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Title:
INHIBITORS OF MACROPHAGE MIGRATION INHIBITORY FACTOR
Document Type and Number:
WIPO Patent Application WO/2010/140902
Kind Code:
A1
Abstract:
The invention provides the use of isothiocyanate and isoselenocyanate compounds for treating diseases and conditions mediated by MIF.

Inventors:
HAMPTON MARK (NZ)
SMITH ROBIN ANDREW JAMES (NZ)
BROWN KRISTEN (NZ)
Application Number:
PCT/NZ2010/000102
Publication Date:
December 09, 2010
Filing Date:
June 02, 2010
Export Citation:
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Assignee:
HAMPTON MARK (NZ)
SMITH ROBIN ANDREW JAMES (NZ)
BROWN KRISTEN (NZ)
International Classes:
A61K31/26; C07C331/24
Domestic Patent References:
WO2008128189A12008-10-23
WO2006075330A22006-07-20
Other References:
DATABASE CAPLUS KOTTKE K. ET AL: "Organic thiocyanate compounds. XLII. Synthesis of thiocyanatoanilines from aminothiophenols", Database accession no. 1969:501813
PRUETT S. B. ET AL: "Sodium Methyldithiocarbamate Inhibits MAP Kinase Activation through Toll-like Receptor 4, Alters Cytokine Production by Mouse Peritoneal Macrophages, and Suppresses Innate Immunity", TOXICOL. SCI., vol. 87, no. 1, 2005, pages 75 - 85
SHARMA A. ET AL: "Targeting Akt3 Signaling in Malignant Melanoma Using Isoselenocyanates", CLIN. CANCER. RES., vol. 15, no. 5, 2009, pages 1674 - 1685
HWANG E.-S. ET AL: "Phenylethyl isothiocyanate and its N-acetylcysteine conjugate suppress the metastasis of SK-Hep1 human hepatoma cells", JOURNAL OF NUTRITIONAL BIOCHEMISTRY, vol. 17, no. 12, 2006, pages 837 - 846, XP024960895, DOI: doi:10.1016/j.jnutbio.2006.02.004
DATABASE CAPLUS Database accession no. 2009:073804
SINGH S. V. ET AL: "Sulforaphane Inhibits Prostate Carcinogenesis and Pulmonary Metastasis in TRAMP Mice in Association with Increased Cytotoxicity of Natural Killer Cells", CANCER RESEARCH, vol. 69, no. 5, 2009, pages 2117 - 2125
NOYAN-ASHRAF M. H. ET AL: "Dietary approach to decrease aging-related CNS inflammation", NUTRITIONAL NEUROSCIENCE, vol. 8, no. 2, 2005, pages 101 - 110
BROWN K. K. ET AL: "Direct Modification of the Proinflammatory Cytokine Macrophage Migration Inhibitory Factor by Dietary Isothiocyanates", J. BIOL. CHEM., vol. 284, no. 47, 23 September 2009 (2009-09-23), pages 32425 - 32433
DATABASE CAPLUS CROSS J. V. ET AL: "Nutrient isothiocyanates covalently modify and inhibit the inflammatory cytokine macrophage migration inhibitory factor (MIF)", Database accession no. 2009:1245586
OUERTATANI-SAKOUHI H. ET AL: "A New Class of Isothiocyanate-Based Irreversible Inhibitors of Macrophage Migration Inhibitory Factor", BIOCHEMISTRY, vol. 48, no. 41, 8 September 2009 (2009-09-08), pages 9858 - 9870, XP002594526, DOI: doi:10.1021/BI900957E
Attorney, Agent or Firm:
ADAMS, Matthew D. et al. (6th FloorHuddart Parker Building Post,Office Square, PO Box 15 Wellington, NZ)
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Claims:
What is claimed is:

1. A method for reducing macrophage migration inhibitory factor (MIF) cytokine or its biological activity or for treating or preventing a disease or condition wherein MIF cytokine or its biological activity is implicated in the disease or condition in a subject in need thereof comprising administering to a subject a therapeutically effective amount of an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof.

2. A method for reducing MIF cytokine or its biological activity or for treating or preventing a disease or condition mediated by MIF in a subject in need thereof comprising administering to a subject a therapeutically effective amount of a compound of formula (I)

R N-

(I)

or a pharmaceutically acceptable salt or prodrug thereof,

wherein X is S or Se; and

R is selected from the group comprising alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, arylalkyl, substituted arylalkyl, allyl, substituted allyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkenyl, substituted arylalkenyl, acyl, substituted acyl, acyloxy, substituted acyloxy, alkyloxycarbonyloxy, substituted alkyloxycarbonyloxy, aryloxycarbonyloxy, substituted aryloxycarbonyloxy, alkoxycarboylacyl, substituted alkoxycarbonylacyl, alkylcarbonylalkyl, substituted alkylcarbonylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl, alkoxy, substituted alkoxy, cycloalkoxy, substituted cycloalkoxy, arylalkoxy, substituted arylalkoxy, alkylsulfϊnylalkyl, and substituted alkylsulfinylalkyl,

wherein R may be substituted with one or more of halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl, -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaRb, -NRaC(=O)ORb, -NRaSO2Rb, ORa, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRb, -OC(=O)NRaRb, -SH, -SRa, -SOR3, -S(=O)2Ra, -OS(=O)2Ra, -S(=O)2ORa, wherein Ra and Rb are the same or different and independently selected from the group comprising hydrogen, alkyl, haloalkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl, wherein when R is a substituted group, the substituent is selected from the unsubstituted groups in the definition of R.

3. A method according to claim 1 wherein R is selected from the group comprising alkyl, substituted alkyl, alkyaryl, substituted alkylaryl, aryl, substituted aryl, alkoxy alkylarly, substituted alkoxy alkylaryl, allyl, substituted allyl, alkylsulfinylalkyl, substituted alkylsulfinylalkyl, alkylamino and substituted alkylamino, wherein when R is substituted the substituent is selected from the group comprising hydrogen, alkyl, aryl, alkylaryl, heteroaryl, acyloxy, alkoxy, and cycloalkyl.

4. A method according to claim 3 wherein R is phenyl (Ci-C6)alkyl or substituted phenyl (C,-C6)alkyl.

5. A method according to claim 2 wherein the compound of formula (I) is selected from the group comprising:

wherein R1, R2 and R3 are independently selected from the group comprising hydrogen, halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl, -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaRb, -NRaC(=O)ORb, -NRaSO2Rb, OR3, -C(=O)R3, -C(=O)OR3, -C(=O)NR3Rb, -OCC=O)NR3R1,, -SH, -SR3, -SORa, -S(=O)2Ra, -OS(O)2R3, -S(=O)2ORa, wherein Ra and Rb are the same or different and independently selected from the group comprising hydrogen, alkyl, haloalkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl;

wherein when R, R2 or R3 are substituted, the substituent is independently selected from the group comprising hydrogen, alkyl, aryl, alkylaryl, heteroaryl, acyloxy, alkoxy, and cycloalkyl;

n = O to 9; and X = S or Se.

6. A method according to claim 5 wherein the compound of formula (I) is selected from the group comprising phenyl isothiocyanate (PITC), 2-(3-(2-aminoethyl)phenyl)ethyl isothiocyanate (amino-PEITC) benzyl isothiocyanate (BITC), 4-hydroxy phenethyl isothiocyanate, phenethylisothiocyanate (PEITC), phenylhexylisothiocyanate, 3,4,5- trimethyloxybenzyl isothiocyanate, allyl isothiocyanate, erucin, erysolin and sulforaphane.

7. A method according to claim 1 wherein the disease or condition is mediated by MIF cytokine or its biological activity is selected from the group comprising rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, inflammatory bowel disease such as Crohn's disease and ulcerative colitis; cardiovascular disease, conjestive heart failure, atherosclerosis, autoimmune myocarditis, autoimmune hepatitis, Alzheimer's disease, Parkinson's disease, muscular dystrophy, granuloma, alopecia, acute pancreatitis, bacterial infection, endotoxemia, glomerulonephritis, inflammatory disease, inflammation, malaria, sepsis, tissue rejection vitreoretinopathy, autoimmune graft versus host disease, multiple sclerosis, endotoxic shock, metastasis, asthma, Cushing's disease, atopic dermatitis, atopy, otitis media, acute respiratory distress syndrome, delayed-type hypersensitivity, contact hypersensitivity type I and II diabetes, endometriosis, psoriasis, sarcoidosis, kidney disease, Guillain Barre syndrome, systemic lupus erythematosus, adult-onset Still's disease and obesity.

8. A method according to claim 7 wherein the disease or condition is mediated by MIF is selected from the group comprising atherosclerosis, rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, sepsis, endotoxic shock, obesity, asthma, acute respiratory distress syndrome, atopy, delayed type hypersensitivity and contact hypersensitivity reactions, systemic lupus erythematusus, psoriasis and sarcoidosis.

9. A method according to any one of claims 1 to 8 wherein X = S.

10. A compound of formula (II)

(H)

or a pharmaceutically acceptable salt of prodrug thereof, wherein n = 0 to 10 and m = 0 to 10.

11. A compound of claim 10 wherein n = m = 2 to 4.

12. A method for reducing MIF activity or for treating a disease or condition is mediated by MIF in a subject in need thereof comprising administering a therapeutically effective amount of a compound of formula (II) as defined in claim 10 or a pharmaceutically acceptable salt or prodrug thereof.

13. A method according to claim 12 wherein n = m = 2 to 4.

14. A method according to claim 12 wherein the disease or condition is mediated by MIF cytokine or its biological activity is selected from the group comprising rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, inflammatory bowel disease such as Crohn's disease and ulcerative colitis; cardiovascular disease, conjestive heart failure, atherosclerosis, autoimmune myocarditis, autoimmune hepatitis, Alzheimer's disease, Parkinson's disease, muscular dystrophy, granuloma, alopecia, acute pancreatitis, bacterial infection, endotoxemia, glomerulonephritis, inflammatory disease, inflammation, malaria, sepsis, tissue rejection vitreoretinopathy, autoimmune graft versus host disease, multiple sclerosis, endotoxic shock, metastasis, asthma, Cushing's disease, atopic dermatitis, atopy, otitis media, acute respiratory distress syndrome, delayed-type hypersensitivity, contact hypersensitivity type I and II diabetes, endometriosis, psoriasis, sarcoidosis, kidney disease, Guillain Barre syndrome, systemic lupus erythematosus, adult-onset Still's disease and obesity.

15. A method according to claim 14 wherein the disease or condition is mediated by MIF cytokine or its biological activity is selected from the group comprising atherosclerosis, rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, sepsis, endotoxic shock, obesity, asthma, acute respiratory distress syndrome, atopy, delayed type hypersensitivity and contact hypersensitivity reactions, systemic lupus erythematusus, psoriasis and sarcoidosis.

16. The method of claim 1, wherein said MIF inhibitor is selected from the compounds listed in Tables 1-8.

17. The method of claim 1, wherein said MIF inhibitor is selected from the group consistsing of Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.

18. The method of claim 17, wherein said compound is Compound 67.

17. A compound of formula (OHPE 9).

(OHPE 9)

or a pharmaceutically acceptable salt of prodrug thereof

18. A compound of formula (OHPE 10).

or a pharmaceutically acceptable salt of prodrug thereof 19. A compound of formula (OHBN 3).

(OHBN 3) or a pharmaceutically acceptable salt of prodrug thereof 20. The method of claim 1 , wherein said MIF inhibitor is OHPE 9, OHPE 10 or OHBN 3.

Description:
INHIBITORS OF MACROPHAGE MIGRATION INHIBITORY FACTOR

Claim of Priority

This patent application claims priority to United States Provisional Patent Application No: 61/183,245 filed 2 June 2009, incorporated herein fully by reference.

Field of the Invention

The invention relates generally to uses of isothiocyanate and isothioselenate compounds that are inhibitors of macrophage migration inhibitory factor (MIF). MIF is a cytokine involved in immunity and inflammation. More particularly, this invention relates to used of isothiocyanate and isothioselenate compounds to treat disorders in which it is desirable to decrease MIF activity.

BACKGROUND

The protein MIF is a cytokine released by many cell types including T-lymphocytes and macrophages. MIF proteins have been identified in several species including mammals and are generally 12-13 kDa in size. MIF levels increase during physiological stress or systemic inflammatory conditions. MIF plays an important role in septic shock and delayed-type hypersensitivity reaction, possibly due to its ability to act as an endogenous regulator of glucocorticoid action within the immune system. Deletion of the MIF gene or immunoneutralisation of MIF protects against septic shock.

Elevated levels of MIF have been observed in a number of disease states including cardiovascular disease, diabetes, sepsis and many cancer types. Furthermore, genetic ablation of MIF has been shown to attenuate various disease states in murine models. While details surrounding the mechanisms of MIF action are still in question, the clinical significance of MIF expression is such that targeted approaches to modulate the biological activities of MIF are currently in development.

MIF is also known to have a proinflammatory role in arthritis and glomerulonephritis. Inhibition of proinflammatory cytokine activity using monoclonal antibodies has been shown to improve disease outcomes in mouse models of rheumatoid arthritis, sepsis, cardiovascular disease, and inflammatory bowel disease. Additional biological activities for MIF include tumour invasion, metastasis and angiogenesis, insulin release, cell growth and apoptosis, regulation of T-cell and macrophage activation and IgE synthesis. MIF also acts as a tautomerase. While the biological significance of this activity is still under debate, the tautomerase site appears to be important for regulating protein-protein interactions that mediate MIF activity.

As MIF is known to be involved in numerous pathological events, inhibition of MIF may have several therapeutic effects. Candidate MIF inhibitors have been obtained from a variety of sources and include antibodies and small molecules. However, there remains a need for alternative MIF inhibitors. Accordingly, it is an object of the present invention to provide the use of further compounds as MIF inhibitors, or to provide the public with a useful choice.

