WO2008074078A1 - Selectively deliverable isatin-based cytotoxic agents - Google Patents

Abstract

The invention relates to compounds comprising a cytotoxic isatin derivative conjugated to a cell targeting moiety via a spacer group. These conjugates allow the cytotoxic isatin derivaties to be targeted to particular cell and tissue types. The invention also relates to novel isatin derivatives, intermediates used in preparing the conjugates and method of using the conjugates.

Description

TITLE OF THE INVENTION Selectively Deliverable Isatin-based Cytotoxic Agents

FIELD OF THE INVENTION The invention generally relates to the development of new cytotoxic agents, and methods of targeting these agents to particular cell and tissue types.

BACKGROUND TO THE INVENTION The rapid developments in pharmaceutical drug discovery have resulted in the emergence of increasing numbers of novel therapeutic drugs for the treatment of a variety of diseases. However, common problems associated with drug administration are still likely to include one or more of: unfavourable bio-distribution of a pharmaceutical throughout the body, lack of specific drug affinity towards a pathological site, non-specific toxicity and side- effects resulting from high doses. For example, although anti-neoplastic agents have for many decades formed the basis of chemotherapeutic treatment for a variety of cancers, a limitation of their use has been that administration of effective doses for a wide variety of these agents may not necessarily be achieved, due to the non-selective effects of these agents on cells other than cancerous ones. As a general rule, the selectivity of chemotherapeutic cytotoxic agents relies on the premise that rapidly proliferating cells are more prone to the cytotoxic effect of these drugs. Generally, off-target toxicity of cytotoxic drugs is one of the more serious problems of cancer chemotherapy.

One attractive strategy to enhance the therapeutic index of cytotoxic drugs is to specifically deliver these agents to defined target cells, keeping them, as largely possible, away from healthy cells. The utilisation of cell-specific receptors to target specific cell types, for example, tumour cells, is an attractive concept because it offers the possibility of minimising non-selective toxic effects. Targeting of drugs specifically to certain cell types, has been the goal of previous studies.

There are, however, difficulties associated with the targeting approach. One difficulty is the identification of suitable receptors or ligands that are present predominantly on the target cells, and are in sufficient density. Another difficulty is that, until recently, little was known about the mechanisms and dynamics of receptor trafficking after binding. In most cases it was not known what modifications of ligands could be tolerated without affecting binding interactions. Furthermore, very little is known about the fate of internalised ligand- receptor complexes. Cytotoxic moieties that can be successfully derivatised so as to be employed in a targeting strategy also need to be identified.

SUMMARY OF THE INVENTION

In one aspect, the invention generally provides a compound comprising (i) at least one derivatised cytotoxic isatin moiety and (ii) a cell-targeting moiety, wherein the at least one or each cytotoxic moiety is linked to the cell targeting moiety through a spacer. Accordingly, a compound of the invention may be represented by formula I:

(R¹X¹^T¹

formula I

wherein:

R¹ is a derivatised cytotoxic isatin moiety;

X¹ represents a spacer;

T¹ is a cell-targeting moiety; and

n is an integer greater than or equal to 1.

When n is greater than one each R¹X¹ unit may be the same or different _. _

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Suitably, the spacer X¹ comprises at least one covalent linkage L² formed with the cell targeting moiety T¹, at least one other covalent linkage L¹ formed with the cytotoxic moiety R¹, and a bridging moiety Z¹, thus providing a compound of formula II:

(R¹L¹Z¹L²^T¹

formula II

wherein R¹, T¹ and n are as defined above.

Desirably, the covalent linkage L² is stable to physiological conditions. Suitably, the covalent linkage L¹ is labile to a physiological environment within or adjacent to the cell to be targeted. As such, the covalent linkage between the cell-targeting moiety and the spacer is typically a covalent linkage stable to physiological conditions and physiological processes, and the covalent linkage between the spacer and the cytotoxic moiety is a linkage that is selected to be labile to different physiological conditions and physiological processes, e.g., enzymes, pH and redox conditions.

In some embodiments the covalent linkage L¹ is an enzymatically cleavable linkage.

In some embodiments the covalent linkage L¹ is an acid labile linkage.

Suitably, the covalent linkage L¹ is stable from at least about pH 6.9 to about pH 7.4.

In some embodiments the covalent linkage L¹ is selected from: =N-, =N-W-, =N- W-C(Y)-, and =N- W-C(Y)-Q- (wherein N is a nitrogen atom and W₅ Y, and Q are independently selected from O, S, CH₂ or NR^N, wherein R^N is selected from hydrogen, hydroxy, amino, optionally substituted C_halky., optionally substituted Ci-salkenyl, optionally substituted Ci-galkynyl, optionally substituted Ci_-4alkylaryl, and optionally substituted aryl), a methyleneoxy group, an ethyleneoxy group, a ketal or a hemi-ketal. Preferably, cleavage of a covalent linkage L¹ generates, from the derivatised cytotoxic isatin moiety R¹, a cytotoxic isatin compound of formula III:

formula III

wherein:

R², R³, R⁴ and R⁵ are independently selected from H; halo; optionally substituted alkyl; optionally substituted cycloalkyl; optionally substituted alkenyl; optionally substituted cycloalkenyl; optionally substituted alkynyl; optionally substituted alkoxy; optionally substituted haloalkyl; optionally substituted hydroxyalkyl; optionally substituted acyl; optionally substituted acyloxy; hydroxyl; optionally substituted aryl or fused aryl; optionally substituted aryloxy; optionally substituted heteroaryl; optionally substituted heteroaryloxy; amino; azido; nitro; nitroso; cyano; carbamoyl; trifluoromethyl; mercapto; optionally substituted alkylamino; optionally substituted dialkylamino; formyl; optionally substituted alkylsulfonyl; optionally substituted arylsulfonyl; optionally substituted alkylsulfonamido; optionally substituted arylsulfonamido; and optionally substituted alkoxycarbonyl; preferably independently selected from H, halo, hydroxy, optionally substituted Ci_-4alkoxy, optionally substituted C].₄alkyl, optionally substituted phenyl, optionally substituted phenoxy, nitro, cyano; more preferably from H and halo, even more preferably from H, Br or Cl and most preferably from H and Br;

alternatively any of R² and R³ or R³ and R⁴ or R⁴ and R⁵, and R⁵ and R⁶ may combine to form an aliphatic or aromatic, heterocyclic or carbocyclic ring system; . . .

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R⁶ is selected from optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted cycloalkyl; optionally substituted heterocycloalkyl - e.g. pyranosyl or furanosyl; optionally substituted alkoxy; optionally substituted aryl; optionally substituted acylaryl; optionally substituted heteroaryl; optionally substituted Ci_-6alkylaryl or optionally substituted Ci_-6alkylheteroaryl; preferably optionally substituted benzyl; optionally substituted methylnaphthyl; optionally substituted phenacyl or optionally substituted phenethyl; and more preferably optionally substituted benzyl (preferably optional substituents include one or more of aryl, C_1-6acyl, C_1-6alkyl, C_{1- 6}alkoxy, C_2-6alkenyl, C_2-6alkynyl, C_1-4alkyloxyC_1-4alkyl; C_2-6alkenylaryl; C_{2- δ}alkenylheteroaryl; C₂-₆alkynylaryl; C_2-6alkynylheteroaryl; C_1-6alkylsulfonyl, arylsulfonyl, -COOH; -C_1-6alkylCOOH; -COO⁺M^'; -C_]-6alkylCOO^'M⁺ - wherein M⁺ is a counter ion, e.g. Na⁺, K⁺, NH₄ ⁺, Ca²⁺; -C(O)OCi_-6alkyl; -C(O)OC _1-6aryl; -C(O)OC_1-6heteroaryl; - C(O)OCi-₆alkylheteroaryl; -C(O)OCi_-6alkylaryl; -d-ealkylC^OQ-ealkyl; -C,. ₆alkylC(O)OCi_-6aryl; -Ci_-6alkylC(O)OCi_-6heteroaryl; -Ci-ealkylC^OCi-ealkylheteroaryl; -C_1-6alkylC(O)OC_1-6alkylaryl; C_1-6alkylsulfonamido, arylsulfonamido, halo, hydroxy, mercapto, trifluoromethyl, carbamoyl, amino, azido, nitro, cyano, C_1-6alkylamino or di(d- ₆alkyl)amino; more preferably halo or trifluoromethyl, even more preferably Cl, Br or trifluoromethyl).

Preferably the derivatised cytotoxic isatin compound is attached to the spacer through the oxo group located at the 3-position of the isatin, optionally via covalent linkage L¹. The 3- oxo group appears to be important for cytotoxic activity and accordingly attachment via this group is believed to lessen or remove the cytotoxic effect of the conjugate relative to the free isatin derivative. An imine or substituted imine linkage is particularly suitable for attaching the spacer to this 3-position of the isatin, because cleavage of such a group under physiological conditions can regenerate the oxo group at the 3-position. The reactivity of the imine group can be manipulated by for example, changing the substituent attached to the imine nitrogen atom or the adjacent phenyl rings. This moderates the pH sensitivity of the imine group and affords control of the hydrolysis behaviour of the linker. However, it is to be understood that other attachment points and linkages are contemplated by the present invention. .

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Suitably, the compound is of formula IV:

formula IV

wherein:

R², R³, R⁴, R⁵, R⁶, L¹, L², Z¹, n and T¹ are as defined above.

In some embodiments the covalent linkage L¹ of formula IV is selected from: =N-, =N-W-, =N- W-C(Y)-, and =N- W-C(Y)-Q- wherein N, W, Y, and Q are as defined above.

In some embodiments T¹ is selected from one of a specific binding pair. Suitably, T is selected from a protein, a peptide, a carbohydrate, a peptidomimetic, or a glycomimetic.

In certain embodiments T¹ is a receptor which binds to a ligand on a cell.

In other embodiments T¹ is a ligand which binds to a receptor on a cell.

Suitably, the bridging moiety Z¹ is of the formula P¹Ar¹V¹ wherein P¹ and V¹ are absent or present and are selected from optionally substituted divalent Ci-ealkyl, optionally substituted divalent C₂-₆alkenyl, optionally substituted divalent C_2-6alkynyl, Ar¹ is selected from optionally substituted divalent aryl or optionally substituted divalent heteroaryl. Compounds of the invention may be prepared by reaction of an intermediate of formula V with a suitable cell targeting moiety:

formula V wherein:

R , R³, R , R⁵, R⁶, L¹ and Z¹ are as defined above; and

L³ is a functional group that reacts with a functional group of a targeting moiety to form the covalent linkage L² as defined above, for example in formula IV, or is a protected or latent functional group that can be converted to a functional group that reacts with a functional group of a targeting moiety to form the linkage L².

Suitably, L³ is selected from -COOH including activated forms thereof; -C_1-6alkylCOO^'M⁺ - wherein M⁺ is a counter ion, e.g. Na⁺, K⁺, NH₄ ⁺, Ca²⁺; -C(O)Cl; -C(O)OC _1-6alkyl; - C(O)OCi_-6aryl; -C(O)OCi_-6heteroaryl; -C(O)OCi_-6alkylheteroaryl; -C(O)OC i_-6alkylaryl; sulfonyl; sulfonylchloride; isothiocyanate, isocyanate, halo, mercapto, amino, azido, cyano, preferably from -COOH including activated forms thereof; -C_1-6alkylCOO^"M⁺ - C(O)Cl; -C(O)OC _]-6alkyl, C(O)OCi_-6aryl and -C(O)OC _]-6alkylaryl.

The intermediate may be of formula VI: .

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formula VI

wherein:

m is an integer from 0 to 4;

R⁷ is selected from Ci_-6acyl, Ci^alkyl, Ci_-6alkoxy, C₂-₆alkenyl, C_2-6alkynyl, C_1- ealkylsulfonyl; aryl, aryloxy; arylsulfonyl, -COOH; -C_1-6alkylCOOH; -COO⁺M^"; -Cj-

₆alkylCOO^"M⁺ - wherein M⁺ is a counter ion, e.g. Na⁺, K⁺, NH₄ ⁺, Ca²⁺; -C_]-6alkylOH;

-C(O)OCi_-6alkyl; -C(O)OCi_-6aryl; -C(O)OCi_-6heteroaryl; -C(O)OCi_-6alkylheteroaryl;

-C(O)OC_1-6alkylaryl; -Ci-₆alkylC(O)OC,_-6alkyl; -C].₆alkylC(O)OCj_-6aryl; -C_1-

₆alkylC(O)OCi.₆heteroaryl; -Ci_-6alkylC(O)OCi_-6alkylheteroaryl; -C_1-6alkylC(O)OC₁. ₆alkylaryl; C_1-6alkylsulfonamido; arylsulfonamido; halo; hydroxy; mercapto; trifluoromethyl; carbamoyl; amino; azido; nitro; cyano; -C^alkylamino or di(Ci-

6alkyl)amino; more preferably -COOH, halo or trifluoromethyl; even more preferably Cl,

Br₅ or trifluoromethyl;

the integer q is selected from 1 to 5; and

r 3

and q are defined as above.

Another preferred intermediate is of formula VII:

formula VII wherein:

R², R³, R⁴, R⁵, V¹, L³, R⁷ and q are defined as above. A further preferred intermediate is of formula VIII:

formula VIII wherein: q is as defined above, R⁷ is selected from Ci_-6alkoxy; nitro; bromo; iodo; Ci-oalkyloxyC^alkyl; Ci-βalkyloxyC]. ₆alkyl; trifluoroniethyl; Ci-₄alkyloxyaryl; COOCi ^alkyl; C_1-4alkyloxyCi_-4alkyl; C₂. ₆alkenylaryl; C2-₆alkenylheteroaryl; C_2-6alkynylaryl; C_2-6alkynylheteroaryl; optionally substituted phenyl; Ci_₄alkyloxyheteroaryl; and

L³ is selected from -COOH; -COO⁺M^"; -Ci_-6alkylCOO^"M⁺ - wherein M⁺ is a suitable counter ion: e.g. Na⁺, K⁺, NH₄ ⁺, Ca²⁺; -C(O)OC_1-6alkyl; -C(O)OC_1-6aryl; -C(O)OC_1- eheteroaryl; -C(O)OC_1-6alkylheteroaryl; -C(O)OC _1-6alkylaryl; -C_1-6alkylC(O)OC_1-6alkyl; - C,_-6alkylC(O)OC_1-6aryl; -C_I-6alkylC(O)OCi_-6heteroaryl; -Ci_-6alkylC(O)OC₁.

₆alkylheteroaryl; -Ci.₆alkylC(O)OCi_-6alkylaryl.

The intermediate may be of formula IX:

formula IX

The compound may be of formula X:

formula X

wherein:

R², R³, R⁴, R⁵, Z¹, L², n, q and T¹ are as defined above;

m is an integer from 0 to 4; and

R⁷ is selected from Ci_-6acyl, Ci_-6alkyl, C_1-6alkoxy, C_2-6alkenyl, C_2-6alkynyl, Cj- ₆alkylsulfonyl, arylsulfonyl, -COOH; -Ci_-6alkylCOOH; -COO⁺M^"; -C_1-6alkylCOO^"M⁺ - wherein M⁺ is a counter ion, e.g. Na⁺, K⁺, NH₄ ⁺, Ca²⁺; -C(O)OC_1-6alkyl; -C(O)OCi_-6aryl; - C(O)OC _1-6heteroaryl; -C(O)OC_1-6alkylheteroaryl; -C(O)OC _1-6alkylaryl; -C_{1- 6}alkylC(O)OCi.₆alkyl; -Ci_-6alkylC(O)OCi_-6aryl; -Ci_-6alkylC(O)OC_1-6heteroaryl; -C_{1- 6}alkylC(O)OCi_-6alkylheteroaryl; -Ci_-6alkylC(O)OCi_-6alkylaryl; C_1-6alkylsulfonamido, arylsulfonamido, halo, hydroxy, mercapto, trifluoromethyl, carbamoyl, amino, azido, nitro, cyano, Ci_-6alkylamino or di(Ci_-6alkyl)amino; more preferably -COOH, halo or trifluoromethyl, even more preferably chloro, bromo, or trifluoromethyl.

Another preferred compound is of formula XI:

formula XI wherein:

R², R³, R⁴, R⁵, V¹, L², T¹, n, q and R⁷ are defined as above.

A further preferred compound is of formula XII:

formula XII wherein:

V¹, L², T¹, n, q and R⁷ are defined as above. A preferred compound is of formula XIII:

formula XIII

wherein T¹ is as defined above and n is an integer from 1 to 12.

Some of the cytotoxic isatin compounds of formula III above are novel and represent a further aspect of the invention.

Accordingly, in a further aspect of the invention there is provided a compound of formula XIV

formula XIV

where R³ and R⁵ are independently selected from Cl, Br, I, CF₃, optionally substituted Ci- ₆alkyl, optionally substituted phenyl and R⁶ is as defined above. _

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Preferably R³ and R⁵ are both Br.

Preferably R⁶ is selected from the R⁶ moieties present in the compounds of formula X, XI, XII and XIII. Such compounds generally correspond to the isatin derivatives released following cleavage from the cell targeting moiety.

The intermediates of formula V, VI, VII, VIII and IX are novel and represent a further aspect of the invention.

The invention also provides a method of preparing a compound for targeting a cell type to induce death of that cell or to inhibit growth of that cell, including the steps of: (i) identifying a cell type;

(ii) identifying a targeting molecule with selectivity for that cell type; and (iii) conjugating that targeting molecule to a derivatised cytotoxic isatin moiety of any of formulae V to IX.

In this aspect of the invention the L¹ moiety is selected such that it is labile to the physiological environment within or adjacent to the cell type to be targeted.

In another aspect the invention provides a method of treating a subject with a compound as broadly described above which comprises administering to the subject a compound as broadly described in formula I.

Suitably, after administration of the compound to the subject, the compound is metabolised at or in a cell type associated with the targeting moiety of the compound of formula I, to provide a compound of formula III. Some compounds of formula III are novel and represent a further aspect of the invention.

In another aspect, the invention provides for the use of a compound of any one of formulae X to XIII which comprises a targeting moiety that binds to a target molecule on a cell type, in the treatment of a condition associated with the presence of the cell type. _ _

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A further aspect of the invention is the use of a compound of any of formulae X to XIII in the manufacture of a medicament for the treatment of a disorder requiring cell-death or the inhibition of cell growth.

The cells to be targeted may be tumour cells or other types of diseased cells types. For example, diseased cells often uniquely express cell surface receptors or ligands that are not usually found on normal (healthy) cells, or are found on normal cells, but to a much lower extent. These unique receptors or ligands represent the targeting molecule to which the targeting moiety binds. The targeting moiety would thus deliver a cytotoxin to the diseased cell causing cell death or inhibition of cell growth. As normal cells express very little to none of the targeting molecule, normal cells would not be targeted efficiently and thus spared.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Shows a graphical comparison of the cytotoxicity of four JV-alkylisatins against the commercially available chemotherapeutic 5-fluorouracil (5FU). Figure 1 shows viability of U937 cells after treatment with increasing concentrations of ■ compound 15, ▲ compound 14, T compound 3, ♦ compound 2 and • 5FU as determined from the MTS cell proliferation assay. Values are means of triplicate samples ±SD and are normalised to DMSO control.

Figure 2: Shows a graphical representation of the activation of caspases 3 and 7 in Jurkat cells (2 x 10^Λ6) over time, after treatment with • 2.5 % DMSO, 6.7 μM (compound 15) and A 2 μM staurosporine. Relative Fluorescence Units (RFLU) were measured using a FLUOStar Optima plate reader (BMG LABTECH) using an excitation (ex) of 480 ran and emmission (em) of 520nm.

Figure 3: Shows a photographic representation of cells (U937) that were treated with 0.39μg/ml of 5,7-dibromo-iV-(p-iodobenzyi)isatin (compound 6) or 5,7-dibromo-iV- cinnamylisatin (compound 20) and viewed under an inverted light microscope after 5 h and 24 h and noted for changes in cell morphology. Magnification x 40.

Figure 4: Shows a photographic representation of U937 and Jurkat cells treated with 0.39μg/ml of 5,7-dibromo-./V-(p-iodobenzyl)isatin (compound 6), 5,6-dibromo-iV-(j!?- methoxybenzyl)isatin (compound 2), 5,7-dibromo-N-isopentylisatin (compound 22) and 5- bromo-./V-(p-methoxybenzyl)-3-phenyliminoisatin (34) and examined for change in cell morphology under an inverted light microscope after 24 h. Magnification x 40.

Figure 5: Shows a photographic representation of U937 cells treated with either 2.5% DMSO (control) or 0.39μg/ml 5,7-dibromo-JV-(j!?-iodobenzyl)isatin (compound 6) for 24 h then nuclei stained using Dif Quick. Briefly, cells (~80,000) were harvested and spun onto slides using a cytospin, and stained as per manufactures instructions. Darker (blue) arrows indicate normal cellular nuclei, lighter (red) arrows indicate abnormal nuclei after treatment. Magnification x 40.

Figure 6: Shows a photographic representation of U937 cells treated with a high (50μg/ml) or low (0.39μg/ml) concentration of 5,7-dibromo-iV-(p-iodobenzyi)isatm (compound 6) and incubated for 5 h, then the nuclei stained were with the vital fluorescent dye propidium iodide (PI). Cells were viewed under a fluorescent light microscope using a FITC filter (ex 490nm, em 510nm). Red fluorescence corresponds to stained nuclei in membrane compromised cells. Magnification x 40.

