(2,6-DIOXO-3-PIPERINYL)AMIDOBENZOIC IMMUNOMODULATORY AND

ANTI-CANCER DERIVATIVES

TECHNICAL FIELD

The present invention is concerned with certain benzoic acid derivatives and their use in the treatment of neoplastic disorders. In particular the present invention is concerned with carboxybenzoyl glutamic acid analogues and their uses.

BACKGROUND

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. Inflammatory cytokines produced within the tumour mass play an essential role in the growth and development of the tumour (Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nature Reviews: Cancer, 2004, 4, 11-22). Inhibition of that process provides a powerful approach to cancer therapy. Potent inhibitors of cytokine biosynthesis are used for the control of inflammatory diseases, and such agents could also be applied for the control of cancer. Such inhibitors of cytokines can of course also be used for immunomodulation generally, where such activity would be beneficial in treatment of symptoms or disease. iV-(o-carboxybenzoyl)glutamic acid imide (CGI) has been shown to inhibit TNF biosynthesis and angiogenesis (US patent application 11/454,420) . CGI is a potentially useful anti-cancer agent which is non-teratogenic and non-sedatory. One disadvantage of CGI however is that it is unstable at low pHs where it converts to products that can be teratogenic. Since low pHs are encountered in the stomach, CGI per se therefore may not be suitable to be given as an oral dose.

There is a need however for novel therapeutic agents and compositions with more desirable and advantageous properties. Compositions that are stable at a broad range of pHs would be significantly advantageous as they could potentially be administered orally without loss of activity. In addition compounds with increased metabolic stability would also be highly desirable, as this would provide longer exposure to the active agent following administration. The object of the present invention is to ameliorate one or more disadvantages of current immunomodulatory and cancer therapies or to provide a useful alternative.

SUMMARY OF THE INVENTION

According to a first aspect there is provided a compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein R ₅ can be selected from -COOH, -COR ₁₁ , -CONR ₁₂ Ri ₃ , -CONR ₁₄ R ₁₅ NR ₁₆ R ₁₇

CONR ₁₈ R ₁₉ OR _2O5 CONR ₂₁ R ₂₂ SO ₂ R ₂₃ , N- ^N ₅ COOR ₂₄ , R ₁ to R ₄ and R ₆ to R ₂₄ can be independently selected from hydrogen, halogen, amino, nitro, cyano, alkyl thioether, alkyl sulfone, alkyl sulfoxide, substituted alkyl, substituted cycloalkyl, hydroxy, alkyl ether, carboxylic acid and acid derivatives such as amide and ester, aryl thioether, aryl sulfone, aryl sulfoxide, aryl ether, NR ₂₅ R _26, aromatic and . heteroaromatic ring,

R ₂₅ and R ₂₆ can be independently selected from hydrogen, substituted alkyl and substituted cycloalkyl, substituted aryl and substituted heteroaryl, R ₁ and R ₂ , or R ₂ and R ₃ , or R ₃ and R ₄ can be fused to generate an additional ring system(s), which may be heterocyclic or carbocyclic (saturated or unsaturated), in the glutarimide ring B two substituents (the same or different) can be attached to a single ring carbon atom, wherein, when R ₁₂ R ₁₃ , Ri ₆ R ₁₇ and R ₂₅ R ₂₆ are alkyl they may be joined together to form a ring system which may additionally contain one or more heteroatoms selected from oxygen, sulphur, nitrogen or selenium and wherein one or more carbonyl groups in the formula may be replaced by thiocarbonyl.

Preferred compounds can be either racemic or comprise a single enantiomer or diastereomer (where this is possible).

The preferred compounds of the present invention can be selected from: 2-{[(2,6-Dioxo-3-piperidinyl)amino]carbonyl}-3,4,5,6-tetrafl uorobenzoic acid, 2-Amino-6-{[(2,6-dioxo-3-piperidinyl)amino]carbonyl}benzoic acid,

3-Ammo-2-{[(2,6-dioxo-3-piperidinyl)amino]carbonyl}benzoic acid, ^-^-(Dimethylam^ethyy-^-Cl^-dioxo-S-piperidiny^-S^jS^-tetraf luoro-N ¹ - methylphthalamide, iV ¹ -(2,6-Dioxo-3-piρeridinyl)-N ² -(2-hydroxyethyl)-N ² -niethylphthalamide, N-(2,6-Dioxo-3-piperidinyl)-2-(lH-tetxaazol-5-yl)benzarnide,

