WO2016028225A1 - Platinum complexes as anticancer agents - Google Patents

Abstract

The present invention relates to platinum compounds of Formula (I) or (II) as defined herein as well as their use for the treatment of cancer, methods for treating cancer in a subject using said compounds, and methods for triggering apoptosis in a cell using said compounds.

Description

PLATINUM COMPLEXES AS ANTICANCER AGENTS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application makes reference to and claims the benefit of priority of a U.S.

Provisional Application for "Platinum Anticancer Agents that Allow Control of Nucleosome Targeting" filed on August 22, 2014, and duly assigned application number US 62/040,768. The content of said application filed on August 22, 2014, is incorporated herein by reference for all purposes, including an incorporation of any element or part of the description, claims or drawings not contained herein and referred to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.

FIELD OF THE INVENTION

[0002] The present invention relates generally to compounds for the treatment of cancer.

More particularly, the invention pertains to monofunctional platinum complexes with DNA intercalating capabilities as anticancer agents.

BACKGROUND OF THE INVENTION

[0003] To date cis-diaminedichloroplatinum(II) (cDDP, cisplatin) and its close analogues carboplatin and oxaliplatin are used to treat about half of all patients receiving chemotherapy for cancer. These platinum drugs achieve their therapeutic efficacy by forming DNA lesions, which interfere with genomic activities and ultimately trigger apoptosis.

[0004] However, these drugs are associated with severe toxicity and intrinsic or acquired resistance effects. The underlying problem is that their small size and square planar geometry allow them to form adducts at most guanine base sites on DNA. Thus, one possibility is to rationally design platinum compounds that can avoid binding to counterproductive sites while still being capable to bind one or more therapeutic targets. Extensive efforts have been made to synthesize and test new platinum-based anticancer agents, with the expectation that compounds with improved antitumor activity and fewer toxic side effects would be discovered. Disappointingly, although thousands of platinum compounds have been synthesized and screened since the entry of the first platinum agent, cisplatin, into the drug market in 1978, only two other platinum drugs have received FDA approval.

[0005] Therefore, there remains an enormous but unmet need to identify or design compounds with similar activity but reduced side effects relative to the platinum drugs currently in use.

SUMMARY OF THE INVENTION

[0006] The inventors of the present invention have found that said need can be met by a novel class of platinum-based anticancer agents, designed by linking monofunctional (diethylenetriamine)-platinum(II) derivatives to DNA intercalating compounds.

[0007] In one aspect, the present invention therefore relates to a compound of Formula (I) or

(II) or a stereoisomer or pharmaceutically acceptable salt thereof,

wherein

X is a leaving group;

R¹, R¹ , R² and R² are each independently hydrogen or methyl;

R³, R³ and R⁴ are each independently selected from the group consisting of hydrogen, CMO alkyl and Ci.₁₀ alkyloxy;

each n is independently an integer of 1 to 10; and

ICA is a DNA intercalating compound.

[0008] In various embodiments, X may be any suitable leaving group, preferably X is selected from the group consisting of halogen, -OH, -SCN, -OR⁵ and -0-C(0)-R⁵, wherein R⁵ is any organic moiety.

[0009] In various embodiments, in the compound of Formula (I), any one or more or all of R¹,

R^{1 '}, R² and R² are hydrogen. In preferred embodiments, all of R¹, R¹ , R² and R² are hydrogen.

Similarly, in various embodiments of the compounds of Formula (II), any one or more or all of R¹, R²,

R² and R⁴ are hydrogen. In preferred embodiments, all of R¹, R², R² and R⁴ are hydrogen.

[00010] In various embodiments, any one or more or all of R³ and R^3' may be hydrogen. In preferred embodiments, all of R³ and R³ are hydrogen.

[00011] In various embodiments, n is 2 to 5, preferably 3. [00012] In various embodiments of the compounds of the invention, the DNA intercalating compound is 1,8-naphthalimide or a derivative thereof. The 1,8-naphthalimide or the derivative thereof are in various embodiments, selected from compounds of formula (III),

wherein R°, R° , R', R' , R°, R^* are each independently selected from the group consisting of hydrogen, Ci-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, Ci-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄ alkylcarbonato, C₆-C₂o arylcarbonato, carboxy, carboxylato, carbamoyl, mono-(C]-C₂₄ alkyl)-substituted carbamoyl, di-(C C₂₄ alkyl)-substituted carbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, mono- and di-(Ci- C₂₄ alkyl)-substituted amino, mono- and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₅-C₂₀ arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato, C]-C₂₄ alkylsulfanyl, arylsulfanyl, Ci-C₂₄ alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, and phosphino, and wherein any two adjacent {ortho) substituents may be linked to form a cyclic structure selected from five-membered rings, six- membered rings, and fused five-membered and/or six-membered rings, wherein the cyclic structure is aromatic, alicyclic, heteroaromatic, or heteroalicyclic, and has 0 to 4 non-hydrogen substituents and 0 to 3 heteroatoms.

[00013] In various embodiments, the DNA intercalating compound is selected from the group consisting of

lH-benzo[i/e]isoquinoline- 1 ,3(2H)-dione (i.e.1 ,8-naphthalimide);

4- nitro- lH-benzo [de] isoquinoline- 1 , 3 (2H)-dione;

5 - nitro- lH-benzo [de] isoquinoline- 1 ,3 (2H)-dione;

6- nitro- 1 H-benzo [de] isoquinoline- 1 ,3 (2H)-dione;

6-amino-5 -nitro- lH-benzo [de] isoquinoline- 1 , 3 (2H)-dione;

6-(piperidin-l-yl)-lH-benzo[ e]isoquinoline-l,3(2H)-dione; 5- nitro-6-(piperidin- 1 -yl)- lH-benzo[i/e]isoquinoline- 1 , 3(2H)-dione;

6- morpholino-lH-benzo[i e]isoquinoline-l,3(2H)-dione;

6-(piperazin-l -yl)-lH-benzo[i/e]isoquinoline-l ,3(2H)-dione;

6-(4-methylpiperazin- 1 -yl)- 1 H-benzo [de] isoquinoline- 1 ,3 (2H)-dione;

5- (piperazin-l -yl)-lH-benzo[de]isoquinoline-l ,3(2H)-dione;

6- (lH-imidazol-l -yl)-lH-benzo[c?e]isoquinoline-l ,3(2H)-dione;

7,8-dihydronaphtho[2, 1 ,8-i/e |isoquinoline-l ,3(2H,6H)-dione;

6,7-dihydro-lH-indeno[6,7,l-rfe ]isoquinoline-l,3(2H)-dione;

4H-benzo[rfe]furo[3,2-g]isoquinoline-4,6(5H)-dione;

4H-benzo [de] furo [2, 3 -g]isoquinoline-4,6(5H)-dione;

4H-dibenzo[(/e,g]isoquinoline-4,6(5H)-dione;

4H-isoquinolino[5,4-^]quinoxaline-4,6(5H)-dione;

lH-dibenzo[<fe,/?]isoquinoline-l,3(2H)-dione;

lH-benzo[i e][2,9]phenanthroline-l,3(2H)-dione;

2,2,2-trichloro-N-((l ,3-dioxo-2,3-dihy(ko-l^

5-phenyl-lH-benzo[i/e]isoquinoline-l ,3(2H)-dione;

5-(p-tolyl)- 1 H-benzo[c/e]isoquinoline- 1 ,3 (2H)-dione;

5-(4-fluorophenyl)-lH-benzo[i/e]isoquinoline-l,3(2H)-dione;

5-(4-(trifluoromethyl)phenyl)-lH-benzo[Je]isoquinoline-l,3(2H)-dione;

5-(3,4-difluorophenyl)-lH-benzo[i/e]isoquinoline-l,3(2H)-dione;

5 -(3 ,4-dinitrophenyl)- 1 H-benzo [ife]isoquinoline- 1 ,3 (2H)-dione;

5- (thiophen-2-yl)-lH-benzo[i e]isoquinoline-l,3(2H)-dione;

6- ((2-(piperazin-l-yl)ethyl)amino)-lH-benzo[^e]isoquinoline-l,3(2H)-dione;

6-((2-hydrazinylethyl)amino)-lH-benzo[<ie]isoquinoline-l,3(2H)-dione;

6-((2-(dimethylamino)ethyl)amino)- lH-benzo [ de] isoquinoline- 1 ,3 (2H)-dione;

6-((2-( henyltMo)ethyl)amino)-lH-benzo[iie]isoquinoline-l,3(2H)-dione;

lH-thioxantheno[2,l,9-i/e ]isoquinoline-l ,3(2H)-dione;

4H-benzo[^e]benzo[4,5]thieno[2,3-g]isoqumoline-4,6(5H)-dione;

9-phenyl-4H-benzo[ e]oxazolo[5,4-g]isoquinoline-4,6(5H)-dione;

4H-benzo[i/e]thieno[2,3-g]isoquinoline-4,6(5H)-dione; and

9-phenylbenzo[i/e]imidazo[4,5-g]isoquinoline-4,6(5H, 10H)-dione.

[00014] In some embodiments, the compound is c/s-chloro[l,2,3-diethylenetriamino-2-N-(3- propyl)-l,8-naphthalimide]platinum(II) chloride or irarcs-chlorofl^^-diemylenetriamino-l-N-P- propyl)-l ,8-naphthalimide]platinum(II) chloride.

[00015] In another aspect, the invention encompasses the compounds disclosed herein for use in a method for the treatment of cancer or for the manufacture of a medicament for the treatment of cancer.

[00016] In a further aspect, the present invention is directed to a method for the treatment of cancer in a subject, the method comprising administering to said subject a therapeutically effective amount of said compound. Said subject is preferably a mammal, more preferably a human being.

[00017] In still another aspect, the invention relates to a method for triggering apoptosis in a cell, preferably a cancer cell, the method comprising contacting said cell with an effective amount of said compound. Said methods may be ex vivo or in vitro methods, such as cell culture methods.

[00018] In another aspect, the invention concerns the use of the compounds described herein for triggering apoptosis in a cell, preferably a cancer cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[00012] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[00013] Figure 1. Scheme for synthesis of cisPfNAP (c5-chloro[l,2,3-diethylenetriamino-2-

N-(3 -propyl)- 1, 8 -naphthalimide]platinum(II) chloride) and trPtNAP (/ra«5-chloro[l,2,3- diethylenetriamino-1 -N-(3-propyl)-l ,8-naphthalimide]platinum(II) chloride).

[00014] Figure 2. DNA sequence, context and site selectivity of cisPfNAP and trPtNAP.

