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7 Platinum Anti-Cancer Drugs 7.1 History of cis-platin

H3N

Pt

H3N

Cl

Cl

cis-platin

cis-diamminedichloroplatinum(II)

1825 Peyrone: first synthesis

1893 Werner: structural characterisation

1965 Rosenberg: Pt electrodes affect bacterial growth

1969 Shown to be active against tumours

1971 Enters clinical trials

1978 Approved for clinical use (USA)

Very successful for testicular and ovarian cancer (>95 % survival rate). Also used for head and neck, bladder, lung and cervical cancers and lymphoma, melanoma and osterosareoma.

7.2 Structure-reactivity relationships

These are a set of loose „rules‟ for anti-cancer activity set out by Cleare and Hoeschele in 1973 (Platinum Metal Rev, 1973, 17, 187). To date all compounds entering clinical trials conform to these rules. However, recently „rule breakers‟ which show some anti-cancer activity have been found (see later).

Platinum drug (PtL2X2) anti-cancer activity requires:

1) cis leaving groups

2) Electrically neutral complex with Pt(II) or Pt(IV) centre

3) Leaving groups should be moderately strongly bound (usually anionic) Highly labile => toxic Too tightly bound => less active

4) Non-leaving group is crucial: amine groups with at least one N-H

The anti-cancer activity is usually tested first in vitro on mouse leukaemia cell lines before testing on animals and then several stages of clinical trials. It has recently been estimated that only 1 in 10,000 screened drugs actually makes it to final approved clinical use.

7.3 Aquation of cis-platin

H3N

Pt

H3N

Cl

Cl

H2O

Cl-

H3N

Pt

H3N

Cl

OH2

+H2O

Cl-

H3N

Pt

H3N

OH2

OH2

2+

40

195Pt and 15N NMR studies have shown the mono-aqua complex to be the active species. The positive charge on this complex means it is attracted to the negative surface of DNA. The 2-3h delay in sensitization after administration of cis-platin is due to the slow formation of this complex.

7.4 Biological targets of cis-platin

Many different cellular components can react with cis-platin, however Pt-DNA binding has been the main focus of studies since:

Diseases where DNA repair processes are deficient are hypersensitive to cis-platin.

Correlations have been shown between Pt-DNA adducts in peripheral blood cells and disease response in cis-platin patients.

Treatment of HeLa cells with high dose of 195mPt radiolabelled cis-platin shows: 1 Pt per 104/105 proteins 1 Pt per 10-1000 RNA 1 Pt per 1 DNA

7.5 Pt binding to DNA

Pt binding to mononucleotides

Theoretical and experimental studies show Guanine N7 (imidazole) to be the most electron rich centre.

G(N7) > A(N7) [ > A(N1), C(N3) - used in base pairing in DNA]

NH

NN

N

O

NH2

DNA

Pt

Cl

H3NN

H H

H

The Pt-G(N7) bond is very stable and can only be broken by a strong nucleophile (cyanide, thiourea etc.). Possibly H-bonding stabilises the adduct too.

This adduct can be readily detected using 1H NMR spectroscopy:

Guanine C8-H δ 7.8 singlet G(N7)-Pt δ 8.8 singlet + 195Pt satellites

This large shift means 1H NMR is a very useful tool in more complex systems along with X-ray crystallography and NMR NOE data.

Plasma [Cl- concentration ≈ 100 mM] very little hydration

cytoplasm [Cl- concentration ≈ 4 mM] slow exchange of Cl- ligands occurs

cell membrane [probably crossed via passive diffusion]

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Pt binding to oligonucleotides

Oligonucleotides are short duplexes (2 to 15 nucleotides long) used for model studies. Cis-platin was found to bind (using X-ray crystallography, 1H,1H NOE NMR and molecular modelling) almost exclusively at the G(N7)-p-G(N7) sites.

