethylene), -CH₂CH₂CH₂- (propylene), and -CH₂CH₂CH₂CH₂- (butylene).

Examples of branched saturated C_1-7 alkylene groups include, but are not limited to, -CH(CH₃)-, -CH(CH₃)CH₂-, -CH(CH₃)CH₂CH₂-, -CH(CH₃)CH₂CH₂CH₂-,

-CH₂CH(CH₃)CH₂-, -CH₂CH(CH₃)CH₂CH₂-, -CH(CH₂CH₃)-, -CH(CH₂CK₃)CH₂-, and -CH₂CH(CH₂CH₃)CH₂-.

Examples of linear partially unsaturated Ci_-7 alkylene groups include, but is not limited to, -CH=CH- (vinylene), -CH=CH-CH₂-, -CH₂-CH=C¾-, -CH=CH-CH₂-CH₂-, -CH=CH-CH₂-CH₂-CH₂-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH₂-, -CH=CH-CH=CH- CH₂-CH₂-, -CH=CH-CH₂-CH=CH-, and -CH=CH-CH₂-CH₂-CH=CH-.

Examples of branched partially unsaturated C_1-7 alkylene groups include, but is not limited to, -C(CH₃)=CH-, -C(CH₃)=CH-CH₂-, and -CH=CH-CH(CH₃)-.

Examples of alicyclic saturated C_1-7 alkylene groups include, but are not limited to, cyclopentylene (e.g., cyclopent-l,3-ylene), and cyclohexylene (e.g., cyclohex-l,4-ylene).

Examples of alicyclic partially unsaturated C_1- alkylene groups include, but are not limited to, cyclopentenylene (e.g., 4-cyclopenten-l,3-ylene), cyclohexenylene (e.g.,

2-cyclohexen-l,4-ylene; 3-cyclohexen-l,2-ylene; 2,5-cyclohexadien-l,4-ylene). These are examples of C_5-6 cycloalkylene groups.

C_1-10 alkylene, Ci_-20 alkyl, C2_-20 alkenyl and C_2-2o alkynyl groups as defined herein are either uninterrupted or interrupted by one or more heteroatoms or heterogroups, such as S, O or N(R") wherein R" is H, Ci_-6 alkyl or aryl (typically phenyl), or by one or more arylene (typically phenylene) or heteroarylene groups. The phrase "optionally interrupted" as used herein thus refers to a C_1-2o alkyl group or an alkylene group, as defined above, which is uninteaupted or which is interrupted between adjacent carbon atoms by a heteroatom such as oxygen or sulfur, by a heterogroup such as N(R") wherein R" is H, aryl or C[-C₆ alkyl, or by an arylene or heteroarylene group.

For instance, a C_1-2o alkyl group such as n-butyl may be interrupted by the heterogroup N(R") as follows: -CH₂N(R")CH₂CH₂CH_3, -CH₂CH₂N(R")CH₂CH₃, or

-CH₂CH₂CH₂N(R")CH₃. Similarly, an alkylene group such as n-butylene may be interrupted by the heterogroup N(R") as follows: -CH₂N(R")CH₂CH₂CH₂-, -CH₂CH₂N(R")CH₂CH₂-, or -CH2CH₂CH₂N(R")CH₂-. Typically an interrupted group, for instance an interrupted C O alkylene, C_1-20 alkyl, C_2-2o alkenyl or C_2-20 alkynyl group, is interrupted by 1, 2 or 3

heteroatoms or heterogroups or by 1 , 2 or 3 arylene (typically phenylene) groups. More typically, an interrupted group, for instance an interrupted C_1-10 alkylene, Ci_-20 alkyl, C_2-20 alkenyl or C₂.₂₀ alkynyl group, is interrupted by 1 or 2 heteroatoms or heterogroups or by 1 or 2 arylene (typically phenylene) groups. For instance, a C_1-2o alkyl group such as n-butyl may - be interrupted by 2 heterogroups N(R") as follows: -CH₂N(R")CH₂N(R")CH₂CH₃.

An arylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, one from each of two different aromatic ring atoms of an aromatic compound, which moiety has from 5 to 14 ring atoms (unless otherwise specified). Typically, each ring has from 5 to 7 or from 5 to 6 ring atoms. An arylene group may be unsubstituted or substituted, for instance, as specified above for aryl. Typically a substituted arylene group carries 1, 2 or 3 substituents, for instance 1 or 2.

A heteroarylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, one from each of two different aromatic ring atoms of a hetero-aromatic compound, which moiety has from 5 to 14 ring atoms (unless otherwise specified). Typically, each ring has from 5 to 7 or from 5 to 6 ring atoms. A heteroarylene group may be unsubstituted or substituted, for instance, as specified above for aryl. Typically a substituted heteroarylene group carries 1, 2 or 3 substituents, for instance 1 or 2.

In this context, the prefixes (e.g., C_5-2₀, C₆.₂₀, C_{5- 1}4, C_5- , Cs-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C_5- arylene," as used herein, pertains to an arylene group having 5 or 6 ring atoms. Examples of groups of arylene groups include Cs_-2o arylene, C6_-20 arylene, C_{5- 1}4 arylene, C_6-14 arylene, C_6-1o arylene, C_5-12 arylene, Cs.jo arylene, C_5-7 arylene, C_5- arylene, C₅ arylene, and C arylene.

The ring atoms may be all carbon atoms, as in "carboarylene groups" (e.g., C₆.₂₀ carboarylene, C6-i₄ carboarylene or C_6-i₀ carboarylene).

Examples of C6-20 arylene groups which do not have ring heteroatoms (i.e., C_6-20 carboarylene groups) include, but are not limited to, those derived from the compounds discussed above in regard to aryl groups, e.g. phenylene, and also include those derived from aryl groups which are bonded together, e.g. phenylene-phenylene (diphenylene) and

phenylene-phenylene-phenylene (triphenylene).

Alternatively, the ring atoms may include one or more heteroatoms, as in

"heteroarylene groups" (e.g., C_5-]o heteroarylene). A heteroarylene group may be unsubstituted or substituted, for instance, as specified above for aryl. Typically a substituted heteroarylene group carries 1, 2 or 3 substituents, for instance 1 or 2.

Examples of heteroarylene groups include, but are not limited to, those derived from the compounds discussed above in regard to heteroaryl groups. Examples of heteroarylene groups include bidentate groups derived from pyridine, pyrazine, pyrimidine, pyridazine, furan, thiofuran, pyrazole, pyrrole, oxazole, oxadiazole, isoxazole, thiadiazole, thiazole, isothiazole, imidazole and pyrazole.

As used herein the terms oxo and keto represent a group of formula: =0

As used herein the term halo represents a group of formula: -X wherein X is a halogen atom. Typically X is F, CI, Br or I.