Existing MIF inhibitors can be classified as non-covalent modifiers, such as ISOl and its analogs, (13, 29, 30) phenyl pyruvic derivatives, ketones, (10) and coumarin derivatives, (12) and as covalent modifiers, including NAPQI, (7) 2-OBP, (31) 4-IPP, (32, 33) PMSF, (34) ITCs, (19, 35,

36) and a hydroxyquinoline(37). These inhibitors have demonstrated complete inhibition of

MIF's tautomerase activity in vitro. In some cases, they have also resulted in the inhibition of glucocorticoids overriding activity7 and have improved survival in an animal model of sepsis (13, 14) in vivo.

SUMMARY

Isothiocyanates are a class of phytochemicals with many well-recognised biological properties.

For example, isothiocyanates are proposed to have chemotherapeutic potential (Conaway, C. C;

Wang, C. X.; Pittman, B.; Yang, Y. M.; Schwartz, J. E.; Tian, D. F.; Mclntee, E. J.; Hecht, S. S.; Chung, F. L. "Phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates inhibit malignant progression of lung adenomas induced by tobacco carcinogens in

A/J mice." Cancer Research, 2005, 65, pp 8548-8557.) and (Chiao, J. W.; Wu, H. Y.;

Ramaswamy, G.; Conaway, C. C; Chung, F. L.; Wang, L. G.; Liu, D. L. "Ingestion of an isothiocyanate metabolite from cruciferous vegetables inhibits growth of human prostate cancer cell xenografts by apoptosis and cell cycle arrest". Carcinogenesis, 2004, 25, pp 1403-1408).

Isoselenocyanates are structurally and electronically similar to isothiocyanates and are also known to have anti-tumor activity (Sharma, A.K. et al. J. Med. Chem., 2008, 51, pp 7820-7826).

Isoselenocyanates, like isothiocyanates, are known to react predominantly with thiols. The active form of these compounds in animals is generally attributed to circulating N-acetyl-L- cysteine conjugates formed by reversible reaction of glutathione with the isothiocyanate or isoselenocyanate. As potential inhibitors of MIF, isothiocyanate and isoselenocyanate compounds have therapeutic applications in conditions in which MIF has a mediating function.

This study has led to the discovery of MIF as a major target of isothiocyanates. This provides a foundation for the development of novel MIF inhibitors to be used in the treatment of cancer and inflammatory diseases, and identifies a previously uncharacterised mechanism through which naturally-occurring isothiocyanates can influence biological systems.

We have unexpectedly discovered that isothiocyanates and isothioselenates can bind to MIF and therefore decrease MIF activity. Thus, isothiocyanates and isothioselenates represent classes of compounds that can be useful in treating disorders in which it is desirable to decrease MIF activity. Such disorders include, for example, cancer and inflammatory diseases.

Accordingly, in one aspect the invention provides a use of an isothiocyanate or isothioselenate compound or pharmaceutically acceptable salt or prodrug thereof for reducing MIF activity.

In one aspect the invention provides an isothiocyanate of isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof for treating a disease or condition mediated- by MIF in a subject in need thereof.

In one aspect the invention provides an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof for reducing MIF activity in a subject in need thereof.

In one aspect the invention provides a method for treating a disease or condition mediated by MIF in a subject in need thereof comprising administering to a subject a therapeutically effective amount of an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof.

In one aspect the invention provides a method for reducing MIF activity in a subject in need thereof comprising administering to a subject a therapeutically effective amount of an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof.

In one aspect the invention provides a use of an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a medicament for treating a disease or condition mediated by MIF. In one aspect the invention provides a use of an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a medicament for reducing MIF activity.

In one aspect the invention provides the use of a compound of formula (I)

R N=C=*

(I)

or a pharmaceutically acceptable salt or prodrug thereof, for reducing MIF activity

wherein X is S or Se; and

R is selected from the group comprising alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, arylalkyl, substituted arylalkyl, allyl, substituted allyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkenyl, substituted arylalkenyl, acyl, substituted acyl, acyloxy, substituted acyloxy, alkyloxycarbonyloxy, substituted alkyloxycarbonyloxy, aryloxycarbonyloxy, substituted aryloxycarbonyloxy, alkoxycarboylacyl, substituted alkoxycarbonylacyl, alkylcarbonylalkyl, substituted alkylcarbonylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl, alkoxy, substituted alkoxy, cycloalkoxy, substituted cycloalkoxy, arylalkoxy, substituted arylalkoxy, alkylsulfinylalkyl, and substituted alkylsulfinylalkyl.

In one embodiment, R may be substituted with one or more of halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl, -NR a R b , -NR a C(=O)R b , -NR a C(=O)NR a R b , -NR a C(=O)OR b , -NR a SO 2 R b , OR a , -C(O)R 8 , -C(O)OR 4 , -C(O)NR 3 R 5 , -OC(=O)NR a R b , -SH, -SR a , -SOR a , -S(O) 2 R 8 , -OS(O) 2 R 3 , -S(O) 2 OR 3 , wherein R 3 and R b are the same or different and independently selected from the group comprising hydrogen, alkyl, haloalkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl. When R is a substituted group the substituent is selected from the unsubstituted groups in the definition of "substituent" herein.

In one embodiment R is selected from the group comprising alkyl, alkylaryl, aryl, alkoxy alkylaryl, allyl and alkylsulfinylalkyl, alkylamino and substituted versions thereof. Preferably, 5 when R is substituted the substituent is selected from the group comprising hydrogen, alkyl, aryl, alkylaryl, heteroaryl, acyloxy, alkoxy, and cycloalkyl.

Preferably, R is arylalkyl, or substituted arylalkyl, preferably phenylalkyl, or substituted phenylalkyl, more preferably phenyl-(Ci to C 6 ) alkyl wherein the phenyl ring is unsubstituted or substituted with 0-3 methoxy groups.

LO In one embodiment the compound of formula (I) is selected from the group comprising:

wherein R 1 , R 2 and R 3 are independently selected from the group comprising hydrogen, halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl,

L5 heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl, -NR a R b , -NR a C(=O)R b , -NR a C(=O)NR a R b , -NR a C(=O)OR b , -NR 3 SO 2 R h , OR 3 , -C(=0)R 3 , -C(=O)OR 3 , -C(=O)NR a R b , -OC(=O)NR a R b , -SH, -SR 3 , -SOR a , -S(=O) 2 R a , -OS(=O) 2 R a , -S(=O) 2 OR a , wherein R a and R b are the same or different and independently selected from the group comprising hydrogen, alkyl, haloalkyl, substituted

>0 alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl; n = 0 to 9; and X = S or Se.

When R is a substituted group the substituent is selected from the unsubstituted groups in the definition of "substituent" herein.

In one embodiment R 1 , R 2 and R 3 are independently selected from the group comprising hydrogen, alkyl, alkoxy and alkylamino. Preferably R 1 is (Q-C^alkyl, (C 1 -C 6 )alkoxy or (C 1 - C 6 )alkylphenyl.

In one embodiment n = 0 to 6.

In one embodiment X = S.

In one embodiment the compound of formula (I) is selected from the group comprising phenyl isothiocyanate (PITC), 2-(3-(2-aminoethyl)phenyl)ethyl isothiocyanate (amino-PEITC) benzyl isothiocyanate (BITC), phenethylisothiocyanate (PEITC), phenylhexylisothiocyanate, 3,4,5- trimethyloxybenzyl isothiocyanate, allyl isothiocyanate, erucin, erysolin and sulforaphane.

In one embodiment the compound of formula (I) is 4-hydroxy phenethyl ITC.

In another aspect the invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt or prodrug thereof for reducing MIF activity.

In another aspect the invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt or prodrug thereof for treating a disease or condition mediated by MIF.

In another aspect the invention provides a method for reducing MIF activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) as defined above or a pharmaceutically acceptable salt or prodrug thereof.

In another aspect the invention provides a method for treating a disease or condition mediated by MIF in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) as defined above or a pharmaceutically acceptable salt or prodrug thereof. In another aspect the invention provides the use of a compound of formula (I) as defined above or a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a medicament for reducing MIF activity in a subject in need thereof.

In another aspect the invention provides the use of a compound of formula (I) as defined above or a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a medicament for treating a disease or condition mediated by MIF.

In one embodiment of the above aspects of the invention, reduction of MIF activity is via binding of the N-terminal proline residue of MIF.

In one embodiment of the above aspects, the disease or condition mediated by MIF is an inflammatory disease. In another embodiment, it is an autoimmune disease. In yet another embodiment it is an infectious disease.

In one embodiment of the above aspects the disease or condition mediated by MIF is selected from diseases or conditions where one or more of the following has been demonstrated;

(a) disease attenuation in an animal study involving genetic ablation of MIF or downstream proteins involved in signalling pathways activated by MIF,

(b) disease attenuation in an animal or human study inducing neutralisation of MIF activity with an antagonistic antibody to MIF or downstream proteins included in signalling pathways activated by MIF,

(c) disease attenuation in an animal or human study involving inhibition of MIF activity with small molecule inhibitors of MIF or downstream proteins involved in signalling pathways activated by MIF, and

(d) increased associated with polymorphisms in the MIF gene and/or its promoter region.

(e) increased MIF associated with mutations or duplications in the MIF gene and/or its promoter region.

In one embodiment of the above aspects, the disease or condition mediated by MIF is selected from the group comprising rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, inflammatory bowel disease such as Crohn's disease and ulcerative colitis; cardiovascular disease, conjestive heart failure, atherosclerosis, autoimmune myocarditis, autoimmune hepatitis, Alzheimer's disease, Parkinson's disease, ir-iscular dystrophy, granuloma, alopecia, acute pancreatitis, bacterial infection, endotoxemia, glomerulonephritis, inflammatory disease, inflammation, malaria, sepsis, tissue rejection vitreoretinopathy, autoimmune graft versus host disease, multiple sclerosis, endotoxic shock, metastasis, asthma, Cushing's disease, atopic dermatitis, atopy, otitis media, acute respiratory distress syndrome, delayed-type hypersensitivity, contact hypersensitivity type I and II diabetes, endometriosis, psoriasis, sarcoidosis, kidney disease, Guillain Barre syndrome, systemic lupus erythematosus, adult-onset Still's disease and obesity.

In one embodiment the disease or condition mediated by MIF is selected from the group comprising atherosclerosis, rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, sepsis, endotoxic shock, obesity, asthma, acute respiratory distress syndrome, atopy, delayed type hypersensitivity and contact hypersensitivity reactions, systemic lupus erythematusus, psoriasis and sarcoidosis.

In one aspect the invention provides a method for treating an inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or pharmaceutically acceptable salt or prodrug thereof.

In one embodiment the inflammatory disease is selected from the group comprising rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, glomerulonephritis and inflammatory bowel disease including Crohn's disease and ulcerative colitis.

In one aspect the invention provides a method for treating an autoimmune disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof.

In one embodiment the autoimmune disease is selected from the group comprising diabetes, multiple sclerosis, Crohn's disease, ulcerative colitis, autoimmune graft versus host disease, systemic lupus erythematosus, rheumatoid arthritis, autoimmune myocarditis and autoimmune hepatitis.

In one aspect the invention provides a method of treating an infectious disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof.

In one embodiment, the infectious disease is selected from the group comprising bacterial infection and endotoxemia. In one aspect the invention provides a method for preventing metastasis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt of prodrug thereof.

In one aspect the invention provides a compound of formula (II)

(H)

or a pharmaceutically acceptable salt or prodrug thereof wherein n = 0 to 10 and m = 0 to 10.

In a preferred embodiment n = m, preferably n = m = 2 to 4, more preferable n = m = 2.

In one embodiment of the above aspects, the isothiocyanate or isothioselenate MIF inhibitor is substantially purified.

In some embodiments, isothiocyanates and isothioselenates of this invention can be ingested in food or as food additives.

It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art. The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such 5 known equivalents are deemed to be incorporated herein as if individually set forth.

BRIEF DESCRIPTION OF THE FIGURES

Aspects of the invention will be described, by way of example only with respect to specific embodiments thereof. Other features of the invention can be appreciated with reference to the following figures:

LO Figure 1 shows the loss of Jurkat cell viability following a 24 hour exposure to PEITC (•) and amino-PEITC (o). Cell viability was assessed by flow cytometric determination of the uptake of propidium iodide, (mean ± SE from three experiments).

Figure 2 shows (A) SDS-PAGE analysis of protein from Jurkat cell lysates eluted from blocked Affi-Gel or Affi-PEITC resins. The highlighted protein band was excised from the gel and

L5 identified by MALDI-TOF mass spectrometry to be the 12.5 kDa cytokine MIF. (B) Matched peptides (underlined) identified by MALDI-TOF mass spectrometry. A 33% coverage of the MIF sequence was positively identified. Cysteine residues and the N-terminal proline are highlighted. (C) Western blot detection of MIF binding to Affi-PEITC. Binding of a polyclonal MIF antibody to increasing amounts of a Jurkat cell lysate (lanes 1-3), an eluate recovered from

»0 blocked Affi-Gel (lane 4), an eluate from Affi-PEITC (lane 5) and an eluate recovered from Affi-PEITC when the lysate was pre-incubated with PEITC (lane 6). All gels are representative of results from at least three experiments.

Figure 3 shows the mass spectroscopy results for: (A) rhMIF before (1) and after (2) incubation with a 10-fold excess of PEITC for 10 minutes. (A) Wild type rhMIF before (i) and after (ii)

»5 incubation with PEITC. A peak with mass 12509, corresponding to MIF (12,345) with addition of one PEITC (163 Da) resulted. In addition a peak with mass 12,248 was also formed. (B) rhMIF with C60S mutation (1), C57S mutation (2) or C81S mutation (3) (12,329 Da) after incubation with PEITC. In all cases a peak with mass 12,492, corresponding to cysteine mutant MIF with addition of one PEITC resulted. In addition a peak with mass 12,231 was also formed.

50 (C) P2A rhMIF before (1) and after (2) incubation with PEITC. Only the mass of the parent protein (12,319) was observed. Figure 4 shows (A) Time course of the inhibition of rhMIF tautomerase activity by 10 μM (•), 20 μM (o) and 30 μM (A) PEITC (results are representative of three experiments). (B) in silico modeling of MIF in the presence of PEITC. (C) Immunoreactivity of rhMIF following a 10 min exposure to 20μM PEITC as measured using a commercial ELISA. Values are mean ± SE of three experiments.