X-ray crystal structure of d(CCTCTG*G*TCTCC)· NMR solution structure d(CCTCTG*G*TCTCC)·

d(GGAGACCAGAGG) containing a cis-GG adduct d(GGAGACCAGAGG) containing a cis-GG adduct

NMR solution structure of d(CTCTAG*TG*CTCAC)· NMR solution structure of d(CATAG*CTATG)· d(GTGAGCACTAGAG) containing a cis-GTG adduct d(CATAG*CTATG) containing a cisplatin interstrand cross-link

(all figures from Chem. Rev., 1999, 99, 2498)

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Pt binding to DNA

A typical experiment for studying cis-platin binding to DNA involves:

1. in-vitro incubation of DNA and cis-platin 2. extraction of DNA from cells 3. digestion of the DNA (enzymes) 4. separation (HPLC) 5. characterisation (1H NMR)

60-65% cis-[Pt(NH3)2(GpG)]

3' p G p G p 5'

5' p C p C p 3'

Pt

H3N NH3

G,G 1,2-intrastrand

20-25% cis-[Pt(NH3)2(ApG)]

3' p A p G p 5'

5' p T p C p 3'

Pt

H3N NH3

G,A 1,2-intrastrand

5-10% cis-[Pt(NH3)2(GMP)2]

3' p G p N p G

5' p C p N p C

Pt

H3N NH3

p

p

5'

3'

3' p G p C p 5'

5' p C p G p 3'

Pt

H3N NH3

1,3-intrastrand interstrand

2-5% cis-[Pt(NH3)2(GMP)(OH)]

3' p G p 5'

5' p C p 3'

PtH3N OH

NH3

monoadduct (time dependant)

< 1% (whole cell studies only) cis-[Pt(NH3)2(GMP)(protein)]

3' p G p 5'

5' p C p 3'

PtH3N protein

NH3

DNA-protein binding (can lead to side effects)

43

It is thought that the 1,2-intrastrand crosslinks are important to anti-cancer activity since:

1. they are the major adducts formed 2. clinically inactive compounds fail to form these cross-links (e.g.,

transplatin)

7.6 Mechanism of reaction of cis-platin with DNA

Kinetics are very important. Aquation of the cis-platin is the rate limiting step. Reaction of the cationic Pt complex with the DNA strand is fast.

Hydrogen bonding from NH3 and OH2 ligands to the phosphate backbone of DNA are possibly important in orientating the Pt complex.

7.7 Damage to DNA on cis-platin binding

Structure shows:

Two Guanine groups in “head to head” configuration

Dihedral angle between rings of approx. 80° (disruption of base stacking)

17 membered ring

H-bonding from NH3 to 5‟-phosphate group

X-ray crystal structure of cis-[Pt(NH3)2{d(pGpG)}]

44

Studies of cis-platin binding to oligonucloetides (see Section 3.5) show that different adducts distort the DNA in different ways. The main observed effects of 1,2-intrastrand cross-links are:

Bending towards major groove

G

Pt GH3N

NH3

40o

Unwinding of duplex (~20°)

Widened shallow minor groove

Distortion of Watson-Crick base-pairing

Duplex destabilization: lower mpt; calorimetric experiments show ~6.37 kcal mol-1 destabilization

All this blocks replication and inhibits transcription. Replication stops at sites corresponding to one nucleotide preceding the first Pt-G residue and at positions opposite the two Pt-G residues.

Gel electrophoresis data obtained during a kinetic study of the effect of a cis-GG adduct on DNA polymerization by HIV-1

reverse transcriptase. Panel A shows fragments generated by enzymatic replication of a 44-bp DNA duplex containing a site-specific cis-{Pt(NH3)2}

2+ cross-link at G(24)/G(25). Polymerization is blocked by platination of the substrate. Panel B depicts

results for an unmodified DNA probe. Image from Biochemistry 1999, 38, 715.

Also some evidence suggests cis-platin induces cells to undergo apoptosis (programmed cell death).

DNA is continually checked for „errors‟ in its sequence by various proteins. These proteins can then activate enzymes to eradicate the errors. Studies show that cells deficient in DNA repair are much more sensitive to cis-platin.

45

How do Pt 1,2-intrastrand cross-links inhibit repair?

Hypothesis: binding of a HMG (High Mobility Group) protein with the 1,2-intrastrand cisplatin–DNA complex shields the DNA from intracellular repair leading to apoptosis (cell death). This binding occurs by means of HMG inserting a phenyl group protruding from its backbone into the notch created when cis-platin forms a complex with DNA. This also increases the bend in the DNA to 60-90°.

7.8 Biotransformation of cis-platin

Platinum is able to bind to sulphur atoms (strong soft-soft interactions). Many biomolecules contain sulphur centres which are potential coordination sites for platinum.

In cells

Intracellular thiols include glutathione (GSH). GSH is present in all cells, typically in concentrations of 3-10 mM.