As used herein the term acyl represents a group of formula: -C(=0)R, wherein R is an acyl substituent, for example, a substituted or unsubstituted C_1-20 alkyl group, a substituted or unsubstituted C3_-2o heterocyclyl group, or a substituted or unsubstituted aryl group. Examples of acyl groups include, but are not limited to, -C(=0)CH₃ (acetyl), -C(=0)CH₂CH₃

(propionyl), -C(=0)C(CH₃)₃ (t-butyryl), and -C(=0)Ph (benzoyl, phenone).

As used herein the term acyloxy (or reverse ester) represents a group of formula: -OC(=0)R, wherein R is an acyloxy substituent, for example, substituted or unsubstituted C_1-20 alkyl group, a substituted or unsubstituted C_3-20 heterocyclyl group, or a substituted or unsubstituted aryl group, typically a C_1-6 alkyl group. Examples of acyloxy groups include, but are not limited to, -OC(=0)CH₃ (acetoxy), -OC(=0)CH₂CH₃, -OC(=0)C(CH₃)₃,

-OC(=0)Ph, and -OC(=0)CH₂Ph.

As used herein the term ester (or carboxylate, carboxylic acid ester or oxycarbonyl) represents a group of formula: -C(=0)OR, wherein R is an ester substituent, for example, a substituted or unsubstituted Ci.₂₀ alkyl group, a substituted or unsubstituted C_3-20 heterocyclyl group, or a substituted or unsubstituted aryl group (typically a phenyl group). Examples of ester groups include, but are not limited to, -C(=0)OCH₃, -C(=0)OCH₂CH₃,

-C(=0)OC(CH₃)₃, and -C(=0)OPh.

As used herein the term amino represents a group of formula -N¾. The term CrC₁₀ alkylamino represents a group of formula -NHR' wherein R^' is a CMO alkyl group, preferably a Ci-6 alkyl group, as defined previously. The term di(C_1-1o)alkylamino represents a group of formula -NR'R^" wherein R^' and R" are the same or different and represent unsubstituted or substituted C_1-10 alkyl groups, preferably unsubstituted or substituted C_1-6 alkyl groups, as defined previously. The term arylamino represents a group of formula -NHR' wherein R^' is an aryl group, preferably a phenyl group, as defined previously. The term diarylamino represents a group of formula -NR'R" wherein R^' and R" are the same or different and represent aryl groups, preferably phenyl groups, as defined previously. The term

arylalkylamino represents a group of formula -NR'R" wherein R^' is a C_1-10 alkyl group, preferably a C_1-6 alkyl group, and R" is an aryl group, preferably a phenyl group.

As used herein the term amido represents a group of formula: -C(=0)NR R , wherein

R and R are independently selected from hydrogen, unsubstituted or substituted C_1-10 alkyl, unsubstituted or substituted C_2-10 alkenyl, unsubstituted or substituted C₂.₁₀ alkynyl, unsubstituted or substituted C_3-20 carbocyclyl, unsubstituted or substituted C_3-20 heterocyclyl, unsubstituted or substituted aryl, and unsubstituted or substituted heteroaryl, provided that R' and R" may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-2o heterocyclyl group or an unsubstituted or substituted heteroaryl group. Examples of amido groups include, but are not limited to, -C(=0)NH₂, -C(=0)NHCH₃, -C(=0)N(CH₃)₂, -C(=0)NHCH₂CH₃, and -C(=0)N(CH₂CH₃)₂, as well as amido groups in which R and R , together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.

As used herein the term thioamido represents a group of formula: -C(=S)NR R , wherein R and R are independently selected from hydrogen, unsubstituted or substituted d. ₁₀ alkyl, unsubstituted or substituted C_2-10 alkenyl, unsubstituted or substituted C2-10 alkynyl, unsubstituted or substituted C₃.₂₀ carbocyclyl, unsubstituted or substituted C_3-20 heterocyclyl, unsubstituted or substituted aryl, and unsubstituted or substituted heteroaryl, provided that R' and R" may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-20 heterocyclyl group or an unsubstituted or substituted heteroaryl group. Examples of thioamido groups include, but are not limited to, -C(=S)NH₂, -C(=S)NHCH₃, -C(=S)N(C¾)₂, -C(=S)NHCH₂CH₃, and -C(=S)N(CH₂CH₃)₂, as well as thioamido groups in which R and R , together with the nitrogen atom to which they are attached, form a heterocyclic structure, for example a piperidino, morpholino, thiomorpholino or piperazino heterocyclic ring. For instance, R and R together may be (CH₂)n, wherein n is 3 to 5, more typically 4 or 5, even more typically 4.

As used herein, the terms "carboxy", "carboxyl" and "carboxylic acid" each represent a group of the formula: -C(=0)OH, or -COOH. As would be understood by the skilled person, a carboxylic acid group (for instance, when employed in the present invention) can exist in protonated and deprotonated forms (for example, -C(=0)OH and -C(=0)0^"), and in salt forms (for example, -C(=0)0^"X⁺, wherein X⁺ is a monovalent cation). As used herein the term acylamido represents a group of formula: -NR^xC(=0)R^y, wherein R^x is an amide substituent, for example, hydrogen, a C^oalkyl group, a C_3-2o heterocyclyl group, an aryl group, preferably hydrogen or a C_1-2o alk l group, and R^y is an acyl substituent, for example, a C_1-20 alkyl group, a C_3-20 heterocyclyl group, or an aryl group, preferably hydrogen or a C_1-20 alkyl group. Examples of acylamide groups include, but are not limited to, -NHC(=0)CH₃ , -NHC(=0)CH₂CH₃, -NHC(=0)Ph, -NHC(=0)C,₅H₃, and -NHC(=0)C₉H₁9. Thus, a substituted C_1-20 alkyl group may comprise an acylamido substituent defined by the formula -NHC(=^:0)-C₁.₂o alkyl, such as -NHC(=0)C₁₅H₃₁ or

R^x and R^y may together form a cyclic structure, as in, for example, succinimidyl, maleimid l, and phthalimidyl:

succinimidyl maleimidyl phthalimidyl

A Ci-io alkylthio group is a said C_1-10 alkyl group, preferably a C_1-6 alkyl group, attached to a thio group. An arylthio group is an aryl group, preferably a phenyl group, attached to a thio group.

A Ci-20 alkoxy group is a said substituted or unsubstituted C_1-2o alkyl group attached to an oxygen atom. A Ci_-10 alkoxy group is a said substituted or unsubstituted Ci-io alkyl group attached to an oxygen atom. A C_\s alkoxy group is a said substituted or unsubstituted C^ alkyl group attached to an oxygen atom. A C_1-4 alkoxy group is a substituted or unsubstituted C alkyl group attached to an oxygen atom. Said Ci_-20, CMO, C_1-6 and C_1-4 alkyl groups are optionally interrupted as defined herein. Examples of alkoxy groups include, -OMe

(methoxy), -OEt (ethoxy), -O(nPr) (n-propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), - O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -0(tBu) (tert-butoxy). Further examples of Ci- 20 alkoxy groups are -0(Adamantyl), -0-CH₂-Adamantyl and -0-CH₂-CH₂-Adamantyl. An aryloxy group is a substituted or unsubstituted aryl group, as defined herein, attached to an oxygen atom. An example of an aryloxy group is -OPh (phenoxy).

Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid or carboxyl group (-COOH) also includes the anionic (carboxylate) form (-COO^"), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-I^HR^²), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-0^"), a salt or solvate thereof, as well as conventional protected forms.

The compounds of formula (la) as defined herein therefore include forms in which one or both of the amino groups are protonated. Thus, the compounds of formula (la) include the following protonated form of those compounds:

wherein R³, R , R⁵ and R⁶ are as defined herein. Specific examples of such compounds of formula (la) include:

Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and 1-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal- forms; a- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers," as used herein, are structural (or constitutional) isomers (i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta- chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., C_1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para- methoxyphenyl). The above exclusion does not pertain to tautomeric forms, for example, keto, enol, and enolate forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enarnine, amide/imino alcohol, amidine/amidine, nitroso/oxime,

thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

keto enol enolate

Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, C, and ¹⁴C; O may be in any isotopic form, including ¹⁶0 and ¹⁸0; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, protected forms and prodrugs thereof.

Examples of pharmaceutically acceptable salts of the compounds for use in accordance with the present invention include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid and phosphoric acid; and organic acids such as methanesulfonic acid, benzenesulphonic acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, butyric acid, isobutyric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, ethanesulfonic acid, aspartic acid, benzoic acid and glutamic acid. Typically the salt is a hydrochloride, an acetate, a propionate, a benzoate, a butyrate or an isobutyrate. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19.

A prodrug of a compound for use in accordance with the present invention is a compound which, when metabolised (e.g., in vivo), yields the desired active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties. The compound of the present invention can be in the free form or the salt form. The compound may also be in prodrug form. The prodrug can itself be in the free form or the salt form.

In another aspect, the present invention provides a method of inhibiting a JMJD2 subfamily histone lysyl demethylase without also inhibiting a 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase, said method comprising:

(a) identifying an inhibitor by a method according to the invention,

(b) contacting a sample comprising at least one JMJD2 subfamily histone lysyl demethylase and at least one 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase with said inhibitor.

In this aspect of the invention, the method may use as an inhibitor any compound of the invention as described above. The method may typically be used in an in vitro assay.

In another aspect, the present invention provides compositions and formulations comprising the compounds of the invention. For example, the invention provides a pharmaceutical composition comprising one or more compounds of the invention, such as one or more compounds identified by a screening method of the invention, formulated together with a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for parenteral, e.g. intravenous, intramuscular or subcutaneous administration (e.g., by injection or infusion). Depending on the route of administration, the modulator may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or more

pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts.

Preferred pharmaceutically acceptable carriers comprise aqueous carriers or diluents.

Examples of suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. These compositions may also contain adjuvants such as

preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Pharmaceutical compositions of the invention may comprise additional active ingredients as well as a modulator of the invention. As mentioned above, compositions of the invention may comprise one or more compounds of the invention. They may also comprise additional therapeutic or prophylactic agents. For example, where a pharmaceutical composition of the invention is intended for use in the treatment of a bleeding disorder, it may additionally comprise one or more agents intended to reduce the symptoms of the bleeding disorder. For example, the composition may comprise one or more clotting factors. The composition may comprise one or more other components intended to improve the condition of the patient. For example, where the composition is intended for use in the treatment of patients suffering from unwanted bleeding such as patients undergoing surgery or patients suffering from trauma, the composition may comprise one or more analgesic, anaesthetic, immunosuppressant or anti-inflammatory agents.

A composition of the invention may be used in the treatment of cancer, for example esophogael cancer, prostate cancer and/or other forms of squamous cell carcinoma. The JMJD2 family has also been shown to interact with and modify gene expression activity of androgen receptors. The composition of the invention may therefore be used to treat any disease associated with androgen receptor malfunction. Prostate cancer is linked with abnormal androgen receptor activity.

Also within the scope of the present invention are kits comprising compounds or compositions of the invention and instructions for use. The kit may further contain one ore more additional reagents, such as an additional therapeutic or prophylactic agent as discussed above. The invention also comprises kits for carrying out a method according to the invention, which will typically comprise one or more reagents including a Zn(II)-binding or chelating agent and instructions for use.

Example

Test compounds

Test compounds la - 16 in Table were identified by the Inventors as compounds with the potential to promote the release of Zn(II) ions from JMJD2 subfamily members and are used in the subsequent experiments. Compound 17 is pyridine-2,4-dicarbox}4ic acid (2,4- PDCA), which is a known Fe(II) -chelating competitive inhibitor of 2-oxoglutarate dependent oxygenases. 2,4-PDCA is known to operate by binding to the active site.

Table 1 Structure

As-^SY^NHR2

s

Test compound la R = Et

Test compound lb R2 = (CH₂)₄

Test compound lc R = CH₂Ph

Test compound 2 " N SNa

Test compound 3

Test compound 4 glutathione (thiol form)

Test compound 5

Test compound 6

Test compound 7

SH

Test compound 8 Η,Ν ^'

Test compound 9 Η,Ν ^'

Test compound 10 PhSNa

Test compound 11 Na₂Se0₃

Test compound 12 PhSeOOH

Test compound 13 PhSeSePh

Test compound 14 PhSeCl

Test compound 15 PhSeH

Test compound 16

Compound 17 pyridine-2,4-dicarboxylic acid

Test compound la is also known as l-(diethylthiocarbamoyldisulfanyl)-N,N-diethyl- methanethioamide or disulfiram. Test compound 16 is also known as 2-phenyl-l, 2- benzisoselenazol-3(2H)-one or ebselen. Synthesis of selected test compounds

Synthesis ofbis(pyrrolidine~thiocarbamoyl) disulfide (compound lb)

Prepared according to a reported procedure (Yin et al, Polyhedron 2008; p663-670). m.p. 119-124 °C; IR v_max ( Br): 2870, 1432, 1151, 954 cm^'1, H NMR (400 MHz, CDC1₃): δ 2.02 (app. quin, J=7.0 Hz, 2H), 2.16 (app. quin, J=7.0 Hz, 2H), 3.97 (app. q, J=7.0 Hz, 4H) ppm; ^I3C NMR (100 MHz, CDC1₃): δ 24.2 (CH₂), 26.5 (CH₂), 50.9 (CH₂), 56.9 (CH₂), 189.1 (CS) ppm; HRMS (ESf): calcd for C₁₀H₁₆N₂NaS₄ (M+Na⁺), 315.0089; found, 315.0085; Elemental analysis: Calcd. for Ci₀Hi₆N₂S₄: C 41.06, H 5.51, N 9.58; found, C 41.11, H 5.63, N 9.64.