Figure 5 shows (A) Time course of the inhibition of cellular MIF tautomerase activity by 1 μM (•), 2 μM (o) and 4 μM (A) PEITC (mean ± SE of three experiments). (B) Immunobinding of MIF monoclonal (mAb) and polyclonal (pAb) antibodies to extracts prepared from Jurkat cells treated with increasing concentrations of PEITC for 50 minutes. Images are representative of results from three experiments. (C) Cells were treated with 15 μM PEITC for 1 h before detectable levels of extracellular MIF (black bars) and intracellular MIF (grey bars) were measured using a commercial ELISA (mean ± SE from three experiments).

Figure 6 shows in silico modeling (I) of PEITC at the tautomerase active site. MIF without PEITC bound is shown in dark grey. The unreacted N-terminal proline is not shown. PEITC is bound to the tautomerase active site of MIF via covalent modification of the N-terminal proline residue. MIF with PEITC bound is shown in light grey).

Figure 7 depicts the concentration-dependence of inhibition of cellular MIF tautomerase activity following a 30 min exposure to PEITC (O), sulforaphane (T) or benzyl isothiocyanate (V). Values are mean ± SE of at least three experiments.

Figure 8 depicts a gel showing progressinve inhibition of MIF binding to PEITC. Jurkat cells were treated with increasing concentrations of PEITC for 30 min. Cell lysates were prepared and the ability of MIF to bind Affi-PEITC was measured. A representative gel is shown.

Figure 9 depicts results of a study in human volunteers. Three volunteers consumed 50 g of watercress. Blood was drawn just prior to eating (0), one hour after eating (1) and two hours (2) after eating. Plasma fractions were prepared immediately after venipuncture. Plasma levels of isothiocyanates and their corresponding dithiocarbamates were measured at all time points in two of the volunteers using a cyclocondensation reaction with 1,2-benzenedithione. Values are mean ± range.

Figure 10 depicts plasma MIF in the volunteer subjects of Figure 9. MIF levels (o) were measured by a commercial ELISA just prior to eating and 2 hours after ingestion of watercress.

Values are mean ± SE. * indicates a significant (p=0.001) difference in the immunoreactivity of plasma MIF before and after watercress consumption as determined by a paired t-test (SigmaStat, SPSS Inc., Chicago, IL, USA).

Figure 11 depicts the chemical reaction between Affi-Gel and Amino PEITC, a derivative of PEITC, to generate the Affi-PEITC probe of this invention.

Figure 12 depicts the tautomerase reaction used to evaluate MIF activity. MIF catalyses the tautomerisation of the chromogenic substrate dopachrome methyl ester to a colourless indole derivative.

Figure 13 depicts In silico modeling (II) of PEITC (yellow) bound to the tautomerase active site of MIF via covalent modification of the N-terminal proline residue. Figure 13 A depicts the MIF homotrimer shown with PEITC docked in one of the tautomerase active sites. MIF without PEITC bound is shown in magenta (pdb3B9S). Figure 13B depicts a close-up image of a portion of Figure 13A, and shows conformational shifts of the catalytic proline by 2 A (arrow 1) and lysine 32 by 1.6 A (arrow 2).

Figure 14 depicts mass spectra of rhMIF-PEITC adduct formation. rhMIF was reacted with 10"- fold excess of PEITC for 10 mintues before analysis of PEITC-adduct formation. Figure 14A is wild type rhMIF before (i) and after (ii) incubation with PEITC. A peak with a mass of 12,508 Da, corresponding to MIF (12,345 Da) with the addition of 1 PEITC (163 Da) resulted. In addition, a peak with a mass of 12.248 Da was also apparent. Figure 14B shows rhMIF with a C60S mutation (i), a C57A mutation (ii) or a C81S mutation (iii) (12,329 Da) after incubation with PEITC. In all cases, a peak with a mass of 12,492 Da, corresponding to cysteine mutant MIF with addition of one PEITC, resulted. In addition, a peak with a mass of 12,232 Da was also formed. Figure 14C shows a P2A rhMIF before (i) and after (ii) incubation with PEITC. Only the mass of the parent protein (12,319 Da) was observed.

Figure 15 depicts mass spectromerty analysis of the modification of MIF by PEITC in the abasence of acid. MIF was reacted with a 10-fold excess of PEITC for 10 min before analysis of the PEITC-adduct formation by mass spectrometry in a formic acid-free buffer. Figure 15A depicts rhMIF before and Figure 15B depicts rhMIF after incubation with PEITC. Figure 15A shows rhMIF in its un-conjugated form with a mass of 12,345 Da. Figure 15B shows a peak with m/z 12,508, corresponding to MIF (m/z 12,346) with addition of one PEITC resulted. Sodiated species (+23 Da) were also formed due to the composition of the buffer. DETAILED DESCRIPTION

Definitions

The term "MIF activity" as used herein means an activity or effect mediated at least in part by MIF and includes, but is not limited to, inhibition of macrophage migration, tautomerase activity, 5 endotoxin induced shock, inflammation, glucocorticoid counter regulation, enhanced proliferation, induction of ERK phosphorylation and MAP kinase activity.

The term "MIF inhibitor" means a molecule (either natural or synthetic) that can alter the conformation of MIF and/or compete with a monoclonal antibody to MIF and can decrease at least one activity of MIF or its export from a cell when compared to the activity or export in the D absence of the inhibitor. An inhibitor alters the conformation, activity or export of MIF if there is a statistically significant change in the amount of MIF measured, MIF activity or in MIF protein detected extracellularly and/or intracellularly in an assay performed with an inhibitor, compared to the assay performed without the inhibitor. MIF inhibitors include the compounds 1- 73 listed herein, as well as others having MIF inhibiting activity.

5 The term "mediated by MIF" as used herein with reference to a condition or disease means a condition or disease in which elevated MIF activity is believed to play a role. Conditions or diseases mediated by MIF are targets for the MIF inhibitors of formula (I) because inhibition of MIF is likely to have a positive effect on the condition or disease. Examples of conditions and diseases mediated by MIF include inflammation, diabetes, sepsis, cardiovascular disease,

D rheumatoid arthritis and the like. Diseases or conditions mediated by MIF also include those where one or more of the following has been demonstrated;

(a) disease attenuation in an animal study involving genetic ablation of MIF or downstream proteins involved in signalling pathways activated by MIF,

(b) disease attenuation in an animal or human study inducing neutralisation of MIF

5 activity with an antagonistic antibody to MIF or downstream proteins included in signalling pathways activated by MIF,

(c) disease attenuation in an animal or human study involving inhibition of MIF activity with small molecule inhibitors of MIF or downstream proteins involved in signalling pathways activated by MIF, and

D (d) increased associated with polymorphisms in the MIF gene and/or its promoter region. (e) increased MIF associated with mutations or duplications in the MIF gene and/or its promoter region.

The term "alkyl," as used herein means, unless otherwise stated, a straight or branched chain, noncyclic or cyclic hydrocarbon radical, which may be fully saturated, mono- or 5 polyunsaturated, including di- and multivalent radicals, and may have the number of carbon atoms designated (i.e. Ci-Ci 0 means one to ten carbons).

Examples of saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n- hexyl and the like. Examples of saturated branched chain alkyls include isopropyl, isobutyl, sec- butyl, test-butyl, isopentyl and the like. Representative saturated cyclic alkyls include

.0 cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. An unsaturated alkyl group is one having one or more double or triple bonds. Examples of unsaturated straight chain alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term "alkyl," unless otherwise stated, is also meant to include those derivatives of alkyl defined

.5 in more detail below as "alkenyl", "alkynyl", "cycloalkyl" and "alkylene."

The term "alkylene" as used herein means, unless otherwise stated, a divalent radical derived from an alkane, as exemplified by -CH 2 CH 2 CH 2 CH 2 -. Typically, an alkylene group will have from 1 to 24 carbon atoms. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

O The term "cycloalkyl" as used herein means, unless otherwise stated, a cyclic version of "alkyl", and includes di-and poly-homocyclic rings such as decalin and adamentane. Examples of cycloalkyls include cyclopentyl, cyclohexyl, 1 -cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.

The term "alkenyl", as used herein means, unless otherwise stated, a hydrocarbon radical having '.5 at least one double bond including, but not limited to, ethenyl, propenyl, 1-butenyl, 2-butenyl and the like.

The term "alkynyl", as used herein means, unless otherwise stated, a hydrocarbon radical having at least one triple bond including, but not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl and the like. The term "aryl," alone or in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) as used herein means, unless otherwise stated, an aromatic carbocyclic moiety which can be a single ring or multiple rings (for example, three rings) which are fused together or linked covalently for example phenyl or napthyl. The rings may each contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Therefore, the term "aryl" unless otherwise stated, is also meant to include those aryl groups containing heteroatoms defined in more detail below as "heteroaryl". The heteroaryl groups can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl groups include phenyl, 1- naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl 4-pyrimidyl, 2-benzothiazolyl, 5- benzothiazolyl, 2-benzoxazolyl, 5-benzoxazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1- isoquinolinyl, 5-isoquinolinyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolinyl, 6-quinolinyl and substituted derivatives of these groups. Substituents for each of the above noted aryl ring systems are selected from the group of acceptable substituents described herein.

The term "heteroaryl," as used herein means, unless otherwise stated, an aromatic heterocycle ring of 5- to 10 members and having at least one heteroatom selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and bicyclic ring systems. Representative heteroaryls include (but are not limited to) furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothioazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl.

The term "aralkyl" or "arylalkyl" as used herein means, unless otherwise stated, an alkyl group having at least one hydrogen atom replaced with an aryl moiety such as a phenyl or naphthyl including mono-, di-, and poly-homocyclic aromatic ring systems, for example, (C 6 - J4 aryl). Preferred arylalkyls comprise a lower alkyl group attached to the aryl group. Non-limiting examples of suitable arylalkyl groups include benzyl, phenylhexyl, 2-phenylethyl and naphthalenylmethyl. The bond to the parent moiety is through the alkyl. The term "heteroarylalkyl," as used herein means, unless otherwise stated, an alkyl group having at least one alkyl hydrogen atom replaced with a heteroaryl moiety, for example, -CH 2 pyridinyl, -CH 2 pyrimidinyl, and the like.

The terms "heterocycle" and "heterocycle ring," as used herein, mean, unless otherwise stated, a 3- to 7-membered monocyclic, or 6- to 14-membered polycyclic, heterocycle ring which is either saturated, unsaturated or aromatic, and which contains from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulphur, and wherein the nitrogen and sulphur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quarternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring as well as tricyclic (and higher) heterocyclic rings. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined above. Thus, in addition to the aromatic heteroaryls listed above, heterocycles also include (but are not limited to) morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The term "heterocyclealkyl", as used herein means, unless otherwise stated, an alkyl having at least one alkyl hydrogen atom replaced with a heterocycle, for example -CH 2 morpholinyl, and the like.

The terms "halo" or "halogen," as used herein mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "fluoroalkyl," are meant to include monofluoroalkyl and poly fluoroalkyl.

The term "haloalkyl" as used herein means, unless otherwise stated, an alkyl group having at least one hydrogen atom replaced with a halogen, for example trifluoromethyl, and the like.

The term "alkoxy", as used herein means, unless otherwise stated, an O-alkyl group wherein "alkyl" is defined herein, for example, methoxy, ethoxy, and the like.

The term "thioalkyl," as used herein means, unless otherwise stated, an alkyl moiety attached through a sulfur bridge (ie., -S-alkyl) such as methylthio, ethylthio, and the like. The term "alkylsulfonyl," as used herein means, unless otherwise stated, an alkyl moiety attached through a sulfonyl bridge (i.e, -SO2-alkyl) such as methylsulfonyl, ethylsulfonyl, and the like.

The term "alkylsulfinyl" as used herein means, unless otherwise stated, an alkyl moiety attached through a sulfinyl bridge (-S(O)-alkyl) where methylsulfinyl, ethylsulfinyl, propylsulfinyl, butylsulfinyl, and the like.

The terms "alkylamino" and "dialkyl amino" as used herein means, unless otherwise stated, one alkyl moiety or two alkyl moieties, respectively, attached through a nitrogen bridge (i.e., -N- alkyl) such as methylamino, ethylamino, dimethylamino, diethylamino, and the like.

The term "hydroxyalkyl," as used herein means, unless otherwise stated, an alkyl substituted with at least one hydroxyl group.

The term "alkylcarbonylalkyl," as used herein means, unless otherwise stated, an alkyl substituted with a -C(=O)alkyl group.

The term "alkylcarbonyloxyalkyl," as used herein means, unless otherwise stated, an alkyl substituted with a -C(=O)Oalkyl group or a -OC(=O)alkyl group.

The term "alkyloxyalkyl," as used herein means, unless otherwise stated, an alkyl substituted with an -O-alkyl group.

The term "alkylthioalkyl," as used herein means, unless otherwise stated, an alkyl substituted with a -S-alkyl group.

The term "mono-or di(alkyl)aminoalkyl," as used herein means, unless otherwise stated, an alkyl substituted with a mono- or di(alkyl)amino.

The term "substituted" as used herein with reference to any of the above defined groups (e.g. alkyl, aryl, arylalkyl, heteroaryl, etc.) means a group or compound wherein at least one hydrogen atom has been replaced by a chemical substituent. In the case of a keto substituent (-(C=O)-), two hydrogen atoms are replaced.

The term "substituent," includes halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle. substituted heterocycle, heteroc"~lealkyl, substituted heterocyclealkyl, -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaRb, -NRaC(=O)ORb, -NRaSO2Rb, -ORa, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRb, -OC(=O)NRaRb, -SH, -SRa, -SORa, -S(=O)2Ra, -0S(=0)2Ra, -S(=0)20Ra, wherein Ra and Rb are the same or different and independently hydrogen, alkyl, haloalkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, 5 substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl or substituted heterocyclealkyl.