Figure 7: Shows a photographic representation of cells (U937) treated with either 2.5% DMSO (control) or 0.79μg/ml 5,7-dibromo-N-(p-trifluoromethylbenzyl)isatin (compound 15) for 24 h then harvested and stained with Tubulin Tracker™ Green (Oregon Green® 488 Taxol, bis-acetate), as per manufacturer's instructions. Fluorescence was visualized by confocal microscopy using an ex 494 nm and em 522 nm.

Figure 8: Shows a graphical representation of the determination of the maximum tolerated dose (MTD) of 5,7-dibromo-iV-(p-methoxybenzyl)isatin (compound 2). The dose was _

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determined in balb/c mice after a single intraperitoneal (IP) injection. Mice were injected with either »50 μLμL 100% DMSO, «25 mg/kg, A35 mg/kg or 50 mg/kg of compound 2 and their weights measured daily and graphed as percent weight change from day zero. Data points are the mean of triplicates ± SD.

Figure 9: Shows a graphical representation of the treatment of nude balb/c mice, inoculated with human breast, MDA-MB-231 cells (right mammary fat pad), and treated with either • 50 μL 100% DMSO, ■ 25 mg/kg (50 μL) compound 15 or A35mg/kg compound 32 using a multiple dose schedule. Tumoured mice were given 5 doses, twice weekly for 2.5 weeks and tumour size monitored twice weekely. Data points are the means of 6 animals per cohort ±SEM.

Figure 10: Shows a schematic and graphical representation of the stability of 5-bromo-3- [m-2'-carboxymethylphenyl)imino-iV-(p-methoxybenzyl)isatin (33) at pH 5.5 and pH 7.4.

Figure 11: Shows a schematic and graphical representation of the stability of compound 32 at pH 5.17 and pH 7.02

Figure 12: Shows a graphical representation of a size exclusion trace from a PD-10 column, indicating protein concentration (measured at 280 nm) over elution volume (mL).

Figure 13: Shows a graphical representation of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) fractionation of conjugated transferrin.

Figure 14: Shows a graphical representation of the effect of N-alkylisatins 14 and 15 on the cell cycle of U937 cells. A) and E) DMSO vehicle control, B) and F) 0.5 μM vinblastine sulfate, C) and G) 0.5 μM 5,7-dibromo-iV-(p-methylbenzyl)isatin (14) or D) and H) 0.5 μM 5,7-dibromo-N-(p-trifluoromethylbenzyl)isatin (15) for 4 h (upper panels) or 24 h (lower panels). Cycle arrest was additionally determined after treatment with high and low concentrations of compounds 14 (panels I and J) and 15 (panels K and L) for 24 h. . .

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Figure 15: Shows a graphical representation of the effect of various N-alkylisatins and commercial anticancer agents on tubulin polymerisation as determined in the in vitro microtubule polymerisation assay. Purified bovine neuronal tubulin was used to assay for microtubule formation in the presence of either: •) vehicle control, •) paclitaxel 10 μM, •) vinblastine sulfate salt 10 μM, •) 5,7-dibromo-iV-(p-methylbenzyl)isatin (14) 10 μM, •) 5,7-dibromo-N-(p-trifluoromethylbenzyl) isatin (15) 10 μM, •) 5,7-dibromo-iV- (cinnamyl)isatin (20) 10 μM or •) 5,7-dibromo-iV-(p-phenylbenzyl)isatin (12) 10 μM at 37⁰C. Changes in fluorescence were measured at an excitation wavelength of 360 ±10 nm and the fluorescence was collected at 440 ±10 nm. Data points are the means of duplicate experiments. SD bars were removed for clarity, and were always less than 12% of the mean.

Figure 16: Shows a graphical representation of ¹²³I labeled compounds 6 (right panels) and 13 (left panels) in F344 Fisher rats bearing 13762 MAT B III mammary adenocarcinoma via SPECT imaging.

Figure 17: Shows a graphical representation of ESI-MS of PAI-2-5-FUdrsucc. Conjugates were prepared for electrospray ionisation mass spectrometry (ESI-MS) in Milli Q water and were made up fresh to a final concentration of 1-10 μM. Numbers displayed on the peaks represent the number of 5-FUdrsucc molecules incorporated into the PAI-2 targeting ligand. Inset: the number of PAI-2-5-Fudrsucc conjugates was plotted against molecular weight (mw). The difference in mass for each species represents the exact weight of 5- FUdrsucc (327 gmol^"1), giving a correlation coefficient (R²) = 1.

Figure 18: Shows a graphical representation of samples fractioned by SDS PAGE showing and their ability of a) unmodified and b) modified PAI-2 to form stable complexes with uPA. Briefly, PAI-2 and PAI-2-5-FUdrsucc were incubated with equimolar amounts of uPA at 37 °C for 30 min. Samples were then fractionated under non-reducing conditions using 12% acrylamide SDS PAGE and visualised by staining with Coomassie blue. Lane 1 : PAI-2, Lane 2: PAI-2: uPA, Lane 3: PAI-2-5-FUdrsucc, Lane 4: PAI-2-5-FUdrsuc: uPA, Lane 5: uPA. Figure 19: Shows a graphical representation of samples fractioned by SDS PAGE and their ability of modified and unmodified PAI-2 to form stable complexes with uPA. Briefly, PAI-2 or PAI-2 conjugated to compound 32 (the PAI-2-CF₃imine conjugate) were incubated with equimolar amounts of uPA at 37 ⁰C for 30 min. Samples were then fractionated under non-reducing conditions using 12% acrylamide SDS PAGE and visualised by staining with Coomassie blue. Lane 1 : PAI-2, Lane 2: PAI-2: uPA, Lane 3: uPA, Lane 4: PAI-2-CF₃imine and Lane 5: PAI-2-CF₃imine: uPA.

Figure 20: Shows a graphical representation of the in vitro cytotoxicity of PAI-2-5- FUdrsucc against the high and low uPA and uPAR expressing breast adenocarcinoma cell lines, MDA-MB-231 and MCF-7. A) Cells (1.0 x 10⁴) were cultured for 48 h in the presence of increasing concentrations of the protein cytotoxin conjugate PAI-2-5-FUdrsucc and percent viability of the cells determined in reference to PBS vehicle controls. B) PAI- 2-5-FUdrsucc at a single concentration of 3.8 μM was incubated with MDA-MB-231 cells or MCF-7 cells pretreated with (+) or without (-) exogenous uPA for 48 h. Percent viability was determined in reference to PBS controls. Data points are the means of triplicate experiments ±SE. *** P <0.001 extremely significant.

Figure 21: Shows a graphical representation of the in vitro cytotoxicity of PAI-2- conjugated to compound 32 (the P AI-2-CF3 inline conjuagte) against U937 and THP-I cell lines. Cells (1.0 x 10⁴) were cultured for 48 h in the presence of 3uM PAI-2 and equivalent protein cone, of PAI-2-CF3imine. The viability of the cells was determined in reference to PBS vehicle controls. Data points are the means of duplicate experiments ±SE. *** P <0.001 extremely significant.

Figure 22: A graphical representation of the UV absorption spectrum of transferrin and transferrin-CF₃imine conjugates under different pH conditions. Comparison of unmodified transferrin with modified transferrin shows three distinct maxima corresponding to the protein (280 ran), the linker (303 ran) and the cytotoxin (440 ran) chromophores. Briefly, protein or protein conjugates were exposed to different pH conditions using 1 M HCl and incubated at RT for 20 h before analysis via scanning UV/Vis spectrophotometry between the wavelengths of 260 and 750 nm.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term "acyl" refers to the functional group which results from removal of a hydroxyl group from a carboxylic acid. Examples of acyl groups include formyl, acetyl, benzoyl, pivaloyl, pentanoyl, propionyl, butanoyl, levulinoyl, biphenylcarbonyl, 2-phenylacetyl and the like. C_1-2, C_1-3, Ci_-4, C_1-6, C_1-8, Ci_-10 and Ci-I₂ acyl groups refer to groups having 1 to 2, 1 to 3, 1 to 4, 1 to 6, 1 to 8, 1 to 10 and q to 12 carbon atoms, respectively.

As used herein, the term "acylamino" refers to an "acyl" group wherein the "acyl" group is in turn attached through the nitrogen atom of an amino group. The nitrogen atom may itself be substituted with an "alkyl" or "aryl" group. Examples of a "acylamino" include hexylcarbonylamino, cyclopentylcarbonyl-amino^ethyl), benzamido, 4-chlorobenzamido acetamido, propylcarbonylamino, 2-chloroacetamido, methylcarbonylamino(phenyl), biphenylcarbonylamino, naphthylcarbonylamino and the like.

As used herein, the term "acylaryl" refers to an acyl group attached to an aryl group. An example of an "acylaryl" group is a phenacyl group. Such a group is attached via the acyl group.

As used herein, the term "acylthio" refers to an "acyl" group wherein the "acyl" group is in turn attached through a sulfur atom. Examples of "acylthio" groups include hexylcarbonylthio, cyclopentylcarbonylthio, benzoylthio, 4-chlorobenzoylthio acetylthio, propylcarbonylthio, 2-chloroacetylthio, biphenylcarbonylthio, naphthylcarbonylthio and the like.

As used herein, the term "acyloxy" refers to an "acyl" group wherein the "acyl" group is in turn attached through an oxygen atom. Examples of "acyloxy" groups include hexylcarbonyloxy, cyclopentylcarbonyloxy, benzoyloxy, 4-chlorobenzoyloxy, acetyloxy, propylcarbonyloxy, 2-chloroacetyloxy, biphenylcarbonyloxy, naphthylcarbonyloxy and the like.

As used herein, the term "alkenyl" refers to groups formed from straight chain, branched or cyclic alkenes (cycloalkenes). Examples of alkenyl include allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2 -butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl,

1-hexenyl, 3-hexenyl, cyclohexenyl, 1,3-butadienyl, l-4,pentadienyl, 1,3-cyclopentadienyl,

1,3-hexadienyl, 1 ,4-hexadienyl, 1,3-cyclohexadienyl and 1,4-cyclohexadienyl. C_2-4, C_2-6,

C_2-8 and C_2-I0 alkenyl groups refer to groups having 2 to 4, 2 to 6, 2 to 8 and 2 to 10 carbon atoms, respectively.

As used herein, the term "alkenylaryl" refers to groups formed from straight chain, branched alkenes attached to an aromatic ring. Examples of alkenylaryl include propenylphenyl and styrenyl.

As used herein, the term "alkenylheteroaryl" refers to groups formed from straight chain, branched alkenes attached to a heteroaryl ring. Examples of alkenylheteroaryl include vinylpyridinyl and propenylpyridyl.

As used herein, the terms "alkoxy" and "alkyloxy" refer to straight chain or branched alkoxy groups having from 1 to 6 carbon atoms. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, cyclohexyloxy, and the different butoxy isomers. C]_-4, Ci_-8, Cj_-6 and Ci_-I0 alkoxy groups, refer to groups having 1 to 4, 1 to 6, 1 to 8, and 1 to 10 carbon atoms, respectively.

The terms "alkoxycarbonyl" or "alkyloxycarbonyl" refer to an alkoxy group attached through a carbonyl group. Examples of "alkoxycarbonyl" groups include methylformate, ethylformate, cyclopentylformate and the like. The terms "alkoxyalkyl" or "alkyloxyalkyl" refer to an alkoxy group attached through an alkyl group. Examples of "alkoxyalkyl" groups include ethoxymethyl, ethoxyethyl, propoxymethyl, methoxymethyl and the like.

As used herein, the term "alkyl", as used alone or as part of a group such as "di(alkyl)amino", refers to straight chain, branched or cyclic alkyl groups. Examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, octyl, cyclopentyl and cyclohexyl. When preceded by, C_1-4, Ci_-6, Ci_-8 and C_1-I0 (for example C_1-4alkyl), the term refers to alkyl groups having 1 to 4, 1 to 6, 1 to 8, and 1 to 10 carbon atoms, respectively.

The terms "alkylamino" or "dialkylamino" refer to one or two alkyl radicals attached to a nitrogen atom: vV-methylamino and

are examples. Similarly, term "Ci_-6alkylamino" refers to a "Ci_-6alkyl" group attached through an amine bridge. Likewise, C_1-4 Ci_-8 and Ci_-I0 alkylamino refers to "Ci_-4alkyl", "C_1-8alkyl", "C_Moalkyl" groups respectively, attached through an amine bridge. Examples of "alkylamino" include methylamino, ethylamino, butylamino and the like. In addition, the term "di(alkyl)amino" refers to two "alkyl" groups having the indicated number of carbon atoms attached through an amine bridge. Examples of "di(alkyl)amino" include diethylamino, N-propyl-N'- hexylamino, iV-cyclopentyl-N'-propylamino and the like.

As used herein, the term "alkylaryl" refers to groups formed from straight chain, branched alkyl groups attached to an aromatic ring. Such groups are attached via the alkyl group. Examples of alkylaryl groups include methylphenyl (benzyl), ethylphenyl, methylnaphthyl, propylphenyl and isopropylphenyl.

As used herein, the term "alkylheteroaryl", refers to an alkyl group attached to a heteroaryl ring as described herein, Examples of alkylheteroaryl include methylpyridinyl, ethylpyridinyl and isopropylpyridinyl, As used herein, the term "alkylsulfonyl" refers to an "alkyl" group attached through a sulfonyl bridge. Examples of "alkylsulfonyl" groups include methylsulfonyl, ethylsulfonyl, isopropylsulfonyl and the like.

As used herein, the term "alkylsulfonamido" refers to an "alkylsulfonyl" group wherein the "alkylsulfonyl" group is in turn attached through the nitrogen atom of an amino group. Examples of "alkylsulfonamido" groups include methylsulfonamido, ethylsulfonamido and the like.

As used herein, the term "alkylthio" refers to straight chain or branched alkyl groups having from 1 to 10 carbon atoms attached through a sulfur bridge. Examples of C_1- loalkoxy include methylthio ethylthio, n-propylthio, isopropylthio, cyclohexylthio, different butylthio isomers and the like. Similarly, C_1-4, Cj_-6 and C_1-8 alkylthio refer to groups having 1 to 4, 1 to 6, and 1 to 8 carbon atoms, respectively.

As used herein, the term "alkynyl" refers to groups formed from straight chain or branched groups as previously defined which contain a triple bond. Examples of alkynyl groups include 2,3-propynyl and 2,3- or 3,4-butynyl. C_2-4, C_2-6, C_2-8 and C_2-10 alkynyl groups refer to groups having 2 to 4, 2 to 6, 2 to 8 and 2 to 10 carbon atoms, respectively.

As used herein, the term "alkynylaryl" refers to groups formed from straight chain or branched alkynes attached to an aromatic ring. Examples of "alkynylaryl" include propargyl benzene (propynylphenyl), and ethynylphenyl.

As used herein, the term "alkynylheteroaryl" refers to groups formed from straight chain or branched alkanes attached to a heteroaryl ring. Examples of alkynylheteroaryl include propynylpyridinyl, and ethynylpyridinyl.

As used herein, the term "amino acid" includes natural and synthetic amino acids and derivatives thereof. As used herein, the term "aryl" refers to optionally substituted monocyclic, bicyclic, and biaryl carbocyclic aromatic groups, of 6 to 14 carbon atoms, covalently attached at any ring position capable of forming a stable covalent bond, certain preferred points of attachment being apparent to those skilled in the art. Examples of monocyclic aromatic groups include phenyl, toluyl, xylyl and the like, each of which may be optionally substituted with Cj_-6acyl, Ci_-6alkyl, Ci_-6alkoxy, Ci_-6alkoxycarbonyl, C_2-6alkenyl, C_2-

₆alkynyl, C_1-6alkylsulfonyl, arylsulfonyl, C_1-6alkylsulfonamido, arylsulfonamido, halo, hydroxy, mercapto, trifluoromethyl, carbamoyl, amino, azido, nitro, cyano, C_1-6alkylamino or di(C₁-₆alkyl)amino. Examples of bicyclic aromatic groups include 1-naphthyl, 2- naphthyl, indenyl and the like, each of which may be optionally substituted with C_1-6acyl, Ci_-6alkyl, C).₆alkoxy, Ci_-6alkoxycarbonyl, C_2-6alkenyl, C_2-6alkynyl, C_1-6alkylsulfonyl, arylsulfonyl, Ci._δalkylsulfonamido, arylsulfonamido, halo, hydroxy, mercapto, trifluoromethyl, carbamoyl, amino, azido, nitro, cyano, Cj-βalkylamino or CU(C₁. ₆alkyl)amino. Examples of biaryl aromatic groups include biphenyl, fluorenyl and the like, each of which may be optionally substituted with C_1-6acyl, C_1-6alkyl, C_1-6alkoxy, C_{2- 6}alkenyl, C₂.₆alkynyl, C_1-6alkylsulfonyl, arylsulfonyl, C_1-6alkylsulfonamido, arylsulfonamido, halo, hydroxy, mercapto, trifluoromethyl, carbamoyl, amino, azido, nitro, cyano, Ci_₆alkylamino or di(Ci_-6alkyl)amino.

As used herein the term "arylalkyl" refers to an aromatic ring to which is attached a straight chain/branched alkyl group. Such groups are attached via the aryl group (cf alkylaryl). Examples of arylalkyl groups include phenylmethyl, phenethyl and the like.

As used herein, the term "arylalkyloxy" refers to an "arylalkyl" group attached through an oxygen bridge. Examples of "arylCi_-6alkyloxy" groups are benzyloxy, phenethyloxy, naphthylmethyleneoxy, biphenylmethyleneoxy and the like.

As used herein, the term "arylsulfonyl" refers to an "aryl" group attached through a sulfonyl bridge. Examples of "arylsulfonyl" groups include phenylsulfonyl, 4- methylphenylsulfonyl, 3-fluorophenylsulfonyl, 4-nitrophenylsulfonyl, naphthylsulfonyl, biphenylsulfonyl and the like. As used herein, the term "arylsulfonamido" refers to an "arylsulfonyl" group wherein the "arylsulfonyl" is in turn attached through the nitrogen atom of an amino group. Examples of "arylsulfonamido" groups include phenylsulfonamido, 4-methylphenylsulfonamido, 3- fluorophenylsulfonamido, 4-nitrophenylsulfonamido, naphthylsulfonamido, biphenylsulfonamido and the like.

As used herein, the term "aryloxy" refers to an "aryl" group attached through an oxygen bridge. Examples of aryloxy substituents include phenoxy, biphenyloxy, naphthyloxy and the like.

The term "aryloxycarbonyl" refers to an aryloxy group attached through a carbonyl group. Examples of "aryloxycarbonyl" groups include naphthyloxycarbonyl, and pheny loxycarbonyl .

The term "arylalkyloxy carbonyl" refers to an arylalkyloxy group attached through a carbonyl group. Examples of "arylalkyloxy carbonyl" groups include benzyloxycarbonyl and fluorenylmethyloxycarbonyl.

As used herein, the term "arylthio" refers to an "aryl" group attached through a sulfur bridge. Examples of arylthio include phenylthio, naphthylthio and the like.

The term "counter ion" refers to an ion, the presence of which allows the formation of an overall neutrally charged species with respect to a charged moiety. Alkali or alkaline earth metals or organic amines are examples of chemical species that may be used as counter ions for anionic moieties such as carboxylates, phosphates, sulfonates and the like. Examples of metals include sodium, potassium, magnesium, calcium and the like. Examples of suitable amines that can be used as counter ions include N₁N'- dibenzylethylenediamine, diethanolamine, ethylenediamine, JV-methylglucamide and the like. A "cell-targeting moiety" is any chemical moiety that specifically binds to, or interacts with, a particular cell or tissue type. A cell targeting moiety may, for example, be a member of a specific binding pair. The selection of cell targeting moiety will depend upon the particular cell or tissue to be targeted. For example, the targeting moiety may be a "synthetic" or a "native" ligand that interacts with a cell surface receptor. Alternatively, the targeting moiety may be a "native" or "modified" receptor that interacts with a cell surface ligand. As such, the targeting moiety may be selected from antibodies, proteins (including glycoproteins, lipoproteins and peptides), steroids, hormones, carbohydrates, lipids, glycolipids, synthetic analogues of a naturally occurring ligands (such as glyco- and peptidomimetics), or any another molecule or ligand that shows specificity for a particular cell or tissue type.

The term "cytotoxic" is descriptive of substances that are directly toxic to cells, preventing their reproduction or growth. It shall be understood that the term "cytostatic", is included within the scope of substances that inhibit, hinder or suppress cellular growth and multiplication.

As used herein the term "derivatised cytotoxic isatin moiety" and variations such as "derivatised isatin moiety" refers to derivatives of isatin (indole-2,3-diones)₅ for example, substituted and functionalised isatins, that have cytotoxic properties. An isatin derivative may generally be considered a chemical derivative that comprises an indole-2,3-dione substructure, wherein one or both of the indole ring system, or the carbonyl functionalities, is further substituted, modified or functionalised. For example, selected atoms of the indole ring substructure may be substituted for other atoms, for example (and as chemically appropriate), carbon atoms of the phenyl ring may be substituted with nitrogen atoms. Other examples of the derivatisation of isatin, include: alkylation or acylation of the nitrogen atom of the five-membered dihydropyrrolyl-type substructure of the indole ring; substitution of the indole phenyl moiety with suitable substituents; and formation of fused carbo- or heterocyclic ring structures together with atoms of the indole phenyl moiety.