2- { [(3 -Fluoro-2,6-dioxo-3 -piperidinyl)amino]carbonyl}benzoic acid,

6- { [(2,6-Dioxo-3 -piperidinyl)arαino]carbonyl} - 1 H-indole-7-carboxylic acid,

7-{[(2,6-Dioxo-3-piperidinyl)amino]carbonyl}-4,5-difluoro-lJ ϊ-indole-6-carboxylic acid, 2-{[(2,6-Dioxo-3-piperidmyl)amino]carbothioyl}-3,4,5,6-tetra fluorobenzoic acid, 2,3,4,5-Tetrachloro-6-{[(2,6-dioxo-3-piperidmyl)amino]carbon yl}benzoic acid, tert-Butyl 2-{[(2,6-dioxo-3-piperidinyl)amino]carbonyl}-3,4,5,6-tetrafl uorobenzoate, 3,6-Dichloro-2-{[(2,6-dioxo-3-piperidinyl)amino]carbonyl}ben zoic acid, 4,5-Dichloro-2-{[(2,6-dioxo-3-piperidinyl)aniino]carbonyl}be nzoic acid, 2-{[(2,6-Dioxo-3-piperidinyl)amino]carbonyl}-3 ₅ 6-difluorobenzoic acid, 2- { [(2,6-Dioxo-3 -piperidinyl)amino] carbonyl} -3 -nitrobenzoic acid, 3-{[(2,6-Dioxo-3-piperidinyl)amino]carbonyl}-2 -naphthoic acid, iV-(2,6-Dioxo-3-piperidinyl)-2-(l -pyrrolidinylcarbonyl)benzamide, or pharmaceutically acceptable salts thereof. According to a second aspect there is provided a composition comprising a compound according to a first aspect in combination with a pharmaceutically acceptable carrier.

According to a third aspect there is provided a method of modulating the immune system comprising administering to a subject a compound according to the first aspect or a composition according to the second aspect.

According to a fourth aspect there is provided a method of inhibiting angiogenesis comprising administering to a subject requiring such treatment a compound according to the first aspect or a composition according to the second aspect.

According to a fifth aspect there is provided a method of treating a neoplastic disorder comprising administering to a subject requiring such treatment a compound according to the first aspect or a composition according to the second aspect.

- A -

Preferably the neoplastic disorder is of haematopoietic origin na d most preferably it is multiple myeloma. The neoplsatic disorder may also be a tumour, for example a vascularised tumour and the like.

According to a sixth aspect there is provided a method of inhibiting cytokine production by contacting a cell capable of producing one or more cytokines with a compound according to the first aspect or a composition according to the second aspect.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Inhibition by FCGI of TNF production induced with lipopolysaccharide - (LPS) or DMXAA in mice

Figure 2: A comparison of FCGI with CGI to inhibit LPS-induced TNF production in mice

Figure 3: Stability of FCGI in solution at different pHs

Figure 4: Inhibition of angiogenesis in vitro by CGI, FCGI and compound 19a

Figure 5: Inhibition of IL-6 production by human multiple myeloma cells by CGI, FCGI and compound 19a

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is based in part on studies which demonstrate the ability of the compound 2-{[(2,6-Dioxo-3-piperidinyl)amino]carbonyl}benzoic acid (1), also referred to as CGI, to modulate cytokines and inhibit cancer growth.

The present invention concerns analogues of CGI, which are useful in cancer therapy and as immunomodulators. The compounds of the present invention have a general formula as follows:

or a pharmaceutically acceptable salt thereof, wherein R ₅ can be selected from -COOH, -COR ₁₁ , -CONR ₁₂ Ri ₃ , -CONR ₁₄ R ₁₅ NRi ₆ R ₁₇ ,

CONRi ₈ R ₁₉ OR ₂₀ , CONR ₂ iR ₂ 2SO ₂ R ₂ 3 , ^{N N} , COOR ₂₄ , R ₁ to R ₄ and R ₆ to R ₂₄ can be independently selected from hydrogen, halogen, amino, nitro, cyano, alkyl thioether, alkyl sulfone, alkyl sulfoxide, substituted alkyl, substituted cycloalkyl, hydroxy, alkyl ether, carboxylic acid and acid derivatives such as amide and ester, aryl thioether, aryl sulfone, aryl sulfoxide, aryl ether, NR ₂₅ R ₂₆₅ aromatic and heteroaromatic ring. R ₂₅ and R ₂₆ can be independently selected from hydrogen, substituted alkyl and substituted cycloalkyl, substituted aryl and substituted heteroaryl. Furthermore two substituents R ₁ and R ₂ , or R ₂ and R ₃ , or R ₃ and R ₄ can be fused to generate an additional ring system(s), which may be heterocyclic or carbocyclic (saturated or unsaturated). When R ₁₂ R ₁₃ , Ri ₆ Ri ₇ and R ₂₅ R ₂₆ are alkyl they may be joined together to form a ring system which may additionally contain one or more heteroatoms such as oxygen, sulphur, nitrogen or selenium.