Synthesis ofbis(dibenzylthiocarbamoyl) disulfide (compound lc)

Prepared according to a reported procedure (Yin et al, Polyhedron 2008; p663-670). m.p. 130-131 °C; IR v_max (KBr): 2905, 1452, 1231, 921, 696 cm^"1, ^!H NMR (400 MHz, CDCI3): δ ppm 5.20 (br. s., 4 H, G¾), 5.38 (br. s., 4 H, CH₂), 6.86 - 7.73 (m, 20 H, ArCH) ppm; ¹³C NMR (100 MHz, CDCI3): δ ppm 54.9 ( H₂), 59.0 ( H₂.), 127.5 (ArCH), 127.9 (ArCH), 128.3 (ArCH), 128.9 (ArCH), 129.1 (ArCH), 134.2 (ArCH), 134.9 (ArCH), 196.3 (C=S) ppm; HRMS (ESI+): calcd for C₃₀H₂₈N₂NaS₄ (M+Na⁺), 567.1028; found, 567.1028; Elemental analysis: Calcd. for C₃₀H₂₈N₂S₄: C 66.14, H 5.18, N 5.14; found: C 66.18, H 5.09, N 5.18.

Synthesis of sodium diethylcarbamodithioate (compound 2)

Prepared according to a reported procedure (Yin et al, Polyhedron 2008; p663-670). m.p. 80-84 °C; IR v_max (KBr): 2978, 2074, 1672, 1615, 1476 cm^'1, 1H NMR (400 MHz, DMSC ¾): δ 1.08 (t, J=7.0 Hz, 6H), 3.97 (q, J=7.0 Hz, 4H) ppm; ¹³C NMR (100 MHz, DMSO-i¾): δ 12.6 (CH₃), 46.1 (CH₂), 212.0 (CS) ppm; Elemental analysis: Calcd. for C₅H₁₀NnaS₂: C 35.07; H 5.89; N 8.18; found, C 35.18; H 5.88; N 8.12.

Synthesis of octane- 1,8-dithiol diacetate (compound 7)

Acetic anhydride (0.64 ml, 6.72 mmol) was added dropwise to a solution of 1 ,8- octanedithiol (0.31 ml, 1.68 mmol), triethylamine (1.17 ml 8.40 mmol) and DMAP (20 mg, 0.17 mmol) in dry DCM (10 ml) at room temperature under an atmosphere of nitrogen. The mixture was stirred for 4 h before the addition of sodium hydrogen carbonate solution (15 ml sat. aq.). The layers were separated and the aqueous phase extracted with DCM (2 10 ml). The combined organic extracts were washed with water (30 ml) and brine (30 ml) before being dried over MgS0 and concentrated in vacuo to give an off-white solid (0.58 g). The crude product was purified by flash chromatography eluting with 1-10% Et₂0/petrol to give diacetate 7 as a white solid (0.43 g, 98%); R_f = 0.59 (4% Et₂0/petroI); mp 37-38 °C; v_max (KBr): 2923 (CH), 2854 (CH), 1687 (CO) cm^'1; δ_Η (400 MHz, CDC1₃) 1.29-1.37 (8H, m, 3- ¾, 4-H₂, 5-H₂ and 6-H₂), 1.56 (4H, br. quint., J 7.5, 2-¾ and 7-¾), 2.33 (6H, s, 2 -OAc), 2.86 (4H, t, J7.5, 1-H₂ and 8-H₂); 5_C (100 MHz, CDC1₃) 28.7 (CH₂), 28.9 (CH₂), 29.1 (CH₂), 29.4 (CH₂), 30.6 (CH₃), 196.1

(ESI) 285 ([MNa]⁺, 100%), 263 ([MH]⁺, 50%).

Methods

Methods for MALDI-TOF MS turnover assay

Assay as reported in Rose et al; J Med Chem 2008, p7053-6.

Expression and purification of JMJD2A and PHD2 were according to standard methods in the art (See, for example, methods reported in Ng et al, Nature 2008 p87-91 and Chowdhury et al, Structure 2009. p981-9). JMJD2A, disodium 2-OG (2-oxoglutarate), ascorbic acid and [ARK(me3)STGGK-NH2] peptide solutions were made up in 50 mM HEPES buffer, pH 7.5. Fe²⁺ solution was prepared from (NH₄)₂Fe(S0₄)₂ dissolved in 20 mM HC1 to make 400 mM stock solution, which was then diluted to the final concentration using MilliQ water.

Together, these were incubated with inhibitor (small molecules were dissolved in DMSO, final in-assay volume was always 5% of total assay mix) for 30 min at 37 °C, before 1 :1 quenching with methanol. When DMSO was present in the sample, methanol quenching was followed by addition of four volumes of 20 mM triammonium citrate. 1 of the diluted assay mixture was then mixed with 1 of recrystallized a-Cyano-4-hydroxycinnamic acid (CHCA, Laser BioLabs) and spotted onto the MALDI-TOF-MS plate before analysis. Preincubation experiments were carried out with JMJD2A, Fe²⁺, ascorbate and test inhibitor pre- incubated for 15 min at 37°C, prior to the addition of peptide and 2-OG. Relative proportions of a particular peptide species (i.e. trimethylated, dimethylated or monomethylated peptide) were calculated by taking the ratio of one methylation state's peak intensity in the mass spectrum to the sum of all three methylation states' peak intensities. %me₃ = 100 x ^

Percentage demethylation at varied inhibitor concentrations was then used to calculate IC50S for inhibitors. Methods for Zn ejection assay

A Novostar spectrophotometer (BMG Labtechnologies) was used for measurements of fluorescence intensity of a Zn-specific fluorophore. In this case, FluoZin™-3 (Invitrogen) was used. Before each experiment, a calibration curve for the fluorophore (between 0-2 μΜ Zn²⁺) was obtained with varied concentrations of Zn²⁺ dissolved in MilliQ water. For assays, lmM solution of FluoZin™-3 was prepared in 50 mM HEPES buffer, pH 7.5, and then diluted in the same buffer to 10 μΜ stock solution. 10 μϋ, of the stock solution was pre-mixed with 10 μΐ, of 20 μΜ enzyme and 30 yL buffer to make the enzyme mix. A solution of compound in DMSO (5

was mixed with 45 )iL buffer. The compound solution was then mixed with the enzyme-fluorophore solution to give a total volume of 100 μΐ,, with final concentrations of FluoZin™-3, enzyme and DMSO of 1 μΜ, 2 μΜ and 5%, respectively. Mixing of the enzyme mix and the Zn-ejector solution took place immediately before the plate was inserted into the spectrophotometer for the first reading. The assay plate was shaken automatically for 5 seconds after each reading. Readings were taken for 90 cycles at a rate of 20 seconds per cycle.