HO2C CH

NH2

CH2 CH2 C

O

NH CH

CH2

SH

C

O

NH CH2 CO2H glu-cys-gly

GSH binds to Pt to give a high M.W. polymer with a Pt:GSH ratio of 1:2

GSH-Pt binding results in depletion of Pt from circulation

The GS-X pump can pump out GS-Pt from tumour cells

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In kidneys

Metallothionen (MT) is a low-molecular-weight protein (MW ~6-7k) which in which one-third of the residues are cysteines. These cysteine residues bind and store metal ions and are used by the kidneys for detoxification of heavy metals.

MT reacts with cis-platin to give Pt7-10MT containing PtS4 units

7.9 Why is trans-platin inactive?

H3N

Pt

H3N

Cl

Cl

H3N

Pt

Cl

Cl

NH3

cis-platin trans-platin

~2.8Å ~4.0Å

The longer distance between the two Cl leaving groups results in trans-platin being unable to form 1,2-intrastrand DNA adducts similar to cis-platin. However, trans-platin can form 1,3-intrastrand (GpNpG), 1,4-intrastrand and interstrand links as well as monoadducts.

Structure of trans-[Pt(NH3)2{d(A*pGpG*CpCpT)}]

The lack of anti-cancer activity for trans-platin is due to:

1. Trans-platin lesions are more easily repaired since they lead to more radical distortion of DNA. They don‟t bind HMG proteins as strongly as cis-platin lesions, possibly due to the lack of an appropriate space for intercalation of the HMG protein phenyl group.

2. Trans-platin is more readily intercepted by sulphur-containing species (e.g., glutathione) leading to removal of Pt from the cancer cells.

Cl Pt

NH3

NH3

ClGSH

GS Pt

NH3

NH3

Cl

Cl > NH3 in the trans effect so cis-platin is less susceptible to this reaction.

23-membered ring

47

3. Trans-platin monoadducts are more easily displaced by the action of trans-labilizing nucleophiles such as glutathione or thiourea.

N7 Pt

NH3

NH3

Cl+ SC(NH2)2

-Cl-N7 Pt

NH3

NH3

SC(NH2)2+ SC(NH2)2- N7

(H2N)2CS Pt

NH3

NH3

SC(NH2)2

7.10 Side effects of cis-platin

Short Term Emetogensis – severe vomiting and nausea Hair loss Ototoxicty – hearing loss / tinnitus Myelosupression – low white blood cell count Nephrotoxicity – kidney damage Neurotoxicity - depression

Chronic Effects Kidney damage Hearing impaired Peripheral neuropathy – loss of feeling in limbs Psycho-sexual difficulties

These side effects are associated with DNA and protein bound platinum (similar to other heavy metal poisoning). This limits the maximum dose of cis-platin to ~100mg per day for up to 5 consecutive days.

The toxic side-effects can be partially controlled by inhibiting the formation of Pt-protein complexes or by „rescuing‟ Pt from these Pt-protein complexes:

RS-, RSH complexation is inhibited by high Cl- concentration. Hence cis-platin is administered in a saline drip. This reduces kidney damage dramatically.

Rescue agents such as:

Et2N C

S- Na+

S

(HOCH2CH2)N C

S- Na+

S

can be administered 3-4 hours after cis-platin treatment. These agents displace Pt from sulphur containing biomolecules but do not affect Pt-DNA complexes.

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7.11 Resistance to cis-platin

Some tumours have natural resistance to cis-platin whilst others develop resistance after initial treatment.

There are three major mechanisms of resistance:

A Decreased intracellular transport

B Increase interception by thiol rich species

C Improved repair

Overcome resistance by:

1. Develop new Pt drugs (see later)

2. Use higher dosage of Pt drugs

3. Use combination chemotherapy with other active anti-tumour drugs (e.g., Taxol)

4. Use in combination with other pharmacological agents capable of modulating know mechanisms of resistance to cis-platin

7.12 Second generation drugs

Carboplatin

H3N

Pt

O

H3N O

O

O

Carboplatin was the first of the second generation Pt anti-cancer drugs to be approved for worldwide clinical use (since 1987 in UK).

Carboplatin is less toxic than cis-platin (although myelosuppression can still be problematic). This means higher doses can be used (up to 2000 mg per day) and outpatient treatment is possible.

The lower toxicity is mainly due to the added stability of carboplatin in the bloodstream, which prevents proteins from binding to it.