In the glutarimide ring B two substituents (the same or different) can be attached to a single ring carbon atom. Preferred compounds can be either racemic or comprise a single enantiomer or diastereomer (where this is possible). One or more carbonyl groups in the formula may be replaced by thiocarbonyl. The activities and/or physical properties as a drug of the analogues of the present invention can be significantly improved over the native CGI compound by the incorporation of appropriate substitutions around the benzene ring and/or the glutarimide ring. In particular, addition of one or more halogen atoms, preferably fluorine to the benzene ring has dramatically improved the potency of CGI. The tetrafiuoro-substituted derivative (2), discussed in more detail below, is especially active. Incorporation of amino functionality to the benzene ring is also beneficial. In particular, compounds such

as (3) and (4) show improved activity. The carboxylic acid group in (1) may be replaced by other functionality generally known in the field of medicinal chemistry as "acid isosteres", such as acyl sulfonamide, tetrazolyl, sulfonyltriazolyl, and the like. Alternatively, prodrugs of the acid group, such as ester derivatives may be prepared to modulate physical properties.

Examples of preferred compounds of the present invention include but are not limited to:

2-{[(2,6-Dioxo-3-piperidinyl)amino]carbonyl}-3,4,5,6-tetrafl uorobenzoic acid, 2-Amino-6-{[(2,6-dioxo-3-piperidinyl)amino]carbonyl}benzoic acid, 3-Amino-2-{[(2,6-dioxo-3-piperidinyl)amino]carbonyl}benzoic acid, iV ¹ -[2-(Dimethylamino)ethyl]-iV ² -(2,6-dioxo-3-piperidmyl)-3,4,5,6-tetrafluoro-iV ¹ - methylphthalamide,

N ¹ -(2,6-Dioxo-3-piperidinyl)-iV ² -(2-hydroxyethyl)-iV ² -methylphthalamide, iV-(2,6-Dioxo-3-piperidinyl)-2-(li/-tetraazol-5-yl)benzamide , 2-{[(3-Fluoro-2,6-dioxo-3-piperidinyl)amino]carbonyl}benzoic acid,

6- { [(2,6-Dioxo-3 -piperidinyl)amino] carbonyl} - lif-indole-7-carboxylic acid, 7-{[(2,6-Dioxo-3-piperidinyl)amino]carbonyl}-4,5-difluoro-lH -indole-6-carboxylic acid, 2-{[(2,6-Dioxo-3-piperidinyl)amino]carbothioyl}-3,4,5,6-tetr afluorobenzoic acid, 2,3,4,5-Tetrachloro-6-{[(2,6-dioxo-3-piperidinyl)amino]carbo nyl}benzoic acid,

?e7"t-Butyl 2-{[(2,6-dioxo-3-piperidinyl)amino]carbonyl}-3,4,5,6-tetrafl uorobenzoate, 3,6-Dichloro-2-{[(2,6-dioxo-3-piperidinyl)amino]carbonyl}ben zoic acid, 4,5-Dichloro-2-{[(2,6-dioxo-3-piperidinyl)amino]carbonyl}ben zoic acid, 2- { [(2,6-Dioxo-3 -piperidinyl)amino] carbonyl} -3 ,6-difluorobenzoic acid, 2-{[(2,6-Dioxo-3-piperidinyl)amino]carbonyl}-3-nitrobenzoic acid, 3-{[(2,6-Dioxo-3-piperidinyl)amino]carbonyl}-2 -naphthoic acid, iV-(2,6-Dioxo-3-piperidinyl)-2-(l -pyrrolidinylcarbonyl)benzamide, or pharmaceutically acceptable salts thereof.

The above compounds can also be represented by the following structural formulae:

19d 19e 21

One of the analogues, namely FCGI (2), was designed to have a reduced capacity to be metabolised, hence slower pharmacokinetics and greater efficacy. Initial tests with FCGI have shown that this analogue is at least as potent as, and more effective than CGI in inhibiting TNF production induced with LPS (Fig.2). FCGI is also more stable at pH ^" 3 than CGI (Fig. 3)

The present invention employs well recognized and accepted cell culture systems to demonstrate the cytokine modulatory activities of the CGI analogues. The relevant methodology is also described in US patent application 11/454,420, which is incorporated in its entirety herein by reference. The compounds and compositions of the present invention are suitable for treatment of neoplastic disorders as well as modulating cytokine production and the immune system.

In the context of the present invention, the term "neoplastic disorder" is intended to encompass any benign or malignant condition or tumour, including vascularised solid tumours. The term "vascularised solid tumour" is used to describe any solid tumour, whether benign or malignant, which relies on ample blood supply for its establishment and growth. Thus, the tissue or organ location, or tissue origin, of such tumours is not material to the compositions and methods of the present invention as long as there is reliance by the tumour on its blood supply. For example _o the compositions and methods of the present invention may be used to treat colon cancers, breast cancers, liver cancers, lung cancers, brain tumours, ovarian tumours, prostate cancers, testicular cancers, uterine tumours and the like. It may also include solid tumours that arise as a consequence of underlying neoplastic conditions which are not normally classified as solid tumours, for example various leukaemias and the like. The term "neoplastic disorder" is also intended to encompass benign or malignant conditions of haematopoietic origin, such as different haematopoietic malignancies. For example, the compounds of the present invention may be used for the treatment of multiple myeloma, a variety of leukaemias and similar malignancies.

