DNA Intercalators and Topoisomerase Inhibitors

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Contents 1. DNA Intercalation and Its Consequences 200

2. Monofunctional Intercalating Agents 201

2.1. Ellipticine and its analogues 201

2.2. Actinomycins 204

2.3. Fused quinoline compounds 204

2.4. Naphthalimides and related compounds 205

2.5. Chartreusin, elsamicin A, and related compounds 206

3. Bifunctional Intercalating Agents 206

4. DNA Topoisomerases 206

4.1. Topoisomerase I mechanism 209

4.2. Topoisomerase II mechanism 209

5. Topoisomerase II Poisons 211

5.1. Acridine derivatives 213

5.2. Anthracyclines and related compounds 213

5.3. Non-intercalating topoisomerase II poisons 214

6. Topoisomerase II Catalytic Inhibitors 216

6.1. Inhibitors of the binding of topoisomerase II to

DNA: Aclarubicin 218

6.2. Merbarone 219

6.3. Bis(dioxopiperazines) 219

7. Specific Topoisomerase I Inhibitors 219

7.1. Camptothecins 220

7.2. Non-CPT topoisomerase I inhibitors 221 References 225

Medicinal Chemistry of Anticancer Drugs © 2008 Elsevier B. V.

DOI: 10.1016/B978-0-444-52824-7.00007-X All rights reserved.

1. DNA INTERCALATION AND ITS CONSEQUENCES

Many anticancer drugs in clinical use (e.g. anthracyclines, mitoxantrone, dactinomy-cin) interact with DNA through intercalation, which can be defined as the process by which compounds containing planar aromatic or heteroaromatic ring systems are inserted between adjacent base pairs perpendicularly to the axis of the helix and without disturbing the overall stacking pattern due to Watson-Crick hydrogen bonding. Since many typical intercalating agents contain three or four fused rings that absorb light in the UV-visible region of the electromagnetic spectrum, they are usually known as chromophores. Besides the chromophore, other substitu-ents in the intercalator molecule may highly influence the binding mechanism, the geometry of the ligand-DNA complex, and the sequence selectivity, if any.

The intercalation process1 starts with the transfer of the intercalating molecule from an aqueous environment to the hydrophobic space between two adjacent DNA base pairs. This process is thermodynamically favoured because of the positive entropy contribution associated to disruption of the organized shell of water molecules around the ligand (hydrophobic effect). In order to accommodate the ligand, DNA must undergo a conformational change involving an increase in the vertical separation between the base pairs to create a cavity for the incoming chromophore. The double helix is thereby partially unwound,2 which leads to distortions of the sugar-phosphate backbone and changes in the twist angle between successive base pairs (Fig. 7.1). Once the drug has been sandwiched

FIGURE 7.1 Deformation of DNA by an intercalating agent.

between the DNA base pairs, the stability of the complex is optimized by a number of non-covalent interactions, including van der Waals and p-stacking interactions,3 reduction of coulombic repulsion between the DNA phosphate groups associated with the increased distance between the bases because of helix unwinding, ionic interactions between positively charged groups of the ligand and DNA phosphate groups, and hydrogen bonding. Generally speaking, cationic species are more efficient DNA intercalators because they interact better with the negatively charged DNA sugar-phosphate backbone in the initial stages and also because intercalation releases counterions associated to phosphate group, such as Na+, leading to the so-called polyelectrolyte effect. This is a very important driving force for intercalation, since it diminishes repulsive interactions between the closely spaced charged counterions. In fact, most intercalating agents are either positively charged or contain basic groups that can be protonated under physiological conditions.

DNA intercalators are less sequence selective than minor groove binding agents, and, in contrast with them, show a preference for G-C regions. This selectivity is mainly due to complementary hydrophobic or electrostatic interactions, which are due to substituents attached to the chromophore within the major or minor grooves. DNA intercalation is also governed by the nearest-neighbour exclusion principle, which states that both neighbouring sites on each site of the intercalation remain empty, that is, they bind, at most, between alternate base pairs.4 This is an example of a negative cooperative effect, whereby binding to one site induces a conformational change that hampers binding to the adjacent base pair.

Intercalation of a drug molecule into DNA is only the first step in a series of events that eventually lead to its biological effects.5 Structural changes induced in DNA by intercalation lead to interference with recognition and function of DNA-associated proteins such as polymerases, transcription factors, DNA repair systems, and, specially, topoisomerases. The role of these enzymes in the design of antitumor drugs will be discussed in Sections 4 and 5.