Methods for non-denaturing ESI-MS binding assay

JMJD2A was desalted using a Bio-Spin 6 Column (Bio-Rad, Hemel Hempstead, UK) in 300 mM ammonium acetate (pH 7.5). Fe²⁺ solution was prepared as described above. The protein (final concentration 15 μΜ) was mixed with 1 eq of Fe²⁺ and 10 eq of inhibitor and incubated for 20 minutes at room temperature prior to ESI-MS analysis. Data were acquired on a Q-TOF mass spectrometer (Q-TOF micro, Micromass, Altrincham, UK) interfaced with a Nanomate (Advion Biosciences, Ithaca, NY, USA) with a chip voltage of 1.70 kV and a delivery pressure 0.25 psi (1 psi = 6.81 kPa). The sample cone voltage was either 80 V or 200 V with a source temperature of 40 °C and with an acquisition/scan time of lOs/l s. Calibration and sample acquisition were performed in the positive ion mode in the range of 500-5,000 mlz. The pressure at the interface between the atmospheric source and the high vacuum region was fixed at 6.60 mbar. Data were processed with MASSLYNX 4.0 (Waters). Methods for Differential Scanning Fluorimetry

Method as reported in Niesen et al, Nat. Protocols 2007, p2212-2221. DSF was used to determine T_m values for JMJD2A in the presence of small molecule Zn ejectors. It was performed using MiniOpticon™ Real-Time PCR Detection System (Bio-Rad). SYPRO orange (Invitrogen) dye was used for unspecifing binding to hydrophobic residues and its increase in fluorescence monitored as a function of time. For measurements of the effect of small molecules on the stability of proteins, compounds were dissolved to appropriate concentration in DMSO, such that the final volume of DMSO in a sample was 2.5 μΐ, (5%, v/v); 2.5 μΐ compound in DMSO, 10

of 20 μΜ protein, 10 of 5x SYPRO orange (diluted 1 : 1000 in 50 mM HEPES buffer from the stock solution in DMSO supplied) and 27.5 μΐ, buffer were mixed on ice. The chamber was cooled to 4 °C before inserting the sample plate and the readings of fluorescence were taken between 4-95 °C, increasing the

temperature linearly for 30 minutes. FAM (492 nm) and ROX (610 nm) filters were used for excitation and emission, respectively. Software automatically performed global minimum subtraction. The T_m is calculated using the Boltzmann equation y = LL + ^"^- , where LL

1 + e ⁰

and UL are values of minimum and maximum intensities and a corresponds to the slope of the curve within l_m. All measurements were performed in triplicate.

Results

Test compounds la - 16 were screened for inhibition of JMJD2A using the MALDI-TOF MS turnover assay. The tested compounds as set out in Table 1 include disulfiram analogues (lb-2), as well as compounds with carbamothioate (3), thiol (4, 8), disulfide (5, 9), thioanhydride (6), thioester (7), and phenyl sulfhydryl (10) functional groups. A variety of selenium derivatives (1 1-16), including ebselen 16, which has been tested clinically for treatment of acute ischaemic stroke. IC₅₀ values were determined and were as follows (values in μΜ):

Test compound la = 15.3, lb = 3.3, lO lOOO, 2 > 1000, 3 > 1000, 4 > 1000, 5 > 1000, 6 > 1000, 7 > 1000, 8 > 1000, 9 = 233, 10 > 1000, 1 1 > 300, 12 = 9.2, 13 > 1000, 14 = 26.1 , 15 > 300, 16 = 10.6. Compound 17 = 1.4.

The thioester, thioanhydride, carbamothioate and phenyl sulfhydryl -containing compounds (3, 6, 7, 10) were inactive (at 1 mM) as inhibitors. However, disulfiram (la) inhibited JMJD2A with IC₅₀ = 15.3 μΜ. Of the three disulfiram analogues (lb, lc, 2), only that with pyrrolidinyl substituents (lb) inhibited, and was found to be more potent than disulfiram, whilst the dibenzyl derivative (lc) did not inhibit JMJD2A. Sodium

diethylthiocarbamate (2), a reduced form of disulfiram, also did not inhibit. Cystamine (9), a biologically relevant disulfide cysteine derivative, also inhibited JMJD2A in the high- micromolar range. However, its reduced analogue, cysteamine (8), did not inhibit, suggesting that the disulfide moiety is necessary for inhibition. Glutathione (GSH) was inactive in both reduced (4) and oxidized (5) forms. Because the concentration of GSH in a cell is 0.1-10 mM, it is perhaps unsurprising that it does not inhibit histone demethylases. Of the selenium derivatives tested, compounds 12, 14 and 16 were identified as inhibitors of JMJD2A, with IC₅o values of 9.2 μΜ, 26.1 μΜ and 10.6 μΜ respectively.

Having shown that sulfur- and selenium-containing Zn-ejectors inhibit JMJD2A, the spectrophotometric Zn-ejection assay was used to investigate the mechanism of inhibition by detecting ejected Zn(II) ions. The amount of Zn(II) released from JMJD2A upon inhibitor treatment was determined by measuring the increase in fluorescence of the Zn(II)-chelating fluorophore (Figure 4). Of the sulfiir-containing compounds (la-10), only disulfiram (la) and its pyrrolidine analogue (lb) led to Zn-ejection, as monitored by an increase in fluorescence. In agreement with the catalytic assay results, lc and 2 showed no Zn-ejection. Cysteamine (9), despite inhibiting JMJD2A (Table 1), showed no Zn-ejection, suggesting it inhibits via a different mechanism.

All the selem^'um-containing compounds tested (11-16) caused varying levels of Zn- ejection. Consistent with the inhibition assay results, benzene seleninic acid (12) and ebselen (16) were the most potent Zn-ejectors (Figure 4). In contrast, pyridine-2,4-dicarboxylic acid (2,4-PDCA; compound 17, a potent JMJD2A inhibitor (IC₅₀ 1.4 μΜ) that binds to the Fe(II) in the active site, did not eject Zn(II). Titrations of compounds lb and 16 against JMJD2A in the presence of the Zn(II)-chelating fluorophore demonstrated dose-dependent Zn-ejection for both compounds (Figure 2), at rates comparable with the rates of JMJD2A inactivation as measured by the catalytic turnover assay (Figure 5).