The compounds of the present invention may be formulated into pharmaceutical preparations by any number of processes well known in the art and may include injectable, topical, oral, slow release and other suitable dosage forms and preparations.

Routes of administration include, but are not limited to, intravenous (iv), intraperitoneal, subcutaneous, intracranial, intradermal, intramuscular, intraocular,

intrathecal, intracerebral, intranasal, transmucosal, or by infusion orally, rectally, via iv drip, patch and implant. Intravenous routes are particularly preferred.

Compositions suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The prevention of the action of micro-organisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirnerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the composition in accordance with the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by, for example, filter sterilization or sterilization by other appropriate means. Dispersions are also contemplated and these may be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders, for the preparation of sterile injectable solutions, a preferred method of preparation includes a vacuum drying and freeze-drying technique that yields a powder of the active ingredient together with any additional desired ingredient from a previously sterile-filtered solution. When the composition in accordance with the invention is suitably protected, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets. For oral therapeutic administration, the composition may be incorporated with excipients and used in the form of an ingestible tablet, a buccal tablet, a troche, a dragee, a capsule, an elixir, a suspension, a syrup, a wafer and the like. Such preparations should contain at least 0.01 % by weight, more preferably 0.1% by weight, even more preferably 1% by weight of a composition in accordance with the invention. The percentage of the preparations may, of course, be varied and may conveniently be

between about 1 to about 99%, more preferably about 2 to about 90 %, even more preferably about 5 to about 80% of the weight of the unit. The amount of the therapeutically useful composition is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ng and 2000 mg of the composition in accordance with the present invention.

The ingestible tablet, buccal tablet, troche, dragee, capsule, elixir, suspension, syrup, wafer or the like may also contain the components as listed hereafter: a binder, for example, gum, acacia, corn starch or gelatin; an excipient, for example, dicalcium phosphate; a disintegrating agent, for example, corn starch, potato starch, alginic acid and the like; a lubricant, for example, magnesium stearate; and a sweetening agent, for example, sucrose, lactose or saccharin and a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, a tablet, a pill, or a capsule may be coated with shellac, sugar or both. A syrup or elixir may contain, in addition to the composition in accordance with the invention, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the composition in accordance with the present invention may be incorporated into a sustained-release preparation or formulation.

The present invention also extends to forms suitable for topical application such as creams, lotions and gels.

The formulation may further comprise one or more excipients suitable for nasal or pulmonary delivery. The excipients may be sugars, for example, lactose, mannitol, xylitol, trehalose, dextrose or other pharmaceutically acceptable sugars. Further excipients may include one or more surfactants, preferably a surfactant which is solid at ambient temperatures, for example, 25 ⁰ C. A carboxylic acid, for example, oleic acid may be employed. Alternative surfactants include lecithin and sorbitan esters. Desiccant excipients may be employed. Delay release excipients may also be used to provide for release of the composition over

- H - a longer period of time in vivo. An excipient of low water solubility may be used, for example, a pharmaceutically acceptable polymer such as a cellulose derivative, polyvinyl pyrollidone or a sugar derivative.

Further excipients may be selected from pH stabilisers, antioxidants and flavouring agents. These may be chosen, if necessary, from standard pharmaceutical anti-oxidants, for example, alpha-tocopherol or ascorbyl palmitate, or pH modifiers, for example, citric acid or tris buffer or physiologically acceptable sodium salts, to enable the long tenn stability of the composition to be improved over a composition without these excipients. A dosage form in accordance with the present invention may incorporate an external excipient for improved flow characteristics of the dry powder composition. Examples of suitable external excipients include lactose, mannitol, trehalose or other sugars or mixtures thereof.

The compositions of the present invention may also include an antimicrobial preservative which can be selected from the group consisting of benzalkonium chloride, methylparaben, sodium benzoate, benzoic acid, phenyl ethyl alcohol, mixtures thereof, and the like. In another preferred embodiment of the invention, the surfactant is selected from the group consisting of: polysorbate 80 NF, polyoxyethylene 20 sorbitan monolaurate, polyoxyethylene 4 sorbitan monolaurate, polyoxyethylene 20 sorbitan monopalmitate, polyoxyethylene 20 sorbitan monostearate, polyoxyethylene 4 sorbitan monostearate, polyoxyethylene 20 sorbitan tristearate, polyoxyethylene 5 sorbitan monooleate, polyoxyethylene 20 sorbitan trioleate, polyoxyethylene 20 sorbitan monoisostearate, sorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan trilaurate, sorbitan trioleate, sorbitan tristearate, mixtures thereof, and the like. In yet another preferred embodiment of the invention, the tonicity agent is selected from the group consisting of: dextrose, lactose, sodium chloride, mixtures thereof, and the like. In a further preferred embodiment of the invention, the suspending agent is selected from the group consisting of: microcrystalline cellulose, carboxymethylcellulose sodium NF, polyacrylic acid, magnesium aluminum silicate, xanthan gum, mixtures thereof, and the like. Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or

agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved and (b) the limitations inherent in the art of compounding.