Preferences of cisPtNAP and trPtNAP adduct formation with respect to double helix state, structure and sequence (native nucleosome core particle (NCP) structure, upper; treated NCP structure, lower; PtNAP adducts in structure, green). Orange and black lettering for the DNA sequence designate regions where the minor as opposed to the major groove, respectively, face inward toward the histone octamer in the NCP. In the native NCP, locations of DNA stretching around SHL ±2 (blue arrows) result in severe kinks into the major groove (left half, SHL -1) or into the minor groove (right half, SHL +1.5) and a shift in the histone-DNA register, depicted as a gap in the sequence. The footprinting analysis of PtNAP-treated naked DNA (D) and nucleosomal DNA (N), shown in Fig. 3, is summarized with exonuclease stop sites depicted as arrowheads, adjacent to the terminal 3' nucleotide, pointing towards the platinum adduct.

[00015] Figure 3. Exonuclease digest analysis shows PtNAP adduct formation profiles.

DNA samples, consisting of either NCP (N) or the corresponding naked DNA (D) treated with trPtNAP or cisPfNAP (10-, 20- or 50-fold molar excess of trPtNAP; 5-, 10- or 50-fold molar excess of cisPtNAP), are shown with purine sequencing standards (m). Numbers represent nucleotide position relative to the nucleosome centre (black arrow), and green asterisks designate the locations of guanine nucleotides. Red arrows indicate regions of DNA stretching populated in the native NCP crystal structure (solid) and alternative configurations available (dashed). [00016] Figure 4. Crystallographic analysis shows trPtNAP and cisPtNAP DNA binding in the nucleosome core. Carbon atoms of the PtNAP ligands are shown in green. Anomalous difference electron density maps appear in magenta (contoured at 9a, 8σ, 7σ and 10σ for trPtNAP/SHL-1.5, trPtNAP/SHL+1.5, cisPtNAP/SHL-1.5 and cisPtNAP/SHL+1.5, respectively). Hydrogen bonds between ligands and DNA are indicated with dashed lines. Arrows depict rotation of the platinum head group from a putative axially coordinated, pre-reaction, complex to the adducted state, which coincides with the swivel-like rotational freedom of the methylene linker for the cisPtNAP, but not for trPtNAP, intercalated configuration.

[00017] Figure 5. Experimental electron density corresponding to trPtNAP and cisPtNAP binding in the nucleosome core. F₀-F_c omit electron density maps (grey; contoured at 2.7σ, 2.0σ, 2.7σ and 2.5σ for trPtNAP/SHL-1.5, trPtNAP/SHL+1.5, cisPfNAP/SHL-1.5 and cisPtNAP/SHL+1.5), calculated with trPtNAP and cisPtNAP ligands (green carbon atoms) removed from the model, and anomalous difference electron density maps (magenta; contoured at 5.5a, 5.0o, 5.5a and 4.5σ for trPtNAP/SHL-1.5, trPtNAP/SHL+1.5, cisPtNAP/SHL-1.5 and cisPtNAP/SHL+1.5, respectively; peak values displayed) are superimposed onto the refined models.

[00018] Figure 6. Stereo view of the experimental electron density corresponding to cisPtNAP binding at SHL +1.5 in the nucleosome core. An F₀-F_c omit electron density map (grey; contoured at 2.5a; cisPtNAP ligands omitted from the model) and an anomalous difference electron density map (magenta; contoured at 4.5σ) are superimposed onto the refined model. The view is identical to that described in Figure 5 (lower right hand panel).

[00019] Figure 7. A non-adducted as well as an adducted binding mode is observed for trPtNAP interaction with the nucleosome core. Carbon atoms of the trPtNAP ligands are shown in olive green and bright green for the non-adducted and adducted states, respectively. Hydrogen bonds between ligands and DNA are indicated with black dashed lines. The platinum atom of the non- adducted configuration is situated, along the axial coordination position on the same side ('inside') as the intercalator group, 3.7 A distant from the 06 atom of guanine -15.

[00020] Figure 8. Evidence of non-adducted trPtNAP binding at SHL -1.5, analogous to that observed at SHL +1.5. An anomalous difference electron density map contoured at 3.5σ (magenta; peak values displayed) is superimposed onto the refined model. Weak platinum atom density situated around 3.4 A adjacent to the 06 atom of the 5' guanine (-15) is consistent with an additional non- adducted intercalative binding mode.

[00021] Figure 9. DNA conformational parameters for the cisPtNAP-NCP and trPtNAP-

NCP structures. Base pair step conformational parameters are shown for the cisPtNAP (red), trPtNAP (green) and native NCP (blue) models. DNA sections where the major groove faces inward towards the histone octamer are shaded grey and sections with the minor groove facing inward are clear. The GG=CC dinucleotide steps of SHL ±1.5 within which PtNAP intercalation occurs are highlighted in gold.

[00022] Figure 10. Impact of trPtNAP and cisPtNAP agents on cell viability and function.

Apoptosis/necrosis profiles (top) and cell cycle profiles (bottom) were derived from flow cytometric analysis of cultured A2780 cells. Day 1 measurements were made immediately after switching cells to agent-free media (subsequent to 24-hour incubation with agent), and Day 2 and Day 3 measurements correspond to cells residing in agent-free media for an additional one or two days (mean±s.d., n=3).

DETAILED DESCRIPTION OF THE INVENTION

[00023] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural, and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[00024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control.

[00025] The present invention provides a class of novel platinum-based compounds generated by linking (diethylenetriamine)-platinum(II) derivatives to DNA intercalating compounds.

[00026] The term "(diethylenetriamine)-platinum(H)", as used herein, refers to the coordination complex formed by diethylenetriamine and the platinum (Il)-ion.

[00027] The term "derivative", as used herein, refers to a substance which comprises the same basic carbon skeleton and functionality as the parent compound, but can also bear one or more substituents or substitutions of the parent compound.

[00028] The term "DNA intercalating compounds", as used herein, is well-known in the art and commonly understood to refer to those compounds capable of non-covalent insertion between the base pairs of a nucleic acid duplex and being specific for double-stranded (ds) portions of nucleic acid structures including those portions of single-stranded nucleic acids which have formed base pairs, such as in "hairpin loops". The nucleic acid structures can be dsDNA, dsRNA or DNA-RNA hybrids. The term is also used to describe the insertion of planar aromatic or heteroaromatic compounds between adjacent base pairs of dsDNA, or in some cases dsRNA.

[00019] The rationale underlying the design of these compounds was that by linking a DNA- adduct forming platinum group to a DNA intercalating compound, a compound with improved DNA site selectivity and reduce side effects can be generated. Further, it was found that a DNA intercalating compound with a certain degree of DNA sequence or motif specificity can target the platinum group conjugated thereto to certain hotspots in DNA, whereby the compound can display a favourable cytotoxicity profile, strikingly distinct from that of cisplatin, carboplatin or oxaliplatin.

[00020] In one first aspect, the present invention thus relates to a compound of Formula (I) or

(II) or a stereoisomer or pharmaceutically acceptable salt thereof,

wherein

X is a leaving group;

R\ R^r, R² and R² are each independently hydrogen or methyl;

R³, R³ and R⁴ are each independently selected from the group consisting of hydrogen, C^o alkyl and C_M0 alkyloxy;

each n is independently an integer of 1 to 10; and

ICA is a DNA intercalating compound.

[00029] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and can include more than one element.

[00030] The term "stereoisomer", as used herein, refers to at least two compounds having the same molecular formula and connectivity of atoms, but having a different arrangement of atoms in a three-dimensional space.

[00031] The term "pharmaceutically acceptable salt" as used herein means those salts of a compound of interest that are safe and effective for administration to a mammal and that possess the desired biological activity. [00032] The term "leaving group", as used herein, refers to groups readily displaceable by a nucleophile, such as an amine, alcohol, phosphorous or thiol nucleophile or their respective anions. Such leaving groups are well known and include carboxylates, N-hydroxysuccinimide, N- hydroxybenzotriazole, halogen (halides), triflates, tosylates, mesylates, alkoxy, thioalkoxy, phosphinates, phosphonates and the like. Other potential nucleophiles include organometallic reagents known to those skilled in the art.

[00033] The term "alkyl", as used herein, refers to a linear, branched, or cyclic saturated hydrocarbon group. The term "Ci.io alkyl" indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it. Examples of Ci.₁₀ alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2 -propyl, 2-methyl-I -butyl, 3 -methyl- 1 -butyl, 2-methyl-3- butyl, 2,2-dimethyl-l -propyl, 2-methyl-l-pentyl, 3-methyl-l-pentyl, 4-methyl-l-pentyl, 2-methyl-2- pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l -butyl, 3,3-dimethyl-l -butyl, 2-ethyl-l -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, cyclohexyl, heptyl, octyl, nonyl and decyl.

[00034] The term "alkyloxy", as used herein, refers to oxygenated straight or branched chain radicals, as illustrated by, but not limited to, methoxy, ethoxy, butoxy and the like. The term "C_MO alkyloxy" indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it.

[00035] It is common general knowledge that platinum-based drugs form highly reactive and charged platinum complexes which bind to nucleophilic groups such as GC-rich sites in DNA to form DNA adducts.

[00036] The term "DNA adducts", as used herein, refers to covalent bonds formed between the platinum atom and the DNA. For example, when cisplatin, carboplatin or oxaliplatin enters the low chloride anion environment within the cell, the chloride or carboxylate leaving groups can undergo aquation, generating reactive aqua-species. Initial attack on DNA coincides with formation of a single platinum-purine bond, corresponding to the monofunctional adduct, followed by potential chelation to yield a bifunctional adduct (cross-link). Drug reaction occurs at the N7 nitrogen atoms of purine bases, generating predominantly 1,2 intrastrand cross-links at GG, and less frequently at AG dinucleotides, in addition to a minor fraction of GNG 1,3 intrastrand and other DNA adducts. The primary mechanism of action appears to relate to local dinucleotide kink distortions and double helix deformations that cause transcriptional arrest through stalling RNA polymerase.

[00037] The compound of the present invention, however, is monofunctional, as each compound has only one leaving group, and intrinsically differs from the clinically used bifunctional species in that it forms only one DNA adduct. This distinction translates into pronounced differences in terms of cytotoxicity, DNA repair pathways and drug resistance properties.

[00038] hi various embodiments of the compounds described herein, the leaving group X can be any leaving group that can undergo aquation in the intracellular environment and thus generate the reactive species of the platinum compound. The leaving group can be any leaving group having this property and falling within the above definition, but preferably is selected from the group consisting of halogen, -OH, -SCN, -OR⁵ and -0-C(0)-R⁵, wherein R⁵ is any organic moiety.

[00039] The term "halogen", as used herein, refers to fluoro, chloro, bromo, and iodo. Preferred halogen substituents X are chloro and bromo substituents, more preferably chloro substituents.