The term "therapeutically-effective amount" as used herein, means that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic or prophylactic effect, commensurate with a O reasonable benefit/risk ratio.

The term "treatment" as used herein in the context of treating a condition or disease, relates generally to treatment and therapy, whether of human or animal, in which some desired therapeutic effect is achieved, for example, the inhibition of progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the 5 condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included.

"Treatment" also includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, a therapeutically effective amount of a compound of formula (I) could be combined with or used O in conjunction with radiation therapy or chemotherapy in the treatment of cancer.

The term "prodrug" as used herein means, unless otherwise stated a compound that undergoes chemical conversion in the body to become an active pharmacological agent with the defined chemical properties. For example, a prodrug of a compound of formula (I) is metabolised or otherwise converted to a compound of formula (I) as defined above. Examples of prodrugs 5 within the scope of the invention include ester and amide derivatives that are hydrolysed to form isothiocyanate and isothioselenate compounds of formula (I) in the body. Also included are N- acetylcysteine conjugates of the isothiocyanate and isothioselenate compounds. These dithiocarbamate compounds are hydrolysed in the body to release N-acetylcysteine and the isothiocyanate or isothioselenate compound, including compounds of formula (I).

O The term "pharmaceutically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms and the like, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, exipient, etc., must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

The term "subject" as used herein refers to a human or non-human mammal. Examples of non- human mammals include livestock animals such as sheep, horses, cows, pigs, goats, rabbits, deer, ostriches and emus; and companion animals such as cats, dogs, rodents, and horses. Preferably, the subject is a human.

The term "comprising" as used herein means "consisting at least in part of. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

A certain compound may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, 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; (+) and (-) forms; keto-, enol-, and enolate- forms; α- 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").

Some compounds of formula (I) have at least one asymmetrical carbon atom and therefore all isomers, including enantiomers, stereoisomers, rotamers, tautomers and racemates of the compounds are contemplated as being part of this invention. The invention includes d and 1 isomers in both pure form and in admixture, including racemic mixtures. Isomers can be prepared using conventional techniques, either by reacting optically pure or optically enriched starting materials or by separating isomers of a compound of the invention. Isomers may also include geometric isomers, e.g., when a double bond is present.

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, -OCH 3 , is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH 2 OH. Similarly, a reference to ortho — chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a preference to a class of structures may well include structurally isomeric forms falling within that class (e.g., Ci -7 alkyl includes w-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and/?αrø-methoxyphenyl).

5 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, imine/enamine, amide/imino alcohol, amidine/amidine nitroso/oxime, thioketone/enethiol, N- nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term "isomer" are compounds with one or more isotopic LO substitutions. For example, H may be in any isotopic form, including H, H (D), and H (T); C may be in any isotopic form, including 12 C, 13 C, and 14 C; O may be in any isotopic form, including 16 O and 18 O; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including racemic and other mixtures thereof. Methods for the preparation (e.g., L5 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 the methods taught herein in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, hydrate, and protected forms of thereof, for example, as discussed below.

!0 If the compound is cationic, or has a functional group which may be cationic (e.g., -NH may be -NH 3 + ), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include, but are not limited to, anions from the following

!5 organic acids: acetic, propionic, succinic, gycolic, stearic, lactic, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetyoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and valeric.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to refer to a

10 complex of solute (e.g., active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

It may be convenient or desirable to prepare, purify, and/or handle the active compound in a chemically protected form. The term "chemically protected form," as used herein, pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions, that is, are in the form of a protected or protecting group (also known as masked or masking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic J Synthesis (T. Green and P. Wuts, Wiley, 1991).

DESCRIPTIONS OF EMBODIMENTS

Introduction

Isothiocyanates are a class of phytochemicals with widely reported anti-cancer and anti- inflammatory activity. However, knowledge of their activity at a molecular level is limited. This invention relates to isothiocyantes and their uses to treat disease. In one aspect, this invention uses phenethyl isothiocyanate (PEITC) to determine which biological targets are affected by isothiocyanates.. An analogue of PEITC, amino-PEITC was synthesised to enable conjugation to a solid-phase resin. Surprisingly, the pleiotropic cytokine macrophage migration inhibitory factor (MIF) was found the major protein captured by the amino-PEITC from cell lysates. Site-directed mutagenesis and mass spectrometry showed that PEITC covalently modified the N-terminal proline residue of MIF. This resulted in complete loss of catalytic tautomerase activity and disruption of protein conformation, as determined by impaired recognition of a monoclonal antibody directed to the region that receptors and interacting proteins bind to MIF. The conformational change was supported by in silico modeling. Monoclonal antibody binding to plasma MIF was disrupted in humans consuming watercress, a major dietary source of PEITC. The isothiocyanates have significant potential for development as MIF inhibitors, and this activity may contribute to the biological properties of these phytochemicals.

Although the mechanisms of action of isothiocyanates are not completely known, they represent a class of phytochemicals with recognised anti-cancer activity. They can act in a chemopreventive capacity via inhibition of carcinogen-activating phase I enzymes (1) and induction of phase II detoxification enzymes (2). Isothiocyanates are also active in the post- initiation phase of tumorigenesis and are therefore proposed to have chemotherapeutic potential (3,4). Isothiocyanate-mediated disruption of cancer progression is achieved by a variety of mechanisms including modulation of cell growth (5), inhibition of angiogenesis (6), suppression 5 of metastasis (7) and induction of apoptosis (8,9). Isothiocyanates can also modulate inflammatory pathways via inhibition of the transcription factor nuclear factor-κB (NF-κB) (10).

The electrophilic carbon residue in the isothiocyanate moiety (-N=C=S) is capable of reacting with biological nucleophiles such as cysteine in proteins and the tripeptide glutathione (11,12). Binding of isothiocyanates to Kelch-like ECH-associated protein 1 (Keapl) (13), transient 0 receptor potential (TRP) channels (14), MEKKl protein kinase (15) and tubulin (16) has been demonstrated to occur via covalent modification of cysteine. Reaction with amines to form stable thiourea derivatives can also occur. However, this is generally considered to be a less favourable reaction at physiological pH (11).

To elucidate the major cellular targets of biologically active isothiocyanates we have utilised an

5 affinity-based target identification approach. An amine linker was added to phenethyl isothiocyanate (PEITC) without compromising cytotoxicity, and the molecule was immobilized to a solid phase resin. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) was identified as a major biological target of PEITC. Using mass spectrometry and site-directed mutagenesis we identified the N-terminal proline of MIF as the target residue and have shown

1 O that conjugation disrupts the catalytic tautomerase activity of MIF and conformational integrity of the protein in vitro and in vivo.

Isothiocyanate MIF Inhibitors

Isothiocyanates can be advantageously used to treat a variety of disorders associated with MIF activity. Thus, in broad aspects, this invention includes use of known isothiocyanates and novel

5 isothiocyanate compounds of this invention. Table 1 below depicts several isothiocyanates useful for inhibiting MIF. In some cases, the chemical names and corresponding structures are known. For convenience, we have supplied informal names to many of the compounds disclosed herein. It can be appreciated that use of the informal names is intended to represent the chemicals depicted in the structural drawings and by their respective chemical names. It can also

■0 be appreciated that isomers, tautomers, enantionmers and other related compounds disclosed herein are intended to be included within the scope of this invention. Table 1

4-((S)-3-(4-chlorophenyl)- 1 - isothiocyanatopropan-2-yl)phenol Informal name: Compound 4

4-((R)- 1 -(3,4-dichlorophenyl)-2- isothiocyanatoethyl)phenol Informal name: Compound 5

Informal name: Compound 6

Informal name: Compound 7

Table 2 below depicts other compounds and their sources useful as inhibitors or precursors of inhibitors of MIF.

Table 2

Table 3 below below depicts other compounds and their sources useful as inhibitors or precursors of inhibitors of MIF.

Table 3

Chemical Name Company Name City State or Country

Province

Benzenemethanol, α-ethyl-4- AKos Consulting Steinen Germany hydroxy- and Solutions

Informal name: Compound GmbH

28

Benzenemethanol, α-ethyl-4- Ambinter Paris France hydroxy-

Informal name: Compound

29

Benzenemethanol, α-ethyl-4- Ambinter Paris France hydroxy-

Informal name: Compound

30

4-(l-Hydroxypropyl)phenol Amfinecom Inc. Petersburg VA USA

Informal name: Compound

31

Benzenemethanol, α-ethyl-4- Aurora Fine San Diego CA USA hydroxy- Chemicals LLC

Informal name: Compound

32 4-( 1 -hydroxypropyl)phenol Aurora Fine San Diego CA USA

Informal name: Compound Chemicals LLC

33

Benzenemethanol, α-ethyl-4- Aurora Fine Graz Austria hydroxy- Chemicals Ltd

Informal name: Compound

34

4-( 1 -hydroxypropyl)phenol Aurora Fine Graz Austπa

Informal name: Compound Chemicals Ltd

35

4-( 1 -hydroxypropyl)phenol Bio-Farma Ltd. Bolton Greater United

Informal name: Compound Mancheste Kingdom

36 r

4-( 1 -hydroxypropyl)phenol Bio-Farma Ltd. Bolton Greater United

Informal name: Compound Mancheste Kingdom

37 r

4-( 1 -hydroxypropyl)phenol Bio-Farma Ltd. Bolton Greater United

Informal name: Compound Mancheste Kingdom

38 r

4-( 1 -Hydroxypropyl)phenol Carbone London UnitedKin

Informal name: Compound Scientific Co., gdom

39 Ltd.

4-(l -Hydroxypropyl)phenol Shanghai FWD Shanghai People's

Informal name: Compound Chemicals Republic

40 Limited of China

4-( 1 -hydroxypropyl)phenol UkrOrgSynthesis Kiev Ukraine

Informal name: Compound

41

4-( 1 -hydroxypropyl)phenol UkrOrgSynthesis Kiev Ukraine

Informal name: Compound

42

4-( 1 -hydroxypropyl)phenol UkrOrgSynthesis Kiev Ukraine

Informal name: Compound

43 4-( 1 -hydroxypropyl)phenol UkrOrgSynthesis Kiev Ukraine Informal name: Compound 44

Table 4 below depicts other compounds and their sources useful as inhibitors or precursors of inhibitors of MIF.

Table 4

Chemical Name Company Name City State or Country

Province

Phenol, 4-(aminophenylmethyl)- AKos Consulting Steinen Germany Informal name: Compound 45 and Solutions

GmbH

4-(Aminophenylmethyl)-phenol Amatek Chemical Kowloon Hong People's Informal name: Compound 46 Co., Ltd. Kong Republic of China

4-(Aminophenylmethyl)-phenol Amatek Chemical Kowloon Hong People's Informal name: Compound 47 Co., Ltd. Kong Republic of China

Phenol, 4-(aminophenylmethyl)- Ambinter Paris France

Informal name: Compound 48 Phenol, 4-(aminophenylmethyl)- Ambinter Paris France

Informal name: Compound 49 Phenol, 4-(aminophenylmethyl)- Aurora Fine San Diego CA USA

Informal name: Compound 50 Chemicals LLC 4- [amino(pheny l)methyl] phenol Aurora Fine San Diego CA USA

Informal name: Compound 51 Chemicals LLC Phenol, 4-(aminophenylmethyl)- Aurora Fine Graz Austria

Informal name: Compound 52 Chemicals Ltd 4- [amino(phenyl)methyl]phenol Aurora Fine Graz Austria

Informal name: Compound 53 Chemicals Ltd 4-[amino(phenyl)methyl]phenol Enamine Kiev Ukraine

Informal name: Compound 54 4-[amino(phenyl)methyl]phenol Enamine Kiev Ukraine

Informal name: Compound 55

4-[amino(phenyl)methyl]phenol Enamine Kiev Ukraine

Informal name: Compound 56

4-[amino(phenyl)methyl]phenol Enamine Kiev Ukraine

Informal name: Compound 57

4-(aminophenylmethyl)phenol NetChem Company New NJ USA

Informal name: Compound 58 Brunswick

4-(aminophenylmethyl)phenol NetChem Company New NJ USA

Informal name: Compound 59 Brunswick

4-(aminophenylmethyl)phenol NetChem Company New NJ USA

Informal name: Compound 60 Brunswick

4-[amino(phenyl)methyl]phenol Ryan Scientific, Inc. Mt. SC USA

Informal name: Compound 61 Pleasant

Phenol, 4-(aminophenylmethyl)- Ryan Scientific, Inc. Mt. SC USA

Informal name: Compound 62 Pleasant

4-[amino(phenyl)methyl]phenol UkrOrgSynthesis Kiev Ukraine

Informal name: Compound 63

4-[amino(phenyl)methyl]phenol UkrOrgSynthesis Kiev Ukraine

Informal name: Compound 64

4-[amino(phenyl)methyl]phenol UkrOrgSynthesis Kiev Ukraine

Informal name: Compound 65

4-[amino(phenyl)methyl]phenol UkrOrgSynthesis Kiev Ukraine

Informal name: Compound 66

Synthesis of Inhibitors of MIF

Isothiocyanate and isothioselenate inhibitors of MIF that are not commercially available may be prepared using any convenient synthetic process. For example, isothiocyanate compounds can be prepared by reacting a primary amine with thiophosgene.

In Scheme 1 below an arylalkyl compound is converted to its corresponding isothiocyanate by replacement of a leaving group substituent (L) with a nitrile functionality, followed by reduction of the nitrile to form a primary amine. Reaction of thiophosgene with the amine produces the corresponding arylalkyl isothiocyanate. The person skilled in the art would be able to devise a suitable synthetic scheme to obtain the desired isothiocyanate inhibitor for use in the invention. Scheme 1

reduction

Another example of the synthesis of an isothiocyanate compound of formula (I) is provided in Example 1 herein below.