Procedures for the preparation of dosage unit forms and topical preparations are readily available to those skilled in the art from texts such as Pharmaceutical Handbook A Martindale Companion Volume Ed. Ainley Wade Nineteenth Edition The Pharmaceutical Press London, CRC Handbook of Chemistry and Physics Ed. Robert C. Weast Ph D. CRC Press Inc.; Goodman and Gilman 's; The Pharmacological basis of Therapeutics. Ninth Ed. McGraw Hill; Remington; and The Science and Practice of Pharmacy. Nineteenth Ed. Ed. Alfonso R. Gennaro Mack Publishing Co. Easton Pennsylvania.

The inventive concept will now be described in more detail with reference to non-limiting examples.

EXAMPLES

Example 1. Synthesis of FCGI (2)

Phenyl 2,6-dioxo-3-piperidinylcarbamate (15)

N,N-Dimethylaminopyridine (100 mg ₅ 0.89 mmol), followed by 1,1'- carbonyldiimidazole (5.78 g, 0.036 mol) were added to a suspension of N- carbobenzyloxyglutamine (14) (5.00 g, 0.018 mol) in dry p-dioxane (100 ml) and the mixture was stirred at room temperature for 10 min, then refluxed under nitrogen for 17 h. The solvent was removed in vacuo and the residue was partitioned between ethyl acetate and water. The organic portion was washed with water (5 times), saturated aqueous NaHCO ₃ , water and brine. Workup gave an oil which was chromatographed on silica. Elution with ethyl acetate gave the title compound 15 as a white solid (4.04 g, 86%), mp 124-126 ⁰ C. ¹ H NMR (400 MHz, DMSO-D6) δ ppm 10.76 (br s, IH), 7.58 (d, J=8.6 Hz, IH), 7.38-7.30 (m, 5H), 5.06 (s, 2H), 4.36-4.29 (m, IH), 2.78-2.68 (m, IH), 2.52-2.44 (m, IH), 2.01-1.88 (m, 2H).

3-Amino-2,6-piperidinedione hydrochloride (16)

A solution of 15 (4.0Og, 0.015 mol) in methanol (200 ml) and 2N HCl (15 ml) was hydrogenated over 5% Pd-C (100 mg) at 60 psi for 4 h. The catalyst was filtered off and the filtrate concentrated to dryness to give the title compound 16 as a white solid (2.61 g, 100%), mp 245 ⁰ C (dec) (lit. 235 ⁰ C (dec)). ¹ H NMR (400 MHz, DMSO-D6) δ ppm 11.22 (br s _? IH), 8.68 (br s, 3H), 4.20 (dd, J=13.0, 5.3 Hz, IH), 2.77-2.65 (m, IH), 2.64- 2.56 (m, IH), 2.27-2.19 (m, IH) ₅ 2.09-1.97 (m, IH).

2-{f(2,6-Dioxo-3-piperidmyl)ammo1carbonvI}-3,4.,5,6-tetra fluorobenzoic acid FGCB (2)

Triethylamine (0.65 mi, 4.54 mmol) was added to a suspension of 16 (0.37 g, 2.27 mmol) in dry tetrahydrofuran (40 ml) and the mixture was stirred at room temperature for 10 min. Tetrafluorophthalic anhydride (17) (0.50 g, 2.27 mmol) was added and stirring was continued at room temperature for 48 h. The reaction mixture was partitioned between ethyl acetate and water and the aqueous portion was acidified with 2N HCl and extracted with ethyl acetate (5 times). The combined extracts were worked up to give an oil which was dried well in vacuo and then triturated with diethyl ether until crystallization began. The solid was filtered off and further triturated with cold diethyl ether (5 times) to give the title compound 2 as a white powder (0.54 g, 62%), mp 254-258 ⁰ C. ¹ H NMR (400 MHz, DMSO-D6) δ ppm 14.00 (br, IH), 10.85 (s, IH, exch. with D ₂ O), 9.08 (d, J=8.0 Hz, IH, exch. with D ₂ O), 4.75-4.69 (m, IH), 2.79-2.69 (m,

IH), 2.58-2.48 (m, IH), 2.08-2.00 (m, IH) ₅ 1.99-1.90 (m, IH). Found: C, 44.71; H, 2.43; N ₃ 7.82. C ₁₃ H ₈ F ₄ N ₂ O ₅ requires C, 44.84; H, 2.32; N, 8.05%.