The term "glycoprotein" refers to a glycosylated protein. As used herein, the term "halo", as used alone or as part of a group such as "C_3-6IIaIo alkenyl", refers to fluoro, chloro, bromo and iodo groups.

As used herein, the term "heteroaryl" refers to a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing at least one nitrogen, sulfur or oxygen atom, in which a carbon or nitrogen atom is the point of attachment. The rings or ring systems generally include 1 to 9 carbon atoms in addition to the heteroatom(s) and may be aromatic or pseudoaromatic. Examples of 5-membered "heteroaryl" groups include pyrrolyl, furyl, thienyl, pyrolidinyl, imidazolyl, oxazolyl, triazolyl, tetrazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl and examples of 6-membered monocyclic nitrogen containing heterocycles include pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl and triazinyl, piperadinyl, piperazinyl, morpholinyl, each of which may be optionally substituted with Ci_-6acyl, C_1-6alkyl, Ci_-6alkoxy, C_2-6alkenyl, C_2-6alkynyl, Ci_-6alkylsulfonyl, arylsulfonyl, C_1-6alkylsulfonamido, arylsulfonamido, halo, hydroxy, mercapto, trifluoromethyl, carbamoyl, amino, azido, nitro, cyano, Ci.₆alkylamino or di(Ci-₆alkyl)amino. Examples of 9- and 10-membered nitrogen containing bicyclic heterocycles include indolyl, benzoxazolyl, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, indazolyl, benzimidazolyl, purinyl, pteridinyl, indolizinyl, isoquinolyl, isoquinolinyl, quinolinyl, quinoxalinyl, cinnolinyl, phthalazinyl, quinazolinyl, benzotriazinyl and the like, each of which may be optionally substituted with Ci_-6acyl, C_1-6alkyl, C_1-6alkoxy, C_2-6alkenyl, C_2-6alkynyl, C_1-6alkylsulfonyl, Ci_-6arylsulfonyl, Ci_-6alkylsulfonamido, C_1-6arylsulfonamido, halo, hydroxy, mercapto, trifluoromethyl, carbamoyl, amino, azido, nitro, cyano, Ci.₆alkylamino or di(C_1-6alkyl)amino. Examples of heteroaryl groups include (optionally substituted) imidazoles, isoxazoles, isothiazoles, 1,3,4-oxadiazoles, 1,3,4-thiadiazoles, 1 ,2,4-oxadiazoles, 1 ,2,4-thiadiazoles, oxazoles, thiazoles, pyridines, pyridazines, pyrimidines, pyrazines, 1,2,4-triazines, 1,3,5-triazines, benzoxazoles, benzothiazoles, benzisoxazoles, benzisothiazoles, quinolines and quinoxalines. As used herein the term "heterocycloalkyl" refers to a cycloalkyl ring substituted with a heteroatom such as oxygen, nitrogen or sulfur. Examples of heterocycloalkyl groups include: aziridines, epoxides, episulfides, azetidines, oxetanes, furanoses, piperidines, piperazines, pyranoses, morpholines and the like.

As used herein, the term "heteroaryloxy", refers to a heteroaryl ring as described hereinabove, bonded through an oxygen atom.

As used herein, the term "lectin" refers to a type of receptor protein that specifically interact with sugar molecules (carbohydrates) without modifying them. These proteins recognize and bind specifically to carbohydrate structures and are classified by which sugar they recognize. Because of the specificity that each lectin has toward a particular carbohydrate structure, even oligosaccharides with identical sugar compositions can be distinguished or separated. Some lectins will bind only to structures with mannose or glucose residues, while others may recognize only galactose residues. Some lectins require that the particular sugar be in a terminal non-reducing position in the oligosaccharide, while others can bind to sugars within the oligosaccharide chain.

As used herein, the term "optionally substituted" means that a group may include one or more substituents. In some instances, the substituent may be selected to improve certain physico-chemical properties such as solubility under physiological conditions. Examples of optional substituents include halo,

C_2-4alkenyl, C_2-4alkynyl, C].₄alkoxy, haloCi_-4alkyl, hydroxyCi-4alkyl, Ci_-4alkoxy, Ci_-7acyl, Ci_-7acylthio, C_1-7acyloxy, hydroxy, aryl, amino, alkoxyalkyl, azido, nitro, nitroso, cyano, carbamoyl, trifiuoromethyl, mercapto,, acylamino, aryloxy, formyl, carbamoyl, alkylsulfonyl, arylsulfonyl, alkylsulfonamido, arylsulfonamido, alkylamino, di(alkyl)amino, and alkoxycarbonyl.

As used herein the term "PAI-2" refers to a member of the serine protease inhibitor

(serpin) protein family. Serpins interact with their target proteases through an exposed peptide loop, known as the reactive center loop. This acts as a "bait" for the active site of the protease, leading to formation of SDS-stable, equimolar, covalent complexes. PAI-2 is an efficient inhibitor of active but not inactive urokinase plasminogen activator (uPA), whether the latter is in solution or bound to its receptor uPAR.

"Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to oligomers and polymers of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally- occurring amino acid polymers.

As used herein, the term "selectivity" refers to the degree by which a drug interaction produces the desired result, and is relatively free of adverse side effects, or similarly the extent to which a compound hits its intended target relative to unintended targets.

As used herein, the term "spacer" refers to a functional group including rings and chains, which provides a chemical linkage between two chemical moieties and which spaces those moieties from each other or provides a bridge between those moieties. Another name for a spacer is a linker.

As used herein the term "specificity" refers to an index of the degree to which an association between two molecular units or assemblies may be considered unique; in many applications it is judged by the magnitude of an effect (enzyme-substrate, ligand-receptor interaction or drug action). Specificity may, for example, be ligand/receptor concentration dependent, or binding avidity/affinity dependent.

The success of any targeting strategy depends both on the selectivity of the targeting molecule as well as the efficiency of the delivered cytotoxin (pro-drug). Covalent attachment of a cytotoxin to a targeting molecule via a modified prodrug approach, allows for specific delivery of the cytotoxin to targeted cells (for example to cancer specific markers/antigens/receptors) which maximises drug effectiveness whilst minimising side effects. Suitably, for successful delivery of a drug to a targeted site, the cytotoxic moiety that is attached to the targeting moiety does not dominate the transport properties of the complex. That is, the specificity of the targeting moiety interacting with a target is not overshadowed by properties of the cytotoxic drug moiety. Furthermore, once a cytotoxin-targeting moiety complex or conjugate has been successfully internalised into, for example, a tumour cell, any masked biological properties of the cytotoxin need to be revealed for successful completion. In specific embodiments, the use of acid sensitive linkages to link cytotoxins to targeting moieties provides for a successful targeting strategy, particularly when the cytotoxin-targeting complex enters a cell via receptor mediated endocytosis. Cleavage of the acid labile linkage in the acidic environment of the lysosomes or endosomes, once the cytotoxic-targeting complex has been internalised, can provide timely release of a cytotoxin, from the complex, to the cell.

Derivatised

Cytotoxic lsatin |_1— Spacer -L³

Moiety

(a)

Derivatised

Cytotoxic lsatin _. L 1¹J- Spacer Cell-Targeting

Moiety -L³ + L⁴- Moiety

(b)

(C)

Derivatised

Cytotoxic lsatin Cell-Targeting

Spacer 1-1 2_

Moiety Moiety

Scheme 1 In Scheme 1 above, a schematic is shown wherein the steps in the preparation of a cytotoxic targeting moiety complex and the ultimate release of a cytotoxin from that complex are depicted. For example in step (a), a spacer is conjugated to a cytotoxic moiety to a derivatised cytotoxic isatin moiety to provide an isatin-spacer conjugate. In step (b), the isatin-spacer conjugate is then further conjugated to a targeting moiety. The cytotoxic isatin-targeting complex can then be administered, for example intravenously, to cells or tissue in need of cytotoxic treatment such as a tumour. Due to the specificity of the targeting moiety, it will preferentially interact with, or bind to, cell types for which the targeting moiety is specific.

Generally, intracellular delivery into the endosome/lysosome compartments of cells is suitable for linkers that are either acid labile (due to the low pH of these environments), that are subject to proteases or esterases also found in lysosomes, or that are susceptible to cleavage in a reducing environment.

The spacer which provides a chemical bridge between the cytotoxic isatin moiety and the cell-targeting moiety, may therefore be cleaved by biological or chemical processes, or by other physiological conditions, so as to chemically release the cytotoxic isatin moiety from the cytotoxin-targeting conjugate at or in the targeted cells or tissue.

Acid labile functional groups that could be used as the cleavable functional group connecting the cytotoxic moiety to the spacer (and therefore ultimately the targeting moiety) include, for example: imines, hydrazides, hydrazones, acetals, esters and cis- aconitic functionalised linkers. Alternatively, the reducing state of the intracellular environment compared to the extracellular environment would also allow, for example, for disulfide-based antibody-drug conjugates.

Cleavage of acid labile linkers may occur as follows: after interaction with the target cell, the cytotoxic-targeting conjugate complex is internalised and passes into endosomes where it is exposed to a more acidic environment which results in cleavage of the acid labile .-.

- 32 -

bond. For example, the complex may interact with a metastatic breast cancer cell, be therein internalised via endocytosis, at which time in the acidic environment of the lysosomes, the linkage between the cytotoxic moiety and the targeting moiety commences cleaving thereby releasing the cytotoxin.

There may be one cytotoxic moiety for every targeting moiety, or there may be multiple cytotoxic moieties for each targeting moiety. For example: 2, 3, 4, 5, 6, 7, 8, 9, 10 11, or 12 cytotoxic moieties may be coupled via linkers to a single targeting moiety. An example of this is a polypeptide, that is itself a targeting moiety, and that has seven lysine residues. The side chains of these lysine residues can be coupled to cytotoxin-spacer conjugates to provide a cytotoxin-spacer-targeting moiety adduct, that has seven cytotoxic moieties with respect to the targeting moiety.

Examples of protein targeting moieties include: PAI-2 (targets receptor bound uPA), transferrin (targets transferrin receptor and facilitates cellular iron uptake), and Herceptin (humanised monoclonal antibody that recognises ErbB2 receptor).

Examples of potential cancer markers include those described by: Weigelt et al {Nature Reviews, 2005, 5, 591), Ranson et al., (2002); Ranson and Andronicos {Frontiers in Bioscience, 2003, 8, s294); and Qian et al., Pharmacological Reviews, 2002, 54, 561). Suitable examples of markers include urokinase plasminogen activator (uPA), HER2 (ErbB2), and transferrin receptor.

Potential protein based targeting molecules - based on the recognition of tumour specific markers shown to be upregulated or expressed only by malignant tumour cells or neo- angiogenic cells compared to normal/unstimulated cells — include: antibody targeting molecules described by Schrama et al, {Nature Reviews, 2006, 5, 147); protein/peptide or small molecule inhibitors of receptor bound targets or receptors themselves, for example plasminogen activator inhibitor type 2 (PAI-2) which inhibits cell surface bound uPA, and molecular inhibitors of uPA or uPA receptor binding antagonists to deliver cytotoxins to the cells (see for example: Romer J, Nielsen BS and Ploug M. {Curr Pharm Des. 2004, 10, 2359-2376; Ertongur S, Lang S, Mack B, Wosikowski K₅ Muehlenweg B and Gires O. Int J Cancer., 2004, 110, 815-824; wilex molecules, Bruncko et al., bioorganic and Medicinal Chemistry Letters, 2005, 15, 93); transferrin (Qian et al., Pharmacological Reviews, 2002, 54, 561).

Potential carbohydrate based targeting molecules are identifiable by modifications such as neoglycosylation, up-regulation and/or over-expression of carbohydrate binding molecules such as lectins, glycosidases and glycosyltransferases. For example, the carbohydrate based Tn antigen (GalNAcα-0-Ser/Thr) is a specific marker of many human carcinomas. The Tn antigen, expressed in an unmasked form in about 90% of human carcinomas, is one of the most specific human cancer-associated structures. A direct link has been demonstrated between carcinoma aggressiveness (e.g. extent of tissue spread and vessel invasion) and the cell-surface density of the Tn antigen. Several proteins have been described that specifically recognize the Tn determinant, including monoclonal antibodies, plant lectins, human macrophage lectins, and glycosyltransferases. For example, SSL is an acidic (pi 5.5), 60-61 -kDa dimeric glycoprotein composed of apparently identical subunits linked by a single disulfide bond. Lectin SSL is isolated from Salvia sclarea seeds. Moreover, non-mammalian carbohydrate epitopes, for example solarium glycosides such as solasodines, have shown specificity for tumour lectins {Cancer Letters, 1990, 55, 209).

Other examples of potential targeting molecules, include lipids, steroids, hormones, folates and nucleotide phosphates including structural analogues and mimetics of these molecules.

An example of the use of the protein targeting moiety transferrin is as follows: a toxin- spacer-transferrin conjugate (Scheme 2) formed from the reaction of a toxin-spacer with transferrin (step (i), Scheme 2), is upon in vivo or in vitro administration to cells, rapidly internalised {via receptor-mediated endocytosis) into cellular endosomes. The letter "T" refers to the transferrin and the letter "n" refers to the plurality of amino groups available for conjugation and taking part in conjugation with the toxin-spacer unit. The transferrin receptor: toxin-spacer-transferrin complex is passed onto late endosomes in which the pH is mildly acidic (pH ~ 6). Here the acid labile linkage may release the cytotoxin from transferrin (step (ii), Scheme 2). Due to lipophilic character, the drug may then diffuse across the endosomal membrane into the cell cytosol leading to cell death. Diffusion across the plasma membrane may also occur, resulting in death of adjacent tumour cells. From the endosomes, transferrin and its receptor are recycled back to the cell surface where transferrin dissociates in the neutral environment but can participate in another round of endocystosis.

Transferrin-spacer

Toxin-spacer Toxin-spacer-transferrin-conjugate

Cytotoxin

Scheme 2

A target-specific protein, peptide or polypeptide may be engineered to have a non-essential binding region or terminal portion, modified to have a high density of amino acids which are suitable for conjugating to cytotoxin-spacer conjugates.

Spacers carrying cytotoxic moieties may be conjugated, for example, to amino acid side chain functional groups of a protein which is in itself a targeting moiety. Examples of amino acid side chains suitable for this purpose are lysine, glutamic acid, aspartic acid, cysteine, serine, threonine and arginine side chains.

Referring to Scheme 3, a variety of bases can be used for anion formation at the ring nitrogen. For example: NaH, Cs₂CO₃, K₂CO₃, KH, Na₂CO₃ and alkoxides may be used. A preferred base for the synthesis of the iV-alkylisatins is K₂CO₃. The alkylation agent is typically of the form R⁶ -X, wherein X is a leaving group. Preferred leaving groups include halides - particularly chlorides and bromides. Other suitable leaving groups are known to those skilled in the art.

Scheme 3

Referring to Scheme 4, the phenyl ring of isatin can be modified by substitution reactions such as halogenation, for example with bromine in ethanol. Other methods of derivatising the phenyl ring of isatins are known to the art and can be found, for example, in the J. Braz. Chem. Soc, 2001, 12(3), 273.

Scheme 4

Referring to Scheme 5, isatin derivatives can be further derivatised by reaction with amines to form inline type linkages. Methods of forming linkages containing the imine structural feature - such as imines, hydrazines and hydrazones are well known to those skilled in the art. .

- 36 -

Scheme 5

The compound of formula I may be in the form of salts. The salts of the compound of formula I are preferably pharmaceutically acceptable, but it will be appreciated that non- pharmaceutically acceptable salts also fall within the scope of the present invention, since these may be useful as synthetic intermediates. The pharmaceutically acceptable salts may include conventional non-toxic salts or quartenary ammonium salts of these compounds, which may be formed, e.g. from organic or inorganic acids or bases. Examples of such acid addition salts include, but are not limited to, those formed with pharmaceutically acceptable acids such as formic, acetic, propionic, citric, lactic, methanesulfonic, toluenesulfonic, benzenesulfonic, ascorbic, hydrochloric, orthophosphoric, sulfuric and hydrobromic acids. Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium magnesium, ammonium and alkylammonium. Also, basic nitrogen-containing groups may be quaternised with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

The compounds of the invention may be in crystalline form or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.

It will be appreciated that some derivatives of the compound of formula I may have an asymmetric centre, and therefore are capable of existing in more than one stereoisomeric form. The invention extends to each of these forms individually and to mixtures thereof, including racemates. The isomers may be separated conventionally by chromatographic methods or using a resolving agent. Alternatively, the individual isomers may be prepared by asymmetric synthesis using chiral intermediates.

While it is possible that, for use in therapy, a compound of the invention may be administered as the neat chemical, it is preferable to present the active ingredient as a pharmaceutical formulation.

The invention thus further provides pharmaceutical formulations comprising a compound of the invention or a pharmaceutically acceptable salt or derivative thereof together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Pharmaceutical formulations include those suitable for oral, nasal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The compounds of the invention, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids, such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use; or in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. The compounds of the present invention can be administrated in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt of a compound of the invention. For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solutions or in physiologically acceptable buffer solutions.

The compounds according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

When desired, formulations adapted to give sustained release of the active ingredient may be employed. The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. An effective dose is a dosage sufficient to provide for treatment of a condition, and may comprise one or more unit doses. An effective dose will vary depending on the patient and/or the condition. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and solutions in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

The invention will now be described with reference to the following examples which illustrate some preferred aspects of the present invention. However, it is to be understood that the particularity of the following description of the invention is not to supersede the generality of the preceding description of the invention.

EXAMPLES Examples 1 to 34

General: Reactions were carried out using dried glassware and under an atmosphere of nitrogen. All solvents were AR grade except dichloromethane (DCM) which was LR grade and distilled before use. The term petroleum spirit refers to petroleum spirit with the boiling range of 40-60 ⁰C. When necessary, the purification of solvents and starting materials was carried out using standard procedures. Reactions were monitored using thin layer chromatography (TLC) on aluminium backed pre-coated silica gel 60 F₂₅₄ plates (E. Merck). The iV-alkylisatins are highly coloured and can usually be clearly seen on a tic plate; colourless compounds were detected using UV light and/or iodine vapour. Flash chromatography (Still, W. C; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923) was carried out using silica gel 60 (230 - 400 mesh) with the solvent system indicated in the individual procedures. All solvent ratios are quoted as vol/vol. The ¹H and ¹³C NMR spectra were acquired at 300 and 75 MHz on a Varian Unity-300 spectrometer and 500 and 126 MHz on a Varian Inova-500 spectrometer with a probe temperature of 298 K. The NMR spectra are referenced to the residual solvent peak in the solvent stated in the individual procedures. Hydrogen and carbon assignments were made using standard NOE, APT, gCOSY, gHSQC and gHMBC spectroscopic techniques. Low resolution EI mass spectra (LREI-MS) were determined on a Shimadzu QP5050 spectrometer. High resolution EI mass spectra (HREI-MS) were determined on VG Autospec spectrometer operating at 70 e V with a source temperature of 250 ⁰C and were referenced with PFK. Melting points were determined on a Reichert melting point apparatus and are uncorrected. General Method I: N-Alkylation oflsatins:

Alkylation of the isatins was carried out using a general method based on that of Torres (Garden, S. J.; Torres, J. C; da Silva, L. E.; Pinto, A. C. Synth. Commun. 1998, 28, 1679- 1689). The appropriate isatin (1 equiv) was taken up in anhydrous DMF (~ 1 mL per 0.1 mmol of isatin) and cooled on ice with stirring. Solid K₂CO₃ (1.4 equiv) or Cs₂CO₃ (1.4 equiv.) was added in one portion and the dark-coloured suspension was brought to rt and stirred for a further 2 h. The appropriate alkylating agent, typically a halide such as a benzyl halide (1.1 equiv.) and KI (0.2 equiv.) were added and the reaction mixture stirred at 80 °C for 5 - 16 h, until all the isatin starting material had been consumed (tic). The reaction mixture was tipped into HCl (0.5 M) and extracted with ethyl acetate (1 x 50 mL). The ethyl acetate layer was washed with brine and dried over MgSO₄. The solvent was removed and the crude product was purified using flash column chromatography with isocratic elution with DCM unless otherwise stated.

General Method 2: Imine Synthesis:

A general method for the synthesis of the iV-alkylisatin imines was based on the method for the synthesis of isatin imines by Kumar et al (Kumar, R.; Giri, S.; Nizamuddin, J, Agric. Food. Chem,. 1989, 37, 1094). The appropriate isatin (1 equiv) was dissolved in methanol and activated molecular sieves or powdered molecular sieves were added. The appropriate amine (0.95 equiv) was added with a catalytic amount of glacial acetic acid and the mixture refluxed until most of the starting material had been consumed. The mixture was cooled and then filtered and the solid sieves washed with a minimum amount of methanol. The filtrate was concentrated and then left to crystallize. If no precipitate formed overnight, the solvent was removed and the imine purified using flash column chromatography.