[00040] The term "any organic moiety", as used herein in relation to the leaving group -OR⁵ or -0-C(0)-R⁵, refers to any substituted or unsubstituted hydrocarbon moiety, preferably having 1 to 10 carbon atoms. Preferred moieties include, without limitation, C_rC₂4 alkyl, C₂-C₂4 alkenyl, C₂-C₂4 alkynyl, C₅-C ₀ aryl, C₆-C₂₄ alkaryl, and C₆-C₂₄ aralkyl, wherein the respective groups can be substituted or unsubstituted. When substituted, the one or more substituent groups may, for example, be defined as described below in the context of the intercalators.

[00041] In certain embodiments, in the compounds of Formula (I) any one or more or all of R¹,

R¹ , R² and R² are hydrogen. This means that 1, 2, 3 or all 4 of R¹, R¹ , R² and R² are hydrogen, with all being hydrogen being preferred.

[00042] In certain embodiments, in the compounds of Formula (II), any one or more or all of

R¹, R^1', R^2' and R⁴ are hydrogen. This means that 1, 2, 3 or all 4 of R¹, R⁴, R² and R^2'are hydrogen, with all being hydrogen being preferred.

[00043] In certain embodiments of the compound, any one or more or all of R³ and R³ are hydrogen. This means that 1, 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of R³ and R³ are hydrogen, with all being hydrogen being preferred.

[00044] In certain embodiments, n is 2 to 5, preferably 3. Particularly preferred are methylene linkers of the formula -(CH₂)_n- with n being 2-5, preferably 3.

[00045] In various embodiments of the invention, the DNA intercalating compound can be any compound suitable for intercalating into a double-stranded nucleic acid structure, preferably a site-specific intercalator. In preferred embodiments, the intercalator is 1,8-naphthalimide or a derivative thereof. It is understood that the respective intercalator is attached to the diethylentriamine- platinum complex via a covalent bond to any suitable atom of the intercalator. Said point of attachment may, for example, be a nitrogen atom. In the 1,8-naphthalimides described herein, the platinum complex is attached via a covalent bond to the nitrogen atom.

[00046] 1,8-naphthalimide is a DNA intercalating compound that preferentially inserts into the open major groove face of extremely kinked DNA sites on nucleosomes (Davey G.E., et al. Nucleic Acids Res. 2010;38:2081-2088).

[00047] The term "nucleosome", as used herein, refers to the basic unit of chromatin structure and consists of a protein complex of eight highly conserved core histones (comprising of a pair of each of the histones H2A, H2B, H3, and H4). Around this complex is wrapped approximately 146 base pairs of DNA. Another histone, HI or H5, acts as a linker and is involved in chromatin compaction. The DNA is wound around consecutive nucleosomes in a structure often said to resemble "beads on a string" and this forms the basic structure of open or euchromatin. In compacted or heterochromatin this string is coiled and super coiled into a closed and complex structure.

[00048] The majority of cellular DNA is wrapped by histone proteins into nucleosomes, rendering these fundamental repeating units of chromatin an important therapeutic target (Davey G.E., Davey C.A. Chem. Biol. Drug Des. 2008;72:165-170). As a matter of fact, comparison of the extent of nucleotide excision repair by mammalian cell extracts of naked and nucleosomal DNA containing the same platinum-DNA adducts has revealed that the nucleosome can significantly inhibit the repair of the DNA damage caused by cisplatin (Wang D., et al. Biochemistry 2003;42:6747-6753). Thus, it was hypothesized that induction of DNA adducts residing within the nucleosome core may be more efficacious.

[00049] In preferred embodiments, the DNA intercalating compound is 1,8-naphthalimide or a derivative thereof having the general structure represented by Formula (III),

wherein R⁶, R⁶ , R⁷, R⁷ , R⁸, R⁸ are each independently selected from the group consisting of hydrogen, Ci-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄ alkylcarbonato, C6-C₂o arylcarbonato, carboxy, carboxylato, carbamoyl, mono-(C C₂₄ alkyl)-substituted carbamoyl, di-(C C₂₄ alkyl)-substituted carbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, mono- and di-(Ci- C₂₄ alkyl)-substituted amino, mono- and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₅-C₂₀ arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato, Ci-G₂₄ alkylsulfanyl, arylsulfanyl, Ci-C₂₄ alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, and phosphino, and wherein any two adjacent (ortho) substituents may be linked to form a cyclic structure selected from five-membered rings, six- membered rings, and fused five-membered and/or six-membered rings, wherein the cyclic structure is aromatic, alicyclic, heteroaromatic, or heteroalicyclic, and has 0 to 4 non-hydrogen substituents and 0 to 3 heteroatoms. The heteroatoms may be selected from N, S, O, Se, Si, and P.

[00050] The term "alkenyl" as used herein refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, «-propenyl, isopropenyl, rc-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like. Generally, although not necessarily, alkenyl groups herein contain 2 to about 18 carbon atoms, preferably 2 to 12 carbon atoms.

[00051] The term "alkynyl" as used herein refers to a linear or branched hydrocarbon group of

2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, «-propynyl, and the like. Generally, although again not necessarily, alkynyl groups herein contain 2 to about 18 carbon atoms, preferably 2 to 12 carbon atoms.

[00052] The term "alkoxy" as used herein intends an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group may be represented as -O-alkyl where alkyl is as defined above.

[00053] The term "aryl" as used herein, and unless otherwise specified, refers to an aromatic substituent containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Preferred aryl groups contain 5 to 20 carbon atoms, and particularly preferred aryl groups contain 5 to 14 carbon atoms. Exemplary aryl groups contain one aromatic ring or two fused or linked aromatic rings, e.g., phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, benzophenone, and the like.

[00054] The term "aryloxy" as used herein refers to an aryl group bound through a single, terminal ether linkage, wherein "aryl" is as defined above. An "aryloxy" group may be represented as -O-aryl where aryl is as defined above. Preferred aryloxy groups contain 5 to 20 carbon atoms, and particularly preferred aryloxy groups contain 5 to 14 carbon atoms. Examples of aryloxy groups include, without limitation, phenoxy, o-halo-phenoxy, m-halo-phenoxy, p-halo-phenoxy, o-methoxy- phenoxy, m-methoxy-phenoxy, p-methoxy-phenoxy, 2,4-dimethoxy-phenoxy, 3,4,5-trimethoxy- phenoxy, and the like.

[00055] The term "alkaryl" refers to an aryl group with an alkyl substituent, and the term

"aralkyl" refers to an alkyl group with an aryl substituent, wherein "aryl" and "alkyl" are as defined above. Preferred aralkyl groups contain 6 to 24 carbon atoms, and particularly preferred aralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groups include, without limitation, benzyl, 2-phenyl-ethyl, 3 -phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4- benzylcyclohexyl, 4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like. Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl, p-cyclohexylphenyl, 2,7- dimethylnaphthyl, 7-cyclooctylnaphthyl, 3-ethyl-cyclopenta-l,4-diene, and the like. [00056] The term "cyclic" refers to alicyclic or aromatic substituents that may or may not be substituted and/or heteroatom containing, and that may be monocyclic, bicyclic, or polycyclic.

[00057] The term "heteroatom" refers to an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur. Similarly, the term "heteroaromatic" refers to "aromatic" substituents that are heteroatom-containing, and the like.

[00058] By "substituted" as in "substituted alkyl," "substituted aryl," and the like, as alluded to in some of the aforementioned definitions, is meant that in the alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation: functional groups such as halo, hydroxyl, sulfhydryl, Ci-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂4 alkynyloxy, C₅-C₂₀ aryloxy, acyl (including C₂-C₂ alkylcarbonyl (-CO-alkyl) and C₆-C₂₀ arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C₂-C₂₄ alkoxycarbonyl (-(CO)-O-alkyl), C₆-C₂₀ aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)- X where X is halo), C₂-C₂₄ alkylcarbonato (-O-(CO)-O-alkyl), C₆-C₂₀ arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COCT), carbamoyl (-(CO)-NH₂), mono-(C C₂₄ alkyl)-substituted carbamoyl (-(CO)-NH(Ci-C₂₄ alkyl)), di-(Ci-C₂₄ alkyl)-substituted carbamoyl (-(CO)-N(C C₂₄ alkyl)₂), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH₂), carbamido (- NH-(CO)-NH₂), cyano(-C ^i), isocyano (-IsT ^T), cyanato (-0-C i), isocyanato (-0-N⁺≡€^_), isothiocyanato (-S-C≡tvT), azido (-Ν=>Γ=ίΓ), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (- H₂), mono- and di-(C C₂ alkyl)-substituted amino, mono- and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido (-NH-(CO)-alkyl), C₆-C₂₀ arylamido (-NH-(CO)-aryl), imino (-CR=NH where R = hydrogen, Ci-C₂ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino (-CR=N(alkyl), where R = hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R = hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-N0₂), nitroso (-NO), sulfo (-S0₂-OH), sulfonate (-S0₂-0^"), C C₂₄ alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), C C₂₄ alkylsulfinyl (-(SO)-alkyl), C₅-C₂₀ arylsulfinyl (-(SO)-aryl), C_rC₂₄ alkylsulfonyl (-S0₂-alkyl), C₅-C₂₀ arylsulfonyl (-S0₂-aryl), phosphono (-P(0)(OH)₂), phosphonato (-P(0)(0^~)₂), phosphinato (- P(0)(0^")), phospho (-P0₂), and phosphino (-PH₂); and the hydrocarbyl moieties C C₂₄ alkyl (preferably Q-Cig alkyl, more preferably C]-C_]2 alkyl, most preferably C C₆ alkyl), C₂-C₂₄ alkenyl (preferably C₂-C_]8 alkenyl, more preferably C₂-C₁₂ alkenyl, most preferably C₂-C₆ alkenyl), C₂-C₂₄ alkynyl (preferably C₂-C_]8 alkynyl, more preferably C₂-Ci₂ alkynyl, most preferably C₂-C₆ alkynyl), C₅-C₂₀ aryl (preferably C₅-C_I4 aryl), C₆-C₂₄ alkaryl (preferably C₆-C₁₈ alkaryl), and C₆-C₂₄ aralkyl (preferably C₆-Ci₈ aralkyl). In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups.

[00059] Various embodiments of the compounds of Formula (ΙΠ), with differing DNA intercalating capabilities and site selectivity, can be found, for example, in Banerjee, S. et al. Chem. Soc. Rev., 2013;42, 1601, Kamal, A. et al. Expert Opin. ner. Pat. 2013;23:299-317, and PCT international applications No. WO 2005/105753A2 and WO 2006/060533 A2, which are incorporated by reference herein in its entirety.