2. MONOFUNCTIONAL INTERCALATING AGENTS

2.1. Ellipticine and its analogues

Ellipticine, an alkaloid isolated from the leaves of Ochrosia elliptica and other Apocynaceae plants, is the prototype of intercalators based on the pyridocarbazole system and displays a broad spectrum of antitumor activity.6 Although at physiological pH values it can exist both as a neutral species and as a monocation (Fig. 7.2), it is the latter form that seems responsible for DNA intercalation, which leads to RNA polymerase inhibition. Other DNA-related enzymes that are inhibited by ellipticine include DNA polymerase, RNA methylase, and topoisomerase II, although it is not known whether these effects are a consequence of intercalation.

Ellipticine is also known to lead to cytochrome P450 (CYP)-dependent metabolites that are able to bind covalently to DNA.7in vitro experiments employing a peroxidase-H2O2 oxidizing system have shown that one of the metabolites from ellipticine, namely its 9-hydroxy derivative 7.1, oxidizes to quinonimine 7.28 but,

Physiological I 3 + H

CH3 CH3

Neutral ellipticine Protonated ellipticlne

FIGURE 7.2 Neutral and protonated forms of ellipticine.

CH3 7.2

CH3 7.2

9_10 CH3

9_10 CH3

Nu- DNA

FIGURE 7.3 Ellipticine metabolites and their binding to DNA

Nu- DNA

FIGURE 7.3 Ellipticine metabolites and their binding to DNA

in spite of its high electrophilicity, this intermediate seems not to be covalently bonded to DNA. More recent studies have proved that DNA binding is associated to other metabolites, including the N-oxide 7.3 and the hydroxymethyl derivative 7.4, which can be tentatively assumed to react through the intermediacy of stabilized cation 7.5 to give the DNA-alkylated product 7.6, as shown in Fig. 7.3.9 Because of the higher efficiency of cations as intercalating agents, some N-2 quaternized ellipticine analogues were assayed, among which the most interesting was N-methyl-9-hydroxyellipticinium (NMHE). Its quinonimine 7.7 is more reactive than its previously mentioned analogue from ellipticine (7.2) because of the presence of the strongly electron-withdrawing cationic heterocyclic nitrogen atom, and has been shown to react with a variety of biologically relevant nucleo-philes at its C-10 position to give adducts 7.9 (Fig. 7.4). However, a correlation between the in vivo antitumor activity of NMHE and the formation of covalent adducts has not been established; in fact, it has been shown that the extent of

CH3 NMHE

O Nu

CH3 NMHE

.CH3

.CH3

O Nu

HO Nu

HO Nu

-CH3

O Nu

-CH3

O Nu

-CH3

FIGURE 7.4 Reaction of N-metylhydroxyellipticinium with nucleophilic biomolecules.

irreversible binding to DNA is similar in NMHE-sensitive and NMHE-resistant cell lines.10 Compounds related to NMHE have been employed as the basis for the design of bis-intercalating compounds (see Section 4).

S-16020 is another important antitumor pyridocarbazole derivative, bearing a (dimethylamino)ethylcarboxamide side chain that increases its DNA intercalating ability. This drug is a potent stimulator of topoisomerase Il-mediated DNA cleavage and is not affected by resistance mediated by P-gp. Despite its close similarity with ellipticine, both compounds show little cross-resistance. Phase I clinical trials have indicated limited antitumor activity in head and neck cancer.11

Intoplicine is an intercalating compound that can be considered as structurally related to the ellipticines. It behaves as a dual topoisomerase I and II poison at cleavage sites different to those of other known topoisomerase inhibitors.12 From spectroscopic results two types of DNA complexes have been proposed accounting for topoisomerase I- and Il-mediated cleavages, involving a 'deep intercalation mode' or an 'outside binding mode', respectively.13 Because of the high activity of intoplicine in preclinical cancer models, original mechanism of action and acceptable toxicity profile, it was further evaluated in several Phase I studies.14 In these trials, patients developed serious liver toxicity at dose levels below the one believed to be necessary for antitumor activity. This toxicity was considered to be dose-limiting.

-CH3

Dactinomycin Mechanism

2.2. Actinomycins

Actinomycin D (dactinomycin) is a member of the actinomycin family of compounds, which was isolated from several Streptomyces strains. It contains a phe-noxazine chromophore attached to two cyclic depsipeptides containing five amino acid residues. It can be considered as a hybrid compound that behaves both as a DNA intercalator and a minor groove binding agent. Although it differs from most intercalating drugs in that it lacks a positive charge, it has been suggested that this is compensated by its high dipole moment, arising from a non-symmetrical distribution of polar substituents.15 Dactinomycin is used to treat sarcomas, pediatric solid tumors (e.g. Wilms' tumor, a type of renal tumor), germ cell cancers (testicular cancer), and choriocarcinoma.