Example 1

N-allyl-5, 7-dibromoisatin (1) The product was a bright red solid (102 mg, 45%), mp 103 - 105 °C, R_f 0.45 (DCM, silica). ¹H NMR (CDCl₃, 500 MHz) δ 4.79 (d, J= 5 Hz, 2H, CH₂), 5.23 (d, J= 17 Hz, IH,

f CE_cisH_trms), 5.26 (d, J= IO Hz, IH, C#_efeH_trans), 5.96 (ddt, J= 5, 10, 17 Hz, IH, CH), 7.70 (d, J = 2 Hz, IH, H4), 7.86 (d, J = 2 Hz, IH, H6). ¹³C NMR (CDCl₃, 126 MHz) δ 43.1, 104.9, 116.8, 117.6, 121.1, 127.3, 131.4, 145.1, 146.6, 157.7, 181.2. LREI-MS m/z 343/345/347 ([M]⁺/([M+2]⁺/[M+4]⁺) (Garden, S. J.; Torres, J. C; da Silva, L. E.; Pinto, A. C. Synth Commun. 1998, 28, 1679-1689).

Example 2

5, 7-Dibromo-N-(p-methoxybenzyl)isatin (2)

The product was a bright red-orange solid (174 mg, 62%), mp 166 - 168 °C, R_f 0.49 (DCM, silica). ¹H NMR (CDCl₃, 300 MHz) δ 3.76 (s, 3H, OCH₃), 5.33 (s, 2H, benzylic

CH₂), 6.83 (d, J= 8 Hz, 2H, phenyl ArH), 7.18 (d, J= 8 Hz, 2H, phenyl ArH), 7.68 (d, J =

2 Hz, IH, H4), 7.79 (d, J= 2 Hz, IH, H6). ¹³C NMR (CDCl₃, 75 MHz) δ 44.1, 55.2, 105.2,

114.2, 117.0, 121.4, 127.4, 127.5, 128.0, 145.2, 146.7, 158.3, 159.1, 181.3. LREI-MS m/z 423/425/427 ([M]⁺/[M+2]⁺/[M+4]⁺); HREI-MS m/z calcd for [M+2]⁺ Ci₉Hi₇ ⁷⁹Br⁸¹BrNO₂:424.9085, found: 424.9077.

Example 3

5, 7-Dibromo-N-(m-methoxybenzyl)isatin (3)

The product was a bright orange-red solid (237 mg, 85%), mp 110 - 112 °C, R_f 0.32 (DCM, silica) ¹H NMR (CDCl₃, 500 MHz) δ 3.77 (s, 3H, OCH₃), 5.37 (s, 2H, CH₂), 6.77 (s, IH, phenyl ArH), 6.80-6.82 (overlapping m, 2H, phenyl ArH x 2), 7.24 (d, J = 8 Hz, IH, phenyl ArH), 7.71 (d, J= 2 Hz, IH, isatin H) 7.81(d, J= 2 Hz₅ IH, isatin H). ¹³C NMR (CDCl₃, 126 MHz) δ 44.6, 55.2, 105.3, 112.6(2C), 117.2, 118.5, 121.4, 127.5, 129.9,

137.3, 145.3, 146.7, 158.2, 159.9, 181.2. LREI-MS m/z 423/425/427 ([M]⁺/[M+2]⁺/[M+4]⁺); HREI-MS m/z calcd for [M+2]⁺ C₁₉H_π ⁷⁹Br⁸¹BrNO₂:424.9085, found: 424.9079.

Example 4

N-benzyl-5, 7-dibromoisatin (4) The product was a bright red solid (209 mg, 80%) mp 150 - 152 °C (lit.149 - 150 °C: Pummerer, R.; Meininger, F. Ann. (1954), 590, 173), R_f 0.47 (DCM, silica). ¹H NMR (CDCl₃, 300 MHz) δ 5.40 (s, 2H, CH₂), 7.18-7.36 (overlapping m, 5H, phenyl ArH), 7.69 (d, J = 2 Hz, IH, H4), 7.79 (d, J = 2 Hz, IH, H6). ¹³C NMR (CDCl₃, 75MHz) δ 44.6, 105.2, 117.1, 121.3, 126.3, 127.4, 127.7, 128.8, 135.6, 145.2, 146.6, 158.2, 181.2. LREI- MS m/z 393/395/397 ([M]⁺/[M+2]⁺/([M+4]⁺).

Example 5

5, 7-Dibromo-N-(p-bromobenzyl)isatin (S)

The product was a bright orange solid (221 mg, 71%) mp 160-161 ⁰C, R_f 0.50 (DCM, silica). ¹H NMR (CDCl₃, 300 MHz) δ 5.31 (s, 2H, CH₂), 7.11 (d, J = 8 Hz, 2H, phenyl ArH), 7.41 (d, J= 8 Hz, 2H, phenyl ArH), 7.66 (d, J= 2 Hz, IH, isatin ArH), 7.77 (d, J= 2 Hz, IH, isatin ArH). ¹³C NMR (CDCl₃, 75 MHz) δ 44.1, 105.0, 117.2, 121.3, 121.6, 127.5, 128.1, 131.8, 134.7, 145.1, 146.2, 158.1, 180.9. LREI-MS m/z 471/473/475/477 ([M]^"7[M+2]⁺/[M+4]⁺/[M+6]⁺), HREI-MS m/z calcd for [M]⁺ C₁₅H₈ ⁷⁹Br₃NO₂:470.8105, found:470.8113.

Example 6

5, 7-Dibromo-N-(p-iodobenzyl)isatin (6)

The product was a bright orange solid (257 mg, 75%) mp 141 - 142 ⁰C, R_f 0.45 (DCM, silica). ¹H NMR (CDCl₃, 500 MHz) δ 5.32 (s, 2H, CH₂), 6,99 (d, J = 8 Hz, 2H₅ phenyl ArH)₅ 7.63 (d, J= 8 Hz, 2H, phenyl ArH), 7.69 (d, J= 2 Hz, IH, isatin ArH), 7.79 (d, J= 2 Hz, IH, isatin ArH). ¹³C NMR (CDCl₃, 126 MHz), δ 44.2, 93.2, 105.1, 117.3, 121.3, 127.6, 128.3, 135.4, 137.8, 145.2, 146.3, 158.2, 180.9. LREI-MS m/z 519/521/523 ([M]⁺/[M+2]⁺/[M+4]⁺); HREI-MS m/z calcd for [M+2]⁺ Ci₅H₈ ⁷⁹Br⁸¹BrINO₂:520.7946, found: 520.7959.

Example 7

5, 7-Dibromo-N-(p-chlorobenzyl)isatin (7)

The crude product was purified using flash chromatography with 7:3 DCM/pet spirit. The product was a bright orange solid (253 mg, 89%) mp 159 - 161 ⁰C, R_f 0.66 (DCM, silica). ¹H NMR (CDCl₃, 500 MHz) δ 5.36 (s, 2H, CH₂), 7.18 (d, J= 8 Hz, 2H, H3'), 7.30 (d, J = 8 Hz, 2H, H4'), 7.72 (d, J= 2 Hz, IH, H4), 7.78 (d, J= 2 Hz, IH, H6). ¹³C NMR (CDCl₃, 126 MHz) δ 44.1 (CH₂), 105.0, 117.3 (CS), 121.4, 127.6 (C4), 127.9, 129.0, 133.6, 134.2, 145.3 (C6), 146.4 (C7a), 158.2 (C2), 181.0 (C3). LREI-MS m/z 427/429/431 ([M]⁺/[M+2]⁺/[M+4]⁺), HREI-MS m/z calcd for [M]⁺ C₁₅H₈ ⁷⁹Br⁸¹BrClNO₂:428.8590, found:428.8593.

Example 8

5, 7-Dibromo-N-(o~nitrobenzyl)isatin (8)

The product was a bright orange solid (278 mg, 96%) mp 198 - 200 °C, R_f 0.56 (DCM₅ silica). ¹H NMR (CDCl₃, 500 MHz) δ 5.75 (s, 2H, CH₂), 7.22 (d, J= 8 Hz, IH, H3' or 6'), 7.51 (t, J= 8 Hz, IH, H4' or 5'), 7.61 (t, J= 8 Hz₅ IH, H4' or 5'), 7.77 (d, J= 2 Hz, IH, isatin ArH), 7.81 (d, J= 2 Hz, IH₅ isatin ArH)₅ 8.21 (d, J= 8 Hz, IH, H3' or 6'). ¹³C NMR (CDCl₃, 126 MHz)₅ δ 43.5, 105.1, 117.7, 121.3, 125.9, 126.7, 127.8, 128.6, 132.2, 134.3, 145.4, 146.1, 147.2, 158.2, 180.7. LREI-MS m/z 438/440/442 ([M]⁺/[M+2]⁺/[M+4]⁺); HREI-MS m/z calcd for [M]⁺ Ci₅H₈ ⁷⁹Br₂N₂O₄:437.8851, found: 437.8872.

Example 9

6-Broτno-N~(p-trifluoromethylbenzyl)isatin (9)

The crude product was purified using flash chromatography with 7:3 pet. spirit/EtOAc.

The product was a bright orange solid (41 mg, 24 %), mp 134 - 136 ⁰C, R_f 0.50 (7:3 pet. spirit/EtOAc, silica). ¹H NMR (CDCl₃₅ 500 MHz) δ 4.96 (s, 2H₅ Hl'), 6.92 (d, J = 2 Hz₅ IH, H7), 7.28 (dd₅ J= 2, 8 Hz₅ IH₅ H5), 7.45 (d, J= 8 Hz, 2H, phenyl ArH)₅ 7.48 (d, J= 8 Hz₅ IH₅ H4)₅ 7.63 (d, J = 8 Hz, 2H, phenyl ArH). ¹³C NMR (CDCl₃₅ 126 MHz) δ 43.6 (Cl')₅ 114.1, 116.3, 124.0 (d, ¹JcF = 251 Hz, CF₃), 126.2 (q, ³J_CF = 4 Hz, C4'), 125.6, 127.4, 127.6, 130.7 (d, ²J_CF = 33 Hz₅ C5'), 133.7, 138.1, 151.0, 158.1 (C2), 181.5 (C3). LREI-MS m/z 383/385 ([M]⁺/[M+2]⁺), HREI-MS m/z calcd for [M]⁺ C₁₆H₉ ⁷⁹BrF₃NO₂: 382.9769, found: 382.9771.

Example 10

5, 7-Dibromo-N-(p-nitrobenzyl)isatin (10) The product was a bright orange solid (216 mg, 74%) mp 185 - 187 ⁰C₅ R_f 0.34 (DCM, silica). ¹H NMR (CDCl₃, 300 MHz) δ 5.49 (s, 2H₅ CH₂), 7.42 (d, J = 9 Hz₅ 2H₅ phenyl ArH), 7.76 (d, J= 2 Hz, IH, isatin ArH), 7.84 (d, J= 2 Hz, IH, isatin ArH), 8.21 (d, J= 8 Hz, 2H, phenyl ArH). ¹³C NMR (CDCl₃, 75 MHz) δ 44.4, 104.9, 117.8, 121.4, 124.2,

127.2, 127.9, 143.2, 145.3, 146.0, 147,6, 158.2, 180.1. LREI-MS m/z 438/440/42 ([M]⁺/[M+2]⁺/[M+4]⁺); HREI-MS m/z calcd for [M]⁺ C₁₅H₈Br₂N₂O₄:437.8851, found:437.8846.

Example 11

5, 7-Dibromo-N-(p-tertbutylbenzyl)isatin (11)

The product was a bright red-orange solid (224 mg, 75%) mp 159 - 161 °C, R_f 0.62 (DCM, silica). ¹H NMR (CDCl₃, 500 MHz) δ 1.29 (s, 9H, CH₃), 5.38 (s, 2H, CH₂), 7.18 (d, J= 8 Hz, 2H, phenyl ArH), 7.33 (d, J = 8 Hz, 2H, phenyl ArH), 7.70 (d, J = 2 Hz, IH, isatin ArH) 7.81 (d, J= 2 Hz, IH, isatin ArH). ¹³C NMR (CDCl₃, 126 MHz), the pairs of signals due to rotamers are shown in brackets δ 31.3, 34.5, (44.3, 44.4), 105.3, 117.1, 121.4, 125.7,

126.3, (127.4, 127.5), 132.5, (145.3, 145.4), 146.9, 150.8, 158.3, 181.4. LREI-MS m/z 449/451/453 ([M]⁺/[M+2]⁺/[M+4]⁺); HREI-MS m/z calcd for [M]⁺

C,₉Hi₇ ⁷⁹Br₂NO₂:448.9626, found: 448.9626.

Example 12

5, 7-Dibromo-N-(p-phenylbenzyl)isatin (12) The crude product was purified using flash chromatography using gradient elution (1 :1 DCM/pet spirit to 7:3 DCM/pet.spirit). The product was a bright red solid (118 mg, 38%) mp 159 - 160 ⁰C, R_f 0.47 (7:3 DCM/pet.spirit, silica). ¹H NMR (CDCl₃, 500 MHz) δ 5.45 (s, 2H, CH₂), 7.32 (d, J= 8 Hz, 2H, H3'), overlapping 7.34 - 7.35 (m, IH, H8'), 7.42 (t, J = 8 Hz, 2H, H7'), 7.55 (d, J= 8 Hz, 4H, H2' and 6'), 7.72 (d, J= 2 Hz, IH₅ H4), 7.82 (d, J = 2 Hz₅ IH₅ H6). ¹³C NMR (CDCl₃, 126 MHz) δ 44.6 (CH₂), 105.2, 117.2, 121.4, 126.9 (CH), 127.0 (CH), 127.4 (CH), 127.5 (CH), 127.6 (CH), 128.8 (CH), 134.6, 140.4, 140.7, 145.3 (CH), 146.7, 158.3 (C2), 181.2 (C3). LREI-MS m/z 469/471/473 ([M]⁺/[M+2]⁺/[M+4]⁺), HREI-MS m/z calcd for [M]⁺ C₂iH₁₃ ⁷⁹Br⁸¹BrNO₂:470.9293, found:470.9293. Example 13

5-Iodo-N-(p-methoxybenzyl)isatin (13)

The product was a bright red solid (200 mg, 70%) mp 131 - 133 ⁰C, R_f 0.72 (DCM, silica). ¹H NMR (CDCl₃, 500 MHz) δ 3.75 (s, 3H, OCH₃), 4.82 (s, 2H, CH₂), 6.60 (d, J = 8 Hz, IH, H7), 6.83 (d, J= 9 Hz, 2H, phenyl ArH), 7.22 (d, J= 9 Hz, 2H, phenyl ArH), 7.73 (dd, J= 1, 8 Hz, IH, H6) 7.80 (d, J= 1 Hz, IH, H4), ¹³C NMR (CDCl₃, 126 MHz) δ 43.5, 55.2, 86.0, 113.1, 114.5, 119.1, 125.9, 128.8, 133.6, 146.2, 149.9, 157.1, 159.4, 181.9. EI-MS m/z 393 ([M]⁺); HREI-MS m/z calcd for [M]⁺ Ci₆Hi₂INO₃:392.9862, found:392.9830.

Example 14

5, 7-Dibromo-N-(p-methylbenzyl)isatin (14)

The product was a bright red solid (252 mg, 93%), mp 121 - 123 °C, R_f 0.61 (DCM, silica). ¹H NMR (CDCl₃, 500 MHz) δ 2.30 (s, 3H, CH₃), 5.34 (s, 2H, benzylic CH₂), 7.11 (d, J= 8 Hz, 2H, phenyl ArH), 7.13 (d, J= 8 Hz, 2H, phenyl ArH), 7.66 (d, J= 2 Hz, IH, isatin ArH), 7.77 (d, J= 2 Hz, IH, isatin ArH). ¹³C NMR (CDCl₃, 126 MHz) δ 21.0, 44.3, 105.1, 116.9, 121.3, 126.3, 127.3, 129.3, 132.5, 137.3, 145.1. 146.6, 158.2, 181.2. LREI- MS m/z 407/409/411 ([M]⁺/[M+2]⁺/[M+4]⁺); HREI-MS m/z calcd for [M]⁺ Ci₆H,i⁷⁹Br₂NO₂:406.9157, found: 406.9175.

Example 15

5, 7-Dibromo-N-(p-trifluoromethylbenzyl)isatin (15)

The crude product was purified using flash chromatography with 7:3 DCM/pet spirit. The product was a bright yellow-orange solid (137 mg, 45%) mp 131 - 132 ⁰C, R_f 0.64 (CHCl₃, silica). ¹H NMR (CDCl₃, 500 MHz) δ 5.45 (s, 2H, CH₂), 7.36 (d, J = 8 Hz, 2H, phenyl ArH), 7.59 (d, J= 8 Hz, 2H, phenyl ArH), 7.72 (d, J= 2 Hz, IH, H4), 7.81 (d, J= 2 Hz, IH, H6). ¹³C NMR (CDCl₃, 126 MHz) δ 44.4 (CH₂), 105.0, 117.5, 121.4, 123.9 (d, , J_CF = 269 Hz, CF₃), 125.8 (q, J_CF = 4 Hz, C4'), 126.7, 128.0, 129.7 (d, , J_CF = 33 Hz, C5'), 139.8, 146.2, 158.2 (C2), 180.8 (C3). LREI-MS m/z 461/463/465 ([M]⁺/[M+2j7[M+4]⁺), HREI-MS m/z calcd for [M]⁺ C _I6H₈ ⁷⁹Br₂F₃NO₂: 460.8874, found:460.8866, [M+2]⁺ C₁₆H₈ ⁷⁹Br⁸¹BrF₃NO₂:462.8853, found:462.8842, [M+4]⁺ C₁₆H₈ ⁸¹Br₂F₃NO₂:464.8833, found:464.8823. Example 16

5-Bromo-N-(p-methoxybenzyl)i$atin (16)

This compound was made from technical grade 5-bromoisatin which contained 10% isatin and gave a mixture of two N-alkylated products. The major product was the bright orange- red 5-bromoisatin derivative (138 mg, 20% based on the amount of 5-bromoisatin in the starting material), mp 144 - 146 ⁰C, R_f 0.43 (DCM, silica) ¹H NMR (CDCl₃, 500 MHz) δ 3.77 (s, 3H, OCH₃), 4.84 (s, 2H, benzylic CH₂), 6.69 (d, J = 8 Hz, IH, H7), 6.86 (d, J= 8 Hz, 2H, phenyl ArH), 7.23 (d, J = 8 Hz, 2H, phenyl ArH), 7.57 (dd, J= 2, 8 Hz, IH, H6), 7.68 (d, J= 2 Hz, IH, H4). ¹³C NMR (CDCl₃, 126 MHz) δ 43.6, 55.3, 112.7, 114.3, 116.6, 118.8, 125.9, 128.1, 128.8, 140.4, 149.4, 157.4, 159.5, 182.2. EI-MS m/z 345/347 ([M]⁺/[M+2]⁺); HREI-MS m/z calcd for [M]⁺ C₁₆Hi₂ ⁷⁹BrNO₃:345.0001, found:344.9998. N-(p-methoxybenzyl)isatin: The minor product (16a) from isatin was a bright orange solid (25 mg, 27% based on the amount of isatin in the starting material), mp 169 - 171 ⁰C, R_f 0.31 (DCM, silica). ¹H NMR (CDCl₃, 500 MHz) δ 3.78 (s, 3H, OCH₃), 4.86 (s, 2H, benzylic CH₂), 6.80 (d, J= 8 Hz, IH, H7), 6.86 (d, J= 8 Hz, 2H, phenyl ArH), 7.08 (t, J = 8 Hz, IH, H5), 7.27 (d, J= 8 Hz, 2H, phenyl ArH), 7.48 (dt, J= 1, 8 Hz, IH, H6), 7.59 (d, J = 7 Hz, IH, H4). ¹³C NMR (CDCl₃, 126 MHz) δ 43.5, 55.3, 110.O₅ 114.4,117.7, 123.7, 125.3, 126.4, 128.9, 138.2, 150.7, 158.2, 159.4, 183.3. EI- MS m/z 267 ([M]⁺).