[00060] In preferred embodiments, the DNA intercalating compound is selected from the group consisting of

1 ,3(2//)-dione

,3(2//)-dione ,3(2/i)-dione

,3(2H)-dione

5-(3,4-dinitrophenyl)-l /-benzo[i'ifr]isoquinoJine-l,3(2^)-dione

9-phenyl-4/-beiizo[^] xazolo[5.4--g]isoquinoline-4_!6(5/i)-iiione

9-pheny]benzo[rfe]imidazo[4^-^]isoquinoline-4,6(5ii,10H)-dione

[00061] In particularly preferred embodiments of the compound, the compound is cw- chloro[l,2,3-diethylene1rianm o-2-N-(3^ropyl)-l,8-naphthalimide]platinum(II) chloride (cisPtNAP) or traws-chloro[l ,2,3-diethylenetriamino-l -N-(3 -propyl)- 1 ,8-naphthalimide]platinum(II) chloride (trPtNAP).

[00062] In this configuration, firstly the diethylenetriamine ligand can be readily attached to the DNA intercalating compound via classical substitution at either a terminal or the central N atom. Secondarily, the tridentate ligand can effectively occupy 3 coordination sites around the square-planar platinum(II) complex to yield selectively a stable and monofunctional DNA-binding motif. Thirdly, the (diethylenetriamine)-platinum(II) group can interact favourably with guanine of DNA via H- bonding with the exocyclic 06 atom, directing platinum towards the nucleophilic N7 atom, conferring some degree of selectivity over other nucleobases. Finally, the linker can provide a N-N separation of up to 5 A between the (diethylenetriamine)-platinum(II) group and 1,8-naphthalimide, and confers structural flexibility.

[00063] Surprisingly, it has been found that cisPtNAP forms DNA adducts at defined, internal locations within the nucleosome core, while trPtNAP efficiently forms adducts only at the most flexible and conformationally unconstrained sites of DNA, i.e. on naked DNA or at the DNA entry/exit points on the nucleosome. Therefore, these compounds are unique in displaying a significant level of DNA site discrimination, unlike existing platinum drugs.

[00064] Moreover, these two compounds, while each displaying distinct impact on cell function, show promising antitumor activities. In particular, the two agents are selective for cancerous cells and show greater potency than cisplatin. Additionally, the two compounds are effectively non- cross-resistant with cisplatin towards cancer cells, making them both potential therapeutic candidates for antitumor treatments, including in particular cancers that have intrinsic or acquired resistance to cisplatin.

[00065] The present invention therefore also features the compounds as described herein for use in a method for treating cancer. Alternatively, the invention also covers the use of said compounds for the manufacture of a medicament for the treatment of cancer. [00066] The terms "treating" and "treatment", as used herein, refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. For example, treatment of a patient by administration of an anticancer agent of the invention encompasses chemoprevention in a patient susceptible to developing cancer (e.g., at a higher risk, as a result of genetic predisposition, environmental factors, or the like) and/or in cancer survivors at risk of cancer recurrence, as well as treatment of a cancer patient dual by inhibiting or causing regression of a disorder or disease.

[00067] The cancers eligible for the treatment by a compound of the present invention should be determined on a case-by-case basis, but may include, without limitation, breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain cancer, leukemia cancer, lung cancer, colon cancer, CNS cancer, melanoma cancer, renal cancer and cervix cancer.

[00068] Platinum-based drugs act therapeutically by forming DNA adducts which interfere with DNA replication and cell proliferation, and are hence useful for treating hyperproliferative diseases other than cancers. In this context, it is to be understood that treatment of hyperproliferative diseases other than cancers using the instantly disclosed compounds is also within the scope of the present invention. Accordingly, all the methods and uses described herein in relation to cancer treatment can readily be extended to the treatment of other hyperproliferative diseases and disorders.

[00069] The term "hyperproliferative diseases", as used herein, refers to excess cell proliferation that is not governed by the usual limitation of normal growth. The term denotes malignant as well as nonmalignant cell populations. The excess cell proliferation can be determined by reference to the general population and/or by reference to a particular patient, e.g. at an earlier point in the patient's life. Hyperproliferative cell disorders can occur in different types of animals and in humans, and produce different physical manifestations depending upon the affected cells.

[00070] Examples of nontumor hyperproliferative disorders include but are not limited to myelodysplastic disorders; cervical carcinoma-in-situ; familial intestinal polyposes such as Gardner syndrome; oral leukoplakias; histiocytoses; keloids; hemangiomas; inflammatory arthritis; hyperkeratoses and papulosquamous eruptions including arthritis. Also included are viral induced hyperproliferative diseases such as warts and EBV induced disease (i.e., infectious mononucleosis), scar formation, blood vessel proliferative disorders such as restenosis, atherosclerosis, in-stent stenosis, vascular graft restenosis, etc.; fibrotic disorders; psoriasis; glomerular nephritis; macular degenerative disorders; benign growth disorders such as prostate enlargement and lipomas; autoimmune disorders and the like. [00071] In another aspect, the invention is directed to a method for the treatment of cancer in a subject, the method comprising administering to said subject a therapeutically effective amount of said compound.

[00072] In preferred embodiments of the method, the subject is a mammal, preferably a human.

[00073] By the terms "effective amount" and "therapeutically effective amount" of a compound of the invention is meant a nontoxic but sufficient amount of the drug or agent to provide the desired effect.

[00074] A compound of the present invention may be administered in the form of a salt, ester, amide, prodrug, active metabolite, analog, or the like, provided that the salt, ester, amide, prodrug, active metabolite or analog is pharmaceutically acceptable and pharmacologically active in the present context. Salts, esters, amides, prodrugs, active metabolites, analogs, and other derivatives of the active agents may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992). "Pharmacologically active" (or simply "active") as in a "pharmacologically active" derivative or analog, refers to a derivative or analog having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[00075] In addition, those novel compounds containing chiral centres can be in the form of a single enantiomer or as a racemic mixture of enantiomers. In some cases, i.e., with regard to certain specific compounds such as cisPfNAP and trPtNAP, chirality (i.e., relative stereochemistry) is indicated. In other cases, it is not, and such structures are intended to encompass both the enantiomerically pure form of the compound shown as well as a racemic mixture of enantiomers. Preparation of compounds in enantiomerically form may be carried out using an enantioselective synthesis; alternatively, the enantiomers of a chiral compound obtained in the form of the racemate may be separated post-synthesis, using routine methodology.

[00076] The compounds and derivatives and analogs thereof may be prepared using standard techniques known to those skilled in the art of synthetic organic chemistry, or may be deduced by reference to the pertinent literature.

[00077] Prior to being used in the treatment of cancer, pharmaceutical formulations composed of one or more of the compounds in association with a pharmaceutically acceptable carrier may need to be formulated. See Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), which discloses typical carriers and conventional methods of preparing pharmaceutical formulations.

[00078] Depending on the intended mode of administration, the pharmaceutical formulation may be a solid, semi-solid or liquid, such as, for example, a tablet, a capsule, caplets, a liquid, a suspension, an emulsion, a suppository, granules, pellets, beads, a powder, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. Suitable pharmaceutical compositions and dosage forms may be prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts and literature, e.g., in Remington: The Science and Practice of Pharmacy, cited above.

[00079] The compounds of the present invention may be administered orally, parenterally, rectally, vaginally, buccally, sublingually, nasally, by inhalation, topically, transdermally, or via an implanted reservoir in dosage forms containing conventional non-toxic pharmaceutically acceptable carriers and excipients. The term "parenteral" as used herein is intended to include subcutaneous, intravenous, and intramuscular injection. The amount of the compound administered will, of course, be dependent on the particular active agent, the condition or disorder being treated, the severity of the condition or disorder, the subject's weight, the mode of administration and other pertinent factors known to the prescribing physician.

[00080] Γη still another aspect, the invention encompasses a method for triggering apoptosis in a cell, preferably a cancer cell, the method comprising contacting said cell with an effective amount of said compound.

[00081] The term "apoptosis" refers to the process of programmed cell death, with its accompanying cellular morphological changes and loss of cell viability. However, because the effects of a compound on cell viability and fate may vary depending on factors such as cell type treated, concentration of the compound and time of treatment, it is to be understood that the effects of an instantly disclosed compound on induction of any other tumor suppressive mechanisms such as necrosis, cell cycle arrest, senescence, autophagic cell death and differentiation are also encompassed in the present invention.

[00082] In a final aspect, the invention concerns use of said compound for triggering apoptosis in a cell, preferably a cancer cell.

[00083] In said uses and methods, the cells in which apoptosis is triggered may be a cultured cell, for example a cultured cancer cell. Accordingly, the respective methods and uses can be ex vivo or in vitro methods, such as cell culture methods. Such methods and uses may for example be used to test the compounds for the efficacy on a specific cancer cell type, study development of resistance or for comparative purposes.

[00084] The present invention is further illustrated by the following examples. However, it should be understood, that the invention is not limited to the exemplified embodiments.

EXAMPLES

Materials and Methods Determination of cell growth inhibition parameters

[00085] Human ovarian carcinoma cell lines, A2780 and cisplatin-resistant A2780 (crA2780), were purchased from the Health Protection Agency Culture Collections (HPACC, Salisbury, UK) and grown at 37 °C and 5% C0₂ in RPMI 1640 medium containing 10% fetal calf serum with 2 mM glutamine, 100 units mL^-1 penicillin and 100 μg mL^"1 streptomycin. The crA2780 cells were additionally treated with 1 μΜ cisplatin for every 2-3 passages to maintain resistance. HaCaT cells (non-tumorigenic human keratinocytes; ATCC, USA) were generously provided by Andrew N.S. Tan and grown at 37 °C and 5% C0₂ in DMEM medium containing 10% fetal calf serum with 2 mM glutamine, 1 mM pyruvate, 100 units mL^"1 penicillin and 100 μg mL^"1 streptomycin. Cisplatin was purchased from Sigma-Aldrich, USA (P4394-250MG).

[00086] To measure the cytotoxicity from exposure to different agents, cells were seeded in

96-well plates (5000 cells per well) and grown for 24 h. Stock solutions were prepared by dissolving cisplatin in complete medium, and by initially dissolving trPtNAP, cisPtNAP and naphthalimide in DMSO followed by subsequent addition into complete medium. These medium stocks were then subjected to serial dilutions and added to the cells at various concentrations. The DMSO concentration in the medium never exceeded 0.5% and was set constant within a given assay, including control cells.

[00087] Following a 72 h incubation, media were aspirated and 100 μΐ of 10% 3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (TOX1-1KT; MTT kit, Sigma-Aldrich, USA) in RPMI or DMEM complete medium was added to cells, which were incubated for 3 h at 37 °C. Subsequently, 100 μΐ of solubihsation buffer was added to each well with vigorous pipetting in order to dissolve formazan. The resulting optical density was measured at 570 and 690 nm using a multi- well plate reader (Infinite M200 PRO, Magellan data analysis software, TECAN, Switzerland). The ratios of surviving cells were calculated by comparing to the untreated samples, and the IC₅₀ was derived based on at least 3 data sets.