H3C-N a-ring HN NH b-ring N-CH

Actinomycin D (dactinomycin)

Actinomycin D is the paradigm of intercalating compounds with sequence selectivity. X-Ray diffraction16,17 and molecular modelling studies18 have been extensively employed to characterize its complex with DNA. The actinomycin chromophore favours guanine-cytosine pairs and is therefore inserted between the G-C step. Hydrogen bonds are established between the guanine 2-amino group and the carbonyl oxygen of threonine, and also between the guanine N-3 atom and the NH group of the same threonine residue, helping to stabilize the actinomycin-DNA complex. The proline, sarcosine, and methylvaline residues of the pentapeptide side chain are involved in further hydrophobic interactions with the DNA minor groove (Fig. 7.5). Several proposals have been put forward regarding the nature of the preferred flanking base sequences adjacent to the GC intercalation site.19 The formation of this very stable actinomycin-DNA complex prevents the unwinding of the double helix which leads to inhibition of the DNA-dependent RNA polymerase activity and hence transcription.20 Like in the case of other intercalating agents, topoisomerase II inhibition may also be one of the causes of cytotoxicity.

2.3. Fused quinoline compounds

TAS-103 is a dual topoisomerase I and II inhibitor that has marked efficacy against various lung metastatic cancers and a broad antitumor spectrum in human xeno-grafts, and it has reached clinical trials for the treatment of solid tumors.21 DNA

I Pentapeptide I

Chromophore — NH2 PO2 Intercalation

Chromophore — NH2 PO2 Intercalation

b-ring

Minor groove ^ binding a-ring b-ring

Minor groove ^ binding a-ring

FIGURE 7.5 DNA intercalation by actinomycin D.

binding and unwinding assays indicate that TAS-103 intercalates into DNA, although spectroscopic studies show that outside binding is also important.22

(H3C)2N

TAS-103

TAS-103

2.4. Naphthalimides and related compounds

Naphthalimide derivatives bearing an aminoalkyl side chain such as mitonafide and amonafide have shown interesting cytotoxic activity,23 which is due to intercalation and topoisomerase II inhibition.24 Both mitonafide25 and amonafide26 have been extensively tested in clinical trials but have not been employed in therapeutics, although they have been used as leads in the design of bis-intercalators (see below).

R = NO2 Mitonafide

R = NH2 Amonafide

R = NO2 Mitonafide

R = NH2 Amonafide

2.5. Chartreusin, elsamicin A, and related compounds

Chartreusin and elsamicin A are structurally related antitumor antibiotics that were isolated from Streptomyces chartreusis and from an unidentified actinomycete strain, respectively. Chartreusin suffered from unfavourable pharmacokinetic properties (slow oral absorption and biliar excretion) which prevented its clinical development. Semi-synthetic chartreusin analogues with improved pharmacoki-netics have been developed. One of them, IST-622, is under Phase II clinical trials for the oral treatment of breast cancer.27

On the other hand, elsamicin A, one of the most potent known inhibitors of topoisomerase II, has entered Phase I clinical studies for relapsed or refractory non-Hodgkin's lymphoma. The activity was modest, but the compound was nevertheless considered promising because of the absence of myelosuppression.28

H?CH3 0 VH3O

w h3coI

O "Xfeo

OH Chartreusin

O "Xfeo

OH Chartreusin

h3co i

0-C0-CH2-CH2-0-C2H5

OH IST-622

0-C0-CH2-CH2-0-C2H5

HOA^^i CH3

OH IST-622

H3CV

OH Elsamicin A

2.6. Other monofunctional intercalating agents

Some other intercalating agents (acridines and anthracyclines) are discussed in Section 5.

3. BIFUNCTIONAL INTERCALATING AGENTS

In efforts to increase the binding constant of intercalating compounds, bifunc-tional or even polyfunctional compounds have been designed. Bifunctional intercalators (bis-intercalators) contain two intercalating units, normally cationic, separated by a spacer chain that must be long enough to allow double intercalation taking into account the neighbour exclusion principle (Fig. 7.6).