Amino-PEITC [2-(3-(2-aminoethyl)phenyl)ethyl isothiocyanate] (Informal name: Compound 67) was prepared from the diamine precursor [l,3-bis(2-aminoethyl)benzene] (Ruggli, P. & Prijs, B. Helvetica Chimica Acta, 1945, 28, pp 674-90) by selective protection as a mono t-Boc derivative (Callahan, J.F. et al. "Structure-activity relationships of novel vasopressin antagonists containing C-terminal diaminoalkanes and (aminoalkyl)guanidines", Journal of Medicinal Chemistry, 1989, 32, pp 391-396) followed by reaction with thiophosgene (Taylor, M.S. & Jacobsen, E.N. "Highly enantioselective catalytic acyl-Pictet-Spengler reactions", Journal of the American Chemical Society, 2004, 126, pp 10558-10559), and deprotection.

l,3-Bis(2-aminoethyl)benzene was obtained by reduction of l,3-bis(cyanomethyl)benzene (Dewey, T.M., Du Bois, J. & Raymond, K.N. "Ligands for oxovanadium(IV): bis(catecholamide) coordination and intermolecular hydrogen bonding to the oxo atom", Inorganic Chemistry, 1993, 32, pp 1729-1738) with a nickel-modified borohydride reagent (Caddick, S., Haynes, A.K.D.K., Judd, D.B. & Williams, M.R.V. "Convenient synthesis of protected primary amines from nitriles", Tetrahedron Letters, 2000, 41, pp 3513-3516). Such a synthetic scheme can be used to prepare novel inhibitors of MIF.

Isothioselenate compounds can be prepared from their corresponding formamides using methods known in the art. See for example, Barton, D. H. R.; Parekh, S. L; Tajbakhsh, M.; Thodorakis, E.A.; Tse, C. -L. Tetrahedron 1994, 50, pp 639-654; Fernandez-Bolanos, J.G.; Lopez, O.; Ulgar, V.; Maya, L; Fuentes, J., Tetrahedron Lett. 2004, 45, pp 4081-4084. Starting from an amine, formylation followed by reaction with triphosgene and selenium powder in the presence of triethylamine will produce the corresponding isoselenocyanate compound. Included within the scope of the invention are prodrugs of isothiocyanate and isothioselenate inhibitors of MIF. In one embodiment the prodrug is an N-acetylcysteine conjugate formed by reaction of the isothiocyanate or isothioselenate with N-acetylcysteine (see formula (III) where X = S or Se).

(III)

N-acetylcysteine conjugates can be prepared by treatment of the corresponding isothicyanate or isothioselenate with N-acetylcysteine in THF in the presence of NaOH.

In Scheme 2 below isothiocyanates are generated by administering a glucosinolate prodrug and the enzyme thioglucosidase (also known as myrosinase). Scheme 2 describes the general structure of glucosinolates, their intermediate and final degradation products. The stars denote positions in the glucose moiety known to be acylated in certain glucosinolates. The conditions favouring the formation of isothiocyantes include a pH of 7.0.

Scheme 2

Glucosinolate D-Glucose

Epithionitrile Thiocyanates Nitriles Isothiocyanates Oxazolidine-thione

CH 2 -CH- CH^-C≡N R-S-C≡N R-C≡N R-N=C=S GHΛ — CH CH CH g S O NH

\ / C

I ! S Thus, in some embodiments, one can administer a precursor glucosinolate to a patient to be treated. Then, either simultaneously or later, one can administer myrosinase or an equivalent enzyme, thereby producing an isothiocyanate. It can be appreciated that administration into the circulation favors the reaction at pH of about 7, whereas administration into the stomach, which 5 has a resting pH of about 1-2, the reaction favored produces nitriles. However, it is known that after feeding, the pH of the stomach can by near 7, which could, under desired conditions, produce the desired isothiocyanates. In particular, co-administration of an antiacid could favor reactions producing isothiocyanates.

Uses of Isothiocyanate and Isoselenocyanate Inhibitors of MIF

LO Electrophilic isothiocyanates and isoselenocyanates are generally thought to react with biological nucleophiles such as cysteine resides in proteins and the tripeptide glutathione. Reaction with amines to form stable thiourea derivatives is known, but is considered to be a less favourable reaction at physiological pH.

It has now been shown that the isothiocyanate compound phenethyl isothiocyanate (PEITC) L5 binds to the N-terminal proline of MIF, as described in Examples 4 and 5.

To demonstrate binding, an amine linker was used to immobilise PEITC to a solid phase resin to form Affϊ-PEITC. MIF was identified as a major biological target of the Affi-PEITC. Mass spectroscopy and site-directed mutagenesis were used to identify the target residue of MIF and confirm that binding at this site alters the enzymology and conformational integrity of MIF.

to Modification results in inhibition of MIF tautomerase activity and also triggers conformational changes that disrupt protein recognition by a monoclonal antibody as shown in Examples 6 and 7 respectively. Currently one of the best indicators of impaired MIF biological activity is the loss of monoclonal antibody binding. This was first shown by Senter et al. ( Senter, P. D.; Al-Abed, Y.; Metz, C. N.; Benigni, F.; Mitchell, R. A.; Chesney, J.; Han, J.; Gartner, C. G.; Nelson, S.

!5 D.; Todaro, G. J.; Bucala, R. "Inhibition of macrophage migration inhibitory factor (MIF) tautomerase and biological activities by acetaminophen metabolites." Proc. Natl. Acad. ScL USA, 2002, 99, pp 144-149), and is now a major property used in the screening of novel MIF inhibitors. It has now been shown that PEITC disrupts monoclonal antibody binding to MIF (Example 7), clearly indicating that crucial epitopes are disturbed upon binding of the

!0 isothiocyanate. In silico modelling predicts a conformational change upon binding of PEITC to the N-terminal proline of MIF with the catalytic proline shifting 2 A compared to that seen in the unmodified structure (Example 8). In addition lysine 32 which sits above the active site shifted by 1.6 A.

In Example 9 the inhibitory effects of several different isothiocyanate compounds of formula (I) were investigated. All were found to inhibit MIF. Benzylthiocyanate provided a control and showed no inhibition. The results obtained demonstrate that compounds of formula (I) are potent inhibitors of MIF. Therefore, compounds of formula (I) have potential in the treatment of conditions and diseases mediated by MIF.

While MIF is involved in many physiological processes, the person skilled in the art will be able to ascertain whether a disease or condition is one that is "mediated by MIF", as discussed in the definition of this term.

Therapeutic Uses of Isothiocyanates and Isothioselenates

As MIF is known to mediate a variety of conditions and diseases, the isothiocyanate and isoselenocyanate inhibitors of MIF have a variety of uses. For example, MIF is involved in the shock response of mammals. Accordingly, inhibition of MIF may provide protection against lethal shock in animals exposed to high concentrations of endotoxin. As MIF' s role is during the post-acute stage of the response, the MIF inhibitors may be able to be used at late stages where treatments such as anti-TNF therapy are ineffective. Isothiocyanates and isoselenocyanates may also be used to prevent the MIF-dependent migration, anchorage-dependent growth and invasion of tumours (metastasis).

MIF is known to play a role in numersous disorders, including the infectious diseases caused by Pseudomonas aeruginosa, Influenza H5N1, Schistosoma mansoni, Granulomas, malaria.

Additionally, MIF is also important in auto-immune diseases, inflammatory bowel disease, Crohn's disease, multiple sclerosis, autoimmune uveitis, Guillain-Barre syndrome, experimental allergic neuritis (EAN), autoimmune glomerulonephritis (experimental model), systemic lupus erythematosus (SLE), experimental allergic neuritis, autoimmune diabetes mellitus (experimental model), systemic sclerosis (SSc), ANCA-associated vasculitides, sarcoidosis, adult-onset Still's Disease, Cushing's Disease, graft versus host Disease, alopecia, kidney disease, psoriasis, atopic dermatitis, endometriosis, otitis media, rheumatoid arthritis, glomerulonephritis, and vitreoretinopathy.

Moreover, MIF is involved in numerous types of cancers. Therefore, broadly, isothiocyanates and isothioselenates can be used to treat any of the above disorders, or others, in which MIF is involved in the pathogenesis of disease.

Consequently, isothiocyanates and isoselenocyanate compounds can be used to treat diseases and conditions mediated by MIF. Such methods include administering an isothiocyanate or isothioselenate compound, such as a compound of formula (I) to a subject in a therapeutically effective amount. Such methods include systemic administration of an inhibitor of MIF, preferably in the form of a pharmaceutical composition. As used herein, systemic administration includes oral and parenteral methods of administration. For oral administration, suitable pharmaceutical compositions of an inhibitor of MIF include powders, granules, pills, tablets, and capsules as well as liquids, syrups, suspensions, and emulsions. These compositions may also include flavorants, preservatives, suspending, thickening, and emulsifying agents, and other pharmaceutically acceptable additives. For parental administration, the compounds of preferred embodiments can be prepared in aqueous injection solutions that may contain, in addition to the inhibitor of MIF activity, and/or export, buffers, antioxidants, bacteriostats, and other additives commonly employed in such solutions.

The inhibitors of MIF can also be administered in the form of natural plant extracts that contain the inhibitory compounds, provided that the extract contains sufficient concentration of the inhibitor to achieve a therapeutic benefit.

The invention also provides uses of isothiocyanate and isothioselenate compounds to reduce MIF activity. The activity may be in vitro or in vivo. For example, the MIF inhibitor may be used as a research tool in an assay to investigate further aspects of MIF functioning. For example, the inhibitor may be used to investigate the biological role of MIF in intracellular and extracellular signalling pathways, and the role of MIF in disease models.

Pharmaceutical Compositions Comprising Isothiocyanate and Isothioselenate MIF Inhibitors

In one embodiment of the above aspects of the invention, the isothiocyanate and isothioselenate MIF inhibitors, including the compounds of formula (I) may be administered simultaneously or sequentially with one or more additional pharmaceutically active compounds.

Pharmaceutical compositions containing the MIF inhibitors, including compounds of formula (I) or pharmaceutically acceptable salts and prodrugs thereof, can be manufactured according to conventional methods such as by mixing, granulating, coating and dissolving the active agent. The MIF inhibitor or its salt or prodrug is present in the composition in an amount that is effective to treat a particular disease or condition. For example, an amount that is sufficient to achieve MIF inhibition, reduce MIF activity, and/or to decrease or eliminate symptoms of the disease or condition in a subject, preferably with acceptable toxicity to the subject. Where the 5 composition comprises a prodrug of a MIF inhibitor, the prodrug is present in an amount that will result in release of a therapeutically effective amount of the MIF inhibitor compound.

In one embodiment, pharmaceutical compositions of the invention comprising the isothiocyanate or isoselenocyanate MIF inhibitors of formula (I) or salts or prodrugs thereof will contain about

0.01 mg to more than 1000 mg per dose depending on the inhibitor or combination of inhibitors

LO present, the intended rate and frequency of administration, and the subject's disease or condition.

In one embodiment, pharmaceutical compositions of the invention will contain MIF inhibitors of formula (I) or salts or prodrugs thereof in an amount of at least 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15, 20, 30, 40, 50, 60, 70, 80, 90 ,100, 120, 140 ,160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, .5 700, 750, 800, 850, 900, 950 to 1000 mg MIF inhibitor. In certain embodiments lower or higher dosages may be appropriate. Appropriate concentrations and dosages can readily be determined by those skilled in the art.

Suitable pharmaceutically acceptable carriers and/or diluents are familiar to those skilled in the art. For compositions formulated as liquid solutions, acceptable carriers and/or diluents include

!0 saline and sterile water, and may optionally include antioxidants, buffers bacteriostats, and other common additives. The compositions can also be formulated as pills, capsules, granules, tablets (coated or uncoated), (injectable) solutions, solid solutions, suspensions, dispersions, solid dispersions (e.g., in the form of ampoules, vials, creams, gels, pastes, inhaler powder, foams, tinctures, lipsticks, drops, sprays, or suppositories). The formulation can contain (in addition to

[5 one or more MIF inhibitors and other optional active ingredients) fillers, disintegrators, flow conditioners, sugars and sweeteners, fragrances, preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, salts for regulating osmotic pressure, buffers, diluents, dispersing and surface-active agents, binders, lubricants, and/or other pharmaceutical excipients as are known in the art. One skilled in this art may further formulate the inhibitor of MIF in an appropriate

10 manner, and in accordance with accepted practices, such as those described in Remington 's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1990. MIF inhibiting compounds including compounds of formula (I) may be used in combination therapies with other pharmaceutical compounds. In preferred embodiments, the MIF inhibiting compound is present in combination with conventional drugs used to treat diseases or conditions mediated by MIF. For example, drugs for the treatment of various cancers, asthma or other respiratory diseases, sepsis, arthritis, inflammatory bowel disease (IBD), or other inflammatory diseases, immune disorders, or other diseases or disorders mediated by MIF.

Such pharmaceutically active agents include, but are not limited to, for example, steroids, glucocorticoids, nonsteroidal anti-inflammatory drugs, anti-infective drugs, beta stimulants, antihistamines, anti-cancer drugs, asthma drugs, anti-sepsis drugs, anti-arthritis drugs, and immunosuppressive drugs, inhibitors of other inflammatory cytokines (e.g, anti-TNFα antibodies, anti-IL-1 antibodies, anti-IFN-γ antibodies), and other cytokines such as IL-IRA or IL-10, and other MIF inhibitors.

Combination therapies can include fixed combinations, in which two or more pharmaceutically active agents are in the same formulation; kits, in which two or more pharmaceutically active agents in separate formulations are sold in the same package, e.g, with instructions for coadministration; and free combinations in which the pharmaceutically active agents are packaged separately, but instruction for simultaneous or sequential administration are provided. Other kit components can include diagnostics, assays, multiple dosage forms for sequential or simultaneous administration, instructions and materials for reconstituting a lyophilized or concentrated form of a pharmaceutical composition, apparatus for administering the pharmaceutically active agents, and the like.