Non-denaturing electrospray ionization mass spectrometry (ESI-MS) was then used to further investigate the mechanism of inhibition (Figure 3). Mass spectra of JMJD2A (Fig. 3.(I)-(V)) incubated with disulfiram la showed covalent modification of JMJD2A with half of one disulfiram molecule, concomitant with loss of Zn(II) (Fig. 3.(11)). When JMJD2A was incubated with both disulfiram (la) and 2,4-PDCA (17), binding of both 17 and half of la was observed under mild ionisation conditions (80V sample cone voltage, Fig. 3.(IV)), together with loss of Zn(II); however, at higher sample cone voltage (200V) only the binding of disulfiram was retained, indicating covalent modification (Fig. 3.(V)). These data indicate that la and 17 inhibit by different mechanisms, la and 16 probably inhibit JMJD2A by

mechanisms related to that proposed by Loo et al, J Med Chem 1996, p4313-20 for the inhibition of aldehyde dehydrogenase by disulfiram (Figure 6) and by Jacob et al. Biochem Biophys Res Commun 1998, p569-73 for Zn-ejection from metallothionein by ebselen:

disulfiram forms a disulfide bond with one of the Zn-binding Cys residues, causing ejection of Zn(II). A related mechanism operates in the ejection of Zn(II) from p300 by

epidithioketopiperazines (ETPs) (Cook, Hilton, Mecinovic, Motherwell, Figg and Schofield; J Biol Chem 2009 July 9).

Differential scaruiing fluorimetry (DSF) was then used to determine the effect of Zn- ejectors on the structural stability of JMJD2A, as indicated by changes in the melting temperature (T_m). Based on structural analysis, Zn-ejection was expected to destabilise the JmjC fold, whereas the Fe(II)-chelating competitive inhibitor 17, which binds to the active site, was expected to stabilise the protein and lead to an increase in T_m. The T_m values were as follows:

T_m of JMJD2A with no compounds added = 47.4 ± 0.7°C. T_m with each tested compound (in °C): la = 32.0±1.6, lb = 29.9±0.4, 4 = 41.3±0.9, 12 = 27.8±0.6, 16 = 23.4±1.3, T_m with compound 17 = 48.9±2.0°C.

These values indicate that JMJD2A is destabilised by Zn-ejectors (la, lb, 12, 16) and that the extent of destabilisation parallels their inhibitory potency. 2,4-PDCA 17, (used at 100 μΜ) led to an increase in T_m, consistent with the hypothesis that the mechanism of inhibition by Zn-ejectors is different from that of previously reported Fe(II)-binding JMJD2A inhibitors (such as compound 17).

Finally, test compounds lb and 16 were tested for inhibition of the related 2-OG dependent oxygenase, prolyl hydroxylase domain 2 (PHD2), in the MALDI-TOF MS turnover assay. Neither test compound inhibited demonstrating that Zn(II) ejection is a viable method for obtaining selective inhibition of JMJD2 subfamily enzymes over other 2-OG dependent oxygenases. Conclusions

The above results demonstrate that Zn(II) removal can achieve selective inhibition of the JMJD2 subfamily histone demethylases over related 2-OG dependent oxygenases, specifically those which lack a Zn(II) binding site. This work also suggests that some of the cellular effects of Zn-ejecting compounds such as disulfiram and ebselen may be mediated via JMJD2 inhibition. Importantly, it has also demonstrated the potential for inhibiting 20G- dependent oxygenases by methods that do not involve Fe(II) chelators, thereby avoiding the potential side-effects associated with Fe chelation-based therapies.

Claims

1. A method of identifying an inhibitor of a JMJD2 subfamily histone lysyl demethylase, said method comprising the steps of:

2. A method according to claim 1 further comprising the step of:

(c) detenruning whether or not the test compound changes the melting temperature (T_m) of the JMJD2 subfamily histone lysyl demethylase, wherein a reduction in T_m indicates that the compound is an inhibitor of a JMJD2 subfamily histone lysyl demethylase; and/or (d) comparing the amount of Zn(II) released in the presence and in the absence of said test compound, wherein a greater amount of Zn(II) released in the presence of the compound compared with the amount of Zn(II) released in the absence of the compound indicates that the compound is an inhibitor of a JMJD2 subfamily histone lysyl demethylase.

3. A method according to claim 1 or 2 wherein the release of Zn(II) ions is monitored using a Zn(II) binding agent.

4. A method according to claim 3 wherein the Zn(II) binding agent produces a detectable signal when bound to Zn(II), optionally wherein said signal is a fluorescent signal.

5. A method according to any one of the preceding claims wherein the JMJD2 subfamily histone lysyl demethylase is JMJD2A.

6. A method according to any one of the preceding claims wherein the test compound comprises sulphur and/or selenium.

7. A method according to claim 6 wherein the test compound is a derivative of 1- (diemylthiocarbamoyldisulfanyl)-N,N-diethyl-methanethioamide (disulfiram) or 2-phenyl-l, 2-benzisoselenazol-3(2H)-one (ebselen).

8. A compound identified as a JMJD2 subfamily histone lysyl demethylase inhibitor by a method according to any one of the preceding claims.

9. A compound according to claim 8 which does not inhibit a 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase.

10. A compound which promotes the release of Zn(II) ions from a JMJD2 subfamily histone lysyl demethylase for use in the inhibition of a JMJD2-subfamily histone lysyl demethylase, wherein the compound does not inhibit a 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase.

11. A compound which promotes the release of Zn(II) ions from a JMJD2 subfamily histone lysyl demethylase for use in the treatment of cancer, wherein the compound does not inhibit a 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase.

12. A compound according to claim 11 wherein said cancer is squamous cell carcinoma or prostate cancer.

13. A compound according to any one of claims 8 to 12 which reduces the T_m of a JMJD2 subfamily histone lysyl demethylase.

14. A compound according to any one of claims 8 to 12 which inhibits the en2yme JMJD2A and / or which does not inhibit the enzyme PHD2.

15. A compound according to any one of claims 8 to 14 which comprises sulphur and/or selenium.

16. A compound according to any one of claims 8 to 15 which is:

- a disulfide of formula (I)

wherein R¹ and R², which are the same or different, are independently selected from unsubstituted or substituted Ci-20 alkyl, unsubstituted or substituted C_2-20 alkenyl,

unsubstituted or substituted C_2-2o alkynyl, unsubstituted or substituted C_3-2o carbocyclyl, unsubstituted or substituted C3_-2o heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, cyano, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, and a group of formula (lb)

(lb)

wherein Y is O or S, and wherein R and R are the same or different and are independently selected from hydrogen, unsubstituted or substituted C_1-10 alkyl, unsubstituted or substituted C2-10 alkenyl, unsubstituted or substituted C₂-io alkynyl, unsubstituted or substituted C_3-20 carbocyclyl, unsubstituted or substituted C_3-20 heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, carboxy, unsubstituted or substituted ester, and unsubstituted or substituted acyl, provided that R¹² and R¹³ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-2o heterocyclyl group or an unsubstituted or substituted heteroaryl group,

wherein said Ci_-2o alkyl, C_2-2o alkenyl, C_2-20 alkynyl, Cj-io alkyl, C2-10 alkenyl and C_2-io alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C ₄ alkyl;

or a pharmaceutically acceptable salt thereof; an organoselenium compound of formula (II)