Ditercalinium is an interesting bis-intercalator derived from ellipticinium with a novel mechanism of action different from that of its monomer, since topoisomerase II inhibition is not involved. Ditercalinium causes inhibition of enzymes that locate and repair damaged DNA sites, specially the nucleotide excision repair (NER) system,29 due to the unstacking and bending that it induces on DNA because of the rigidity of the linker chain.30

Elinafide is a bis-intercalator derived from the naphthalimide pharmaco-phore31 that exhibited excellent antitumor activity and reached Phase I clinical trials,32 showing anti-neoplastic activity in ovarian cancer, breast cancer, and mesothelioma. Mechanistic studies on elinafide and its analogues are still in progress,33 but this drug suffers from neuromuscular dose-limiting toxicity that has halted its clinical development.

,och3

,och3

Ditercalinium

Elinafide

Elinafide

Ditercalinium

Echinomycin is an antitumor antibiotic isolated from S. echinatus, which consists of two quinoxaline chromophores attached to a cyclic octadepsipeptide ring, with a thioacetal cross-bridge. Because of its potent antitumor activity, this compound has been advanced to several Phase II clinical studies,34,35 although it was eventually withdrawn from further clinical trials because it showed a high toxicity without any marked therapeutic benefit. More recently, echinomycin has been characterized as a very potent inhibitor of the binding of HIF-1 (hypoxia-inducible factor 1) to DNA. This is an interesting feature because HIF-1 is a transcription

Intercalation Dna
FIGURE 7.6 Schematic interaction between a bis-intercalator and DNA.
Dna Intercalator
FIGURE 7.7 Schematic interaction between echinomycin and DNA.

factor that controls genes involved in processes important for tumor progression and metastasis, including angiogenesis, migration, and invasion.36

Several studies have proved that both echinomycin quinoxaline rings bis-intercalate into DNA, with CG selectivity, while the inner part of the depsipeptide establishes hydrogen bonds with the DNA bases of the minor groove region of the two base pairs comprised between the chromophores (Fig. 7.7).37 A calorimet-ric study has proved that the binding reaction is entropically driven, showing that the complex is predominantly stabilized by hydrophobic interactions, although direct molecular recognition between echinomycin and DNA, mediated by hydrogen bonding and van der Waals contacts, also plays an important role in stabilizing the complex.38

4. DNA TOPOISOMERASES

Identical loops of DNA having different numbers of twists are topoisomers, that is, molecules with the same formula but different topologies, and their interconversion requires the breaking of DNA strands. DNA topoisomerases are enzymes that regulate the three-dimensional geometry (topology) of DNA, leading to the interconversion of its topological isomers and to its relaxation. This is related to the regulation of DNA supercoiling, which is essential to DNA transcription and replication, when the DNA helix must unwind to permit the proper function of the enzymatic machinery involved in these processes.

Topoisomerase I breaks a single DNA strand while topoisomerase II breaks both strands and requires ATP for full activity. In both cases, the enzyme is covalently attached to the DNA through tyrosine residues in the active site. These are transient, easily reversible linkages, and for this reason this covalently bound structure is known as the 'cleavable complex'. Afterwards, another DNA strand passes through the transient break in the DNA, and finally the DNA break is resealed. The end result of the reaction is a DNA molecule which is chemically unchanged, but closed in a different topology. The normal catalytic cycle of both types of topoisomerases involves two transesterification steps, one for the cleavage and other for the religation process. In the cleavage reaction, nucleo-philic attack of an active site tyrosine forms a covalent bond with DNA by nucleophilic attack of its hydroxy group to a phosphate group of the phosphodie-ster DNA backbone. In the religation step, the 5'-hydroxyl group from deoxyri-bose attacks the previously formed tyrosine phosphate.

Topoisomerases are crucial for the several DNA functions (e.g. replication and transcription) that require the DNA to be unravelled, a process that generates tension and entanglement in DNA. Drugs that inhibit the topoisomerases include some of the most widely used anticancer drugs.39 On the other hand, topoisomer-ase poisons may trigger chromatid breakage to inactivate the ataxia telangiectasia (AT) gene function, disable cell cycle control, and induce genetic instability.40 In this connection, some alarming studies have been published, suggesting that maternal exposure to low doses of dietary topoisomerase II poisons, including bioflavonoids such as genistein or quercetin, may contribute to the development of infant leukaemia.41

4.1. Topoisomerase I mechanism

In the case of eukaryotic topoisomerase I, a single strand is attacked and a 3'-phosphotyrosyl linkage is formed. Religation takes place through attack of the 5' hydroxyl to the previously formed phosphate group (Fig. 7.8).

Topoisomerase Inhibitor

FIGURE 7.8 Transesterification reactions involved in topoisomerase I (topo I) activity.