In particularly preferred embodiments, one or more MIF inhibiting compounds are present in combination with one or more nonsteroidal anti-inflammatory drugs (NSAIDs) or other pharmaceutical compounds for treating arthritis or other inflammatory diseases. Preferred compounds include, but are not limited to, celecoxib; rofecosib; NSAIDs, for example, aspirin, choline magnesium trisalicylate, diclofenac potassium, diclofenac sodium, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, melenamic acid, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxicam, rofecoxib, salsalate, sulindac, and tolmetin; and corticosteroids, for example, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, lluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.

In particularly preferred embodiments, one or more MIF inhibiting compounds are present in combination with one or more beta stimulants, inhalation corticosteroids, antihistamines,

5 hormones, or other pharmaceutical compounds for treating asthma, acute respiratory distress, or other respiratory diseases. Preferred compounds include, but are not limited to, beta stimulants, for example, commonly prescribed bronchodilators; inhalation corticosteroids, for example, beclomethasone, fluticasone, triamcinolone, mometasone, and forms of prednisone such as prednisone, prednisolone, and methylprednisolone; antihistamines, for example, azatidine,

.0 carbinoxamine; pseudoephedrine, cetirizine, cyproheptadine, dexchlorpheniramine, fexofenadine, loratadine, promethazine, tripelannamine, brompheniramine, cholopheniramine, clemastine, diphenhydramine; and hormones, for example, epinephrine.

In particularly preferred embodiments, one or more MIF inhibiting compounds are present in combination with pharmaceutical compounds for treating IBD, such as azathioprine or .5 corticosteriods, in a pharmaceutical compositon.

In particularly preferred embodiments, one or more MIF inhibiting compounds are present in combination with pharmaceutical compounds for treating cancer, such as paclitaxel, in a pharmaceutical composition.

In particularly preferred embodiments, one or more MIF inhibiting compounds are present in

10 combination with immunosuppressive compounds in a pharmaceutical composition. In particularly preferred embodiments, one or more MIF inhibiting compounds are present in combination with one or more drugs for treating an autoimmune disorder, for example, Lyme disease, Lupus (e.g., Systemic Lupus Erythematosus (SLE)), or Acquired Immune Deficiency

Syndrome (AIDS). Such drugs may include protease inhibitors, for example, indinavir,

'.5 amprenavir, saquinavir, lopinavir, ritonavir, and nelfinavir; nucleoside reverse transcriptase inhibitors, for example, zidovudine, abacavir, lamivudine, idanosine, zalcitabine, and stavudine; nucleotide reverse transcriptase inhibitors, for example, tenofovir disproxil fumarate; non nucleoside reverse transcriptase inhibitors, for example delavirdine, efavirenz, and nevirapine; biological response modifiers, for example, etanercept, infliximab, and other compounds that

(0 inhibit or interfere with tumor necrosing factor; antivirals, for example, amivudine and zidovudine. In particularly preferred embodiments, one or more MIF inhibiting compounds are present in combination with pharmaceutical compounds for treating sepsis, such as steroids or anti- infective agents. Examples of steroids include corticosteriods, for example, cortisone, hydrocortisone, methylprednisone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinalone, triamcinolone acetonide, clobetasol propionate, and dexamethasone. Examples of anti-infective agents include anthelmintics (mebendazole), antibiotics including aninocylcosides (gentamicin, neomycin, tobramycin, antifungal antiobiotics (amphotericin b, fluconazole, griseofulvin, itraconazole, ketoconazole, nystatin, micatin, tolnafitate), cephalosporins cefaclor, cefazolin, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, cephalexin), beta-lactam antibiotics (cefotetan, meropenem), chloramphenicol, macrolides (azithromycin, clarithromycin, erythromycin), penicillins (penicillin G sodium salt, amoxicillin, ampicillin, dicloxacillin, nafcillin, piperacillin, ticarcillin), tetracyclines (doxycycline, minocycline, tetracycline), bacitracin; clindamycin, colistimethate sodium; polymyxin b sulfate; vancomycin; antivirals including acyclovir, amantadine, didanosine, efavirenz, foscarnet, ganciclovir, indinavir, lamivudine, nelfinavir, ritonavir, saquinavir, stavudine, valacyclovir, valganciclovir, zidovudine; quinolones (ciprofloxacin, levofloxacin); sulfonamides (sulfadiazine, sulfisoxazole); sufones (dapsone); furazolidone; metronidazole; pentamidine; sulfanilamidum crystallinum; gatifloxacin; and sulfamethoxazole/trimethoprim.

In the treatment of certain diseases, it may be beneficial to treat the patient with a MIF inhibitor in combination with an anesthetic, for example, ethanol, bupivacaine, chloroprocaine, levobupivacaine, lidocaine, mepivacaine, procaine, ropivacaine, tetracaine, desflurane, isoflurane, ketamine; propofol, sevoflurane, codeine, fentanyl, hydromorphone, marcaine, meperidine, methadone, morphine, oxycodone, remifentanil, sufentanil, butorphanol, nalbuphine, tramadol, benzocaine, dibucaine, ethyl chloride, xylocaine, and phenazopyridine.

The compounds of preferred embodiments can generally be employed as the free acid or the free base. Alternatively, the compounds of the preferred embodiments can preferably be in the form of acid or base addition salts. The term "pharmaceutically acceptable salt" of structures (I), (II), and (III) is intended to encompass any and all acceptable salt forms. While salt forms of the preferred embodiments are preferably pharmaceutically acceptable salts, pharmaceutically unacceptable salts can be employed (e.g., for preparation, isolation, and/or purification purposes).

Modification of proteins is recognised as a key mechanism underlying the biological activity of isothiocyanates, but knowledge of the major intracellular binding targets of these phytochemicals is lacking. We have used an affinity-based approach to identify targets of PEITC. A derivative of PEITC, amino-PEITC, was synthesized and retained biological activity. When coupled to a solid phase matrix amino-PEITC permitted the identification of MIF as the most prominent intracellular binding target of isothiocyanates. PEITC was shown to bind MIF via a proline residue at the N-terminus of the protein. Modification resulted in inhibition of MIF tautomerase activity and also triggered conformational changes.

MIF was originally identified as a lymphocyte-derived factor with cytokine-like activity (28,29). MIF has since been shown to be constitutively expressed in numerous tissues and cell types (30) and have a variety of biological activities, including pivotal roles in the regulation of immune and inflammatory responses and promotion of tumorigenesis (31). Elevated levels of MIF have been observed in a number of disease states including cardiovascular disease (32), arthritis (33), diabetes (34), sepsis (35) and many cancer types (36,37). Furthermore, genetic ablation of MIF has been shown to attenuate various disease states in murine models (38-40). While details surrounding the mechanisms of MIF action are still in question, the clinical significance of MIF expression is such that targeted approaches to inhibit the biological activities of MIF are currently in development (41,42). At least some of the biological activities of MIF are mediated by binding to, and downstream signalling from, the extracellular receptors CD74, CXCR2 and CXCR4 (43,44). However, MIF also possesses two distinct catalytic activities, a thiol protein oxidoreductase (TPOR) activity (45) and the ability to catalyze a tautomerase reaction (46,47). Whether or not either of these catalytic reactions is essential for MIF biological activity remains controversial, although some of the pro-inflammatory activities of MIF are impaired by mutations that affect enzymatic activity (46,48,49).

MIF has three cysteine residues, two of which (C57 and C60) are critical to the TPOR activity of MIF, while a third cysteine residue (C81) is believed to play a role in maintenance of protein conformation (50). When we initially identified MIF as an isothiocyanate target these cysteines were considered prime candidates for direct modification based on the known reactivity of isothiocyanates with thiols. Using mutant rhMIF in which the cysteine residues were isosterically mutated to serine we have shown that PEITC is not dependent on any of these thiols. Instead, the

N-terminal proline was identified as the critical binding site of PEITC. The sensitivity of the proline to attack by isothiocyanates can partly be explained by its unusually low pK a of 5.6, a value which is almost four pH units less than the pK a of free proline (48). The increased nucleophilicity of the N-terminal proline allows it to function as a general base catalyst in the tautomerase reaction (51) and favours reactivity with electrophiles such as isothiocyanates.

Not surprisingly isothiocyanate binding to this proline inhibited MIF tautomerase activity. Due to the lack of an identified physiological substrate, whether tautomerase activity is a biologically relevant function of MIF is a matter of debate. On the negative side, P2 mutants retain MIF-like activity in assays of its pro-inflammatory action (52). However, others argue that the tautomerase active site may be critical for permitting and regulating interactions of MIF with receptors or binding proteins, including CD74 (41). Multiple sequence alignment analysis has revealed that many of the invariant residues in 11 MIF homologues cluster around the N-terminal proline, indicating an evolutionary pressure to preserve this site (48). Indeed, there is evidence that the interaction of MIF with CD74 occurs near the tautomerase active site and that inhibition of tautomerase activity correlates with inhibition of CD74 binding (41). We have shown that PEITC disrupts monoclonal antibody binding to MIF, clearly indicating that crucial epitopes are disturbed upon binding of the isothiocyanate. Results from the in vivo study indicate that ingested isothiocyanates can also bind MIF in the complex milieu of human plasma. As MIF is both pro-inflammatory and pro-tumorigenic we propose that its inhibition in plasma could contribute to the strong correlation between increased dietary intake of isothiocyanates and decreased risk of many types of cancer including lung, breast and prostate cancers (53).

Materials

Affi-Gel ® 10 and D c protein assay kit were purchased from BioRad Laboratories (Hercules, CA, USA). PEITC and Z-3,4-dihydroxyphenylalanine methyl ester were sourced from Sigma Chemical Co. (St Louis, MO, USA). Cell culture materials were from Invitrogen New Zealand Ltd. (Auckland, New Zealand). Complete™ protease inhibitors and CHAPS were from Roche Diagnostics (Mannheim, Germany). SYPRO ® Ruby protein gel stain was obtained from Invitrogen Molecular Probes (Eugene, OR, USA). A goat polyclonal antibody to human MIF, a mouse monoclonal antibody to human MIF and a human MIF ELISA kit were obtained from R & D systems (Minneapolis, MN, USA). Hybond-PVDF membrane and enhanced chemiluminescence (ECL™) western blotting system were from Amersham Biosciences (Buckinghamshire, England). All other chemicals and reagents were from Sigma Chemical Co. (St Louis, MO, USA) and BDH Laboratory supplies (Poole, England). Recombinant human

MIF and MIF mutant proteins were prepare^ from pETl lb vector as previously described (Bernhagen, J.; Mitchell, R. A.; Calandra, T.; Voelter, W.; Cerami, A.; Bucala, R. Purification, Bioactivity, and Secondary Structure-Analysis of Mouse and Human Macrophage-Migration Inhibitory Factor (Mif). Biochemistry 33:14144-14155; 1994.) (Kleemann, R.; Kapurniotu, A.; Frank, R. W.; Gessner, A.; Mischke, R.; Flieger, O.; Juttner, S.; Brunner, H.; Bernhagen, J. Disulfide analysis reveals a role for macrophage migration inhibitory factor (MIF) as thiol- protein oxidoreductase. Journal of Molecular Biology 280:85-102; 1998.) (Kleemann, R.; Rorsman, H.; Rosengren, E.; Mischke, R.; Mai, N. T.; Bernhagen, J. Dissection of the enzymatic and immunologic functions of macrophage migration inhibitory factor. Full immunologic activity of N-terminally truncated mutants. Eur J Biochem 267:7183-93; 2000).

Cell culture

The Jurkat T-lymphocyte cell line was obtained from American Type Culture Collection (Rockville, MD) and was maintained in RPMI- 1640 containing 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were grown at 37°C in a humidified atomosphere with 5% CO 2 . Fresh antibiotic-free medium was added to cells 1 h before treatment. Working solutions of isothiocyanates were prepared in DMSO and added to cells so that the final concentration of DMSO in the media was kept constant at 0.1%. Cells were treated at a density of 1 x 10 6 cells/mL.

EXAMPLES

The following Examples are intended to illustrate embodiments of this invention and are not intended to limit the scope of this invention. Persons of ordinary skill in the art can develop other variants and modifications, based on the teachings herein to arrive at other embodiments that are all considered within the scope of this invention.

Example 1: Synthesis of amino-PEITC I

A novel analogue of PEITC containing an aminoethyl group at the meta position of the aromatic ring (amino-PEITC, formula (IV); Informal name: Compound 67)

(IV) was synthesised to enable coupling to Affi-Gel , a solid-phase resin containing reactive N- hydroxysuccinimide ester groups. The synthesis was achieved in accordance with Scheme 3 below.