Genomic DNA adduct quantification

[00088] Isolation of genomic DNA was carried out using a GenElute Mammalian Genomic

DNA Miniprep Kit (G1N70; Sigma Aldrich, USA). A2780 cells were maintained in culture as described above. For the measurements in Table 2, cells were grown in 60 mm plates to approximately 90% confluency before being incubated with 18 μΜ trPtNAP, 25 μΜ cisPtNAP or 100 μΜ cisplatin for 18 h at 37 °C. The amount of DNA was quantified by UV absorption measurements at 260 nm and subjected to atomic absorption spectroscopic analysis to measure platinum content. Determination of platinum levels on DNA was conducted by pipetting 20 μΐ of sample into the reaction chamber of a Hitachi Z-2000 Polarized Zeeman Atomic Absorption Spectrophotometer in graphite furnace mode.

Cellular death and cell cycle analyses

[00089] Cells were seeded into 35 mm-wells, incubated overnight, and the following day 2 μΜ cisPtNAP or trPtNAP were added into triplicate samples for 24-h incubation. Subsequently, the medium containing compound was removed and replaced with fresh medium. The cells were collected for analysis immediately (day 1) and subsequent to further 24-h (day 2) and 48-h (day 3) incubations in compound-free media.

[00090] Extent of apoptosis/necrosis was assessed utilizing an Annexin-V-FLUOS staining kit

(11988549001; Roche Diagnostics, Germany). Floating cells as well as attached cells were collected, washed in PBS and stained with Annexin V and Propidium Iodide (PI). Cells were then incubated for 15 min in the dark and analysed on an LSR II flow cytometer (BD FACSDiva software, BD Biosciences).

[00091] For the cell cycle analysis, cells were rinsed in PBS, trypsinized, pelleted and washed before they were added in a drop-wise manner into 70% ethanol. After overnight-incubation, cells were washed in PBS and incubated with DAPI dihydrochloride (3μΜ, Life Technologies) for 15 min in the dark before analysing on an LSR II flow cytometer (BD FACSDiva software, BD Biosciences).

[00092] Analysis of cellular data was based on three independent replicates of each experiment. Graphic plots were generated with Excel (Microsoft Corporation). Mahalanobis multivariate analysis (Mahalanobis P.C. Proc. Natl. Inst. Sci. India 1936;2:49-55; Adhireksan Z., et al. Nat. Commun. 2014;5:3462) was carried out using Xlstat (version 2013.1.01; Addinsoft Corporation).

DNA footprinting analysis

[00093] Exonuclease III footprinting experiments for obtaining PtNAP adduct formation profiles, based on digest (3' to 5' direction) termination at DNA lesion sites, were conducted in a similar fashion as described previously for platinum adduct mapping (Wu B., Droge P., Davey C.A. Nat. Chem. Biol. 2008;4:110-112; Wu B., et al. Nucleic Acids Res. 2011;39:8200-8212). 1 mM stock solutions of trPtNAP and cisPtNAP were prepared by dissolving agent in DMSO. 2.5 μΜ naked DNA or NCP in a buffer of 20 mM K-cacodylate (pH 6.0) were incubated at room temperature for 24 hours in the dark with a 10-, 20- or 50-fold molar excess for trPtNAP, and a 5-, 10- or 50-fold molar excess for cisPtNAP. An equal volume of 4 M NaCl was then added to each reaction, and the samples heated at 55 °C for 1 h to dissociate DNA from histones. To precipitate the histones, 0.5 volumes of phenol and CIA (24: 1 chloroform:isoamyl alcohol) were added to the mixture, which was then vortexed for 1 m. The phases were separated by centrifugation at 14,500 rpm for 3 m at room temperature, and the DNA was ethanol-precipitated from the aqueous phase and redissolved in a buffer of 10 mM Tris (pH 7.5) and 0.1 niM EDTA (pH 8.0) to the desired concentration.

[00094] DNA samples were 5' end-labeled by T4 polynucleotide kinase in reactions containing 1.5 μΜ DNA, 0.5 units μ\^Λ T4 polynucleotide kinase, 0.5 μθ μΓ¹ γ³²Ρ-ΑΤΡ, and 1.5X polynucleotide kinase buffer (New England Biolabs, USA), incubated at 37 °C for 60 m. Samples were then purified by centrifugation at 800 g for 3 m with DyeEx 2.0 Spin Kits (Qiagen, Germany). Exonuclease III digestion was typically conducted in 50 μΐ reactions containing 0.15 μΜ DNA, 5 units μΐ ¹ Exonuclease ΠΙ, and IX NEBuffer 1 (New England Biolabs, USA) at 37 °C for 60 m. The digestion was stopped by adding 0.5 volumes of a solution of 0.9 M sodium acetate and 0.09 M EDTA, and 4 volumes of cold 100% ethanol, with a 10 m incubation on ice. The samples were centrifuged at 12,000 rpm for 5 m at room temperature, and the pellets were washed with cold 70% ethanol to remove excess salt. The precipitated DNA fragments were resuspended in 200 μΐ of 1 M thiourea and incubated at 58 °C overnight to deplatinate the DNA. Upon completion of deplatination, 0.1 volumes of 3 M sodium acetate and 3 volumes of cold 100% ethanol were added, and the samples incubated for 10 m on ice to precipitate the DNA. The samples were centrifuged at 12,000 rpm for 5 m at room temperature, and the pellets were washed with cold 70% ethanol to remove any crystalline thiourea. Following a final round of centrifugation at 12,000 rpm for 5 m, the precipitated DNA fragments were dissolved in IX denaturing DNA loading dye (95% v/v formamide, 0.025% w/v Bromophenol blue, 0.025% w/v Xylene cyanol FF, 0.5 mM EDTA), heated at 95 °C for 3 m, immediately chilled on ice, and then analyzed on 9% denaturing PAGE.

[00095] Maxam-Gilbert purine sequencing standards (Maxam A.M., Gilbert W. Meth.

Enzymol. 1980;65:499-560) were prepared by 5' end-labeling unreacted DNA samples with T4 polynucleotide kinase as described above. To the labeled samples, formic acid was added to a final concentration of 57% and allowed to react for 4-6 m at room temperature. The reaction was quenched by adding 6 volumes of 0.3 M sodium acetate, 0.1 M EDTA and 25 μg ml^"1 RNA, and the DNA was precipitated immediately by addition of 3 volumes of cold 100% ethanol. After 10 m incubation on ice, the samples were centrifuged at 12,000 rpm for 5 m, and the pellets were washed with cold 70% ethanol. The pellets were then dissolved in 200 μΐ 10% (v/v) piperidine with 50 mM EDTA and heated at 99 °C for 45 m to cause strand cleavage at formic acid-modified purine bases. The reaction was stopped by ethanol precipitation and the standards used in the analysis described above.

Denaturing polyacrylamide gel electrophoresis

[00096] 9% denaturing polyacrylamide gels were cast in 42.5 cm x 21 cm x 0.75 mm gel plates. The gel was pre-run at 60 W at room temperature until the gel temperature reached 55 °C. Labeled DNA samples with approximately equal radioactive counts in IX denaturing DNA loading dye were heated at 100 °C for 5 m and then loaded into each well, and the gel run at 55 W for 2 or 3 h at room temperature in 0.5X TBE buffer (44.5 mM Tris, 44.5 mM Boric acid, 1 mM EDTA). The gel was harvested onto Whatman filter paper, fixed with 0.5X TBE, 5% methanol and 5% acetic acid, and dried at 65 °C for 2 h by using a Slab Gel Drier Model GD2000. DNA bands were visualized by exposing the dried gel to Imaging Screen-K for 12-72 h, and the screen was scanned by using a Molecular Imager FX machine. Quantity One (Bio-Rad Laboratories) was used to quantify the intensity of gel bands.

Crystallographic analysis of treated nucleosome core particle

[00097] NCP crystals were produced and stabilized in harvest buffer (37 mM MnCl₂, 40 mM

KCl, 20 mM K-cacodylate [pH 6.0], 24% 2-methyl-2,4-pentanediol and 2% trehalose) as previously described (Davey C.A., et al. J. Mol. Biol. 2002;319:1097-1113). The 37 mM MnCl₂ buffer component was subsequently eliminated by gradual replacement with 10 mM MgS0₄ followed by thorough rinsing of crystals with the MgS0₄-containing buffer to remove any residual MnCl₂ (Wu B., et al. Chemistry 2011;17:3562-3566.). PtNAP agents were dissolved in the MgS0₄-containing buffer at varying concentrations, which was used to soak the crystals for various incubation times at room temperature. The NCP-PtNAP crystal structures reported here stem from a 24-h incubation with 0.1 mM trPtNAP and a 23-h incubation with 0.25 mM cisPtNAP.

[00098] Single crystal X-ray diffraction data were recorded, with crystals mounted directly into the cryocooling N₂ gas stream set at -175° C (Adhireksan Z., et al. Nat. Commun. 2014;5:3462), at beam line X06DA of the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) using a Mar225 CCD detector and an X-ray wavelength of 1.07 A (platinum absorption edge). Data were processed with MOSFLM (Leslie A.G. Acta Crystallogr. D Biol. Crystallogr. 2006;62:48-57) and SCALA from the CCP4 package (Bailey S. Acta Crystallogr. D Biol. Crystallogr. 1994;50:760-763).

[00099] Native NCP models (Wu B., et al. Nucleic Acids Res. 2011;39:8200-8212; Davey

C.A., et al. J. Mol. Biol. 2002;319:1097-1113; Vasudevan D., Chua E.Y., Davey C.A J. Mol. Biol. 2010;403:1-10) were used for initial structure solution by molecular replacement. Structural refinement and model building were carried out with routines from the CCP4 suite (Bailey S. Acta Crystallogr. D Biol. Crystallogr. 1994;50:760-763). Models for cisPtNAP and trPtNAP were built by combining crystallographic data files of N-(2,3-epoxypropyl)-l,8-naphthalirnide (CCDC deposition number 756223) (Davey G.E., et al. Nucleic Acids Res. 2010;38:2081-2088) and a platinum- diethylenetriamine complex (CCDC deposition number 663059) (Ndinguri M.W., et al. Inorg. Chim. Acta 2010;363:1796-1804). Data collection and structure refinement statistics are given in Table 3. Dinucleotide step conformational parameters were obtained by using 3DNA (Lu X.J., Olson W.K. Nucleic Acids Res. 2003;31 :5108-5121; Zheng G., Lu X.J., Olson W.K. Nucleic Acids Res. 2009;37:W240-W246). Graphic figures were prepared with PyMOL (DeLano Scientific LLC, San Carlos, CA, USA). [000100] Atomic coordinates and structure factors for the trPtNAP-NCP and cisPtNAP-NCP models have been deposited in the Protein Data Bank under accession codes 4WU8 and 4WU9, respectively.