FIGURE 7.8 Transesterification reactions involved in topoisomerase I (topo I) activity.

Binding site

1. Topoisomerase binding

2. Single strand nicking

Binding site

1. Topoisomerase binding

2. Single strand nicking

Topo l O-Tyr

Topo l O-Tyr

n coils

Cleavable complex fS

Rotation of free 5' end n-1 coils

Topo l

1. Religation

2. Enzyme dissociation Tyr—O 3

n-1 coils

FIGURE 7.9 Mechanism of DNA unwinding by topoisomerase I.

Topoisomerase I acts by making a transient break (nick) of a single strand of DNA, catalyzing the passage of DNA strands through one another and allowing release of the superhelical tension. Topoisomerase I enzymes have been sub-divided into type IA and type IB sub-families based on their reaction mechanism. Type I topoisomerases of the type IA sub-family form covalent linkages to the 5' end of the DNA break, while type IB sub-family enzymes form covalent linkages to the 3' end of DNA break (Fig. 7.9). Eukaryotic DNA topoisomerase I is attached to the 3' DNA end of the break site, and is therefore a type IB topoisomerase. This enzyme is located in areas of active RNA transcription to release superhelical stress generated during mRNA synthesis.

4.2. Topoisomerase II mechanism

Eukaryotic topoisomerase II is a homodimeric enzyme that requires ATP to function. It makes a transient double strand break, where the tyrosines from the active sites of both monomers attack the phosphodiester bond to the 5'-side of the phosphate, leading to a covalent 5'-phosphotyrosyl linkage in each strand

After binding of the enzyme to DNA, a double strand DNA break is produced by nucleophilic attack of both tyrosine residues. These breaks between the strands are not directly opposite to each other; instead, they are separated by a four base pair overhang, generating a space through which another region of intact DNA can be passed. The final steps involve religation of the DNA break, dissociation, and release of DNA from the topoisomerase (Fig. 7.11). Several of these steps require the binding and hydrolysis of ATP.

The catalytic cycle of topoisomerase II is complex and is summarized in Fig. 7.12, together with the names of some drugs that have steps of this cycle as targets.42 The enzyme assumes two different conformations, resembling an open

Base

Topoisomerase Test Condition
DNA

Base

FIGURE 7.10 Transesterification reactions involved in topoisomerase II (topo II) activity.

n coils

Topo II

I c) Strand passage d) Religation e) Enzyme dissociation 5, 3'

FIGURE 7.11 Mechanism of DNA unwinding by topoisomerase II.

I c) Strand passage

Topoisomerase
FIGURE 7.12 Catalytic cyclic of topoisomerase II and its main inhibitors.

clamp in the absence of ATP and a closed clamp in the presence of ATP. The open conformation can bind two segments of DNA, forming the pre-cleavage complex. One of these segments will be nicked by the enzyme (G segment) and another that will be transported (T segment). Afterwards, two ATP molecules are bound, leading to the dimerization of the ATPase domains and hence to a conformational change from the open- to the closed-clamp structure. The nucleophilic reactions that break both strands of the G segment of DNA then take place, generating the post-cleavage complex. This allows the passage of the T segment through the gap thus produced, which requires the hydrolysis of one molecule of ATP. The broken ends of the G segment are then ligated and the remaining ATP molecule is hydrolyzed. Upon dissociation of the two ADP molecules from ATP hydrolysis, the T segment is transported through the opening at the C-terminal part of the enzyme, which is then closed. Finally, the enzyme returns to the open clamp conformation, liberating the G segment.

Some antitumor drugs acting at the topoisomerase level have inhibition of enzymatic activity as their primary mode of action, and are known as 'catalytic topoisomerase inhibitors'.43-45 Other drugs targeting the topoisomerases, including intercalating drugs, interfere with the enzyme's cleavage and rejoining activities by trapping the cleavable complex and thereby increasing the half-life of the transient topoisomerase catalyzed DNA break. Some of the most clinically useful anticancer drugs are of the latter type and are normally referred to as 'topoisomerase poisons' because they convert the topoisomerase enzyme into a DNA-damaging agent.

Because the level and time-course of expression of these enzymes vary in different cell types, and the development of resistance to one type of inhibitor is often accompanied by a concomitant rise in the level of the other enzyme, there is an increasing interest in drugs that can act as dual topoisomerase I and II poi-sons.46,47 Finally, it is important to mention that topoisomerase inhibitors are among the most efficient inducers of apoptosis.48

5. TOPOISOMERASE II POISONS

This class of topoisomerase II inhibitors act by trapping the G strand enzyme intermediate, thus blocking religation and enzyme release and leaving the DNA with a permanent double strand break. Besides the compounds discussed here, many intercalating agents previously mentioned in Section 2 have this property.