Example 2. Synthesis of analogues 19(a-e) of FCGI (2)

a: R=3,6-diCI a: R=3,6-diCI b: R=4,5-diCI b: R=4,5-diCI c: R=3,6-diF c: R=3,6-diF d: R=3-NO ₂ d: R=3-NO ₂ e: R=4,5-benzo e: R=4,5-benzo

3,6-Dichloro-2-{f(2,6-dioxo-3-piperidmyl)amino1carbonvI)b enzoic acid (19a)

Reaction of 3,6-dichlorophtlialic anhydride (18a) with (16) as described above for the preparation of (2) gave the title compound (19a) as a white powder (37%), mp 132-135 ⁰ C. ¹ H NMR (400 MHz, DMS0-D6) δ ppm 13.80 (br, IH), 10.80 (br s, IH), 8.95 (d, J=8.1 Hz, IH), 7.58 (s, 2H), 4.73-4.64 (m, IH) ₃ 2.77-2.65 (m ₃ IH) ₃ 2.58-2.48 (m ₃ IH), 2.07-1.89 (m, 2H). Found: C, 44.44; H, 3.44; N ₅ 7.43. C ₁₃ H ₁₀ Cl ₂ N ₂ O ₅ MeOH requires C, 44.58; H ₃ 3.74; N ₃ 7.43%. 4,5-DichIoro-2-U(2,,6-dioxo-3-piperidinyl)aminolcarboiiyl)be nzoic acid (19b) Reaction of 4,5-dichlorophthalic anhydride (18b) with (16) as described above for the preparation of (2) gave the title compound (19b) as a pale-pink powder (35%), mp >300 ⁰ C. ¹ HNMR (400 MHz ₅ DMSO-D6) δ ppm 14.00 (br, IH), 10.84 (s, IH), 8.82 (d, J=8.0 Hz, IH), 7.96 (s, IH) ₅ 7.70 (s, IH), 4.74-4.67 (m, IH), 2.78-2.72 (m, IH), 2.59-2.49 (m, IH) ₅ 2.05-1.98 (m, 2H). MS (APCI ⁺ ) m/z 345 (M+H). 3,6-Difluoro-2-{F(2,6-dioxo-3-piperidinyI)amino1carbonvUbenz oic acid (19c)

Reaction of 3,6-difluorophthalic anhydride (18c) with (16) as described above for .the preparation of (2) gave the title compound (19c) as a pale-purple powder (15%), mp 251-253 ⁰ C. ¹ H NMR (400 MHz, DMSO-D6) δ ppm 13.50 (br, IH) ₅ 10.82 (s, IH) ₃ 8.93 (d, J=8.0 Hz, IH) ₅ 7.49-7.40 (m ₅ 2H) ₃ 4.74-4.68 (m, IH), 2.78-2.69 (m, IH) ₃ 2.57-2.52 (m ₅ IH) ₃ 2.03-1.94 (m ₅ 2H). MS (APCI ⁺ ) m/z 313 (M+H).

2-U(2,6-dioxo-3-piperidinyl)amino1carbonyI}-3-nitrobenzoi c acid (19d)

Reaction of 3-nitrophthalic anhydride (18d) with (16) as described above for the preparation of (2) gave the title compound (19d) as a white powder (31%), mp 289-291 ⁰ C. ¹ HNMR (400 MHz, DMSO-D6) δ ppm 13.75 (br, IH) ₅ 10.80 (s, IH) ₅ 8.95 (d, J=8.0 Hz ₅ IH), 8.23 (d, J=8.0 Hz ₅ IH) ₅ 8.17 (d, J=8.0 Hz ₅ IH) ₅ 7.75 (t, J=8.0 Hz ₅ IH), 4.77- 4.70 (m, IH) ₅ 2.77-2.68 (m, IH), 2.59-2.50 (m, IH) ₅ 2.25-2.20 (m, IH) ₅ 1.90-1.84 (m, IH). ¹³ C NMR (400 MHz ₅ DMSO-D6) δ ppm 172.93, 171.29, 165.85, 164.00, 147.37, 134.58, 132.31, 132.26, 130.06, 127.11, 49.66, 30.18, 23.66. MS (APCf) m/z 322 (M+H).