Example 1: Synthesis of cisPtNAP and trPtNAP

[000101] The synthesis of the two PfNAPs is outlined in Fig. 1. Naphthalamide precursors 1 and 2 were prepared in accordance to published protocols (Hossain S.U., Sengupta S., Bhattacharya S. Bioorg. Med. Chem. 2005;13:5750-5758.). Ligand 3 was obtained by reacting 2 with neat diethylenetriamine at 70° C for 2 h. Precursor 4 was synthesized by refluxing boc-protected diethylenetriamine with 2 in 1:1 v/v MeCN/CHCl₃ for 2 d. CHC1₃ was added to the reaction medium due to the poor solubility of 2 in MeCN. Precursor 4 was purified by flash column chromatography over silica and deprotected using concentrated HC1 to yield ligand 5. TrPtNAP was obtained by refluxing excess ligand 5 and czs-PtCl₂(DMSO)₂ in MeOH. The reaction mixture was evaporated to dryness and washed sequentially with diethyl ether and CH₂C1₂ to remove unreacted ligand 5. CisPtNAP was synthesized from ligand 3 using a similar approach. The detailed synthetic procedures are given in the following paragraphs.

[000102] /V-(3-hydroxylpiOpyI)naphthalamide, 1. 1,8-naphthalic anhydride (2.50 g, 0.013 mol) was added to 3-amino-l-propanol (1.0 mL, 0.013 mol) in EtOH (20 mL) and refluxed for 1.5 h. The reaction mixture was concentrated under reduced pressure and subsequently cooled at 4°C. The resulting precipitate was filtered and the residue washed with cold EtOH to afford 2 as a pale brown solid (2.59g, 78 %). ¾ NM (CDC1₃, 300MHz): δ 8.62 (d, 2 H, Ar-H), 8.59 (d, 2H, Ar-H), 7.76 (dd, 2H Ar-H), 4.35 (t, 2H, CCH₂0), 3.59 (t, 2H, NCH₂C), 3.12 (br, 1H, OH), 2.01 (m, 2H, CCH₂C).

[000103] V-(3-bromopropyl)naphthalamide, 2. PBr₃ (0.8 mL, 8.51 mmol) was added to a stirred suspension of 1 (1 g, 4.149 mmol) in MeCN (5 mL). The reaction mixture was stirred at room temperature for 20 m, and subsequently heated at 75° C for 1.5 h. The reaction mixture was cooled, added to ice water (15 mL) and extracted with CHC1₃ (3 x 50 mL). The extract was washed with saturated NaHC0₃ (10 mL), followed by water (3 x 50 mL), and the purity of the product was monitored by TLC. The solvent was evaporated under reduced pressure to afford 3 as a pale yellow solid (1.03 g, 78 %). Ή NMR (CDC1₃, 300MHz): δ 8.60 (d, 2 H, Ar-H), 8.22 (d, 2H, Ar-H), 7.76 (t, 2H Ar-H), 4.33 (t, 2H, CCH₂0), 3.50 (t, 2H, NCH₂C), 2.34 (m, 2H, CCH₂C).

[000104] Ligand 3. Compound 2 (500 mg, 1.57 mmol) was added to a stirring mixture of neat diethylenetriamine (2 mL, 18.5 mmol) and triethylamine (0.5 mL, 3.59 mmol) and heated at 70° C for 2 h. The reaction mixture was cooled and added to excess ether (50 mL). The mixture was filtered and the filtrate was evaporated under reduced pressure. The residue was subsequently dissolved in water (7 mL), and the product was extracted with CHC1₃ (3 x 20 mL) to afford ligand 3 as a viscous yellow liquid (444 mg, 83%). ¾ NMR (CDC1₃, 300MHz): δ 8.21 (d, 2H, Ar-H), 7.86 (d, 2H, Ar-H), 7.41 (t, 2H, Ar-H, 2.58 - 2.41 (m, 10, NCH₂), 1.69 (m, 2H, CCH₂C).

[000105] Ligand 5. Boc-protected diethylenetriamine (0.352 g, 1.16 mmol) was stirred in MeCN/CHCl₃ (20 mL, 1 :1 v/v). Triethylamine (0.485 mL, 3.48 mmol) was added to the reaction mixture, which was stirred at room temperature for 30 m. Compound 2 (0.369g, 1.16 mmol) was then added and the reaction was refluxed for 2 d. The solvent was removed and was separated by flash column chromatography over silica to yield 4 as a colorless liquid (0.190 g, 30 %). Compound 4 was directly deprotected in 4 M HCl/dioxane (4 mL) at 60°C for 12 h. The white precipitate was filtered and washed with diethyl ether (3x 5 mL) to afford ligand 5 as a 5-3HC1 salt. ¾ NMR of 4 (CDC1₃, 300MHz): δ 8.52 (d, 2H, Ar-H), 8.15 (d, 2H, Ar-H), 7.69 (t, 2H, Ar-H), 5.30 (br, 2H, NH), 4.16 (t, 2H, NCH₂), 3.15 (d, 4H, NHCH₂₎, 2.53 (m, 6H, NCH₂), 1.85 (m, 2H, CCH₂C), 1.38 (s, 18H, CCH₃). ¾ NMR of 5-3HC1 (D₂0, 300 MHz): δ 7.69 (d, 4H, Ar-H), 7.22 (t, 2H, Ar-H), 3.72 (t, 2H, NCH₂), 3.61 - 3.42 (m, 8H, NCH₂CH₂N), 3.36 (m, 2H, NCH₂), 1.95 (m, 2H, CCH₂C); ¹³C NMR (D₂0, 75.5 MHz): 164.0 (s, CO), 134.9, 130.9, 129.8, 126.6, 125.5, 119.1 (s, Ar-H), 52.0 (s, NC), 49.7 (s, NC), 36.9 (s, NC), 33.5 (s, NC), 22.0 (s, CH₂).

[000106] TrPtNAP. Ligand 5-3HC1 (40 mg, 0.118 mmol) was neutralized with ₂C0₃ (171 mg, 1.24 mmol ) in DMSO:DMF: water (10 mL, 1 :1:1 v/v) at room temperature for 12 h. The reaction mixture was extracted with CH₂C1₂ (3 x 10 mL) and dried over Na₂S0₄. The solvent was removed and 5 was dissolved in MeOH (8 mL). C«-PtCl₂(DMSO)₂ (40 mg, 0.095 mmol) was added, and the solution mixture was refluxed for 2 h. The solvent was removed and residue was washed with diethyl ether (3 x 20 mL) and cold CH₂C1₂ (1 x 10 mL) to afford trPtNAP as a light brown solid (41 mg, 76%). Ή NMR (DMSO, 500 MHz): 8.50 (d, 2H, Ar-H), 8.48 (d, 2H, Ar-H), 7.88 (t, 2H, Ar-H), 5.69 (br, 2H, NH), 5.34 (br, 2H, NH), 4.11 (t, 2H, NCH₂), 3.47 (t, 2H, NCH₂), 3.12 (d, 4H, NCH₂), 2.96 (d, 2H, NCH₂), 2.84 (d, 2H, NCH₂), 1.97 (m, 2H, CCH₂C). 13C NMR (DMSO, 125.8 MHz): 163.6 (s, CO), 134.3, 131.3, 130.7, 127.4, 127.2, 122.0 (s, Ar-C) 60.8 (s, NC), 52.2 (s, NC), 47.8 (s, NC), 37.3 (s, NC), 21.0 (s, CH₂). ESI (MS) m/z = 571 [M]⁺. Anal. Calcd. for trPtNAP, C₁₉H₂₄Cl₂N₄0₂Pt: C, 37.63; H, 3.99; N, 9.24. Found: C, 37.37; H, 3.70; N, 9.15.

[000107] CisPtNAP. Ligand 3 (220 mg, 0.647 mmol) was dissolved in MeOH (10 mL) and m-PtCl₂(DMSO)₂ (200 mg, 0.475 mmol) was added. The reaction was refluxed for 2 h and the solvent was subsequently removed. The residue was washed with diethyl ether (3 x 40 mL) and cold MeOH (1 x 10 mL) to afford cisPtNAP as a light yellow solid (230 mg, 80%). ¾ NMR (DMSO, 500 MHz): 8.47 (m, 4H, Ar-H), 7.87 (t, 2H, Ar-H), 5.50 (br, 0.5 H, NH), 6.67 (br, 0.5H, NH), 5.52 (br, 0.5H, NH), 5.46 (br, 0.5H, NH), 4.07 (t, 2H, NCH₂), 2.97 (br, 2H, NCH₂), 2,79 (m, 2H, NCH₂), 2.65 (br, 2H, NCH₂), 2.58 (br, 2H, NCH₂), 2.26 (m, 1H, CCH₂C), 2.00 (m, 1H, CC¾C), 1 set of NCH₂ peaks overlap with H₂0 peak. 13C NMR (DMSO, 125.8 MHz): 163.5 (s, CO), 134.3, 131.3, 130.7, 127.4, 127.2, 122.0 (s, Ar-C), 57.3 (s, NC), 54.3 (s, NC), 53.1 (s, NC), 50.4 (s, NC), 49.8 (s, NC), 48.6 (s, NC), 37.1 (s, NC), 25.9 (s, CH₂). ESI (MS) m/z = 571 [M]⁺. Anal. Calcd. for cisPtNAP, C₁₉H24Cl2N₄0₂Pt: C, 37.63; H, 3.99; N, 9.24. Found: C, 37.51; H, 4.36; N, 9.00.

Example 2: Potent and specific activity of cisPtNAP and trPtNAP against tumour cells

[000108] Cell growth inhibition assays were conducted for the two platinum-intercalator species, cisPtNAP and trPtNAP, and compared with the activity of cisplatin (Table 1). For the commonly used 3-day agent exposure assays, it was found that cisPtNAP and trPtNAP are approximately 4-fold and 5-fold, respectively, more cytotoxic than cisplatin to human ovarian (A2780) cancer cells. To coriflrm that these low IC₅₀ values, 0.25 μΜ and 0.18 μΜ respectively for cisPtNAP and trPtNAP, were not largely due to the intercalator activity itself, the cell growth inhibitory capacity of 1,8-naphthalimide was tested. It was found that the naphthalimide functionality alone yields a much higher IC₅₀ value (55.3 μΜ), which corresponds to respectively 221-fold and 307-fold lower cytotoxicity than cisPtNAP and trPtNAP.

[000109] Table 1. Cell growth inhibition parameters.