5.1. Acridine derivatives

The intercalation concept was first introduced to explain the non-covalent binding of some acridine derivatives to DNA. Interest in these intercalators has led to the development of amsacrine (mAMSA), a drug used in the treatment of malignant lymphomas and acute non-lymphocytic leukaemia.49,50 The main mechanism of action of mAMSA is the formation of a ternary complex with DNA and topoisomerase II, trapping the cleavable complex, and inhibiting the religation step.51 Besides amsacrine, a large number of natural and synthetic acridines have been tested as anticancer agents and, so far, a few molecules have entered clinical trials and have been approved for chemotherapy.52 For instance, asulacrine is a close analogue with a broader spectrum of activity in experimental tumors but without improved clinical antitumor activity. DACA (XR5000) is an acridinecarboxamide and a mixed topoisomerase I and II poison that has undergone extensive clinical trials.53 Hybrid compounds have also been designed which combine the acridine intercalating moiety with other groups that provide secondary interactions with the DNA minor groove.

h3co

Amsacrine

Amsacrine

H3CO

H3CO

CH3 CO-NH-CH3 Asulacrine

NHSO2CH3

CH3 CO-NH-CH3 Asulacrine

DACA

DACA

The pyrazoloacridone KW-2170 is a topoisomerase II inhibitor of synthetic origin that has entered Phase II clinical trials.54 On the other hand, the related pyrazoloacridine PD-115934 is a dual topoisomerase I and II inhibitor, and has also entered Phase II clinical trials.55

H3CO

H3CO

PD-115934

PD-115934

.CH3

5.2. Anthracyclines and related compounds

The anthracycline antibiotics, which were studied in Section 3 of Chapter 4, also intercalate with DNA. The tetracyclic A-D chromophore of these compounds is oriented with its long axis perpendicular to the long axis of adjacent base pairs at the intercalation site. The daunorubicin-DNA complex is stabilized by the stacking interactions of rings B and C and by hydrogen bonding involving the hydroxyl group at C-9 of ring A, which acts as a donor to N-3 of guanine and as an acceptor from the amino group of the same guanine. Ring D protrudes into the major groove and the amino sugar moiety lies in the minor groove and does not take part in the interaction with DNA, although it is crucial for antitumor activity (see below).

As other antitumor intercalating agents, anthracyclines are topoisomerase II poisons because of the formation of a stable drug-DNA-topoisomerase II ternary complex and consequent inhibition of replication and transcription. The sugar unit is crucial for the stabilization of this complex, and suppression of the C-4 methoxy and C-3' amino groups increases topoisomerase II inhibition.56 The formation of topoisomerase-mediated DNA breaks seem to be too modest to explain the activity of the anthracyclines unless other mechanisms are taken into account (see Chapter 4), but some of these mechanisms are enhanced by anthracycline intercalation and minor groove binding; for instance, intercalation is known to favour DNA propenylation by malondialdehyde.57

In the case of nogalamycin, the presence of two sugar residues at both ends of the chromophore leads to a special way of interaction with DNA, called threading intercalation58 in which one of the sugar units is located at the minor groove and the other at the major groove. The structure of the nogalamycin-DNA complex has been studied by X-ray diffraction.59

Other anthracyclines that act primarily as topoisomerase II catalytic inhibitors, such as aclarubicin, will be mentioned in Section 6.1.

O OH OH

(CH3)2N

i nh2

CH3 OH

HHC3OC4^

3 H3CO OCH3

Nogalamycin

CH3 OH

R = OH Doxorubicin R = H Daunorubicin (daunomycin)

HHC3OC4^

3 H3CO OCH3

Nogalamycin

Mitoxantrone is a simplified analogue of the anthracyclines which has also been discussed in Chapter 4. It has a complex mechanism of action that includes generation of a stable drug-DNA-topoisomerase II ternary complex. Isosteric substitution of one or more carbons of the benzene rings by nitrogen atoms has been employed as a strategy for the design of mitoxantrone analogues with geometries similar to those of the parent compounds, but with increased affinity for DNA due to the presence of sites suitable for hydrogen bonding or ionic interactions. This increased affinity allows the suppression of the phenolic hydro-xyls of mitoxantrone, which are responsible for its chelating properties and therefore for its cardiotoxicity through oxygen radicals generated through Fenton chemistry. Based on this idea, some aza-bioisosters related to the anthracene-9, 10-diones have been synthesized and screened in vitro and in vivo against a wide spectrum of tumor cell lines.60,61