Example 17

4-Bromo-N- (p-methoxybenzyl) isatin (17)

The product was the bright orange solid (142 mg, 65%), mp 173 - 175 ⁰C₅ R_f 0.42 (DCM, silica) ¹H NMR (CDCl₃, 500 MHz) δ 3.77 (s, 3H, OCH₃), 4.85 (s, 2H, benzylic CH₂), 6.76 (d, J= 8 Hz₅ IH, H5 or H7), 6.85 (d, J= 8 Hz, 2H, phenyl ArH)₅ 7.19 (d, J= 8 Hz₅ IH, H5 or H7), 7.24 (d, J= 8 Hz, 2H, phenyl ArH), 7.29 (t, J= 8 Hz, IH₅ H6). ¹³C NMR (CDCl₃, 126 MHz) δ 43.5, 55.2, 109.8, 114.4, 116.4, 121.5, 126.0, 128.4, 128.8, 138.2, 152.2, 157.2, 159.5, 180.6. EI-MS m/z 345/347. ([M]⁺/[M+2]⁺); HREI-MS m/z calcd for [M]⁺ Ci₆Hi₂ ⁷⁹BrNO₃:345.0001, found:344.9998. Example 18

6-Bromo-N-(p~methoxybenzyl)isatin (18)

The product was the bright orange solid (122 mg, 42%), mp 177 - 179 ⁰C, R_f 0.45 (DCM, silica) ¹H NMR (CDCl₃, 500 MHz) δ 3.80 (s, 3H, OCH₃), 4.84 (s, 2H, benzylic CH₂), 6.89 (d, J= 9 Hz, 2H, H4'), 6.98 (d, J= 8 Hz, IH, H7), 7.24 (dd, J= 8, 1 Hz, IH, H5), 7.26 (d, J= 9 Hz, 2H, H3'), 7.45 (d, J= 8 Hz, IH, H4). ¹³C NMR (CDCl₃, 126 MHz) δ 43.7, 55.3, 114.4(7), 114.5(1), 116.3, 125.9, 126.3, 127.0, 128.9, 133.4, 151.5 (C7a), 158.0 (C2), 159.6 (C5'), 182.0 (C3). EI-MS m/z 345/347 ([M]⁺/[M+2]⁺); HREI-MS m/z calcd for [M]⁺ C_]6H,₂ ⁷⁹BrNO₃:345.0001, found:344.9997.

Example 19

7-Bromo-N-(p-methoxybenzyl)isatin (19)

The product was the bright orange-red solid (145 mg, 65%), mp 159 - 161 °C, R_f 0.53

(DCM, silica) ¹H NMR (CDCl₃, 500 MHz) δ 3.78 (s, 3H, OCH₃), 5.37 (s, 2H, benzylic CH₂), 6.85 (d, J= 9 Hz, 2H, phenyl ArH), 6.99 (t, J = 8 Hz, IH, H5), 7.23 (d, J = 9 Hz, 2H, phenyl ArH), 7.61 (d, J= 8 Hz, IH, H4 or H6), 7.66 (d, J= 8 Hz, IH, H4 or H6). ¹³C NMR (CDCl₃, 126 MHz) δ 44.0, 55.2, 104.4, 114.1, 120.9, 124.7, 125.2, 127.9(6), 128.0(5), 144.1, 147.8, 159.0, 159.1, 182.4. EI-MS m/z 345/347 ([M]⁺/[M+2]⁺); HREI-MS m/z calcd for [M]⁺ Ci₆H₁₂ ⁷⁹BrNO₃:345.0001, found:344.9992.

Example 20

5, 7-Dibromo-N-(cinnamyl)isatin (20)

The product was a bright red solid (133 mg, 48%),mp 115 - 117 ⁰C, R_f 0.53 (CH₂Cl₂, silica). ¹H NMR (CDCl₃, 500 MHz) δ 4.92 (dd, J= 1, 6 Hz, 2H, Hl'), 6.29 (dt, J = 6, 16 Hz, IH, H2'), 6.63 (d, J = 16 Hz, IH, H3'), 7.23 (t, J= 7 Hz, IH, H7'), 7.29 (d, J= 7 Hz, 2H, H6'), 7.33 (d, J = 7 Hz, 2H, H5'), 7.67 (d, J= 2 Hz, IH, H4), 7.84 (d, J= 2 Hz, IH, H6). ¹³C NMR (CDCl₃, 126 MHz) δ 42.8 (Cl'), 104.9, 117.0, 121.3, 122.5 (C2')_> 126.4 (C5^!), 127.5 (C4), 128.1 (C7^!), 128.6 (C6')_> 133.7, 135.9, 145.1 (C6), 146.6, 157.8 (C2), 181.3 (C3). LREI-MS m/z 419/421/423 ([M]⁺/[M+2]⁺/[M+4]⁺), HREI-MS m/z calcd for [M+2]⁺ Ci₇H_n ⁷⁹Br⁸¹BrNO₂: 420.9136, found: 420.9130. Example 21

5, 7-Dibromo-N-(2 '-methoxyethyljisatin βl)

The crude product was purified using flash chromatography eluting with CHCl₃. The product was a dark red oil that solidified on standing (84 mg, 35%), mp 112 - 114 ⁰C₅ R_f 0.38 (CH₂Cl₂, silica). ¹H NMR (CDCl₃, 500 MHz) δ 3.34 (s, 3H, CH₃), 3.68 (t, J= 6 Hz₅

2H₅ H2'), 4.40 (t, J= 6 Hz, 2H, Hl'), 7.68 (d, J= 2 Hz, IH, H4), 7.86 (d, J = 2 Hz, IH,

H6). ¹³C NMR (CDCl₃, 127 MHz) δ 40.6 (Cl'), 59.0 (OCH₃), 69.8 (C2^!), 105.0, 116.9,

121.5, 127.5 (C4), 145.1 (C6), 146.8, 158.3 (C2), 181.3 (C3). LREI-MS m/z 361/363/365

([M]⁺/[M+2f/[M+4]⁺); HREI-MS m/z calcd for [M+2]⁺ CnH₉ ⁷⁹Br⁸¹BrNO₃: 362.8929, found: 362.8926.

Example 22

J, 7-Dibromo-N-(3 '-methylbutyl)isatin βl)

The product was a bright red oil (167 mg, 67%), R_f 0.57 (CH₂Cl₂, silica). ¹H NMR (CDCl₃, 500 MHz) δ 0.90 (d, J = 6 Hz, 6H, 2 x CH₃), 1.57 (m, IH₅ H3'), 1.66 (m, 2H,

H3'), 4.09 (t, J= 8 Hz, 2H, Hl '), 7.60 (d, J= 2 Hz, IH, isatin ArH), 7.80 (d, J= 2 Hz, IH, isatin ArH). ¹³C NMR (CDCl₃, 126 MHz) δ 22.4, 26.1, 37.9, 40.3, 104.7, 116.7, 121.4,

127.4, 145.1, 146.9, 157.9, 181.7. LREI-MS m/z 31313151311 ([M]⁺/[M+2]^"7[M+4]⁺);

HREI-MS m/z calcd for [M]⁺ Ci₃Hi₃ ⁷⁹Br₂NO₂: 372.9313, found: 372.9311.

Example 23

4-[(5, 7-Dibromo-2,3-dihydro-2,3-dioxo-lH-indol-l-yl)methyl]benzoic acid methyl ester

The product was a bright red solid (193 mg, 65%), mp 165 - 166 ⁰C, R_f 0.36 (CH₂Cl₂, silica). ¹H NMR (CDCl₃, 500 MHz) δ 3.89 (s, 3H, OCH₃), 5.43 (s, 2H, Hl'), 7.29 (d, J= 8

Hz, 2H, phenyl ArH), 7.72 (d, J = 2 Hz, IH, isatin ArH), 7.79 (d, J = 2 Hz, IH, isatin

ArH), 7.98 (d, J = 8 Hz, 2H, phenyl ArH). ¹³C NMR (CDCl₃, 126 MHz) δ 44.6, 52.1,

105.1, 117.4, 121.4, 126.2, 127.6, 129.7, 130.1, 140.9, 14 5.2, 146.4, 158.2, 166.5, 180.9.

LREI-MS m/z 451/453/455 ([M]⁺/[M+2]⁺/[M+4]⁺); HREI-MS m/z calcd for [M]⁺ Ci₇H_n ⁷⁹Br₂NO₄: 450.9055, found: 450.9071. Example 24

4-[(5, 7-Dibromo-2,3-dihydro-2,3-dιoxo-lH-indol-l-yl)methyl]benzoic acid β4) The methyl ester 23 (70 mg, 0.15 mmol) was dissolved in a mixture of cone. HCl (8 niL), glacial acetic acid (8 niL) and water (2 mL) and stirred at reflux for 1.5 h. The reaction mixture was poured onto ice (100 mL) and extracted with ethyl acetate (2 x 100 mL) and the combined ethyl acetate layers were washed with brine and dried over MgSO₄. The solvent was removed to yield a bright orange solid. The solid was suspended in CH₂Cl₂ (20 mL) and then filtered and the resulting solid washed with CH₂Cl₂ to remove any trace of starting material. The product 21 was a bright red-orange solid (59 mg, 90%) mp > 250 ⁰C₅ R_f 0.13 (9:1 CH₂Cl₂MeOH, silica). ¹H NMR (DMSO-^, 500 MHz) δ 5.30 (s, 2H, CH₂), 7.50 (d, J = 8 Hz, 2H, ArH), 7.79 (d, J = 2 Hz, IH, isatin ArH), 7.88 (d, J = 8 Hz, 2H, ArH), 7.98 (d, J= 2 Hz, IH, isatin ArH), 12.80 (br s, IH, OH). ¹³C NMR (DMSO-^, 126 MHz) δ 44.9, 104.8, 116.4, 123.5, 127.0, 130.1, 130.3, 143.0, 143.9, 146.8, 159.8, 167.8, 181.2. LREI-MS m/z 437/439/451 ([M]⁺/[M+2]⁺/([M+4]⁺). HREI-MS m/z calcd for [M+2]⁺ Ci₆H₉Br⁸¹BrNO₄: 438.8878, found: 438.8880.

Examples 25 and 26

N-allyl-5,6, 7-tribromoisatin (25) and N-allyl-5,6-dibromoisatinβd) A mixture of 5,6-dibromoisatin and 5,6,7-tribromoisatin (97 mg, See Vine et at, Boorganic an 1997 for synthesis details) was allylated with allyl bromide using general method 1. The crude products were bright, red oil which was purified via flash column chromatography on silica, eluting with 8:2 CHCl₃/pet spirit.

The iV-allyl-5,6,7-tribromoisatin (25) formed bright red-orange crystals (24 mg), mp 149- 154°, R_f 0.22 (5:1 CHCl₃/pet spirit, silica). ¹H NMR (CDCl₃, 500 MHz) δ 4.85 (m, 2H,

CH₂), 5.21 (d, J= 17Hz, IH, CH_cisi-W, 5-26 (d, J= 10Hz, IH, C^A™), 5.96 (ddt, J=

5, 10, 17Hz, IH₅ CH), 7.82 (s, IH, H4). ¹³C NMR (CDCl₃, 126 MHz) δ 43.6(Cl'),

109.3(C7), 117.8(C3'), 119.6, 120.9, 128.0(C4), 131.7(C2')₅ 140.8(C9), 148.0, 158.1,

180.9. EI- MS m/z 423 ([M+2]⁺), HREI-MS m/z calcd for [M+2]⁺ C_nH₆ ⁷⁹Br₂ ⁸¹BrNO₂:422.7928, found: 422.7913. The N-allyl-5,6-dibromoisatin (26) was a bright red-orange solid (13 mg), mp 153-157°, R_f 0.16 (5:1 CHCl₃/pet spirit, silica). ¹H NMR (CDCl₃, 500 MHz) δ 4.35 (d, J = 5Hz, 2H, CH₂), 5.33 (d, J= 17Hz, IH, CH_cis/W>, 5.34 (d, J= 10Hz, IH, CH_C(SH_trans), 5.82 (ddt, J= 5, 10, 17Hz, IH, CH), 7.20 (s, IH, H7), 7.82 (s, IH, H4). ¹³C NMR (CDCl₃, 126 MHz) δ 42.7, 116.3, 117.5, 119.3, 119.5, 129.5, 129.6, 135.4, 149.5, 157.1, 181.1. EI- MS m/z 343/345/347 ([M]⁺/([M+2]⁺/[M+4]⁺), HREI-MS m/z calcd for [M]⁺ C₁iH₇ ⁷⁹Br⁸¹BrNO₂:344.8823, found:344.8820.

Examples 27 and 28 N-(p-methoxybenzyl)-5,6, 7-tribromoisatin (11) and N-(p-methoxybenzyl)-5,6- dibromoisatin (28)

A mixture of 5,6,7-tribromoisatin and 5,6-dibromoisatin (90 mg) was alkylated with p- methoxybenzyl chloride using general method 1. The combined crude products were a yellow-orange residue that was purified using flash column chromatography (silica), eluting with DCM.

The major product was 7V-(p~methoxybenzyl)-5,6,7-tribrornoisatin (27) which formed a bright yellow solid (174 mg) mp 166-168°, R_f 0.49 (DCM, silica). ¹H NMR (CDCl₃, 300 MHz) δ 3.77 (s, 3H, OCH₃), 5.41 (s, 2H, benzylic CH₂), 6.84 (d, J = 9Hz, 2H, phenyl ArH), 7.15 (d, J = 9Hz, 2H, phenyl ArH), 7.82 (s, IH, H4); ¹³C NMR (CDCl₃, 75 MHz) δ 44.5, 55.2, 109.5, 114.3, 119.8, 121.0, 127.7, 127.8, 128.0, 140.9, 140.0, 158.6, 159.1, 180.9. EI-MS m/z 501/503/505 ([M]⁺/[M+2]⁺/[M+4]⁺); HREI-MS m/z calcd for [M]⁺ Ci₆Hi₀Br₃NO₃ :424.9085, found:424.9077.

The 5,6-dibromo-iV-(p-methoxybenzyl)isatin (28) was a bright red-orange solid (83 mg), ¹H NMR (CDCl₃, 300 MHz) δ 3.76(s, 3H, OCH₃), 5.33(s, 2H, benzylic CH₂), 6.83(d, J = 8Hz₅ 2H₅ phenyl ArH), 7.18(d, J = 8Hz, 2H, phenyl ArH), 7.68(d, J = 2Hz, IH, H4) 7.79(d, J = 2Hz, IH, H6), ¹³C NMR (CDCl₃, 75MHz) δ 44.1, 55.2, 105.2, 114.2, 117.0, 121.4, 127.4, 127.5, 128.0, 145.2, 146.7, 158.3, 159.1, 181.3. EI-MS m/z 425 ([M]⁺); HREI-MS m/z calcd for [M]⁺ Ci₆H] ,Br₂NO₃ :424.9085, found:424.9077. Example 29

5, 7-Dibromo-3-(phenylimino)-2-indolinone (29)

The crude product was recrystallized from ethanol to yield a mixture of the E and Z isomers (4:1 EiZ) as bright orange needles (127 mg, 48%), mp 223-224 °C, R_f 0.57 (9:1 DCM:methanol). ¹H NMR (OMSO-d₆, 500 MHz) major (E) isomer δ 6.32 (d, J = 1 Hz, IH, H4), 6.99 (d, J = 8 Hz, 2H, H2'), 7.29 (t, J = 8 Hz, IH, H4'), 7.49 (t, J = 8 Hz, 2H, H3'), 7.81 (d, J = 1 Hz, IH, H6), 11.40 (br s, IH, NH), minor (Z) isomer δ 7.06 (d, J= 8 Hz, 2H, H2'), 7.14 (t, J = 8 Hz, IH, H4'), 7.32 (t, J - 8 Hz, 2H, H3'), 7.68 (d, J = 1 Hz, IH, H4), 7.88 (d, J = 1 Hz, IH, H6), 11.40 (br s, IH, NH). ¹³C NMR (DMS0-.4 126 MHz) major isomer δ 105.8, 113.9, 117.8 (CT), 119.3, 126.2 (C3')_> 127.2 (C4), 130.4, 138.7 (C6), 146.2, 150.5, 154.4 (C3), 163.9 (C2); minor isomer δ 104.9, 114.9, 120.2, 124.8, 125.5, 126.0, 129.0, 138.3, 144.8, 148.8, 152.3 (C3), 158.8 (C2). HREI-MS m/z calcd for [M+2]⁺ Ci₄H₈ ⁷⁹Br⁸¹BrN₂O: 379.8983; found: 379.8978.

Example 30

Isatin-3-phenylhydrazone (30)

A mixture of isatin (100 mg, 0.67 mmol) phenylhydrazine hydrochloride (108 mg, 0.75 mmol) and sodium acetate (112 mg, 1.36 mmol) in absolute ethanol (100 mL) were heated at reflux for 16 h. The reaction mixture was allowed to cool and the resulting precipitate filtered from the solution. The crude product was purified using flash chromatography eluting with DCM. The product was a bright yellow solid (159 mg, 75%), mp 214-216 °C (lit.³⁷ 213-214⁰C) (Popp, F.D; J Heterocyclic Chem. 1984, (6), 1641-5) R_f 0.50 (silica, 3:7 ethyl acetate/pet spirit). ¹H NMR (CDCl₃, 500 MHz) δ 6.91 (d, J= 8 Hz, IH, H4 or H7), 7.03 and 7.04 (2 overlapping t, 2 x IH, H4 and H5 or H6), 7.24 (dt, J= 1, 8 Hz, IH, H5 or H6), 7.36 (t, J= 8 Hz, 2H, H3'), 7.42 (d, J= 8 Hz, 2H, H2'), 7.54 (d, J= 7 Hz, IH, H4 or H7), 11.00 (br s, IH₅ NH), 12.74 (s, IH, NH). ¹³C NMR (CDCl₃, 126 MHz) δ 110.5, 114.0, 118.6, 121.1, 121.8, 122.9, 127.7, 128.5, 129.4, 139.8, 142.5, 163.1 (C2). LREI-MS m/z 237 ([M]⁺).

Example 31

5-bromo-3-phenylimino-N-(p-rnethoxybenzyl)isatin βl) Synthesis of compound 34 was accomplished using General Method 2 with 5-bromo-iV-(p- methoxybenzyl)isatin (16), (100 mg, 0.29 mmol) and freshly distilled aniline (27 mg, 0.29 mmol) and heating under reflux for 16 h. The solvent was removed and the bright yellow residue was purified using flash column chromatography, eluting with DCM. The product was a bright yellow solid (101 mg, 83 %), R_f 0.18 (DCM), LREI-MS m/z 420/422 [M]⁺/[M+2]⁺.

Example 32 5, 7-dibromo~3-[3-carboxymethylphenyl)imino-N-(p-trifluoromethyl-benzyl)isatin (32) Synthesis of compound 32 was accomplished using General Method 2 with 5,7-dibromo- N-(p-trifluoromethylbenzyl)isatin (15) (100 mg, 0.215 mmol), 3-aminophenylacetic acid (31 mg, 0.205 mmol) with refluxing for 5 h. The solvent was removed and the bright orange/red residue was purified using flash column chromatography, eluting with DCM to remove excess isatin and then 98:2 DCM/methanol to elute the imine. The product was a bright red-orange glass that slowly crystallized at room temperature and decomposed on heating (90mg, 70 %), R_f 0.37 (9:1 DCM:methanol). The product was a mixture of the E and Z isomers (3:1 E:Z). ¹H NMR (DMSO-cfo, 500MHz) major (E) isomer δ 3.64 (s, 2H, H2), 5.43 (s, 2H, Hl"), 6.62( J= 2 Hz, IH, H4'), 6.99 (d, J= 2 Hz, IH, H4'), 6.93 (d, J= 8 Hz, IH₅ H6 or H8), 6.95 (s, IH, H4), 7.21 (d, J = 8 Hz, IH, H6 or H8), 7.47 (t, J= 8 Hz, IH, H7), 7.57 (d, J= 8 Hz, 2H, H3" or H4"), 7.71 (d, J= 8 Hz, 2H, H3" or H4"), 7.80 (d, J = 2 Hz, IH, H6'); minor (Z) isomer δ 3.56 (s, 2H, H2), 5.27 (s, 2H, Hl"), 7.03-7.09 (overlapping m, 3H, H4, H6 and H8), 7.27 (t, J = 8 Hz, IH, H7), 7.52 (d, J = 8 Hz, 2H, H3" or H4"), 7.67 (d, J= 8 Hz, 2H, H3" or H4"), 7.83 (d, J= 2 Hz, 2H, H4' or H6'), 7.89 (d, J= 2 Hz, 2H, H4' or H6'). LREI-MS m/z 594/596/598 [M]^"7[M+2]⁺/[M+4]⁺.

Example 33

5-bromo-3-[m-2 '-carboxymethylphenyl)imino-N-(p-methoxybenzyl)isatin βi) Synthesis of compound 33 was accomplished using General Method 2 with 5-bromo-iV-(p- methoxybenzyl)isatin (16), (100 mg, 0.29 mmol), 3-aminoρhenylacetic acid (44 mg, 0.29 mmol) with heating under reflux for 5 h. The solvent was removed and the bright red residue was purified using flash column chromatography, eluting with DCM to remove unreacted 16 and then 98:2 DCM/methanol to elute the imine. The product was a bright red solid (75 mg, 52 %), R_f 0.11 (95:5 D CM methanol), LREI-MS m/z 492/494 [M]⁺/[M+2]⁺.

Example 34

5, 6-Dibromoisatin (34) was a bright red-orange solid (135 mg, 50%), mp >265 ⁰C (287-290 ⁰C), R_f 0.49 (silica, DCM:MeOH, 9:1). ¹H NMR (DMSO-αk, 500 MHz) δ 7.24 (s, IH, H7), 7.82 (s, IH, H4), 11.24 (br s, IH, NH). ¹³C NMR (DMSO-J₆, 126 MHz) δ 116.7, 116.8 (C7), 118.8, 128.6 (C4), 133.1, 149.8, 159.0 (C2), 182.3 (C3). HREI-MS m/z calcd for [M+2]⁺ C₈H₃ ⁷⁹Br⁸¹BrNO₂: 304.8510; found: 304.8514.