A2780³ crA2780^a Resistance^b Non-tumor^{a d}

Cisplatin 1.00±0.05^c 14.0±0.3^C 14.0 3.58±0.87

cisPtNAP 0.25±0.02 0.48±0.02 1.9 18.4±2.2

24-hour 3.58±0.33

trPtNAP 0.18±0.05 0.32±0.08 1.8 26.8±0.8

24-hour >40.0

Naphthalimide 55.3±3.6 96.3±9.5 1.7 ^a IC50, μΜ; based on 72 -hour treatment, unless indicated otherwise; ^b crA2780:A2780 IC₅₀ value ratio (fold resistance); ^c From Adhireksan Z., et al. Nat. Commun. 2014;5:3462; ^d HaCaT (non- tumorigenic human keratinocytes); Mean±s.d., n=3.

[000110] Alongside the A2780 cell assays, the activity of cisPtNAP and trPtNAP on inhibiting the growth of a cisplatin-resistant A2780 (crA2780) cell line (Behrens B.C., et al. Cancer Res. 1987;47:414-418) was tested. The crA2780 cells are 14-fold resistant to cisplatin (IC₅₀=14 μΜ) relative to the A2780 cells. However, the cytotoxicity of cisPtNAP (IC₅₀=0.48 μΜ) and trPtNAP (IC₅o=0.32 μΜ) to these resistant cells is only 1.9-fold and 1.8-fold, respectively, below that observed for the A2780 cells. This corresponds to a basal level of cross-resistance, common to DNA damaging agents completely unrelated to cisplatin (Adhireksan Z. et al. Nat. Commun. 2014;5:3462; Behrens B.C., et al. Cancer Res. 1987;47:414-418).

[000111] In order to assess the therapeutic potential of cisPtNAP and trPtNAP in terms of selective activity against tumour cells, the impact on a model for healthy tissue, HaCaT cells, which are a non-tumorigenic human keratinocyte line that is well suited for tissue culture, was also tested. Interestingly, cisplatin is still appreciably toxic to the HaCaT cells, with a 3.6 μΜ IC₅₀, which corresponds to an only 3.6-fold increase over that for the A2780 cells. On the other hand, the respective HaCaT IC₅₀ values for cisPtNAP and trPtNAP, 18.4 μΜ and 26.8 μΜ, are much higher than for the A2780 cells, corresponding to diminished cytotoxicity on the order of 74-fold and 149- fold respectively.

Example 3: Proficient accumulation of DNA adducts in cells treated with cisPtNAP or trPtNAP

[000112] Given the chemical nature of cisPtNAP and trPtNAP, one would assume their very high cytotoxicity to tumour cells is stemming from DNA adducts. To establish DNA adduct formation potential, cells were treated with agent at concentrations proportional to their 3 -day IC₅₀ values for 18 hours, isolated the genomic DNA and quantified platinum adduct levels (Table 2). This analysis showed that cisPtNAP and trPtNAP form similar levels of adducts, namely approximately 8 and 6 adducts, respectively, per 10⁴ base pairs (bp). This corresponds to about one-half the adduct levels found for analogous treatment with cisplatin, which yields ~12 adducts per 10⁴ bp. On the other hand, relative to the agent concentration used in the cell treatment, cisPtNAP and trPtNAP yield almost identical adduct levels, -31 adducts per 10⁶ bp per μΜ, which is in fact a genomic DNA adduct formation activity that is roughly 2.5-fold greater than that of cisplatin (-12 adducts per 10⁶ bp per μΜ).

[000113] Table 2. Platinum adduct levels on genomic DNA. adducts cisPtNAP trPtNAP Cisplatin

Per 10⁶ bp^a 787±144 557±110 1235±50

Relative (nM^'1)^b 31.5 30.9 12.3 ^a Platinum atoms per 10 base pairs (bp); Agent concentrations used in cell treatments, 25 μΜ cisPtNAP, 18 μΜ trPtNAP, 100 μΜ cisplatin; ^b Platinum atoms per 10⁶ bp per μΜ of compound used in cell treatment; Mean±s.d., n=3.

Example 4: Isomer-dependent DNA site selectivity of cisPtNAP and trPtNAP

[000114] With the very similar activity of cisPtNAP and trPtNAP observed from the cellular cytotoxicity and genomic DNA adduct formation assays described above, it would seem to follow that the two compounds have similar DNA site selectivity. However, DNA footprinting analysis of agent- treated nucleosome core particle (NCP) and the corresponding naked DNA show that this is not the case (Fig. 2 & 3). For this method, as well as for the X-ray crystallographic analysis, a palindromic 145 bp DNA fragment was utilized and nucleosome core assembled with recombinant histones (Ong M.S., Richmond T.J., Davey C.A. J. Mol. Biol. 2007;368:1067-1074; Davey C.A., et al. J. Mol. Biol. 2002;319:1097-1113; Wu B., et al. Chemistry 2011;17:3562-3566).

[000115] In the footprinting analysis, although both isomers show a clear discrimination to form adducts at only guanine nucleotides, they display highly distinct preferences for specific DNA sequence elements— that is the sequence context of the guanine sites. Moreover, the site discrimination seen towards the nucleosomal substrate is dramatically different, with trPtNAP forming substantial adducts only at the guanine nucleotides nearest the DNA termini of the nucleosome core. In fact, this agent has a striking preference for a palindromic CTGCAG element (SHL ±5.5/6), where it efficiently forms adduct at nucleotide -58 (underlined) on both the naked DNA and NCP substrates. The selectivity for this particular sequence element is so pronounced that it is the sole site of significant adduct formation at low trPtNAP concentrations.

[000116] In contrast to the selectively of trPtNAP towards specific DNA sequence elements, cisPtNAP avidly forms adducts at internal nucleosomal sites and in particular at the location of superkinking at SHL ±1.5. Thus, cisPtNAP is effective at targeting the highly deformed DNA site in the nucleosome for which it was designed, whereas adduct formation by trPtNAP at this location is barely detectable in the solution state.

Example 5: Stereochemical reactivity control in cisPtNAP and trPtNAP

[000117] In order to understand the basis for the profound difference in site selectivity displayed by cisPtNAP and trPtNAP, X-ray crystallographic experiments were conducted in which NCP crystals that are composed of the same DNA fragment used in the footprinting studies were derivatized. Many trials were carried out to find the best compromise between adduct occupancy and resolution, as longer duration and higher concentration treatments yield progressively higher site occupancy albeit correspondingly lead to greater disordering of the crystals and thus lower resolution data.

[000118] Treatment strategies that allowed us to acquire 2.6 A and 2.45 A resolution data sets for cisPtNAP and trPtNAP, respectively, were determined, with which accurate atomic models for DNA adducts formed by each isomer could be built (Table 3). For both compounds, adducts only at the locations of extreme DNA kinking in both symmetry-related halves of the nucleosome (SHL -1.5 and +1.5; Fig. 2 & 4) are observed, with one exception relating to cisPtNAP, which is observed to also associate with one of the NCP DNA termini. It is not clear whether this corresponds to an adducted state, but in any case it is an artefact of (non-physiological) DNA termini, which loosely stack between neighbouring NCPs in the crystal and thus promote intercalation of the naphthalimide group.

[000119] Table 3. Data collection and refinement statistics for NCP treated with PtNAP NCP-trPtNAP NCP-cisPtNAP

Data collection*

Space group ¥2 2_\2_\ P2₁2₁2₁

Cell dimensions

a (A) 106.41 106.57

b (A) 109.63 109.33

c (A) 183.01 181.28

Resolution (A) 2.45-54.8 2.60-70.3

(2.45-2.58) (2.60-2.74)

R_merge (%) 5.6 (49.2) 5.6 (48.6)

Il ol 17.5 (2.7) 14.7 (1.5)

Completeness (%) 93.4 (68.4) 89.0 (55.7)

Redundancy 6.6 (4.8) 5.6 (2.2)

Refinement

Resolution (A) 2.45-54.8 2.60-70.3

No. reflections 72,422 57,156

^work tf_free (%) 21.4 / 26.3 21.6 / 27.4

No. atoms 12,128 12,110

Protein 6,064 6,064

DNA 5,939 5,939

Solvent 46 29

Adduct 78 78

5-factors (A²) 72 102

Protein 47 69

DNA 97 135

Solvent 44 69

Adduct 132 170

R.m.s. deviations

Bond lengths (A) 0.008 0.007

Bond angles (°) 1.48 1.41

Single-crystal data; Values in parentheses are for the highest-resolution shell; R.m.s., root mean square.

[000120] To pinpoint the locations of the platinum atoms and to detect adduct sites also with low occupancy, diffraction data at the X-ray absorption edge for platinum were collected. The anomalous difference electron density maps calculated with this data show that for cisPtNAP, there are actually three distinct platinum atom locations around the GG dinucleotide element of SHL ±1.5 (Fig. 4, 5 & 6). The major adduct configuration of these three corresponds to platinum coordination to the 3 ' guanine N7 group and is sufficiently populated to allow construction of an atomic model. The naphthalimide group is nearly fully intercalated within the GG step and the cisPtNAP amine group connecting the naphthalimide group forms a hydrogen bond with the 06 atom of the coordinating guanine base. The two additional, minor, platinum atom configurations correspond to alternate coordination to the 5' guanine base of the GG element and what appears to be an alternate intercalative mode associated with a distinct stretching configuration of the double helix.

[000121] For trPtNAP, there are two distinct configurations in the crystal that are sufficiently populated to allow atomic modelling (Fig. 4, 5, 7 & 8). One of these is observed clearly at both symmetry-related sites (SHL -1.5 and +1.5) and entails adduct formation at the same site as for cisPtNAP. The trPtNAP and cisPtNAP adduct structures are similar, however the different configuration of linkage between the intercalator and platinum head groups results in differential orientation of the naphthalimide plane and triamine ligand. The triamine group in the trPtNAP adducts is oriented with an inclination that allows for hydrogen bonding with both the 5' phosphate group and guanine 06 atoms on the opposing side. The other binding configuration of trPtNAP, seen with sufficient clarity to allow model building only for SHL +1.5, corresponds to a mode of intercalation in which the platinum atom is not coordinated to the DNA (Fig. 7). The naphthalimide group resides within the GG bp step element as in the case for the adducts, but the platinum head group is localized instead adjacent to the 5' guanine with the platinum atom situated axially -3.7 A from the 06 atom. Although this non-adductive binding mode must be largely promoted by the hydrophobic interactions of intercalation, it seems to be additionally stabilized by hydrogen bonding between one of the amine ligands and the N7 group of the adjacent adenine to the 5' side. This non- adducted binding mode is also apparent, albeit with poor clarity either because of structural disorder or low occupancy, in the symmetry-related site at SHL -1.5 (Fig. 8).