Among these compounds, pixantrone has a high level of activity in blood-related tumors and is currently being studied in Phase III trials for the treatment of non-Hodgkin's lymphoma.62,63 Interestingly, pixantrone was curative in some models of lymphoma and leukaemia where currently marketed anthracyclines only prolonged survival, but it showed no measurable cardiotoxicity compared to them at equi-effective doses in animal models.64 Another potential application of this drug is as an immunosuppressant in multiple sclerosis patients.65

The mechanism of action of pixantrone involves intercalation with DNA and interaction with topoisomerase II, causing breaks in DNA strands.66

HO O HN

HO O HN

Mitoxantrone

Mitoxantrone

O HN

O HN

Pixantrone

O HNV

Pixantrone

5.3. Non-intercalating topoisomerase II poisons 5.3.1. Etoposide and its analogues

Podophyllin resin derivatives have been used as folk medicines for centuries, its main active ingredient being podophyllotoxin. In the 1950s, a search began to identify a more effective podophyllotoxin derivative67 that eventually resulted in the development of a new class of anti-neoplastic agents which target topoisomerase II. The most important compounds are etoposide and teni-poside, two semi-synthetic derivatives of 4-epipodophyllotoxin. Etoposide (VP-16)68 is used mainly to treat testicular cancer which does not respond to other treatment and as a first-line treatment for small cell lung cancers. It is also used to treat chorionic carcinomas, Kaposi's sarcoma, lymphomas, and malignant melanomas. A phosphate pro-drug of etoposide (etopophos) has been used for ADEPT therapy and will be discussed in Section 3 of Chapter 11. Teniposide is used less frequently, especially to treat lymphomas.

rho^ "oho h3co y och och3

Podophyllotoxin

OCH3

R = CH3 Etoposide R = 2-Thienyl Teniposide

OCH3

R = CH3 Etoposide R = 2-Thienyl Teniposide

Etoposide and teniposide activity is cell cycle dependent and phase specific, with maximum effect on the S and G2 phases of cell division. They cause DNA damage through inhibition of topoisomerase II, and their mechanism of action has been studied specially for the case of etoposide. DNA religation inhibition by this compound seems to be due to inhibition of the release of ADP from the hydrolysis of ATP69 and to its activation through oxidation-reduction reactions to produce derivatives that bind directly to DNA. It has been shown that the O-demethylated metabolite of etoposide 7.10 has the same potency as the parent drug. This etoposide catechol is subsequently oxidized to an ortho-quinone metabolite 7.12 which is also a potent inhibitor of the topoisomerase II-DNA cleavable complex.70 It has been proposed that the presence of free radical intermediates such as semi-quinone 7.11 contribute to DNA strand breakage, which seems to be supported by the fact that the 4'-OH group of etoposide is essential for its activity as shown by the inactivity of its 4'-OMe derivative. On the other hand, etoposide is a substrate of myeloperoxidase, an enzyme with tyrosinase activity that catalyses a one-electron oxidation to form the phenoxyl radical 7.13 (Fig. 7.13). However, the formation of radicals 7.11 and 7.13 has been proposed to be related to the increased risk of secondary myeloid acute leukaemia induced by long-term etoposide treatment.71,72

Other interesting epipodophyllotoxins under clinical assay are TOP-53 and tafluposide. TOP-53,73 which bears a basic aminoalkyl side chain that improves its solubility while allowing its association with phosphatidylserine resulting in selective accumulation in lung and is in Phase I trials. The phosphate pro-drug tafluposide74 is a lipophilic perfluorinated epipodophyllotoxin that has a dual topoisomerase I and II inhibitory activity, has shown high in vivo activity and has entered Phase I clinical trials for solid tumours.

OCH3

H3CO

OH TOP-53

N(CH3)2

H3CO

OCH3

OH TOP-53

Tafluposide

h3co" "och3

Tafluposide

5.3.2. Salvicine

Salvicine is a semi-synthetic diterpenoid quinone compound obtained by structural modification of a natural lead isolated from the Chinese medicinal herb Salvia prionitis. This compound is a non-intercalative topoisomerase II poison with a potent, broad spectrum in vitro and in vivo antitumor activity, and is currently in Phase II clinical trials. Salvicine is also an inhibitor of several resistance mechanisms (see Chapter 12), including multi-drug resistance (MDR) by down-regulating the expression of MDR-1 mRNA via the activation of c-jun, and DNA repair by the DNA protein kinase (DNA-PK) enzyme.