Scheme 3

A solution of the bistrifluoroacetate salt of l,3-bis(2-aminoethyl)benzene (17) (291 mg, 0.74 mmol) and triethylamine (205 μL, 1.48 mmol) in dry methanol (15 niL) was stirred at room temperature for 10 min then a solution of di-t-butyl dicarbonate (41.3 mg, 0.19 mmol) in methanol (10 mL) was added drop wise over 5 min and the solution stirred at room temperature for 24 hrs. The solvents were removed in vacuo and the yellow residue washed successively with diethyl ether, ethyl acetate and dichloromethane (5mL). The combined organic extracts were washed with saturated aqueous sodium bicarbonate and evaporated to give the mono t-Boc amine (67 mg) as a yellow oil. 1 H NMR (500 MHz, chloroform-^) δ 7.24 (IH, t, J = 7.5 Hz, ArH), 7.1-7.0 (3H, m, ArH), 3.35 (2H, m, CH 2 NHCO), 2.96 (2H, t, J = 7 Hz, CH 2 NH 2 ), 2.78 (2H, broad t, J = 6.5 Hz, CH 2 CH 2 NHCO), 2.73 (2H, t, J = 7 Hz, CH 2 CH 2 NH 2 ), 1.43 (9H, s, C(CH 3 ) 3 ) ppm

The crude t-Boc amine (67 mg) was dissolved in dichloromethane (6 mL) and partitioned with saturated aqueous sodium bicarbonate (3 mL) at 0 C for 5 min. Thiophosgene (19.3 μL, 0.25 mmol) was then added to the organic layer and the mixture stirred at O 0 C for 20 min. The dichloromethane was then separated and evaporated in vacuo to give a yellow oil (69 mg) which was filtered through a column of silica gel (2g) with 1 :1 petroleum ether/diethyl ether and evaporated to give the t-Boc isothiocyanate as a clear oil (56 mg). 1 H NMR (500 MHz, chloroform-di) δ 7.28 (IH, t, J = 7.5 Hz, ArH), 7.12-7.0 (3H, m, ArH), 3.72 (2H, t, J=6.5 Hz, 5 CH 2 NCS), 3.38 (2H,m, CH 2 NHCO), 2.97 (2H, t, J = 7Hz, CH 2 CH 2 NCS), 2.81 (2H, m, CH 2 CH 2 NHCO), 1.44 (9H, s, C(CH 3 ) 3 ) ppm

The crude t-Boc isothiocyanate (56 mg) was dissolved in dichloromethane (3 mL) and stirred with trifluoroacetic acid (0.5 mL) for 3 h. Evaporation of the solvents at room temperature in vacuo gave the product, as the trifluoroacetate salt, in the form of an orange oil (53 mg). HPLC

.0 one peak detected at 270 nm on a Jasco PU 980 HPLC system with a reverse phase HPLC column (Prodigy 5 μ Cl 8 column, 250 x 4 mm, Phenomenex, San Jose, CA, USA) eluted with 50% acetonitrile/50% water containing 0.1% trifluoroacetic acid at a rate of 1 niL/min. 1 H NMR (500 MHz, chloroform-di) δ 7.6 (3H, broad, NH 3 ), 7.33 (IH, t, J = 7.5 Hz, ArH), 7.14 (2H, broad t, J= 8 Hz, ArH), 7.09 (IH, broad s, ArH), 3.72 (2H, t, J=6.5 Hz, CH 2 NCS), 3.30 (2H,m,

.5 CH 2 NH 3 ), 3.0 (4H, m, CH 2 CH 2 NCS + CH 2 CH 2 NH 3 ) ppm. IR (film) v max 2186, 2113 (N=C=S). 1780 (C=O) cm "1 . ESMS (Bruker Daltonics MicroTOF, positive ion) m/e found 207.0946 CnHi 5 N 2 S requires 207.0950.

Example 2 : Cytotoxicity of Amino-PEITC I

The cytotoxicity of amino-PEITC was tested. Amino PEITC retained cytotoxicity but with 0 slightly reduced potency compared to the parent PEITC. Cytotoxicity was indicated by plasma membrane integrity, as monitored using propidium iodide (PI) staining. After a 24 h treatment with each isothiocyanate, 5 μg PI was added to cells and samples were allowed to incubate in the dark for 10 min. Cell fluorescence was measured using a FC500 MPL Flow Cytometry system (Beckman Coulter Inc., Fullerton, CA). The results are shown in Figure 1.

5 Example 3: Preparation of Affi-PEITC I

100 μL of Affi-Gel ® 10 activated immunoaffϊnity support was thoroughly washed with 0.1 M NaHCO 3 and resuspended in 500 μL of the same buffer. The Affi-Gel suspension was reacted with 7 μL of amino-PEITC (100 mg/mL in DMSO) for 1 h at room temperature with constant rotation. Remaining reactive ester groups were blocked by addition of 50 μL of 1 M i0 ethanolamine (pH 8.0). The suspension was then incubated for a further 1 h at room temperature with constant rotation. An unreactive resin (blocked Affi-Gel) was prepared by incubating 100 μL AffiGel-10 in 500 μL 0.1 M NaHCO 3 containing 50 μL of 1 M ethanolamine (pH 8.0) for 2 h with constant rotation.

The resulting Affϊ-PEITC and blocked Affi-Gel preparations were thoroughly washed with binding/wash buffer (0.1 M KCl, 20 mM HEPES, pH 7.6, 0.1 M EDTA, 0.1% NP-40, 0.25 rnM PMSF) before use.

Example 4: Confirmation of Binding of Affϊ-PEITC to MIF I

Jurkat cells were collected and lysed in a buffer consisting of 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl 2 , 1 mM EDTA, 10% glycerol and Complete™ protease inhibitors. Protein concentration was determined using a D c protein assay and if necessary adjusted to 2 mg/mL with additional buffer. Affi-PEITC and blocked Affi-Gel prepared in Example 3 were incubated with 1 mL of cell lysate for 1 h at room temperature with constant rotation. Following incubation the resins were thoroughly washed with binding/wash buffer. Bound protein was eluted by boiling the resin in the presence of 100 μL of reducing sample buffer (62.5 mM Tris-HCl, pH 6.8, 10 % glycerol, 2% SDS, 0.025% bromophenol blue and 700 mM β-mercaptoethanol). The resin was pelleted and the resulting supernatant was resolved by SDS-PAGE. Total protein was stained with SYPRO ® Ruby and visualised using a Molecular Imager ® FX (BioRad Laboratories, Hercules, CA, USA). Bands of interest were excised from the gel, digested and the resulting peptide fragments were analysed by MALDI-TOF mass spectrometry (Centre for Protein Research, University of Otago, Dunedin, New Zealand). Figure 2 depicts results of these studies. A prominent band with an apparent molecular weight of 12 kDa was consistently captured from the lysates (Figure 2A). This band was absent when lysates were incubated with an unreactive resin (blocked Affi-Gel) indicating that the interaction was not due to non-specific binding to the matrix itself.

MALDI-TOF mass spectrometry analysis of the excised band identified the protein as the 12.5 kDa cytokine MIF (Figure 2B). To confirm the identity of the target protein, eluates were resolved by SDS-PAGE and immunoblotted with a polyclonal anti-MIF antibody. Samples were transferred to PVDF membrane which were then blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 2 o (TBST 20 ). Blots were probed with goat anti-human MIF antibody (0.2 μg/mL) or mouse anti-human MIF antibody (2 μg/mL) in TBST 20 containing 2% skim milk. Immunoblotted proteins were visualised using horseradish-peroxidase conjugated secondary antibodies and the ECL system. Images were obtained using a ChemiDoc XRS system (BioRad Laboratories, Hercules, CA, USA). While antibody binding was absent in the elution fraction from blocked Affi-Gel resin, MIF was clearly shown to associate with the Affi-PEITC resin (Figure 2C). Pre-treatment of lysates with PEITC, prior to incubation with Affi-PEITC, prevented the capture of MIF indicating that isothiocyanate modification was sufficient to inhibit subsequent binding of protein to the resin.

Example 5: Site of Binding of PEITC to MIF I

To assess the stoichiometry and site of isothiocyanate binding, recombinant human MIF (rhMIF) and mutant rhMIF proteins were reacted with a 10-fold excess of PEITC for 20 min and adduct formation was monitored by mass spectroscopy. Samples were passed through spin columns pre- equilibrated with water and then diluted 1 :1 with acetonitrile containing 0.1% formic acid. Mass spectrometry was performed using an LC Q™ DECA χp plus ion trap instrument (ThermoFinnigan, San Jose, CA). Samples were directly infused using a Hamilton syringe at a flow rate of 5 μL/min. A full scan for the mass range 100-2000 m/z was monitored. Data was collected for 1 min before deconvolution using BioworksBrowser 3.1 SRl (ThermoFinnigan).

Co-incubation of rhMIF with PEITC resulted in a species with mass corresponding to the addition of one PEITC (Figure 3A). MIF has three cysteines (see Figure 2B), but rhMIF proteins with C57S, C60S and C81S mutations were still able to bind PEITC, suggesting an alternate site of modification (Figure 3B). In each of the samples a second peak was observed having a mass 97KDa less than the parent protein. PEITC was found not to bind to rhMIF in which the N-terminal praline was mutated to Alanine (P2A). The truncated product was also absent (Figure 3C) suggesting that in native MIF the N-terminal proline is cleared in an Edman degradation reaction.

Example 6: Inhibition of Tautomerase Activity by PEITC I

The N-terminal proline plays a critical role as a catalytic residue in the tautomerase active site of MIF (Bendrat, K.; AlAbed, Y.; Callaway, D. J. E.; Peng, T.; Calandra, T.; Metz, C. N.; Bucala, R. Biochemical and nutational investigations of the enzymatic activity of macrophage migration inhibitory factor. Biochemistry 36:15356-15362; 1997). The tautomerase activity of rhMIF was investigated using a dopachrome tautomerization assay.

A I-dopachrome methyl ester solution was prepared just before use by combining 72 μL of a sodium periodate stock (20 mM) with 108 μL of I-3,4-dihydroxyphenylalanine methyl ester solution (4 mM) in 1620 μL of sodium phosphate buffer (10 mM sodium phosphate, 1 mM

EDTA, pH 6.2). rhMIF was diluted to 1 μM with sodium phosphate buffer and 20 μL aliquots were transferred to a 96 well plate. Appropriate dilutions of PEITC (0.5 μL) were added to the rhMIF and incubated at room temperature for 5, 10 or 30 min at which point 180 μL of dopachrome solution was added to all wells and the change in absorbance at 475 nm due to dopachrome tautomerization was monitored for 2 min. A blank containing sodium phosphate buffer and 0.5 μL of DMSO was included in all experiments.

Figure 4 depicts results of these studies. Co-incubation of rhMIF with PEITC resulted in a dose- and time-dependent loss of MIF tautomerase activity (Figure 4A). While the biological significance of inhibiting MIF tautomerase activity is controversial, a previous study recognized that covalent modification of the N-terminal proline residue can disrupt the integrity of epitope(s) critical to the biological activity of MIF, as measured by the ability of an monoclonal anti-MIF antibody to bind the protein (Senter, P. D.; Al-Abed, Y.; Metz, C. N.; Benigni, F.; Mitchell, R. A.; Chesney, J.; Han, J. L.; Gartner, C. G.; Nelson, S. D.; Todaro, G. J.; Bucala, R. Inhibition of macrophage migration inhibitory factor (MIF) tautomerase and biological activities by acetaminophen metabolites. Proceedings of the National Academy of Sciences of the United States of America 99: 144-149; 2002.

Inhibition of MIF tautomerase activity was further examined in Jurkat cells. Following treatment, 1.5 χ 6 Jurkat cells were collected and resuspended in 60 μL of lysis buffer (40 mM HEPES, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.6 mg/niL Complete™ protease inhibitors and 1% CHAPS). Insoluble material was removed by centrifugation at 15 000 x g for 4 min. Protein extracts (25 μL) were transferred to a 96 well plate in duplicate. 200 μL of dopachrome solution was added to each well and the change in absorbance at 475 nm was monitored for 2 min. A blank containing lysis buffer was included in all experiments. PEITC was shown to inhibit cellular MIF tautomerase activity in a time- and concentration-dependent manner with an IC 50 of 1.9 μM (± 0.1 μM) following a 30 min exposure to PEITC (Figure 5A).

Example 7: Reduction of Immonoreactivity of rhMIF by PEITC

PEITC was shown to reduce the immunoreactivity of rhMIF as measured by an ELISA using an anti-MIF monoclonal antibody (Figure 4C). rhMIF was reacted with PEITC for 10 min before detectable MIF levels were determined by a commercial ELISA according to manufacturer's instructions. 10 6 Jurkat cells (10 6 cells/mL) were resuspended in fresh media and incubated for 4 h at 37 °C before addition of 15 μM PEITC for 1 h. A control sample was prepared by resuspending Jurkat cells in media and incubating for 5 h at 37 0 C. Following treatment cells were pelleted by centrifugation at 10 000 x g for 1 min. Media was collected and stored before cells were lysed in 1 mL of water and clarified by centrifugation at 15 000 x g for 4 min. Detectable levels of both extracellular and intracellular MIF were determined by a commercial ELISA according to manufacturer's instructions. This clearly indicates that isothiocyanate modification of MIF imparts a conformational change in the protein. Disruption of MIF immunoreactivity was confirmed by ELISA with both intracellular and extracellular MIF being susceptible to isothiocyanate modification (Figure 5C).

In addition, the immunoreactivity of intracellular MIF was compromised when Jurkat cells were treated with PEITC as observed by western blotting with an anti-MIF monoclonal antibody (Figure 5B) while no change in total MIF protein levels, as determined by binding of a polyclonal antibody, was observed.

Example 8: In silico Modeling of PEITC bound to MIF I

Isothiocyanates were built within sybyl8.0.3 using sketcher and minimised using MMFF forcefield with 1000 iterations of conjugate gradient method. Modified isothiocyanate compounds were docked into an active site of the homotrimer of MIF covalently bound to inhibitor 4-iodo-6-phenylpyrimidine (4-IPP) (pdb3B9S) using GOLD4.0.1 using the covalent bond constraint.

Modeling of PEITC at the tautomerase active site revealed a conformational change with the catalytic proline shifting 2 A compared to that seen in the unmodified structure. In addition lysine 32 which sits above the active site shifted by 1.6 A (Figure 4B).

Example 9: Inhibition of Tautomerase Activity by Isothiocyanate Compounds I

The inhibitory effects of a range of isothiocyanate compounds of formula (I) was investigated using the tautomerase assay described in Example 6. Benzylthiocyanate was used as a control. 1 μM rhMIF was treated with the isothiocyanate/thiocyanate for 5 minutes before the tautomerase activity was measured. The results are shown in Table 5 below. Table 5

Isothiocyanate Compounds

Based on the core structures of isothiocyanate compounds and analysis of available chemicals and starting materials the following compounds useful in this invention include the following examples.

Example 10: (OHPE 2) Informal name: Compound 68

OHPE 2 Amine precursor

OHPE 2 can be prepared from the amine using conventional chemical methods, which need not be further disclosed herein.

Example 11: (OHPE 9): Informal name: Compound 69

Amine precursor OHPE 9 is a novel compound and can be prepared from a corresponding amine. The synthesis of this compound is complex, but can be achieved using conventional methods.

Example 12: (OHPE 10): Informal name: Compound 70

OHPE lO Amine precursor

OHPE 10 is a novel compound that can be prepared from an amine which is available from Aldrich Chemical Company.

O Example 13: (OHBN 1): Informal name: Compound 71

OHBN 1 is available commercially from Santa Cruz Biotechnology Inc.