[000122] The adducts formed by cisPtNAP and trPtNAP at the locations of superkinking are seen to result in nearly identical structures between the two symmetry-related sites for a given isomer (Fig. 2, 4 & 9). This is notable since the DNA conformations in the native state are highly distinct between the regions spanning SHL -1 to -2 and SHL +1 to +2 where the double helix stretching distortions occur in the two symmetry-related halves of the nucleosome. In the 'minus' half, the stretch-associated extreme kink resides at a CA=TG step of SHL -1, whereas due to differences in crystal contacts, the kink instead occurs at the GG=CC step of SHL +1.5 in the 'plus' half of the NCP. In spite of this difference, intercalation is seen to occur in either case within the unstacked GG=CC step, and the resulting DNA conformational parameters are very similar between all the adduct sites. This emphasizes the dynamic nature of exchange between available DNA stretching configurations in the nucleosome, analogous to what have been observed in the past with different DNA attacking agents (Davey G.E., et al. Nucleic Acids Res. 2010;38:2081-2088; Wu B., et al. Nucleic Acids Res. 2011 ;39:8200-8212; Adhireksan Z., et al. Nat. Commun. 2014;5:3462), but it also indicates that the final conformation resulting from adduct formation by cisPtNAP and trPtNAP is highly constrained. This correspondingly suggests that conformational constraints on reactive intermediates may also be very pronounced, providing a rationale for the site selectivity distinction between these two structural isomers.

Example 6: Isomer-specific impact of cisPtNAP and trPtNAP on cancer cells

[000123] Given the striking differences in DNA site selectivity between trPtNAP and cisPtNAP, experiments were conducted on the A2780 tumour cell line to shed more light on how differences in genomic site targeting could influence cellular behaviour. Cells were treated with an equal concentration of either agent for 24 hours, which yields roughly equivalent genomic DNA adduct levels in the cell, after which cells were switched to agent-free media and quantified cell survival and cell cycle arrest immediately (1-day) and then subsequently 24 and 48 hours later (2-day, 3-day; Fig. 10).

[000124] Beyond their general tumour cell growth inhibitory properties, the two compounds display distinct attributes of impact on cell function. In particular, cisPtNAP has much more rapid cancer cell killing activity compared to trPtNAP, with approximately 4-fold more cell death after 1- day exposure to agent (relative to control cells; fraction of dead cells: 12.8% control, 20.3% trPtNAP, 41.0% cisPtNAP). This is consistent with cisPtNAP displaying greater than 1 1 -fold higher cytotoxicity to cancer cells in the short-term (for a 1-day as opposed to the 3-day exposure cell growth inhibition assays; Table 1). Nonetheless, subsequent to removal of free compound from the cell media, exposure to either agent continues to kill a greater fraction of the cell population, and by day 3 the cell death profile of trPtNAP has become similar to that of cisPtNAP. This is also consistent with the fact that the 3 -day cytotoxicities of both compounds are very similar.

[000125] Given the different rate of cisPtNAP versus trPtNAP tumour cell killing, one would anticipate an analogous differential impact on the cell cycle pattern, but in fact the influence that is observed is in an opposing sense. More specifically, after one day exposure of cancer cells to agent, the degree of cell cycle S-phase arrest (stalling at DNA replication stage) induced by trPtNAP is pronounced, but diminishes over the following two days. For cisPtNAP, the degree of S-phase arrest at day 1 is only slight, but then increases by day 3 to a level similar to that seen for trPtNAP at day 1. Moreover, although trPtNAP induces a slight increase in G2/M arrest by day 2, that seen for cisPtNAP is especially pronounced by days 2 and 3.

[000126] The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. All documents listed are hereby incorporated herein by reference in their entirety.

[000127] The invention has been described broadly and generically herein. Each of the narrower species and subgenenc groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[000128] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

CLAIMS What is claimed is:

1. Compound of Formula (I) or (Π) or a stereoisomer or pharmaceutically acceptable salt thereof,

wherein

X is a leaving group;

R¹, R^1', R², and R² are each independently hydrogen or methyl;

R³, R³ and R⁴ are each independently selected from the group consisting of hydrogen, Ci_i₀ alkyl and C]_io alkyloxy;

each n is independently an integer of 1 to 10; and

ICA is a DNA intercalating compound.

2. The compound of claim 1 , wherein X is selected from the group consisting of halogen, -OH, - SCN, -OR⁵ and -0-C(0)-R⁵, wherein R⁵ is any organic moiety. .

3. The compound of claim 1 or 2, wherein in Formula (I) R¹, R¹ , R² and R² are hydrogen.

4. The compound of claim 1 or 2, wherein in Formula (II), R¹, R¹ , R² and R⁴ are hydrogen.

5. The compound of any one of claims 1 to 4, wherein any one or more or all of R , and R³ are hydrogen.

6. The compound of any one of claims 1 to 5, wherein n is 2 to 5, preferably 3.

7. The compound of claim 1 -6, wherein the DNA intercalating compound is 1 ,8-naphthalimide or a derivative thereof.

8. The compound of claim 7, wherein the 1 ,8-naphthalimide or a derivative thereof is a compound of formula (III),

wherein R⁶, R⁶ , R⁷, R^r, R⁸, R^8' are each independently selected from the group consisting of hydrogen, Ci-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂4 alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, Ci-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂o aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄ alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl, mono-(Ci-C₂₄ alkyl)- substituted carbamoyl, di-(Ci-C₂₄ alkyl)-substituted carbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, mono- and di-(C C₂ alkyl)-substituted amino, mono- and di-(C₅-C₂₀ aryl)- substituted amino, C₂-C₂ alkylamido, C₅-C₂₀ arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato, C]-C₂₄ alkylsulfanyl, arylsulfanyl, C C₂₄ alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, and phosphino, and wherein any two adjacent {ortho) substituents may be linked to form a cyclic structure selected from five-membered rings, six-membered rings, and fused five- membered and/or six-membered rings, wherein the cyclic structure is aromatic, alicyclic, heteroaromatic, or heteroalicyclic, and has 0 to 4 non-hydrogen substituents and 0 to 3 heteroatoms. he compound of claim 8, wherein the DNA intercalating compound is selected from the group consisting of

lH-benzo[de]isoquinoline-l ,3(2H)-dione (i.e.l,8-naphthalimide);

4- nitro- IH-benzo [de] isoquinoline- 1 , 3(2H)-dione;

5 -nitro- 1 H-benzo [de] isoquinoline- 1 ,3 (2H)-dione;

6-nitro-lH-benzo[£ e]isoquinoline-l,3(2H)-dione;

6-amino-5 -nitro- 1 H-benzo [de] isoquinoline- 1 ,3 (2H)-dione;

6-(piperidin-l -yl)-lH-benzo[i e]isoquinoline-l ,3(2H)-dione;

5- nitro-6-(piperidin- 1 -yl)- 1 H-benzo [de] isoquinoline- 1 ,3 (2H)-dione;

6- mo holino-lH-benzo[ί/e]isoquinoline-l ,3(2H)-dione;

6-(piperazin-l -yl)-lH-benzo[i e]isoquinoline-l ,3(2H)-dione;

6-(4-methylpiperazin- 1 -yl)- 1 H-benzo[i/e] isoquinoline- 1 , 3 (2H)-dione;

5 -(piperazin- 1 -yl)- 1 H-benzo [de] isoquinoline- 1 ,3(2H)-dione;

6-(lH-imidazol-l -yl)-lH-benzo[(/e]isoquinoline-l ,3(2H)-dione;

7,8-dihydronaphtho[2, 1 ,8-i/e ]isoquinoline-l ,3(2H6H)-dione;

6,7-dihydro-lH-indeno[6,7,l -t/e ] isoquinoline- 1 ,3(2H)-dione;

4H-benzo[ e]furo[3,2-g]isoquinoline-4,6(5H)-dione;

4H-benzo [ de] furo[2,3 -g] isoquinoline-4,6(5H)-dione;

4H-dibenzo[i e,g]isoquinoline-4,6(5H)-dione;

4H-isoquinolino[5,4-^]quinoxaline-4,6(5H)-dione;

1 H-dibenzo [de, h] isoquinoline- 1 ,3 (2H)-dione;

lH-benzo[ii?e][2,9]phenanthroline-l,3(2H)-dione;

2,2,2-tricMoro-N-((l ,3-dioxo-2,3-dihydro-lH-benzo[i/e]isoquinolin-5-yl)carbamoyl)acetamide; 5 -phenyl- 1 H-benzo [de] isoquinoline- 1 ,3 (2H)-dione;

5-(p-tolyl)-lH-benzo[i e]isoquinoline-l,3(2H)-dione;

5-(4-fluorophenyl)-lH-benzo[i e]isoquinoline-l,3(2H)-dione;

5-(4-(trifluoromethyl)phenyl)-lH-benzo[i e]isoquinoline-l ,3(2H)-dione;

5- (3,4-difluorophenyl)-lH-benzo[6?e]isoquinoline-l,3(2H)-dione;

5 -(3 ,4-dinitrophenyl)- IH-benzo [t/e]isoquinoline- 1 ,3 (2H)-dione;

5 -(thiophen-2-yl)- lH-benzo[t/e] isoquinoline- 1 , 3 (2H)-dione;

6- ((2-(piperazin- 1 -yl)ethyl)amino)- 1 H-benzo [de] isoquinoline- 1 , 3 (2H)-dione;

6-((2-hydrazinylethyl)amino)- 1 H-benzo [de] isoquinoline- 1 ,3 (2H)-dione;

6-((2-(dimethylamino)ethyl)amino)- 1 H-benzo [de] isoquinoline- 1 , 3 (2H)-dione;

6-((2-(phenylthio)ethyl)amino)- IH-benzo [de] isoquinoline- 1 ,3(2H)-dione;

lH-thioxantheno[2,l,9-t/e ]isoquinoline-l,3(2H)-dione;

4H-benzo[ e]benzo[4,5]thieno[2,3-g]isoquinoline-4,6(5H)-dione; 9-phenyl-4H-benzo[i/e]oxazolo[5,4-g]isoquinoline-4,6(5H)-dione;

4H-benzo[i e]thieno[2,3-g]isoquinoline-4,6(5H)-dione; and

9-phenylbenzo[i e]imidazo[4,5-g]isoquinoline-4,6(5H, 10H)-dione.

10. The compound of any one of claims 1 to 9, wherein the compound is cw-chloro[l,2,3- diethylenetriamino-2-N-(3-propyl)-l,8-naphthalimide]platinum(n) chloride or

diethylenetriamino-l-N-(3^ropyl)-l,8-naphthalimide]platinum(lT) chloride.

11. The compound of any one of claims 1 to 10 for use in a method for treating cancer.

12. Method for the treatment of cancer in a subject, the method comprising administering to said subject a therapeutically effective amount of the compound of any one of claims 1 to 10.

13. The method according to claim 12, wherein the subject is a mammal, preferably a human.

14. Method for triggering apoptosis in a cell, preferably a cancer cell, the method comprising contacting said cell with an effective amount of the compound of any one of claims 1 to 10.

15. Use of the compound of any one of claims 1 to 10 for triggering apoptosis in a cell, preferably a cancer cell.