CH3 HO' S-"CH3 Salvicine Oh

CYP3A4

H3Ca QCH3 QH

Etoposide

CYP3A4

H3Ca QCH3 QH

Etoposide

QH 7.10

7.11

Myeloperoxidase

H3CH0

H3CH0

ROS generation DNA and protein damage

FIGURE 7.13 Reactive species generated in etoposide metabolism.

Salvicine inhibits the catalytic activity of topoisomerase II with weak DNA cleavage action in contrast to the classic topoisomerase II poison etoposide. It stabilizes DNA strand breaks through interactions with the enzyme by trapping the DNA-topoisomerase II complex.75 Molecular modelling studies predicted that salvicine binds to the ATP pocket in the ATPase domain and superimposes on the phosphate and ribose groups, while competition with ATP was confirmed experimentally.76

H3C0

6. TOPOISOMERASE II CATALYTIC INHIBITORS

This group of topoisomerase II inhibitors differ from topoisomerase poisons in that they do not stabilize the cleavable complex, but act on other steps of the catalytic cycle. Catalytic inhibitors and topoisomerase II poisons can exert synergic or antagonistic effects, depending on the treatment schedule. When cells are treated with high concentrations of drugs for short periods of time, competition is observed between both types of inhibitors because all available enzyme molecules are occupied by one of them, which brings about competition for the other. On the other hand, synergistic effects are observed after continuous exposure of cells to low concentrations of both types of inhibitors because under these conditions not all the available enzyme molecules are occupied by one of the drugs, and some of them are therefore available to the other. These results resemble those observed under clinical conditions and for this reason additive or synergistic effects are normally observed for both types of inhibitors under clinical settings. The therapeutic use of catalytic topoisomerase II inhibitors as anticancer agents is limited to aclarubicin and sobuzoxane.44

6.1. Inhibitors of the binding of topoisomerase II to DNA: Aclarubicin

Although most anthracyclines act as specific topoisomerase II poisons, some of them, such as aclarubicin (aclacinomycin A), can act by different mechanisms. This drug is clinically used in the treatment of acute myelocytic leukaemia and it behaves as a strong intercalating agent that prevents the binding of topoisomerase II to DNA and hence as a topoisomerase II catalytic inhibitor. Subsequent studies have shown that aclarubicin is also a topoisomerase I inhibitor at biologically relevant concentrations.77

CO2CH3 OH

CO2CH3 OH

HC^O

Aclarubicin (aclacinomycin A)

6.2. Merbarone

Merbarone is a derivative of thiobarbituric acid that was discovered in the course of a study of a large number of barbituric acid analogues by the NCI. This compound has been shown to inhibit the induction of DNA-topoisomerase II cleavable complexes and has been tested clinically against a large number of tumors,78 although it showed nephrotoxicity and poor anticancer activity.

Merbarone

Merbarone

6.3. Bis(dioxopiperazines)

As mentioned in Section 3 of Chapter 4, this class of drugs were introduced as chelating agents, since they behave as pro-drugs to EDTA amides, and are useful as cardioprotectors when associated with anthracyclines. They have subsequently been shown to inhibit topoisomerase II at a point upstream to the formation of the cleavable DNA-enzyme complex by stabilizing the closed-clamp form of topoisomerase II as a post-passage complex. This is achieved by inhibiting the ATPase activity of the enzyme after interaction with its N-terminal domain. The main bis(dioxopiperazines)79 are shown below.

Compound

R

R'

ICRF-154

H

H

ICRF-159

H

CH,

(dexrazoxane)

ICRF-193

CH3

Sobuzoxane (MST-16)

7. SPECIFIC TOPOISOMERASE I INHIBITORS

Compounds that inhibit topoisomerase I can be divided into following two categories:80

a. Topoisomerase I suppressors, which are those compounds that inhibit the enzyme but do not stabilize the intermediate DNA-topoisomerase I covalent complex.

b. Topoisomerase I poisons, which act after DNA cleavage by inhibiting religa-tion. This can be achieved through three different mechanisms, involving (1) binding of the enzyme to the previously formed drug-DNA binary complex, (2) recognition of the enzyme-DNA binary complex by the drug, and (3) interaction of DNA with the drug-enzyme complex.81

15A^0

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Responses

  • Hattie
    Do topoisomerase inhibitors preventing release of supercoiling and separation of the DNA strands?
    2 months ago
  • Mustafa
    How does dna intercalation cause strand break?
    13 days ago

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