^{^}Se R²¹ (II)

wherein

R²⁰ is unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C_3-2o carbocyclyl, unsubstituted or substituted C3_-20 heterocyclyl, unsubstituted or substituted C_1-2o alkyl, unsubstituted or substituted C₂-₂₀ alkenyl,

unsubstituted or substituted C_2-2o alkynyl, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester, or unsubstituted or substituted acyl, wherein said Ci_-20 alkyl, C_2-20 alkenyl and C_2-20 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or Q-₄ alkyl; and R²¹ is hydrogen, halo, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C3_-20 carbocyclyl, unsubstituted or substituted C_3-2o heterocyclyl, unsubstituted or substituted C_1-2o alkyl, unsubstituted or substituted C_2-20 alkenyl, unsubstituted or substituted C_2-20 alkynyl, cyano, amino, hydroxyl, thiol,

unsubstituted or substituted C^o alkylamino, unsubstituted or substituted di(C_1-1o)alkylamino, unsubstituted or substituted arylamino, unsubstituted or substituted diarylamino, unsubstituted or substituted arylalkylamino, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, unsubstituted or substituted acyloxy, unsubstituted or substituted Cj.io alkoxy, unsubstituted or substituted aryloxy, unsubstituted or substituted C^o alkylthio, unsubstituted or substituted arylthio, or -Se-R , wherein R is as defined above for R , wherein said C[.2₀ alkyl, C_2-2o alkenyl and C_2-20 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or Q-₄ alkyl;

or a pharmaceutically acceptable salt thereof;

- a seleninic acid derivative of formula (III)

O II

_/Se R25

R²⁴ CT (HI)

wherein

R²⁴ is unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C_1-20 alkyl, unsubstituted or substituted C2_-20 alkenyl,

unsubstituted or substituted C₂.₂₀ alkynyl, unsubstituted or substituted C_3-20 carbocyclyl, unsubstituted or substituted C₃.₂₀ heterocyclyl, wherein said C_1-20 alkyl, C₂.₂o alkenyl and C_2-2o alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C_r4 alkyl; and

R²⁵ is hydrogen, unsubstituted or substituted C_1-2o alkyl, unsubstituted or substituted

C_2-2o alkenyl, unsubstituted or substituted C_2-20 alkynyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C_3-2o carbocyclyl, unsubstituted or substituted C₃.₂₀ heterocyclyl, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, unsubstituted or substituted amido, unsubstituted or substituted thioamido or -Se(0)R²⁶, wherein R²⁶ is as defined above for R²⁴;

or a pharmaceutically acceptable salt thereof; - a cyclic selenium compound of formula (IV)

wherein

Ar is an unsubstituted or substituted aryl or heteroaryl ring;

L¹ is -C(O)- or unsubstituted or substituted alkylene; and

X is N( ²⁷), O, S, C(0) or -C(R²⁷)(R²⁸)-, wherein R²⁷ and R²⁸, which are the same or different when both are present, are independently selected from hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted Ci- alkyl, unsubstituted or substituted C_2-1o alkenyl, unsubstituted or substituted C_2-10 alkynyl, unsubstituted or substituted C_3-2o carbocyclyl, unsubstituted or substituted C_3-2o heterocyclyl, cyano, amino, halo, nitro, hydroxyl, thiol, unsubstituted or substituted CMO alkylamino, unsubstituted or substituted di(Ci-i₀)alkylamino, unsubstituted or substituted arylamino, unsubstituted or substituted diarylamino, unsubstituted or substituted arylalkylamino, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, unsubstituted or substituted acyloxy, unsubstituted or substituted Ci-io alkoxy, unsubstituted or substituted aryloxy, unsubstituted or substituted Ci_-10 alkylthio, and unsubstituted or substituted arylthio, wherein said CMO alkyl, C_2-10 alkenyl and C_2-10 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C ₄ alkyl;

or a pharmaceutically acceptable salt thereof; or

- a selenite salt.

17. A compound according to claim 16 wherein the disulfide of formula (I) is a thiuram disulfide of formula (la)

wherein

R³, R⁴, R⁵ and R⁶ are the same or different and are independently selected from hydrogen, unsubstituted or substituted Q-io alkyl, unsubstituted or substituted C -i₀ alkenyl, unsubstituted or substituted C2-io alkynyl, unsubstituted or substituted C_3-2o carbocyclyl, unsubstituted or substituted C_3-2o heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, carboxy, unsubstituted or substituted ester, and unsubstituted or substituted acyl, wherein said C_1-10 alkyl, C_2-10 alkenyl and C_2-10 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or Ci- alkyl, provided that R³ and R⁴ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C3_{- 0} heterocyclyl group or an unsubstituted or substituted heteroaryl group, and

provided that R⁵ and R⁶ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C_3-2o heterocyclyl group or an unsubstituted or substituted heteroaryl group.

18. A compound according to claim 16 or claim 17 wherein, in the

organoselenium compound of formula (II),

R²⁰ is unsubstituted or substituted aryl or heteroaryl; and

R²¹ is hydrogen, halo, unsubstituted or substituted C_1-20 alkyl, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, or -Se-R²², wherein R²² is as defined for R²⁰ wherein said C_1-20 alkyl is optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or d-₄ alkyl.

19. A compound according to any one of claims 16 to 18 wherein, in the seleninic acid derivative of formula (III),

R²⁴ is unsubstituted or substituted aryl, or unsubstituted or substituted C_1-10 alkyl; and R²⁵ is hydrogen.

20. A compound according to any one of claims 16 to 19 wherein the cyclic selenium compound of formula (IV is a compound of formula (IVa)

wherein

L¹ is -C(O)- or unsubstituted or substituted Ci_-2 alkylene; and

X is N(R ), wherein R is hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, or unsubstituted or substituted Cno alkyl.

21. A compound according to any one of claims 16 to 20 wherein the selenite salt is a selenite salt of formula (V)

[Xⁿ⁺]p[Se0₃ ²-]_{q (v)} wherein

X is a cation having a charge n+ wherein n is a positive integer; and

p and q are positive integers wherein n multiplied by p is equal to 2q.

22. A compound according to any one of claims 16 to 21 which is:

1 -(diethylthiocarbamoyldisulfanyl)-N,N-diemyl-methanethioamide (compound 1 a; disulfiram), bis(pyrrolidine-thiocarbamoyl) disulfide (compound lb), Na?Se0₃ (compound 11), PhSeOOH (compound 12; benzene seleninic acid), PhSeSePh (compound 13), PhSeCl (compound 14), PhSeH (compound 15), or:

(compound 16; ebselen).