Examples 35 to 47

General Method 3: Preparation of iV-phenethyl derivatives and N-naphthylmethyl derivatives

The procedure for the synthesis of the N-phenethyl derivatives was based on a combination of literature methods. Briefly, 5,7-dibromoisatin was treated with a base such as NaH or K₂CO₃ in DMF which formed an intense purple colored anion that was subsequently reacted with the appropriate aryl alkyl halide to give the iV-phenethyl derivatives in moderate yields. High temperatures are usually required in these syntheses to drive the reactions to completion because the reaction mixtures are prone to crystallization of the impure product at low temperatures. JV-Benzylisatin derivatives had been obtained previously by heating at 80 C, however a temperature of 50 ⁰C was used to to reduce the risk of substituted styrenes forming as side products from the base catalyzed elimination of the phenethyl bromides. The isatin anionic intermediate is also an ambidentate anion which could undergo N- or (9-alkylation, however no evidence for O-alkylation was found through ¹H and ¹³C NMR spectroscopic studies. The 7V-naphthylmethyl analogues were prepared in a similar manner to the N-phenethyl derivatives. General Method 4: Preparation of iV-phenacyl derivatives

The synthesis of the iV-phenacyl derivatives was difficult. Attempts to synthesize these compounds included various alkylation protocols and protecting group strategies. A possible competing reaction was a Darzens condensation involving the C3 carbonyl group of the 5,9-bromo isatin. While the Darzens condensation occurs when a ketone or aldehyde reacts with a haloester to form an epoxy ester, it also proceeds with halogenomethylsulfones and halogenoketones such as phenacyl halides. It is has been reported that phenacyl halides preferentially yield a Darzens product rather than the corresponding phenacylisatin, although in the current study this was not observed in the NMR spectra. The desired compounds were obtained in a pure form but in low yields, which were not optimized as the required compounds were obtained in sufficient quantity for cytotoxicity screening.

Example 35

5, 7-Dibromoisatin (35).

Yield: 2.9O g, (28 %) as bright red/orange needles, m.p. 251-253 ⁰C (lit.²³ 248-250 ⁰C), Rf 0.63 (silica, DCM:MeOH, 9:1). ¹H NMR (DMSO-^₅ 500 MHz): δ 7.66 (d, J = 1.5 Hz, IH, H4), 8.02 (d, J= 1.5 Hz, IH, H6), 11.43 (bs, IH, NH). ¹³C NMR (DMSO-^₅ 126 MHz): δ 105.7, 114.5, 121.3, 125.9, 141.1, 148.6, 159.4, 182.5. LREI-MS: m/z 303; 305; 307 [M⁺]⁷⁹Br⁷⁹Br; ⁷⁹Br⁸¹Br; ⁸¹Br⁸¹Br.

Example 36

5, 7-Dibromo-l-phenethyl-lH-indole-2,3-dione (36). A mixture of 5,7-dibromoisatin (200 mg, 0.66 mmol) and K₂CO₃ (128 mg, 0.92 mmol) or NaH (36.8 mg, 0.92 mmol) was dissolved in anhydrous DMF (4 mL) and stirred under nitrogen at 4 ⁰C for 3 h (or 20 min at RT for NaH) before the addition of KI (22.0 mg, 0.13 mmol) and (2-bromoethyl)benzene (270 mg, 0.2 mL, 1.46 mmol). The reaction mixture was heated at 50 °C and stirred at this temperature for 18 h. To the resulting solution was added water (80 mL) and 1 M HCl (2 mL) to acidify to pH 1. The suspension was filtered and the precipitate washed with water. The resulting solid was purified by flash chromatography on silica gel (CHCI₃) to yield the title compound (86.3 mg, 32 %) as bright red crystals, m.p. 165-168 °C, R_f 0.71 (silica, DCM). ¹H NMR (500 MHz): δ 3.03 (t, J= 8 Hz, 2H, H2'), 4.39 (t, J= 8 Hz, 2H, Hl '), 7.30 (m, 5H, H2"- H6"), 7.69 (d, J= 1.5 Hz, IH, H4), 7.89 (d, J= 1.5 Hz, IH, H6). ¹³C NMR (126 MHz): δ 35.0, 42.4, 104.5, 116.6, 121.1, 126.7, 127.2, 128.4^a, 128.6^a, 136.7, 144.8, 146.4, 157.5, 181.1. HREI- MS: m/z calcd for Ci₆H_nNO₂ ⁷⁹Br⁸¹Br [M⁺]: 406.9157; found 406.9166.

Example 37

5, 7-Dibromo-l-[2-(3-bromophenyl)ethyl]-lH-indole-2, 3-dione (37). The compound was prepared according to the method for 36 using 5,7-dibromoisatin (100 mg, 0.33 mmol) and 3-bromophenethyl bromide (193 mg, 0.11 mL, 0.73 mmol) as starting materials. The resulting solid was purified by flash chromatography on silica gel (DCM) to yield the title compound (67.1 mg, 42 %) as bright red/orange crystals, m.p. 188-190 ⁰C, Rf 0.54 (silica, DCM). ¹H NMR (Acetone-^, 500 MHz): δ 3.09 (t, J = 8 Hz, 2H, H2'), 4.38 (t, J= 8 Hz, 2H, Hl'), 7.27 (t, J= 8 Hz, IH, H5"), 7.34 (d, J= 8 Hz, IH, H6"), 7.42 (d, J= 8 Hz, IH, H4"), 7.51 (s, IH, H2"), 7.72 (d, J= 1.5 Hz₅ IH, H4), 8.06 (d, J= 1.5 Hz, IH, H6). ¹³C NMR (Acetone-^, 126 MHz): δ 34.9, 42.4, 104.7, 110.0, 116.0, 122.3, 126.8, 128.2, 130.0, 130.8, 132.0, 140.9, 144.4, 147.5, 158.4, 181.6. HREI- MS: m/z calcd for Ci₆Hi₀NO₂ ⁷⁹Br⁸¹Br⁸¹Br [M⁺]: 488.8221; found 488.8207.

Example 38

5, 7-Dibromo-l-[2-(4-bromophenyl)ethyl]-lH-indole-2, 3-dione (38)

The compound was prepared according to the method for 36 using 5,7-dibromoisatin

(100 mg, 0.33 mmol) and 4-bromophenethyl bromide (193 mg, 0.11 mL, 0.73 mmol) as starting materials. The resulting solid was purified by flash chromatography on silica gel (CHCl₃) to yield the title compound (81.1 mg, 50 %) as bright red crystals, m.p. 189- 190 ⁰C, Rf 0.48 (silica, DCM). ¹H NMR (300 MHz): δ 2.99 (t, J = 8.4 Hz, 2H, H2'), 4.35 (t, J= 8.4 Hz, 2H, Hl '), 7.13 (d, J= 8.7 Hz, 2H, H2", H6"), 7.42 (d, J= 8.7 Hz, 2H, H3", H5"), 7.69 (d, J= 1.8 Hz, IH, H4), 7.89 (d, J= 1.8 Hz, IH, H6). ¹³C NMR (126 MHz): δ 35.1, 42.6, 104.9, 117.3, 121.2, 121.6, 127.9, 130.8⁸, 132.1^a, 136.2, 145.4, 146.8, 158.0, 181.4. HREI-MS: m/z calcd for Ci₆Hi₀NO₂ ⁷⁹Br⁸¹Br⁸¹Br [M⁺]: 488.8221; found 488.8214.

Example 39 5, 7-Dibromo-l-[2-(3-methoxyphenyl)ethyl]-lH-indole-2, 3-dione (39).

The compound was prepared according to the method for 36 using 5,7-dibromoisatin (100 mg, 0.33 mmol) and 3-methoxyphenethyl bromide (157 mg, 0.11 mL, 0.73 mmol) as starting materials. The resulting solid was purified by flash chromatography on silica gel (DCM) to yield the title compound (65.0 mg, 45 %) as bright red/orange crystals, m.p. 179-180 ⁰C, R_f 0.56 (silica, DCM). ¹H NMR (500 MHz): δ 3.00 (t, J = 8 Hz, 2H, H2'), 3.79 (s, 3H, OCH₃), 4.38 (t, J= 8 Hz, 2H, Hl '), 6.76 (d, J= 7.5 Hz, IH, H4"), 6.80 (s, IH, H2"), 6.84 (d, J= 7.5 Hz, IH, H6"), 7.22 (t, J= 7.5 Hz, IH, H5"), 7.69 (d, J= 2 Hz, IH, H4), 7.89 (d, J= 2 Hz, IH, H6). ¹³C NMR (126 MHz): δ 35.3, 42.6, 55.2, 104.8, 112.2, 114.8, 116.9, 121.2, 121.4, 127.5, 129.8, 138.5, 145.1, 146.7, 157.8, 159.8, 181.3. HREI- MS: m/z calcd for C]₇H₁₃NO₃ ⁷⁹Br⁸¹Br [M⁺]: 438.9242; found 438.9244.

Example 40

5, 7-Dibromo-l-[2-(4-methoxyphenyl)ethyl]-lH-indole-2, 3-dione (40).

The compound was prepared according to the method for 36 using 5,7-dibromoisatin (100 mg, 0.33 mmol) and 4-methoxyphenethyl bromide (157 mg, 0.11 mL, 0.73 mmol) as starting materials. The resulting solid was purified by flash chromatography on silica gel

(CHCl₃) to yield the title compound (89.5 mg, 62 %) as bright red/orange crystals, m.p.

176-178 ⁰C, R_f 0.53 (silica, DCM). ¹H NMR (500 MHz): δ 2.96 (t, J = 8 Hz, 2H, H2'),

3.78 (s, 3H, OCH₃), 4.34 (t, J= 8 Hz, 2H, Hl'), 6.83 (d, J= 8.5 Hz, 2H, H3", H5"), 7.16 (d, J= 8.5 Hz, 2H, H2", H6"), 7.68 (d, J= 2 Hz, IH, H4), 7.89 (d, J= 2 Hz, IH, H6).

¹³C NMR (126 MHz): δ 34.7, 43.1, 55.5, 105.0, 114.4^a, 117.1, 121.6, 127.8, 129.2, 130.1^a,

145.3, 147.0, 158.1, 158.8, 181.6. HREI-MS: m/z calcd for C_nHi₃NO₃ ⁷⁹Br⁸¹Br [M⁺]:

438.9242; found 438.9241.

Example 41

5, 7-Dibromo-l-(l-naphthylmefhyl)-lH-indole-2, 3-dione (41). A mixture of 5,7-dibromoisatin (101 mg, 0.33 mmol) and NaH (18.0 mg, 0.46 mmol) was dissolved in anhydrous DMF (2.5 mL) and stirred under nitrogen at RT for 20 min before the addition of KI (l l.O mg, 0.066 mmol) and 1 -chloromethylnaphthalene (128 mg, 0.11 mL, 0.73 mmol). The reaction mixture was heated at 60 °C and stirred at this temperature for 19 h. After cooling, ethyl acetate (5O mL) was added and the resulting solution was extracted with 0.5 M HCl (50 mL) followed by brine (50 mL). The orange organic layer was dried over MgSO₄ and the solvent was removed to yield a sticky red/orange residue. The resulting solid was purified by flash chromatography on silica gel [DCM:PS (3:2)] to yield the title compound (90.8 mg, 62 %) as dark red crystals, m.p. 218-219 ⁰C₅ R_f 0.35 (silica, DCM). ¹H NMR (500 MHz): δ 5.81 (s, 2H, Hl'), 7.06 (d, J= 7 Hz, IH, H2"), 7.34 (t, J= 7.5 Hz₅ IH, H3"), 7.53 (t, J= 7 Hz₅ IH₅ H6"), 7.58 (t, J= 7 Hz₅ IH, H7"), 7.76 (m, 3H₅ H4, H6, H4") 7.88 (d, J= 8.5 Hz, IH, H5"), 7.93 (d₅ J= 8.5 Hz₅ IH₅ H8"). ¹³C NMR (126 MHz): 5 42.8, 105.5, 117.1, 121.3, 121.4, 122.I₅ 125.3, 126.0, 126.6, 127.5, 128.0, 129.0, 129.8, 130.7, 133.8, 145.3, 146.8, 158.1, 181.2. HREI-MS: m/z calcd for C₁₉H_nNO₂ ⁷⁹Br⁸¹Br [M⁺]: 444.9136; found 444.9131.

Example 42

5, 7-Dibromo-l-(2-naphthylmethyl)-lH-indole-2, 3-dione (42).

The compound was prepared according to the method for 41 using 5,7-dibromoisatin (50.5 mg, 0.16 mmol) and 2-bromomethylnaphthalene (80.1 mg, 0.36 mmol) as starting materials. The resulting red solid was purified by flash chromatography on silica gel

[DCM:PS (3:2)] to yield the title compound (46.6 mg, 64 %) as dark red crystals, m.p.

140-142 ⁰C₅ R_f 0.58 (silica, DCM). ¹H NMR (500 MHz): 5 5.56 (s, 2H, Hl'), 7.36 (dd,

J = 2 Hz, 8 Hz, IH, H3"), 7.47 (m, 2H, ArH x 2), 7.63 (s, IH, Hl"), 7.74 (d, J = 2 Hz₅ IH, H4), 7.76 (m, IH, ArH), 7.79 (d, J = 2 Hz, IH, H6), 7.81 (m, 2H, ArH x 2). ¹³C NMR

(126 MHz): 8 45.I₅ 105.5, 117.5, 121.7, 124.6, 125.3, 126.4, 126.7, 127.8, 128.0, 128.0,

129.1, 133.0, 133.3, 133.5, 145.6, 147.0, 158.6, 181.5. HRMS: m/z calcd for C₁₉H_nNO₂

⁷⁹Br⁸¹Br [M⁺]: 444.9136; found 444.9135.

Example 43

5, 7-Dibromo-l-(2-oxo-2-phenylethyl)-lH-indole-2, 3-dione (43). A mixture of KI (113 mg, 0.68 mmol) and phenacyl bromide (68.0 nig, 0.34 mniol) was dissolved in anhydrous DMF (0.5 mL) and stirred at -5 ⁰C under nitrogen for 5 h, followed by cooling in a freezer at -18 °C for 20 h.²⁴ A mixture of the 5,7-dibromoisatin (103 mg, 0.34 mmol) and K₂CO₃ (47.0 mg, 0.34 mmol) or NaH (13.7 mg, 0.34 mmol) was dissolved in anhydrous DMF (5 mL) and stirred under nitrogen at 4 ⁰C for 3 h. This anion solution was added in portions (0.5 mL) to the phenacyl iodide maintained at -2 °C such that each portion had reacted before the addition of the next portion (monitored by TLC). The yellow/brown reaction mixture was stirred at RT for 25 h but no change in colour intensity was observed after 2 h. To the resulting solution was added water (60 mL) and 1 M HCl (2 mL) to acidify to pH 1. The suspension was filtered and the precipitate washed with water. The resulting solid was purified by flash chromatography on silica gel (DCM) to yield the title compound (13.5 mg, 9 %) as bright red/orange crystals, m.p. 173-175 ⁰C, R_f 0.58 (silica, DCM). ¹H NMR (500 MHz): δ 5.64 (s, 2H, Hl'), 7.54 (t, J = 7.5 Hz₅ 2H, H3", H5"), 7.67 (t, J= 7.5 Hz, IH, H4")₅ 7.75 (d, J= 2 Hz, IH, H4), 7.80 (d, J= 2 Hz, IH, H6), 8.00 (d, J= 7.5 Hz, 2H, H2", H6"). ¹³C NMR (126 MHz): δ 48.2, 105.4, 117.1, 121.3, 126.5, 127.6, 128.3% 129.1^a, 134.4, 144.8, 146.9, 158.2, 180.8, 191.3. HREI-MS: m/z calcd for C₁₆H₉NO₃ ⁷⁹Br⁸¹Br [M⁺]: 422.8929; found 422.8928.

Example 44 5, 7-Dibromo-l-[2-(3-bromophenyl)-2-oxo-ethyl]-lH-indole-2,3-dione (44).

This compound was prepared according to the method for 43 using 5,7-dibromoisatin (305 mg, 1.00 mmol) and 3-bromophenacyl bromide (278 mg, 1.00 mmol) as starting materials. The reaction mixture was stirred at 100 ⁰C for 16 h.²⁵ The resulting solid was purified by flash chromatography on silica gel (DCM) and subsequent preparative TLC (silica, DCM) to yield the title compound (10.6 mg, 2 %) as bright red/orange crystals, m.p. 160-162 ⁰C, R_f 0.54 (silica, DCM). ¹H NMR (500 MHz): δ 5.60 (s, 2H, Hl'), 7.44 (t, J = 8 Hz, IH, H5"), 7.77 (d, J= 2 Hz, IH, H4), 7.80 (d, J= 8.5 Hz, IH, H4"), 7.81 (d, J= 2 Hz, IH₅ H6), 7.93 (d, J= 8.5 Hz, IH, H6"), 8.13 (s, IH, H2"). ¹³C NMR (126 MHz): δ 48.0, 105.3, 117.3, 121.3, 123.5, 126.6, 127.7, 130.7, 131.2, 135.4, 137.3, 144.8, 146.6, 158.0, 180.6, 190.2. HREI-MS: m/z calcd for C₁₆H₈NO₃ ⁷⁹Br⁷⁹Br⁸¹Br [M⁺]: 500.8034; found 500.8037. Example 45

5, 7-Dibromo-l-[2-(4-bromophenyl)-2-oxo-ethyl]-lH-indole-2, 3-dione (45). 5,7-Dibromoisatin (153 mg, 0.5 mmol) and NaH (20.0 mg, 0.5 mmol) was dissolved in anhydrous DMF (1.25 mL) and stirred at RT under nitrogen for 20 min before the addition of freshly distilled trimethylsilyl chloride (81.0 mg, 0.095 mL, 1.5 mmol).²⁶ The reaction mixture was heated at 50 ⁰C and stirred at this temperature for 1 h before the addition of 4-bromophenacyl bromide (139 mg, 0.5 mmol) and further heating at 100 °C for 1.5 h. Upon cooling, water (15 mL) was added, the suspension was filtered and the precipitate washed with hot water (90 ⁰C) to yield a rust coloured compound. The product was recrystallized from glacial AcOH, filtered and washed with ice cold water to yield the title compound (11.2 mg, 5 %) as a light yellow powder, m.p. 184-186 °C, R_f 0.65 (silica, DCM). ¹H NMR (500 MHz): δ 5.60 (s, 2H, Hl '), 7.70 (d, J = 8.5 Hz, 2H, H3", H5")₅ 7.76 (d, J= 2 Hz, IH, H4), 7.80 (d, J= 2 Hz, IH, H6), 7.87 (d, J= 8.5 Hz, 2H, H2", H6"). ¹³C NMR (126 MHz): δ 47.9, 105.3, 117.2, 121.3, 127.6, 129.6^a, 129.8, 132.5^a, 132.5, 144.7, 146.7, 158.1, 180.7, 190.4. HREI-MS: m/z calcd for C]₆H₈NO₃ ⁷⁹Br⁸¹Br⁸¹Br [M⁺]: 502.8013; found 502.8007.

Example 46 5, 7-Dibromo-l-[2-(3-methoxyphenyl)-2-oxo-ethyl]-lH-indole 2, 3-dione (46).

This compound was prepared according to the method for 43 using 5,7-dibromoisatin (306 mg, 1.00 mmol) and 3-methoxyphenacyl bromide (230 mg, 1.00 mmol) as starting materials.²⁷ The resulting solid was purified by flash chromatography on silica gel (DCM) to yield the title compound (43.5 mg, 10 %) as bright orange crystals, m.p. 155-157 ⁰C₃ R_f 0.50 (silica, DCM). ¹H NMR (500 MHz): δ 3.88 (s, 3H, OCH₃), 5.62 (s, 2H, Hl '), 7.20 (d, J= 8 Hz, IH₅ H4"), 7.45 (t, J= 8 Hz, IH, H5"), 7.51 (s, IH, H2"), 7.58 (d, J= 8 Hz, IH, H6"), 7.75 (d, J= 2 Hz, IH, H4), 7.81 (d, J= 2 Hz, IH, H6). ¹³C NMR (75 MHz): δ 48.1, 55.5, 105.4, 112.5, 117.1, 120.5, 120.7, 121.3, 127.5, 130.1, 135.0, 144.7, 146.9, 158.1, 160.1, 180.8, 191.2. HREI-MS: m/z calcd for C_nH₁₁NO₄ ⁷⁹Br⁷⁹Br [M⁺]: 450.9055; . found 450.9048. Example 47

5, 7-Dibromo-l-[2-(4-methoxyphenyl)-2-oxo-ethyl]-lH-indole-2, 3-dione (47). This compound was prepared according to the method for 46 using 5,7-dibromoisatin (111 mg, 0.36 mmol) and 4-methoxyphenacyl bromide (82 mg, 0.36 mmol) as starting materials. The reaction mixture was then stirred at RT for 51 h. The suspension was filtered and the precipitate washed with water. The resulting solid was purified by flash chromatography on silica gel (DCM) to yield the title compound (18.2 mg, 11 %) as bright orange crystals, m.p. 205-207 ⁰C, R_f 0.50 (silica, DCM). ¹H NMR (500 MHz): δ 3.91 (s, 3H, OCH₃), 5.59 (s, 2H, Hl '), 7.00 (d, J= 8 Hz, 2H, H3", H5"), 7.74 (d, J= 2 Hz₅ IH, H4), 7.80 (d, J= 2 Hz, IH, H6), 7.98 (d, J= 8 Hz, 2H, H2", H6"). ¹³C NMR (126 MHz): δ 47.9, 55.9, 105.7, 114.6^a, 117.2, 121.6, 126.9, 127.7, 130.7^a, 145.0, 147.3, 158.5, 164.7, 181.2, 186.9. HREI-MS: m/z calcd for C₁₇HnNO₄ ⁷⁹Br⁸¹Br [M⁺]: 452.9034; found 452.9048.