5 Example 14: (OHBN 2): Informal name: Compound 72

OHBN 2 Hydroxyl precursor

O OHBN 2 can be made from commercially available hydroxyl precursor such as 4-(l- hydroxypropyl) phenol which is available from UkrOrgSynthesis. Example 15: (OHBN 3): Informal name: Compound 73

OHBN 3 Amine precursor

OHBN 3 can be prepared from commercially available amine precursor such as 4- [amino(phenyl)methyl] phenol which is available from UkrOrgSynethesis.

Example 16: Synthesis of Amino-PEITC II

Amiήo-PEITC [2-(3-(2-aminoethyl)phenyl)ethyl isothiocyanate] was prepared as in Example 1 from the diamine precursor [l,3-bis(2-aminoethyl)benzene] (17) by selective protection as a mono t-Boc derivative (18) followed by reaction with thiophosgene (19) and deprotection. 1,3- Bis(2-aminoethyl)benzene was obtained by reduction of l,3-bis(cyanomethyl)benzene (20) with a nickel-modified borohydride reagent (21). Nuclear magnetic resonance (NMR) spectra were obtained on a Varian Unity 500 spectrometer.

Example 17: Culture of Jurkat T-Lymphocytes.

The Jurkat T-lymphocyte cell line was obtained from American Type Culture Collection (Rockville, MD) and was maintained in RPMI 1640 containing 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen New Zealand Ltd., Auckland, New Zealand). Cells were grown at 37°C in a humidified atmosphere with 5% CO 2 .

Example 18: Preparation of Affi-PEITC II

An unreactive resin (blocked Affϊ-Gel) was prepared by incubating 100 μL AffiGel-10 in 500 μL 0.1 M NaHCO 3 containing 50 μL of 1 M ethanolamine (pH 8.0) for 2 hr with constant rotation. As in Example 3, the resulting Affi-PEITC and blocked Affi-Gel preparations were thoroughly washed with binding buffer (0.1 M KCl, 20 mM HEPES, pH 7.6, 0.1 M EDTA, 0.1% NP-40, 0.25 mM PMSF) before use. Example 19: Mass Spectrometry of Purified MIF

Recombinant human MIF and MIF mutant proteins were expressed, purified, and renatured from pETl lb vector as previously described (22). 10 μg recombinant human MIF and mutant MIF proteins were reacted with a 10-fold molar excess of PEITC (Sigma Chemical Co., St Louis, MO, USA) for 20 min. Samples were passed through spin columns pre-equilibrated with water and then diluted 1:1 with acetonitrile containing 0.1% formic acid. Mass spectrometry was performed using an LCQ™ DECA χp pIus ion trap instrument (ThermoFinnigan, San Jose, CA, USA). Samples were directly infused using a Hamilton syringe at a flow rate of 5 μL/min. A full scan for the mass range 100-2000 m/z was monitored. Data was collected for 1 min before deconvolution using BioworksBrowser 3.1 SRl (ThermoFinnigan, San Jose, CA, USA).

Example 20: Immunoreactivity of MIF by Immunoblotting

1 x 10 6 Jurkat cells were treated with PEITC for 1 hr before cells were lysed in a buffer consisting of 40 mM HEPES, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.6 mg/mL Complete™ protease inhibitors and 1% CHAPS (Roche Diagnostics, Mannheim, Germany). Insoluble material was removed by centrifugation at 15 000 x g for 4 min. Samples were diluted in reducing sample buffer and resolved by SDS-PAGE before immunoblot analysis as described above.

Example 21: Ex Vivo Immunoreactivity of MIF Following Ingestion of Watercress

Blood was obtained from healthy human volunteers under approval of the Upper South A Regional Ethics Committee. Volunteers consumed 50 g of fresh watercress and blood was drawn into heparinized tubes just prior to and 1 and 2 hr after ingestion. Plasma was prepared by immediate centrifugation of the blood samples at 1000 x g for 10 min and stored at -80°C. Plasma MIF levels were determined by a commercial ELISA according to manufacturer's instructions (see Figures 9 & 10).

Example 22: Quantitative Determination of Plasma Isothiocyanate/Dithiocarbamate Levels

Plasma isothiocyanate and dithiocarbamate derivatives were determined by a cyclocondensation reaction as previously described (23). Fractions were analysed using a Waters 2690 HPLC system (Waters, Milford, MA 5 USA) with a reverse phase HPLC column (Luna 5 μ Cl 8 column, 250 x 4.6 mm, Phenomenex, San Jose, CA, USA) and eluted with 80% methanol/20% water at a rate of 0.6 mL/min. The l,3-benzodithiole-2-thione was eluted at approximately 12 min, and the peak was detected and integrated by a Photodiode array detector (Waters Model 996) at 365 run. The instrument was calibrated with pure l,3-benzodithiole-2-thione and plasma samples spiked with known concentrations of isothiocyanates were included to ensure completion of the 5 cyclocondensation reaction (see Figure 9).

Example 23: Identification of Protein Targets for PEITC

A novel analogue of PEITC, amino-PEITC [2-(3-(2-aminoethyl)phenyl)ethyl isothiocyanate], containing a meta aminoethyl group was synthesised to enable coupling to Affi-Gel , a solid- phase resin containing reactive iV-hydroxysuccinimide ester groups (Figure 11). Amino PEITC

.0 retained cytotoxicity, albeit with slightly reduced potency compared to PEITC (Figure 1). Amino-PEITC was conjugated to Affi-Gel ® and lysates from Jurkat T-lymphoma cells were incubated with the resin. Proteins that bound to the matrix were eluted and separated by SDS- PAGE. A prominent band with an apparent molecular weight of 12 kDa was consistently captured from the lysates (Figure 2A). This band was only faint in the whole cell lysate,

.5 indicating considerable concentration during capture, and it was absent when lysates were incubated with an unreactive resin (blocked Affi-Gel), indicating that the interaction was not due to non-specific binding to the matrix itself. MALDI-TOF mass spectrometry analysis of the excised band identified the protein as the 12.5 kDa pro-inflammatory and pro-tumorigenic cytokine MIF (Figure 2B). As confirmation, eluates were resolved by SDS-PAGE and

:0 immunoblotted with a polyclonal anti-MIF antibody. Whereas antibody binding was absent in the elution fraction from blocked Affi-Gel resin (Figure 2C), MIF was clearly shown to associate with the Affi-PEITC resin. Pre-treatment of lysates with PEITC prior to incubation with Affi-PEITC prevented the capture of MIF indicating that isothiocyanate modification was sufficient to inhibit subsequent binding of protein to the resin. Pre-treatment of lysates with the

:5 isothiocyanate sulforaphane prior to incubation with Affi-PEITC also prevented the capture of MIF (data not shown).

Example 24: Covalent Modification of MIF by PEITC

To assess the stoichiometry and site of isothiocyanate binding to MIF, recombinant human MIF (rhMIF) was incubated with PEITC and adduct formation was monitored by mass spectrometry. .0 Co-incubation of rhMIF with PEITC resulted in a species with mass corresponding to the addition of one PEITC (Figure 14A). MIF has three cysteine residues (Figure 2B), but rhMIF proteins with C57S, C60S and C81S mutations were still able to bind PEITC (Figure 14B), suggesting an alternate site of modification. In all of the isothiocyanate-treated samples a second peak was present with a mass 97 Da less than the parent protein. We proposed that this resulted from loss of proline, and tested the ability of PEITC to bind rhMIF in which the N-terminal proline was mutated to alanine (P2A). P2A rhMIF did not bind PEITC and the truncated product was absent (Figure 14C), suggesting that in native MIF the N-terminal proline is cleaved in an Edman degradation reaction. The cleavage itself is likely to be an in vitro artifact driven by acidification in the buffer used for positive ion mass spectrometry. Indeed, in the absence of acid a single peak with a mass equivalent to PEITC -modified rhMIF was observed (Figure 15).

Example 25: Effect of Isothioicyanate Adduct Formation of MIF Tautomerase Activity

MIF possesses a catalytic tautomerase activity and can convert the methyl ester of Z-dopachrome to an indole derivative (Figure 12). The N-terminal proline lies within the tautomerase active site and plays a critical role as a catalytic residue in the tautomerase reaction (24). Co-incubation of rhMIF with PEITC resulted in a dose- and time-dependent loss of MIF tautomerase activity (Figure 4A). PEITC also inhibited MIF tautomerase activity in Jurkat cells in a time- and concentration-dependent manner with a half-maximal inhibitory concentration (IC 5 o) of 2 μM following a 30 min treatment (Figure 5A). Additional naturally-occurring isothiocyanates were shown to inhibit cellular MIF tautomerase activity with IC 50 values of 1.3 μM for sulforaphane and 0.6 μM for benzyl isothiocyanate (Figure 7). When cells were pre-incubated with PEITC prior to affinity capture, a dose-dependent inhibitionOf MIF binding was observed (Figure 8). The concentrations at which capture was disrupted were very similar to the concentrations required to inhibit tautomerase activity. Consistent with the purified system, these results indicate that PEITC directly modifies cellular MIF and inhibits its tautomerase activity.

Example 26: Isothiocyanate Binding and Conformational Integrity of MIF

While the biological significance of inhibiting MIF tautomerase activity is controversial, a previous study recognized that covalent modification of the N-terminal proline residue can disrupt the integrity of epitope(s) critical to the biological activity of MIF (25). In silico modeling of PEITC at the tautomerase active site highlighted a conformational change with the catalytic proline shifting 2 A compared to that seen in the unmodified structure. In addition, lysine 32, which sits above the active site, was shifted by 1.6 A. This is a direct consequence of the formation of a thiourea adduct with the N-terminal proline where the active site must accommodate the planar thiourea group (R 2 NC=SNR) including the planar (sp 2 hybridisation) proline nitrogen. Covalent modification of MIF by acetaminophen metabolites has previously been shown to affect immunorecognition of MIF by a monoclonal antibody (26). In the same study loss of immunorecognition was associated with impaired MIF biological activity. A short incubation of rhMIF with PEITC reduced the immunoreactivity of MIF (Figure 4C) as measured by an ELISA

5 using a neutralizing monoclonal anti-MIF antibody (clone 12302). Loss of antibody recognition is consistent with isothiocyanate modification disrupting the conformational integrity of MIF epitope(s) as predicted by in silico modeling. The immunoreactivity of cellular MIF was also compromised when Jurkat cells were treated with PEITC as observed by immunoblotting with the monoclonal anti-MIF antibody (Figure 5B). No change in total MIF protein levels, as

.0 determined by binding of a polyclonal anti-MIF antibody, was observed. Disruption of MIF immunoreactivity was confirmed by ELISA with both intracellular and secreted (extracellular) MIF being susceptible to isothiocyanate modification (Figure 5C).

Example 27: MIF as an In Vivo Target of Isothiocyanates

To determine whether MIF is a biologically relevant target of dietary isothiocyanates, a pilot

.5 study was designed to measure the immunoreactivity of plasma MIF in three human volunteers following ingestion of watercress {Nasturtium officinale), a rich dietary source of PEITC (27).

Consumption of 50 g (wet weight) of watercress led to a time-dependent increase in plasma levels of isothiocyanates and their corresponding thiocarbonyl derivatives (dithiocarbamates) and a concomitant decrease in immunoreactive plasma MIF. Two hours after ingestion plasma

:0 immunoreactive MIF had decreased by approximately 45% indicating that isothiocyanates can target MIF in complex biological systems and at physiologically achievable concentrations.

Example 28: Effect of Substitutions of Aromatic Isothiocyanates on MIF Inhibition

Molecular modelling showed that a hydroxyl derivative in the para position of aromatic isothiocyanates would enhance MIF binding through hydrogen bonding. Substitution at other 5 positions is not optimal.

Synthesis

1 μM rhMIF was treated with the isothiocyanate for 5 min before the tautomerase activity was measured. Experiments were performed as described in Example 6, with one difference, the incubation of rhMIF with the isothiocyanate was undertaken at pH 7.4 before MIF activity was .0 measured at pH 6.2. The results are shown in Table 6 below. Table 6

Example 29: Inhibition of Cellular MIF by Isothiocyanates

It is possible that differences in the efficacy of isothiocyanates observed with purified MIF may be altered in complex biological mixtures due to altered accessibility and cellular scavenging. This information will be important for optimizing therapeutic potential.

Jurkat cells (1 x 10 6 /ml) were treated with varying doses of selected isothiocyanates for 30 min. The cells were then harvested, and 1.5 x 10 6 resuspended in 60 μL of lysis buffer consisting of 40 mM HEPES, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.6 mg/mL Complete™ protease inhibitors and 1% CHAPS. Insoluble material was removed by centrifugation at 15,000 x g for 4 min. Cell extracts (25 μL) were transferred to a 96 well plate in duplicate. 200 μL of the dopachrome methyl ester solution was added to each well and the change in absorbance at 475 nm was monitored for 2 min. A blank containing lysis buffer was included in all experiments. Table 7 below depicts the IC50s of some selected isothiocyanates.

Table 7

Example 30: In sttico Fitness Scores of Isothiocyanates Docking to MIF

Isothiocyanates were built within SYBYL8.0 using sketcher and minimized MMFF forcefield with 1000 iterations of conjugate gradient method. Modified isothiocyanate compounds were docked into an active site of the homotrimer of MIF covalently bound to inhibitor 4-iodo-6- phenylpyrimidine (4-IPP) (pdb3B9S) using GOLD4.0.1 using the covalent bond constraint. Figure 6 depicts an example of results of such modeling. Figure 6A depicts the MIF molecule with an isothiocyanate compound bound thereto. Figure 6B depicts an enlarged image of a portion of the image shown in Figure 6 A.

The Goldscore fitness functions generated from the docking shows a definite trend positively correlating to the biological assay IC 50 values in Examples 9, 28 and 29. There is a slight inconsistency where the Goldscore of Benzyl ITC is lower than the score of Phenethyl ITC, however some variability between in silico and in vitro experiments can be expected and the procedure appears valid in principle. Table 8 below depicts Goldscores for selected isothiocyanates or related compounds.

Table 8

References

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