3-{f(2,6-Dioxo-3-piperidinvI)aminolcarbonyl>-2-naphthoic acid (19e) Reaction of 2,3-naphthalenedicarboxylic anhydride (18e) with (16) as described above for the preparation of (2) gave the title compound (19e) as a white powder (57%), mp >300 ⁰ C. ¹ H NMR (400 MHz, DMSO-D6) δ ppm 12.93 (br, IH), 10.82 (br s, IH), 8.76 (d, J=8.2 Hz, IH), 8.40 (s, IH) ₅ 8.12-8.05 (m, 2H), 8.04 (s, IH), 7.71-7.64 (m, 2H), 4.76 (dd, J=16.9, 8.4 Hz ₅ IH) ₅ 2.82-2.72 (m ₅ IH) ₅ 2.60-2.54 (ddd, J=I 6.9, 4.0, 3.9 Hz, IH), 2.10-2.04 (m, 2H). Found: C, 62.32; H, 4.53; N ₅ 8.42. C ₁₇ H ₁₄ N ₂ O ₅ requires C ₅ 62.57; H ₅ 4.32; N ₅ 8.59%. Example 3. Synthesis of the bisamide (21)

2-(l-PyrroIidinylcarbonyI)benzoic acid (20)

Pyrrolidine (5.6 ml, 0.067 mol) was added in portions to a refluxing suspension of phthalic anhydride (10.0 g, 0.067 mol) in dry benzene. The resulting solution was refluxed a further 15 min and then concentrated to dryness in vacuo. The residue was slurried with ethyl acetate (2x) to leave the title compound (20) as a white solid (6.31 g, 43%), mp 131-133 ⁰ C. ¹ HNMR (400 MHz, DMSO-D6) δ ppm 13.02 (br, IH), 7.89 (dd, J=7.8, 1.1 Hz, IH), 7.62 (ddd, J=7.8, 7.5, 1.3 Hz ₅ IH), 7.49 (ddd, J=7.8, 7.5, 1.3 Hz, IH) ₅ 7.31 (dd ₅ J=7.5, 1.1 Hz, IH), 3.43 (t, J=6.7 Hz, 2H), 3.01 (t, J=6.7 Hz ₅ 2H) ₅ 1.88- 1.73 (m ₅ 4H). JV-(2,6-Dioxo-3-piperidinyl)-2-(l-pyrroIidinylcarbonyI)benza inide (21)

Pivaloyl chloride (0.62 ml, 5.01 mmol) was added dropwise at 5 ⁰ C to a solution of (20) (1.0Og, 4.56 mmol) and triethylamine (0.70 ml, 5.02 mmol) in dichloromethane (20 ml) and the solution was stirred at this temperature for 1 h. Triethylamine (1.40 ml, 0.01 mol) was added, followed by the powdered salt (16) (0.79 g, 4.79 mmol). The solution was stirred at room temperature for 16 h and the crude product was adsorbed directly onto silica by removal of the solvent, and chromatographed. Ethyl acetate eluted fore fractions, while 5-10% methanol/ethyl acetate eluted crude (21), which was triturated with diethyl ether to leave the title compound (21) as a hygroscopic white powder (0.53 g, 35%), mp 106-109 ⁰ C. ¹ H NMR (400 MHz, DMSO-D6) δ ppm 10.81 (br s, IH), 8.59 (d, J=8.3 Hz, IH), 7.68 (dd, J=7.5, 1.0 Hz, IH), 7.55 (ddd, J=7.4, 7.4, 1.1 Hz, IH) ₅ 7.49 (ddd, J=7.5, 7.4, 1.1 Hz, IH), 7.32 (dd, J=7.5, 1.1 Hz, IH), 4.75-4.69 (m, IH), 3.42-3.36 (m, 2H), 3.15-3.06 (m, 2H), 2.82-2.73 (m, IH), 2.57-2.50 (m, IH), 2.06-1.92 (m, 2H), 1.88-1.74 (m, 4H). Found: C, 61.84; H, 6.10; N, 12.07. C ₁₇ Hi ₉ N ₃ O ₄ .1/4Et ₂ O requires C, 62.15; H, 6.23; N, 12.08%. Other relevant methodologies and details can be found in publications such as

Zahn H. and Folsche E.T. Notiz uber die Bildung von Benzyloxycarbonyl-α- aminoglutarimid aus Benzyloxycarbonyl-L-glutamin. Chem. Ber., 1969, 102, 2158- 2159, incorporated in its entirety herein by reference.

Example 4. Modulation of TNF Production by FCGI in Mice Mice (C57B1/6 bred at the Animal Resources Unit, University of Auckland, New

Zealand), were divided into groups of 3. One group was untreated. Other groups were treated with LPS (300 μg/mouse) alone; LPS (300 μg/mouse) plus FCGI (10 mg/kg); LPS (300 μg/mouse) plus FCGI (100 mg/kg); DMXAA (50 mg/kg) alone; DMXAA (50 mg/kg) + FGCI (10 mg/kg); DMXAA (50 mg/kg) + FGGI (100 mg/kg). One hour after treatment with LPS, or 4 hours after DMXAA treatment mice were anaesthetized with halothane (NZ Pharmacology Ltd., Christchurch, New Zealand), and blood was collected from the ocular sinus. Blood was coagulated overnight at 4 ⁰ C, and then centrifuged for 30 min at 2,000 x g and 4 ⁰ C, and the serum was removed and stored at - 80 ⁰ C until use. TNF levels in serum were assayed for TNF using a commercially available ELISA kit (OpEIA murine TNF kit, PharMingen, San Diego, CA, USA), according to the manufacturer's instructions. Briefly, samples (100 μl/well) in duplicate together with a serial dilution of TNF (31.25 - 2000 pg/ml) for the standard curve, were