Examples 48

Synthesis ofN~(p-methoxybenzyl)-5-(tribιιtylstannyl)isatin (48)

A mixture of 5-bromo-iV-(p-methoxyphenyl)-isatm (90 mg, 0.26 mmol), hexabutyldistannane (0.30 mL, 0.59 mmol) and tetrakis(triphenyl-phosphine)palladium (10 mg, 0.01 mmol) in dry toluene (20 mL) was heated at reflux for 6.5 h under N₂. The solvent was removed at reduced pressure and the crude product was purified by flash chromatography, eluting with DCM, to afford the stannylated isatin as a bright orange oil (60 mg, 41%), R_f 0.35 (DCM, silica). ¹H NMR D 0.87 (d, J = 7 Hz, 9H, CH₃), 1.03 (m, 6H, CH₂), 1.30 (m, 6H, CH₂), 1.48 (m, 6H, CH₂), 3.78 (s, 3H, OCH₃), 4.84 (s, 2H, benzylic CH₂), 6.78 (d, J= 8 Hz, IH, H7), 6.87 (d, J= 9 Hz, 2H, phenyl ArH), 7.28 (d, J= 9 Hz, 2H, phenyl ArH), 7.54 (dd, J = 1, 8 Hz, IH, H6), 7.66 (d, J = 1 Hz, IH, H4). ¹³C NMR δ 9.7, 13.6, 27.3, 28.9, 43.5, 55.3, 110.8, 114.3, 117.4, 126.7, 128.9, 132.6, 137.1, 146.2, 150.7, 158.2, 159.4, 183.9. LREI-MS m/z 557 ([M]⁺); HREI-MS m/z calcd for [M]⁺ C₂₈H₃₉NO₃ ¹¹⁶Sn: 553.1940, found 553.1940.

Example 49

Synthesis of 5, 7-Dibromo-N-[4 '-(tributylstannyl)benzyl]isatin (49) 5,7-Dibromoisatin (100 mg, 0.33 mmol) and K₂CO₃ (63 mg, 0.46 mmol) were suspended in dry DMF (5 mL) and the mixture stirred at 0-5 ⁰C for 30 min. 4-(Tributylstannyl)benzyl chloride (150 mg, 0.36 mmol) and KI (11 mg, 0.07 mmol) were added and the suspension stirred at 80 °C for 16 h. The reaction mixture was poured into HCl (0.5 M, 25 mL) and then extracted with ethyl acetate (1 x 25 mL) and the ethyl acetate layer washed with brine and dried over MgSO₄, The solvent was removed and the residue was purified using flash column chromatography, eluting with 1 : 1 DCM/pet spirit. The product was a bright red semi-solid that solidified on standing (97 mg, 43%), mp 111 - 113 ⁰C, R_f 0.39 (1 :1 DCM/pet spirit, silica). ¹H NMR D 0.87 (t, J = 7 Hz, 9H, H4"), 1.03 (m, 6H, Hl"), 1.31 (m, 6H, H3"), 1.52 (m, 6H, H2"), 5.39 (s, 2H, Hl '), 7.19 (d, J= 8 Hz₅ 2H, H3'), 7.41 (d, J = 8 Hz, 2H, H4'), 7.71 (d, J= 2 Hz, IH, H4), 7.81 (d, J= 2 Hz, IH₃ H6). A minor signal at D7.41 (dd, J= 8, 37 Hz, H4') due to coupling of H4' with Sn was also observed. ¹³C NMR δ 9.6 (Cl"), 13.6 (C4"), 27.3 (C3"), 29.0 (C2"), 44.7 (Cl'), 105.3, 117.1, 121.4, 125.9 (C3'), 127.5, 135.2, 136.9 (C4'), 141.6, 145.3, 146.8, 158.3 (C2), 181.3 (C3). LREI-MS m/z 570 ([M-C₈H₁₈]⁺); HREI-MS m/z calcd for [M]⁺ C₂₇H₃₅ ⁸¹Br₂NO₂ ¹¹⁸Sn: 685.001, found: 685.000.

Example 50

Synthesis ofN-(p-methoxybenzyl)-5-(¹²³I)iodoisatin (50) To a solution of N-(p-methoxybenzyl)-5-(tributylstannyl)isatin in EtOH (3.6 mM, 100 μL) was added 3.3 mCi of Na¹²³I in aqueous NaOH (20 mM, 20 μL) and peracetic acid solution (37%, 100 μL). The resulting mixture was then shaken for 5 min at RT and quenched with aqueous NaS₂O₅ (300 mM, 100 μL) and neutralised with aqueous NaHCO₃ (595 mM, 100 μL). To the reaction mixture, mobile phase (ACN/H₂O 65/35 + 0.01% TFA, 400 μL) was added and 2.2 mCi (1 mL) was injected into a HPLC (Phenomenex Bondclone Cl 8 300 x 7.8 mm, 2 niL/min 65/35 ACN/H₂O + 0.01% TFA). The ¹²³I labeled product was collected at 14.8 min (1.0 mCi) in a glass vial which was then evaporated to dryness under high vacuum. Identity confirmation was done using a HPLC trace comparison with unlabeled iV-(p-inethoxybenzyl)-5-iodoisatin; retention time 14.8 min, radiochemical purity 37% (Phenomenex Bondclone C18 300 x 7.8 mm, 2 mL/min 65/35 ACN/H₂O + 0.01% TFA, UV 254 nm). Example 51

Synthesis of 5, 7-dibromo-N-[4'-('²³I)iodobenzyl]isatin (Si) a) Method 1. To a solution of 5,7-dibromo-iV-[4'-(tributylstannyl)-benzyl]isatin in EtOH (2.9 niM, 100 μL) was added 4.0 mCi of Na¹²³I in aqueous NaOH (20 niM, 15 μL) and peracetic acid solution (37%)/acetic acid (1 :10 v/v, 100 μL). The resulting mixture was then shaken for 5 min at RT and quenched with aqueous NaS₂O₅ (300 mM, 100 μL) and neutralised with aqueous NaHCO₃ (595 mM, 100 μL). To the reaction mixture, mobile phase (ACN/H₂O 80/20 + 0.1% TFA, 300 μL) was added and 3.5 mCi (1 mL) was injected into a HPLC (Phenomenex Bondclone Cl 8 300 x 7.8 mm 2 mL/min 80/20 ACN/H₂O + 0.1% TFA, UV 254 nm). The ¹²³I labeled product was collected at 13.3 min (1.3 mCi) in a glass vial which was then evaporated to dryness under high vacuum. Identity confirmation was done by HPLC (Phenomenex Bondclone Cl 8 300 x 7.8 mm 2 mL/min 80/20 ACN/H₂O + 0.1% TFA, UV 254 nm) comparison with unlabeled 5,7-dibromo-N-[4'- (iodo)benzyljisatin; retention time 13.3 min, radiochemical purity 37%.

b) Method 2. To a solution of 5,7-dibromo-N-[4'-(tributylstannyl)-benzyl]isatin in EtOH (2.9 mM, 100 μL) was added 4.2 mCi of Na¹²³I in aqueous NaOH (20 mM, 15 μL) and peracetic acid solution (37%)/acetic acid (1:1 v/v, 100 μL). The resulting mixture was shaken for 5 min at RT and then quenched with aqueous NaS₂O₅ (300 mM, 100 μL) and neutralised with aqueous NaHCO₃ (595 mM, 100 μL). To the reaction mixture, mobile phase (ACN/H₂O 80/20 + 0.1% TFA, 300 μL) was added and 3.8 mCi (1 mL) was injected into a HPLC (Phenomenex Bondclone Cl 8 300 x 7.8 mm 2 mL/min 80/20 ACN/H₂O + 0.1% TFA, UV 254 nm). The ¹²³I labeled product was collected at 13.3 min (1.3 mCi) in a glass vial which was then evaporated to dryness under high vacuum. Identity confirmation was done by HPLC (Phenomenex Bondclone Cl 8 300 x 7.8 mm 2 mL/min 80/20 ACN/H₂O + 0.1% TFA, UV 254 nm) comparison with unlabeled 5,7-dibromo-JV-[4'- (iodo)benzyl]isatin; retention time 13.3 min, radiochemical purity 34%. The QC data indicated that there was no product breakdown in the mobile phase at RT after 2 h. Examples 52 and 53

Characterisation of Protein-Cytotoxin Conjugates a Electrospray Ionisation Mass Spectrometry (ESI-MS)

The protein conjugate ratio was determined by ESI-MS. Samples were desalted by centrifugation using 3OkDa cut-off microconcentrators for ESI-MS. Protein and protein conjugates were washed 5 times with milliQ water by centrifugation (~6000 x g, 5 min) before being made up to a final protein concentration of 1-10 μM. Samples were injected into the Micromass Q-TOF Ultima mass spectrometer (Waters, Wyntheshawe, UK) and run at 30 cone volts and a resolution power of 5000 Hz. The electrospray ion series was transformed to a mass scale using the MaxEnt deconvolution algorithm (See Figure 17).

b PAI-2: uPA Complex Formation

The ability of PAI-2 to retain activity and form stable complexes with uPA after modification by conjugation was compared to unmodified PAI-2 as previously described (Ranson, M.; Tian, Z.; Andronicos, N. M.; Rizvi, S.; Allen, B. J. In vitro cytotoxicity of bismuth-213 (213Bi)-labeled-plasminogen activator inhibitor type 2 (alpha-PAI-2) on human breast cancer cells. Breast Cancer Res. Treat. 2002, 71, 149-159). Briefly, conjugated and unconjugated PAI-2 were incubated with equimolar amounts of uPA in PBS (pH 7.4) for 30 min at 37 ⁰C and complex formation visualised by Coomassie blue staining of samples fractionated by SDS-PAGE under non-reducing conditions (see Figures 18 and 19).

Example 52

Conjugation of 2'-deoxy-5-fluoro-3'-O-(3-carbonylpropanoyl)uridine (5-FUdrsucc) to PAI-2 (52; a) Activation of the ester

The active ester of 5-FUdrsucc was prepared using a modification to the method described previously (Goerlach, A.; Krauer, K. G.; McKenzie, I. F.; Pietersz, G. A. In vitro antitumor activity of 2'-deoxy-5-fluorouridine-monoclonal antibody conjugates. Bioconjug. Chem. 1991, 2, 96-101). Briefly, 5-FUdrsucc, 1.73 mg, 5 μmol) was dissolved in dry DMF (70 μL). N-hydroxysuccinimide (NH S, 0.59 mg, 5.1 μmol) in DMF (17 μL) and dicyclohexylcarbodiimide (DCC, 6.18 mg, 30 μmol) in DMF (50 μL) were then added and the reaction mixture kept at RT for 3 h and then at 4 °C overnight. The crude colourless product (R_f 0.55, silica, 100% DCM) was used in future conjugation experiments without further purification.

b) Conjugation to PAI-2

In two separate experiments, a 20 and 50 fold molar excess of the activated ester (71) were incubated with PAI-2 (ca 2 mg/mL) in PBS (pH 7.4) at RT, with shaking. After 3 h the reaction mixture was centrifuged (maximum speed for 5 min) to remove any precipitate. The conjugate was then purified by gel filtration (PD-IO column), with PBS (pH 7.4) as eluant. Fractions corresponding to the protein peak (as determined by UV/Vis spectrophotometry, 280 nm) were pooled, sterile filtered (0.22 μM) and stored at 4 °C for future cytotoxicity studies. Protein concentration was determined using the standard Lowery protein determination assay and the amount of 5-FUdrsucc bound to protein determined by electrospray ionisation mass spectrometry (ESI-MS) analysis (see Figure 17). The protein yield was > 85%.

Example 53

Conjugation of 5, 7-dibromo-3-[m-(2'-carboxymethyl)-phenylimino)-N-(p-trifluoro- methyl)isatin to PAI-2 (53) a) Activation of the ester

5,7-Dibromo-3-[m-(2'-carboxymethyl)-phenylimin-o)-N-(p-trifluoromethyl)isatin (1.73 mg, 5 μmol) was dissolved in dry DMF (70 μL). iV-hydroxysuccinimide (NHS, 0.59 mg, 5.1 μmol) in DMF (17 μL) and dicyclohexylcarbodiimide (DCC, 6.18 mg, 30 μmol) in DMF (50 μL) were then added and the reaction mixture shaken at RT between 15 min and 3 h and then 4 ⁰C overnight. The crude orange product was then used in conjugation experiments without further purification; R_f 0.43 (100% DCM, silica).

b) Conjugation to PAI-2 A 20-fold molar excess of the activated ester was incubated with PAI-2 (2.89 mg/mL) in PBS (pH 8.2) at RT, with shaking. After 1 h 15 min the reaction mixture was centrifuged (maximum speed for 5 min) to remove any precipitate. The conjugate was then purified by gel filtration (PD-IO column), eluting with PBS (pH 8.2). Fractions corresponding to the protein peak (as determined spectrophotometrically at 280 nm) were pooled, sterile filtered (0.22 μM) and stored at 4 ⁰C for future cytotoxicity studies. Protein concentration was determined using the standard Lowery protein determination assay and the amount of isatin bound to protein determined by UV/Vis spectrophotometry (ε₄₃₂ = 2140 M^-1Cm^"1) and electrospray ionisation mass spectrometry (ESI-MS) analysis. Protein yield was > 80%.

Example 54

In vitro Cytotoxicity Evaluation ofN-alkyl/aryl Isatin Derivatives f54) The cytotoxic activities of the various isatin derivatives were screened for activity against human lymphoma (U937), leukemic (Jurkat), breast carcinoma (MDA-MB-231, and MCF- 7), prostate carcinoma (PC-3) and colorectal carcinoma (HCT-116) cancer cell lines and freshly prepared human peripheral blood lymphocytes (PBL) in vitro. Cytotoxicity was determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS), in 96-well microplates as described previously (Cory et al., 1991). Briefly, cells (1.0 x 10⁴) in a total volume of 90 μL of complete media were seeded into 96-well microtitre plates and incubated for 24 h (37 ⁰C, 95% humidity, 5% CO₂) prior to the addition of test compounds. Test compounds were made up fresh on the day of testing in dimethyl sulfoxide (DMSO, 100%) at a final concentration of 4 mg/mL and diluted in complete media to give a final DMSO concentration of 25%. Compounds of various concentrations (10 μL) were then added in triplicate to the wells of a 96-well microtitre plate, containing cells, to give a final volume of 100 μL. Solvent controls containing 2.5% DMSO and background controls containing test compounds, but no cells, were also prepared. Cells were incubated for a further 24 h (37 ⁰C, 95% humidity, 5% CO₂). The MTS assay substrate (20 μL) was then added to each well and cells incubated for 3 h to allow colour development. The absorbance at 490 nm was measured using the Spectromax 250 UV plate reader (Molecular Devices, USA) utilising Softmax Pro^® software. All cell proliferation assays were performed using the number of cells which fell in the linear range. Results for each compound are reported as the concentration (μM) required to inhibit the metabolic activity of 50% of the cell population (IC₅₀) in comparison to vehicle- treated (DMSO) control cells. These values were calculated from logarithmic sigmoidal dose response curves using the variable slope parameter, generated from GraphPad Prism V. 4.02 software (GraphPad Software Inc.).

The results are shown in Table 1 below:

Table 1

OC

IC50 = concentration at which 50% cell metabolic activity is inhibited

A-I = U937 human monocyte-like, histiocytic lymphoma.A-2 = PC3 human epithelial prostate adenocarcinoma. A-3 = MDA-MB-231 human epithelial, mammary gland adenocarcinoma (metastatic). A-4 = HCTl 16 human epithelial colorectal carcinoma. A-5 = Fresh PBL peripheral human blood lymphocytes.

A-6 = Jurkat human leukemic T- cell line.A-7 = MCF-7 human epithelial, mammary gland adenocarcinoma (non-metastatic^").

Example 55

Comparison of in vitro cytotoxicity of N-alkylisatins and the commercial anticancer agent 5-Fluorouracil (5-FU). Briefly, cells (1 x 10^Λ6) were incubated for 72 h at 37 ⁰C (95% humidity, 5% CO₂) with increasing concentrations of compounds (■ 15, A 14, T 3, ♦ 2 and • 5FU), then analysed for a change in metabolic activity and expressed as percent viability in reference to the DMSO control. Each data point is a mean of triplicates ± SE of one representative experiment. Figure 1 shows the viability of U937 cells after treatment with increasing concentrations of ■ 15, A 14, ▼ 3, ♦ 2 and • 5FU (see Figure 1).

Example 56

The Stability oflmine Compounds at neutral and acidic pH

Hydrolysis of the imine bond can be monitored by measuring the absorbance at 435 nm because of the large difference in E₄₃₅ of the imine and the liberated isatin (see Figures 10 and 11). For example, Figure 11 shows a graph depicting the behaviour of the imine 32, in sodium acetate buffer at pH 7.02 and 5.17. In this example the calculated endpoints (or values for A₄₃₅ at complete hydrolysis of the imine) were 0.11 for the pH 5.17 solution and 0.06 for the pH 7.02 solution. The imine bond of compound 32 was stable at neutral pH (7.02) over an 8 h period, but is almost entirely hydrolysed in less than 4 h in pH 5.17 buffer at 18°C. TLC and MS of the solution after the time course showed that the isatin cytotoxin (compound 15) was released intact at pH 5.17 and that the imine (compound 32) was unchanged at neutral pH.

Example 57

Preparation of an Isatin-Spacer-Transferrin Conjugate

a): Activation of the Isatin Ester.

All manipulations were carried out under a blanket of dry nitrogen gas. 5,7-dibromo-7V-(p- trifluormethylbenzyl)isatin imine (3.0 mg, 5 μmol, compound 32) was dissolved in 70 μL dry DMF followed by the addition of solutions of JV-hydroxysuccinamide (NHS, 0.6 mg,

5.0 μmol, in 17 μL of dry DMF) and iV.iV-dicyclohexylcarbodimide (DCC, 6.0 mg, 30 μmol, in 50 μL of dry DMF). The mixture was left to react for 1 h or 24 hours at room temperature to form the activated (NHS) ester. The molar ratio of compound 32:NHS:DCC (1 :1:6).

b): Conjugation of activated compound 32 to Transferrin.

(i) 50 fold excess A Transferrin (5 mg) stock solution was made up to 2 mL (PBS pH 8.2) from a lyophilized powder to give a final concentration of 2.5 mg/mL. 21.7 μL of activated compound 32 (50 fold molar excess) was added to transferrin (500 μL), wrapped in foil and shaken for 1 h at room temp.

(U) 12 fold excess

5 μL of activated isatin (12 fold molar excess) was added to transferrin (500 μL), wrapped in foil and shaken for 1 h at room temp. After conjugation, any precipitated protein was removed by spinning on a benchtop centrifuge at max speed for ~ 5 min. The sample was then loaded onto a PD-IO size exclusion column pre-equilibrated with PBS pH 8.4, The 500 μL sample was then loaded and left to absorb into the gel bed followed by 2.5 mL PBS (pH 8.2) and 0.5 mL fractions collected thereafter. 24 fractions were collected for both proteins in total. The protein peak was visualized by reading 100 μL of each fraction at 280280 nm in a 96 well quartz plate. Fractions 5, 6 and 7 were pooled from the first conjugation (50 fold) and 5 and 6 from the second conjugation (12 fold) the protein cone, determined in the Lowry Assay. (5 μL of sample was added to the wells of a 96 well microtitre plate followed by 25 μL reagent A and 200 μL reagent B. Cone was determined by interpolation from a BSA standard curve).

The total protein yield for the conjugation reactions was 87.2% (using 50 fold excess) and 82.3% (using 12 fold excess). See Figure 12.

c): Analysis of protein integrity after conjugation

The integrity of the protein after conjugation was assessed by SDS PAGE. Approximately 5 μg of protein was loaded onto a 12% acrylamide gel and run at 120 V under non- reducing conditions. The molecular weight of transferrin is ~78, 500 Da. (See Figure 13).

d): Conjugation efficiency

The average number of cytotoxins per protein molecule was calculated by using uv-vis spectroscopy.

(i) The difference in absorbance between the conjugate from the 50 fold reaction and transferrin = 0.049 - 0.028 = 0.021 (ii) (The difference in absorbance between the conjugate from the 12 fold reaction and transferrin = 0.034 - 0.028 = 0.006.

Using: the relationship A = εC x 0.3 (where 0.3 is the path length of a well of a 96-well quartz plate containing 100 μL of sample).

Then for (i), the concentration of isatin is 0.021 = 2140 x C x 0.3 = 32.7 μM and the concentration of transferrin is 0.671 mg/ml / 78500 g/mol = 8.5 μM and therefore the ratio of protein to isatin in (i) is 32.7 μM / 8.5 μM = 1 :3.8. Using the same method of calculation the ratio of protein to isatin in (ii) is μM9.3 μM / 12.3 μM = 1 : 0.75.