23. A method of identifying a compound that promotes the release of Zn(II) ions from a JMJD2 subfamily histone lysyl demethylase, said method comprising the steps of:

(a) contacting a subfamily JM JD2 histone lysyl demethylase with a test compound; and

24. A pharmaceutical composition comprising

(a) an inhibitor of a subfamily J JD2 histone lysyl demethylase identified by a method of any one of claims 1 to 7 or 23; or

(b) a compound as defined in any one of claims 8 to 22

and a pharmaceutically acceptable carrier or diluent.

A composition according to claim 24 for use in the treatment of cancer.

26. A method of inhibiting a JMJD2 subfamily histone lysyl demethylase without also inhibiting a 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase, said method comprising:

(a) identifying an inhibitior by a method according to any one of claims 1 to 7 or 23; and

(b) contacting a sample comprising at least one JMJD2 subfamily histone lysyl demethylase and at least one 2-oxoglutarate dependent oxygenase that is not a JMJD2 subfamily histone lysyl demethylase with the inhibitor of step (a) or with a compound as defined in any one of claims 8 to 22.

27. A compound which is:

- a disulfide of formula (I)

_/S ^ ^R²

R¹ S (I)

unsubstituted or substituted C_2-2₀ alkynyl, unsubstituted or substituted C3-2₀ carbocyclyl, unsubstituted or substituted C3_-20 heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, cyano, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, and a group of formula (lb)

wherein Y is O or S, and wherein R and are the same or different and are independently selected from hydrogen, unsubstituted or substituted Ci-io alkyl, unsubstituted or substituted C_2-1o alkenyl, unsubstituted or substituted C_2-10 alkynyl, unsubstituted or substituted C₃.₂₀ carbocyclyl, unsubstituted or substituted C₃.₂₀ heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, carboxy, unsubstituted or substituted ester, and unsubstituted or substituted acyl, provided that R¹² and R¹³ may together form, with the nitrogen atom to which they are bonded, an unsubstituted or substituted C₃.₂₀ heterocyclyl group or an unsubstituted or substituted heteroaryl group, wherein said C_1-20 alkyl, C_2-2o alkenyl, C₂.₂₀ alkynyl, C^o alkyl, C2-10 alkenyl and C_2-1o alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C1-4 alkyl;

or a pharmaceutically acceptable salt thereof;

- an organoselenium compound of formula (II)

R²⁰

^Se R²¹ (II)

wherein-

R²⁰ is unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C3-2₀ carbocyclyl, unsubstituted or substituted C3-20 heterocyclyl, unsubstituted or substituted C1.20 alkyl, unsubstituted or substituted C_2-20 alkenyl,

unsubstituted or substituted C2-20 alkynyl, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester, or unsubstituted or substituted acyl, wherein said C1.20 alkyl, C2_-2o alkenyl and C2_-2o alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or d-4 alkyl; and R²¹ is hydrogen, halo, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C3_-2o carbocyclyl, unsubstituted or substituted C3J20 heterocyclyl, unsubstituted or substituted C_1-2o alkyl, unsubstituted or substituted C₂-₂₀ alkenyl, unsubstituted or substituted C2.20 alkynyl, cyano, amino, hydroxyl, thiol,

unsubstituted or substituted CMO alkylamino, unsubstituted or substituted di(C_1-10)alkylarnino, unsubstituted or substituted arylamino, unsubstituted or substituted diarylamino, unsubstituted or substituted arylalkylamino, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, unsubstituted or substituted acyloxy, unsubstituted or substituted C O alkoxy, unsubstituted or substituted aryloxy, unsubstituted or substituted C_1-10 alkylthio, unsubstituted or substituted arylthio, or -Se-R , wherein R is as defined above for R , wherein said C_1-20 alkyl, C_2-20 alkenyl and C2_-20 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C_R4 alkyl;

or a pharmaceutically acceptable salt thereof;

- a seleninic acid derivative of formula (III)

wherein

R is unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C_1-20 alkyl, unsubstituted or substituted C₂.₂₀ alkenyl,

unsubstituted or substituted C2-20 alkynyl, unsubstituted or substituted C_3-2₀ carbocyclyl, unsubstituted or substituted C3_-20 heterocyclyl, wherein said C_1-20 alkyl, C_2-20 alkenyl and C_2-20 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C ₄ alkyl; and

R²⁵ is hydrogen, unsubstituted or substituted C_1-20 alkyl, unsubstituted or substituted C_2-20 alkenyl, unsubstituted or substituted C_2-20 alkynyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted C₃.₂₀ carbocyclyl, unsubstituted or substituted C_3-2₀ heterocyclyl, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, unsubstituted or substituted amido, unsubstituted or substituted thioamido or -Se(0)R²⁶, wherein R²⁶ is as defined above for R²⁴;

or a pharmaceutically acceptable salt thereof;

- a cyclic selenium compound of ormula (IV)

wherein

Ar is an unsubstituted or substituted aryl or heteroaryl ring;

L¹ is -C(O)- or unsubstituted or substituted C\ alkylene; and

X is N(R²⁷), 0, S, C(O) or -C(R²⁷)(R²⁸)-, wherein R²⁷ and R²⁸, which are the same or different when both are present, are independently selected from hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted Cno alkyl, unsubstituted or substituted C_2-io alkenyl, unsubstituted or substituted C_2-io alkynyl, unsubstituted or substituted C₃.₂₀ carbocyclyl, unsubstituted or substituted C_3-2o heterocyclyl, cyano, amino, halo, nitro, hydroxyl, thiol, unsubstituted or substituted CMO alkylamino, unsubstituted or substituted di(C_1-i₀)alkylamino, unsubstituted or substituted arylamino, unsubstituted or substituted diarylamino, unsubstituted or substituted arylalkylamino, unsubstituted or substituted amido, unsubstituted or substituted thioamido, carboxy, unsubstituted or substituted ester, unsubstituted or substituted acyl, unsubstituted or substituted acyloxy, unsubstituted or substituted C O alkoxy, unsubstituted or substituted aryloxy, unsubstituted or substituted C_1-10 alkylthio, and unsubstituted or substituted arylthio, wherein said C M O alkyl, C_2-i₀ alkenyl and C_2-10 alkynyl groups are optionally interrupted by O, S, N(R"), arylene or heteroarylene, wherein R" is H, aryl or C1-4 alkyl;

or a pharmaceutically acceptable salt thereof; or

- a selenite salt, provided that the compound is other than: 1 -(diethylthiocarbamoyldisulfanyl)-N,N- diethyl-methanethioamide (compound la; disulfiram); bis(pyrrolidine-thiocarbamoyl) disulfide (compound lb); bis(dibenzylthiocarbamoyl) disulfide (compound lc); glutathione (disulfide form) (compound 5); cystamine (compound 9); Na₂Se0₃ (compound 11);

PhSeOOH (compound 12; benzene seleninic acid); PhSeSePh (compound 13); PhSeCl (compound 14), PhSeH (compound 15); and

(compound 16; ebselen).