added to flat-bottom 96-well plates pre-coated with immobilised monoclonal anti-TNF antibody and incubated at room temperature for 2 h. The wells were then washed, biotinylated polyclonal antibody to TNF was added, followed by peroxidase-labelled streptavidin, and incubated at room temperature for 1 h. The wells were washed again, and the substrate, tetramethylbenzidine and hydrogen peroxide was added and after 10 min the reaction was stopped with 2 N sulphuric acid. The absorbance at 450 nm was determined using a microtitre plate reader.

The results in Figure 1 show that FCGI inhibited TNF production induced by LPS or DMXAA. Example 5. Increased potency of FGCI compared with CGI to inhibit LP S-induced TNF production in mice

To compare the dose-potency of FCGI to that of CGI to inhibit LPS-induced TNF production, C57B1/6 mice in groups of three were treated with LPS (300 μg/mouse) alone; or with varying doses of FCGI or CGI (0.1-10 mg/kg). After 1 hour, mice were bled, serum prepared and assayed for TNF as described above in section 2. Results in Figure 2 indicate a greater inhibition of LPS-induced TNF production by FCGI than CGI over a similar dose range. 6. Stability of FCGI over a range ofpHs

To examine the stability of FCGI, the drug was firstly dissolved in DMSO to make a 1 mM stock, and then added to a 10 mM phosphate buffer at different pHs (3.0, 7.0 and 8.0), to give a final concencentration of FCGI of 100 μM. Aliquots (100 μl) were automatically loaded onto a Agilent 1100 chromatogragh (Aigilent Technologies, Waldbronn, Germany). Compounds were separated by reversed phase chromatography with UV detection at 240 and 270nm. The chromatography column used was a Luna 5μ phenylhexyl (100mm x 4.6mm) and the mobile phase consisted of solvent A: acetonitrile in water (80%v/v) and solvent B: ammonium formate buffer (45mM; ph 3.5) at 0.6ml/min. A linear gradient was used to separate FCGI and the internal standard, phenacetin. The mobile phase conditions were: 95% solvent B (0-25min), changing to 100% solvent A over 25-30 min, returning to 95% solvent B over 30-35min. FCGI was found to be completely stable over 24 hours at pH 3.0, but unstable at pH 8.0 (Figure 3).

Example 7. Inhibition ofangiogenesis in vifto

Since inhibition of angiogenesis could be important mechanism for the anticancer activity of the analogues, inhibition of tube formation by endothelial cells on matrigel matrix was used was used as an in vitro model to investigate the anti- angiogenic activity of CGI, FCGI and 19a. ECV304 human endothelial-like cells (from ATCC, CRL-1998) were plated at a concentration of IxIO ⁵ in a final volume of ImI of M- 199 medium supplemented with 10% FBS, containing FCGI, CGI and 19a at varying concentrations (2 - 500 μg/ml) and was plated onto the solidified matrigel matrix (100 μl, diluted 1:3, Becton Dickinson, Bedford, USA) in 24-well plates and incubated at 37 ⁰ C, 5% CO ₂ . Development of capillary-like networks was evaluated 18 hours after plating. The plates were photographed using 5.0 megapixel professional digital compact camera (Olympus Camedia c-5050 zoom). The total surface area covered by the tubes was measured using Image J Bioimaging software (National Institute of Mental Health, Maryland, USA) and was divided by the total area of the field (mm/mm ² ). Inhibition of tube formation increased with the addition of increasing concentrations of CGI, FCGI and 19a with complete inhibition observed at concentrations of 250 μg/ml or higher (Figure 4). CGI, FCGI and 19a were equipotent in inhibiting tube formation by ECV 304 cells in vitro. Example 8. Inhibition of IL-6 production by human multiple myeloma cell-line The analogues were tested for their ability to inhibit the production of IL-6, a growth factor required by multiple myeloma cells to survive. RPMI 8226 (from ATCC, CCL-1555) human multiple myeloma cells (10 per well) were cultured with lipopolysaccharide (0.1 μg/ml) and varying concentrations of each analogue in RPMI medium supplemented with 10% FBS in 96-well plates for 20 hours. Culture supernatants were assayed for IL-6 concentrations using ELISA kit for human IL-6

(Catalogue number 555220, BD Biosciences Pharmingen, San Diego, CA). The results for CGI, FCGI and 19a are shown in Figure 5, with FCGI showing the greatest inhibition.

Although the present invention has been described with reference to certain preferred embodiments, it will be understood that variations and alternatives, which are in keeping with the spirit of the invention, are also within its scope.