Example 58 Cytotoxic Activity and SAR

The N-phenethyl compounds 36 to 40 and iV-naphthylmethyl derivatives 41 and 42 were initially tested for cytotoxicity using the MTS cell proliferation assay against three human cancer cell lines including a lymphoma (U937), leukemia (Jurkat) and metastatic breast adenocarcinoma cell line (MDA-MB-231). Since the iV-phenacyl derivatives 43-47 were less active than the others on U937 they were not tested on other cell lines. The results are shown in Table 2.

Table 2.

Initial cytotoxicity screening (IC₅₀ μM) of novel iV-substituted isatin derivatives 36-42^a

Compound U937^b Jurkat⁰ MDA-MB-231^α

35 10.5^e 14.3^e 42.3^e

36 0.78 1.52 4.35

37 0.78 1.21 2.72

38 0.88 1.15 2.01

39 1.07 2.00 5.24

40 2.35 2.66 4.51

41 0.19 0.91 2.49

42 0.74 0.41 2.56

43 9.97 nt^f nt

44 6.36 nt nt

45 9.18 nt nt

46 4.70 nt nt

47 5.32 nt nt

Vinblastine 6.88^g nt nt

Values are the mean of triplicates of at least two independent experiments. b Human monocyte-like histiocytic lymphoma cell line. c Human leukemic T-cell line. d Human metastatic mammary gland adenocarcinoma cell line. e Vine, et al. Vine, K. L.; Locke, J. M.; Ranson, M.; Benkendorff, K.; Pyne, S. G.; Bremner, J. B. Bioorg. Med, Chem. 2007, 15, 931. f Not tested. g Vine, et al. Vine, K. L.; Locke, J. M.; Ranson, M.; Pyne, S. G.; Bremner, J. B. J. Med, Chem. 2007, JO, 5109.

The four most potent derivatives 37, 38, 41, and 42, (Table 2) were selected for further cytotoxic screening against a panel of other adherent human tumor cell lines: colorectal (HCT- 116), prostate (PC-3), non-metastatic breast (MCF-7) and melanoma (A375). The results are shown below in Table 3.

Table 3. Further cytotoxicity screening (IC₅₀ μM) of derivatives 37, 38, 41 and 42 against a panel of human adherent tumor cell lines. ^a

Compound HCT-116^b PC-3^C MCF-7^d A375^e

37 1.41 2.09 4.04 3.44

38 1.62 2.21 6.02 4.34

39 1.15 1.44 7.71 3.95

40 1.51 2.29 3.50 6.56 a Values are the mean of triplicates of at least two independent experiments. Human colorectal carcinoma cell line. c Human prostate adenocarcinoma cell line. Human non-metastatic mammary gland adenocarcinoma cell line. e Human malignant melanoma cell line.

Example 59

2.3 Mode of Action Studies While performing in vitro cytotoxicity testing, it was observed that the isatin derivatives 36-47 caused the U937, and to a lesser extent, the Jurkat cells to undergo elongation. The elongated morphology was more pronounced when the cells were treated with iV-naphthylm ethyl derivatives. This morphological change was also observed in U937 cells treated with vinblastine, a microtubule destabilizer. This suggested that these isatin analogues may interfere with microtubule dynamics, which are imperative for the maintenance of cell shape.

To further investigate the effects of the aforementioned analogues on microtubule formation, a cell-free in vitro tubulin polymerization assay was performed. The two naphthyl derivatives were chosen due to their in vitro potency against U937 and Jurkat cells, as well as two representative phenethyl compounds, 36 and 38. Vinblastine sulfate and paclitaxel were used as a known microtubule destabilizer and stabilizer respectively. Consistent with literature reports, at 10 μM paclitaxel stabilized microtubules while vinblastine sulfate was a potent microtubule destabilizer. The test compounds, in particular the 2-naphthyl derivative 42, appeared to be potent microtubule destabilizers at 10 μM.

The three most potent destabilizers 36, 41 and 42, were chosen for further studies on tubulin polymerization. AU of these compounds inhibited the rate of tubulin polymerization in a dose-dependent manner and the IC_5O values are reported in Table 4. Vinblastine displayed similar inhibition in this assay to that reported previously.

Table 4.

Inhibition of tubulin polymerization (IC₅₀ μM) of compounds 36, 41 and 42 and vinblastine at varying time points. ^a

Compound 20 min 60 min

36 4.04 6.60

41 4.71 12.0

42 2.87 6.47

Vinblastine 0.95 1.66 a Values are the mean of duplicates in one experiment.

The compound 42 displayed potent destabilizing effects at higher concentrations and stabilized microtubules at lower concentrations. This phenomenon has also been reported in the case of vinblastine and suggests that these compounds may bind to more than one binding site on tubulin with different affinities.

Example 60

Single Photon Emission Computed Tomography (SPECT) Imaging of Rat Mammary

Tumour Models (60)

The radiotracers were prepared fresh on the day of imaging (as described above in Examples 50 and 51) and 100 μL (in 0.9% saline and 5% EtOH) injected via the tail vein.

Between 35- 45 min post injection (p.i.), animals were anaesthetised using inhalant isoflurane with 200 mm/min oxygen via a nose cone fitted to the animal bed of the X- SPECT^® SPECT/CT imaging system. Once the animal was no longer responsive to stimuli (pinching between toes) the animal was placed on the bed lying on its stomach and the bed driven into the imaging tunnel. The HRES (5" x 5") collimators were fitted and rotated to be horizontal above and below the animal, and moved in as close as possible to the animal without touching it (Head 1 was at 0° above animal). Acquisitions were taken to obtain images of the head and/or body at the time points indicated in Table 5. Afterwards, the animals were removed from anaesthetic, woken and observed for recovery between each imaging time point and had access to food and water. Animals were sacrificed by CO₂ suffocation followed by cervical dislocation at the last imaging time point. The results are shown in Figure 16.

Table 5

Protocol for SPECT imaging of radiotracers in F344 Fisher rats bearing 13762 MAT B III mammary adenocarcinoma.⁸

Time Point Acquisition Type Acquisition Time Camera Position

(Post Injection) (min)

45 - 60 min Dynamic 15 Lower body

3 frames

(5 min per frame)

I h Static 5 Head and upper body

3 h Dynamic 15 Lower body

3 frames

(5 min per frame)

3 h 20 min Static 5 Head and upper body

6 h Static 10 Lower body

6 h 15 min Static 5 Head and upper body

24 h Static 20 Lower body

48 h Static 40 Lower body

72 h^b Static 40 Lower body ^a Rats were injected with 5 μCi/μL of either 50 or 5I₅ 11 days post tumour cell inoculation. ^b This additional time point was conducted on freshly euthanised rats.

Example 61 In vitro Cytotoxicity Evaluation

The cytotoxic activities of protein alone (PAI-2), protein-cytotoxin conjugates (PAI-2-5- FUdrsucc and PAI-2-CF₃imine (i.e. compound 32 conjugated to PAI-2)) as well as unconjugated cytotoxins (5-FUdr, 5-FUdrsucc, 5,7-dibromo-N-(p- trifluoromethylbenzyl)isatin (15) and 5,7~dibromo-3-[rø-(2'-carboxymethyl)-phenylimino)- iV-(p-trifluoromethyl)isatm (32) were determined against two human breast adenocarcinoma cell lines (MDA-MB-231 and MCF-7) which vary in their expression levels of uPA (Andronicos, N. M.; Ranson, M. The topology of plasminogen binding and activation on the surface of human breast cancer cells. Br J Cancer 2001, 85, 909-916); Ranson et al. (Ranson, M.; Tian, Z.; Andronicos, N. M.; Rizvi, S.; Allen, B. J. In vitro cytotoxicity of bismuth-213 (213Bi)-labeled-plasminogen activator inhibitor type 2 (alpha- PAI-2) on human breast cancer cells. Breast Cancer Res. Treat. 2002, 71, 149-159). Cytotoxicity was determined using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS), in 96-well microplates. Test compounds were incubated with different cell lines, in triplicate, at increasing concentrations for 48 h prior to the addition of MTS reagent. Cytotoxic activity was then determined spectrophoto-meterically at 490 nm. Results for each compound are reported below in Table 6 as the concentration (μM) required to inhibit the metabolic activity of 50% of the cell population (IC50) in comparison to PBS or DMSO treated cells. These values were calculated from logarithmic sigmoidal dose response curves using the variable slope parameter, generated from GraphPad Prism V 4.02 software (GraphPad Software Inc.). Table 6

The effect of PAI-2-5-FUdrsucc and unconjugated cytotoxins 5-FUdr and 5-FUdrsucc on two mammary adenocarcinoma cell lines varying in their expression levels of uPA.

IC₅₀ (μM)^a

Cell line uPA^b 5-FUdr 5-FUdrsucc PAI-2 PAI-2-5-FUdrsucc

MCF-7 low >203 >144 NA^C >3.2 MDA-MB-231 high 0.27 2.8 NA 1.0 a IC_5O values were calculated based on moles of cytotoxin from sigmoidal dose response curves (variable slope), generated using GraphPad Prism V. 4.02 (GraphPad Software Inc.). Values are the mean of triplicate experiments. b Relative expression levels of uPA (Andronicos and Ranson 2001; Ranson et ah, 2002). ^c NA: Not active, even at the highest concentration tested (i.e. 1.1 μM).

a) Addition of Exogenous uPA

Assays were performed using MTS reagent as described above, however, prior to the addition of PAI-2-5-FUdrsucc (4 μM protein equiv.), MCF-7 cells were pre-incubated with 18 nJVI exogenous uPA (+ uPA) or PBS (- uPA) for 10 min at RT. Cells were then washed 3 xwith RPMI-1640 (containing 5% FCS).

Statistical Analyses

Statistical significance of treatment groups as compared to control groups were determined using an unpaired students T-test (GraphPad Prism V 4.02). P values < 0.05 were considered statistically significant.

Example 62

Flow cytometry analysis of cellular DNA content was performed using a modification to the method described previously (Chen, Z.; Xiao, Z.; Chen, J.; Ng, S. C; Sowin, T. et al.

Human Chkl expression is dispensable for somatic cell death and critical for sustaining G2

DNA damage checkpoint. MoI. Cancer Ther. 2003, 2, 543-548). Briefly, cells (2.0 * 10⁴) were harvested by centrifugation (1500 rpm, 5 min) and fixed with ice-cold ethanol (70%) for 30 min to overnight at -20 ⁰C. The ethanol was then removed by centrifugation and cells were stained with PI master mix (100 μg/mL RNase A, 40 μg/mL PI, PBS pH 7.4) for 30 min at 37 °C. DNA content was then measured using a Becton Dickinson BD™ LSR II FACSort flow cytometer (BD Biosciences, USA) and the proportion of cells in G0/G1, S and G2/M phases of cell cycle were calculated on the basis of DNA distribution histograms using Flow Jo software (V7.1, Tree Star Inc., USA). The results are shown in figure 14.

Example 63

Compounds were tested for their anti-mitotic ability in reference to vinblastine sulfate and paclitaxel positive controls in a fluorescent based Tubulin Polymerisation Assay adapted from the method described by Bonne et al., (Bonne, D.; Heusele, C; Simon, C; Pantaloni, D. 4',6-Diamidino-2-phenylindole, a fluorescent probe for tubulin and microtubules. J. Biol. Chem. 1985, 260, 2819-2825). Briefly, 50 μL tubulin reaction mix (2 mg/mL purified bovine brain tubulin in 80 mM PIPES pH 6.9, 2.0 mM MgCl₂, 0.5 mM EGTA, 1.0 mM GTP and 20% glycerol was added to duplicate wells of a half area 96-well black plate containing 5 μL of either vehicle control, paclitaxel, vinblastine sulfate or test compounds all at a final concentration of 3 μM or 10 μM. The rate of polymerisation was followed for 1 h at 37 °C using an excitation wavelength of 360 ±10 nm and the fluorescence was collected at 440 ±10 nm. The results are shown in figure 15.

Claims

CLAIMS:

1. A compound of formula I:

(R¹X¹^T¹

formula I

wherein: R¹ is a derivatised cytotoxic isatin moiety; X¹ represents a spacer; T¹ is a cell-targeting moiety; and n is an integer greater than or equal to 1.

2. A compound according to claim 1 of formula II:

(R¹L¹Z¹L^_nT¹ formula II

wherein R¹, T¹ and n are as defined in claim 1 and wherein L¹ represents a covalent linkage formed with R¹; L² represents a covalent linkage formed with T¹; and Z¹ represents a bridging moiety.

3. A compound according to claim 2 wherein L² is stable to physiological conditions and L¹ is labile to a physiological environment within an adjacent to the cell to be targeted by T¹.

4. A compound according to claim 2 or claim 3 wherein L¹ is an enzymatically cleavable linkage or an acid labile linkage.

5. A compound according to claim 4 wherein L¹ is stable from at least about pH 6.9 to pH 7.4.

6. A compound according to any one of claims 2 to 5 wherein L¹ is selected from =N-, =N~W~, =N- W-C(Y)-, and =N- W-C(Y)-Q- (wherein N is a nitrogen atom and W, Y, and Q are independently selected from O, S, CH₂ or NR^N, wherein R^N is selected from hydrogen, hydroxy, amino, optionally substituted Ci.galkyl, optionally substituted C^alkenyl, optionally substituted C_1-8alkynyl, optionally substituted C_1-4alkylaryl, and optionally substituted aryl), a methyleneoxy group, an ethyleneoxy group, a ketal or a hemi-ketal.

7. A compound according to any one of claims 2 to 6 wherein cleavage of covalent linkage L¹ from the derivatised cytotoxic isatin moiety R¹ releases a cytotoxic isatin compound of formula III:

formula III wherein:

R², R³, R⁴ and R⁵ are independently selected from H; halo; optionally substituted alkyl; optionally substituted cycloalkyl; optionally substituted alkenyl; optionally substituted cycloalkenyl; optionally substituted alkynyl; optionally substituted alkoxy; optionally substituted haloalkyl; optionally substituted hydroxyalkyl; optionally substituted acyl; optionally substituted acyloxy; hydroxyl; optionally substituted aryl or fused aryl; optionally substituted aryloxy; optionally substituted heteroaryl; optionally substituted heteroaryloxy; amino; azido; nitro; nitroso; cyano; carbamoyl; trifluoromethyl; mercapto; optionally substituted alkylamino; optionally substituted dialkylamino; formyl; optionally substituted alkylsulfonyl; optionally substituted arylsulfonyl; optionally substituted alkylsulfonamido; optionally substituted arylsulfonamido; and optionally substituted alkoxycarbonyl; or

alternatively any of R² and R³ or R³ and R⁴ or R⁴ and R⁵, and R^sand R⁶ may combine to form an aliphatic or aromatic, heterocyclic or carbocyclic ring system;

R⁶ is selected from optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted cycloalkyl; optionally substituted heterocycloalkyl; optionally substituted alkoxy; optionally substituted aryl; optionally substituted acylaryl; optionally substituted heteroaryl; optionally substituted C_1-6alkylaryl or optionally substituted Ci_-6alkylheteroaryl.

8. A compound according to claim 7 wherein R to R are independently selected from H and halo.

9. A compound according to claim 8 wherein R² and R⁵ are independently selected from H and Br.

10. A compound according to claim 7 or claim 8 wherein R⁶ is selected from optionally substituted benzyl; optionally substituted methylnaphthyl; optionally substituted phenacyl or optionally substituted phenethyl.

11. A compound according to claim 10 wherein R⁶ is optionally substituted benzyl.

12. A compound according to claims 10 and 11 wherein the optional substituents are selected from aryl, Ci_-6acyl, Ci-βalkyl, Ci_-6alkoxy, C_2-6alkenyl, C_2-6alkynyl, Ci_-4alkyloxyCi_-4alkyl; C_2-6alkenylaryl; C_2-6alkenylheteroaryl; C_2-6alkynylaryl; C_2-6alkynylheteroaryl; Ci_-6alkylsulfonyl, arylsulfonyl, -COOH; -C_]-6alkylCOOH; -COO⁺M^"; -Ci_-6alkylCOO^"M⁺, wherein M⁺ is a counter ion; -C(O)OCi_-6alkyl; -C(O)OCi_-6aryl; -C(O)OCi_-6heteroaryl; -C(O)OC i_-6alkylheteroaryl; -C(O)OC_)-6alkylaryl; -Ci_-6alkylC(O)OCi_-6alkyl; -C_1-6alkylC(O)OC_1-6aryl; -Ci_-6alkylC(O)OC_1-6heteroaryl; -C_1-6alkylC(O)OCi.₆alkylheteroaryl; -C₁.₆alkylC(O)OC_1-6alkylaryl; C_1-6alkylsulfonamido; arylsulfonamido; halo; hydroxy; mercaptol; trifluoromethyl; carbamoyl; amino; azido; nitro; cyano; Ci_-6alkylamino or di(Ci_-6alkyl)amino.

13. A compound according to claim 12 wherein the optional substituents are selected from Cl, Br and trifluoromethyl.

14. A compound according to claim 7 wherein R² to R⁵ are independently from H and Br.

15. A compound according to any one of claims 1 to 14 wherein the derivatised cytotoxic isatin compound is attached to the spacer through the 3-oxo group of the isatin compound.

16. A compound according to claim 15 of formula IV :

formula IV wherein:

R >2% r

R> 6^b, T L 1¹, T 1/ 2, Z n,\¹, n and T¹ are as defined in claim 7.

17. A compound according to claim 15 of formula X:

formula X

wherein:

R², R³, R\ R⁵, Z¹, L² and n are as defined in claim 7; q is an integer selected from 1 to 5; T¹ is a cell-targeting moiety; m is an integer from 0 to 4; and

R⁷ is selected from C_1-6acyl; Ci.₆alkyl; Ci_-6alkoxy; C_2-6alkenyl; C₂-6alkynyl;

C_1-6alkylsulfonyl; aryl; aryloxy; arylsulfonyl; -COOH; -C_1-6alkylCOOH; -COO⁺M^"; -Ci-

₆alkylCOO^"M⁺, wherein M⁺ is a counter ion, e.g. Na⁺, K⁺, NH₄ ⁺, Ca²⁺; -C_1-6alkylOH -C(O)OCi_-6alkyl; -C(O)OC i_-6aryl; -C(O)OC_1-6heteroaryl; -C(O)OCi_-6alkylheteroaryl; -

C(O)OCi_-6alkylaryl; -Ci_-6alkylC(O)OC_]-6alkyl; -C_1-6alkylC(O)OCi_-6aryl; -Ci-

₆alkylC(O)OCi_-6heteroaryl; -Ci_-6alkylC(O)OC_1-6alkylheteroaryl;

₆alkylaryl; Ci_-6alkylsulfonamido; arylsulfonamido; halo; hydroxy; mercaptol; trifluoromethyl; carbamoyl; amino; azido; nitro; cyano; -C_1-6alkylamino or di(Ci_ ₆alkyl)amino.

18. A compound according to claim 17 wherein R⁷ is selected from Cl, Br and trifluoromethyl.

19. A compound according to claim 15 of formula XI:

formula XI

wherein:

R², R³, R⁴, R⁵, L², n and R⁷ are defined in claim 7;

V¹ is selected from optionally substituted divalent Ci_-6alkyl, optionally substituted divalent C_2-6alkenyl, optionally substituted divalent C_2-6alkynyl; q is an integer selected from 1 to 5; and T¹ is a cell targeting moiety.

20. A compound according to claim 15 of formula XII:

formula XII wherein:

L², T¹, n and R⁷ are as defined in claim 7;

V¹ is selected from optionally substituted divalent Ci_-6alkyl, optionally substituted divalent C_2-6alkenyl, optionally substituted divalent C_2-6alkynyl; q is an integer selected from 1 to 5; and T is a cell-targeting moiety.

21. A compound according to claim 15 of formula XIII:

formula XIII wherein T¹ is a cell-targeting moiety and n is an integer from 1 to 12,

22. A compound of formula XIV :

formula XIV where R³ and R⁵ are independently selected from Cl, Br, I, CF₃, optionally substituted alkyl, optionally substituted phenyl, and R⁶ is as defined in claim 7.

23. A compound of claim 22 wherein R³ and R⁵ are both Br.

24. A compound according to claim 22 or 23 wherein R corresponds to the moiety attached to the ring nitrogen (1 -position) of the compounds of any one of claims 17 to 21.

25. A method of preparing a compound for targeting a cell type to induce death of that cell or to inhibit growth of that cell, including the steps of:

(iv) identifying a cell type;

(v) identifying a targeting molecule with selectivity for that cell type; and

(vi) conjugating that targeting molecule to a derivatised cytotoxic isatin moiety of any one of formulae V to IX.

26. A method of targeting a cytotoxic isatin compound to a cell-type comprising conjugating the cytotoxic isatin compound via a spacer to a cell-targeting moiety capable of targeting that cell type, and administering the resultant conjugate to a subject in need thereof.

27. A method of inducing death in cells in a subject comprising administering to the subject an effective amount of a compound of any of claims 1 to 21, wherein the cell- targeting moiety is selected to target the cells.

28. A method of claim 27 wherein the cells are associated with a tumour or disease state.