Carboplatin is less potent that cis-platin and approximately 4x the dose is required compared to cis-platin for similar effectiveness. In addition, it is approximately 10x as expensive.

Similar to cis-platin, carboplatin is administered via intravenous infusion. It is thought to be only active against same range of tumours as cis-platin.

This is because carboplatin forms the same adducts with DNA as cis-platin does.

A

B

C

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Nedaplatin

H3N

Pt

O

H3N OO

Nedaplatin has similar clinic properties to carboplatin, and is predominately used in Japan for treatment of testicular, ovarian and cervical cancers.

Oxaliplatin

NH2

H2N

Pt

O

O

O

O

Oxaliplatin was recently approved for clinical use in 2004 under its trade name Eloxatin. It is used for the treatment of stage 3 colorectal cancers after surgery to remove the tumour. Eloxatin is administered (via an IV drip) in combination with 5-fluorouracil and leucovorin for increased effectiveness.

7.13 ‘Third generation’ Pt anti-cancer drugs

The next generation of anti-cancer drugs (many of which are currently undergoing clinical trials) have decreased toxicity, increased activity against cis-platin resistant cells and / or the ability to be administered orally.

This has been achieved by the design of a range of new platinum coordination compounds with decreased reactivity towards nucleophiles, better bio-distribution properties, different bonding modes to DNA and / or increased water solubility and stability.

(1) Sterically hindered Pt-complexes

N

Pt

Cl

NH3

ClH3C

Steric hindrance leads to decreased substitution reaction rates (slower attack from nucleophiles). Crucially, slower reaction rates with sulphur containing biomolecules leads to fewer side-effects and can potentially overcome type B resistance.

ZD0473 is currently undergoing clinical trials for lung and ovarian cancers. The 2-methyl group sits above the plane of the molecule leading to axial steric hindrance. ZD0473 has been observed to form 1,2-intrastrand links with DNA similar to those formed by cis-platin.

ZD0473

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(2) Pt(IV) Complexes

NH2

Pt

Cl

H3N Cl

OCCH3

OCCH3

O

O

NH2

H2N

Pt

Cl

Cl

Cl

Cl

Me2CHNH2

Me2CHNH2

Pt

Cl

Cl

OH

OH

Satraplatin (JM-216) Tetraplatin (Ormaplatin) Iproplatin

Pt(IV) centres are more inert to ligand substitution than Pt(II) centres. Therefore Pt(IV) compounds are less likely to be intercepted by biomolecules and have potentially fewer side-effects and are also active against some cis-platin resistant cells. Although some studies have shown that these complexes can form Pt(IV) adducts with DNA resulting in a large kinks in the DNA, it is perhaps more likely that they are slowly reduced to Pt(II) complexes by extracellular and intracellular agents and form the same adducts with DNA as shown by cis-platin.

Satraplatin, tetraplatin and iproplatin are all currently undergoing clinical trials. Satraplatin is an orally administered compound which is soluble in water and in lipids (OAc groups allow passage though the stomach lining). It is currently undergoing phase III clinical trials for prostrate cancer and some ovarian and lung cancers. (3) Complexes bearing a second DNA binding function

Cl

Pt

Cl

H2N NH

doxorubicinanilinoacrinide

R =

N

NH

R(CH2)n

Cl

Pt

Cl

H2N NH2

HO

By attaching DNA-intercalators such as doxorubicin or anilioacrinide to the active complex it is envisaged that these compounds will localise on the DNA even before aquation of the platinum centre occurs. In addition, these binding functions can stabilise the DNA adducts after Pt binding at guanine.

An alternative approach to achieve the same effect is the addition of hydrogen bonding groups to the complex, which will bind to the DNA phosphate backbone, or attachment of proteins or other molecules which are known to bind in the minor groove of B-DNA.

(4) Trans-Pt complexes

Pt

ClN

NCl

Pt

ClHN

HNClMe

MeO

Me

OMe

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Sterically hindered ligands are used to reduce the kinetic activity of trans-platinum complexes resulting in aquation rates approaching those of cis-platin. This overcomes many of the problems encountered with trans-platin being too easily incepted by other biomolecules (see Section 3.9).

It is envisaged that the different adducts formed by trans-Pt complexes compared to cis ones will result in activity against cells which show type C resistance.

(5) Multinuclear Pt complexes

Pt

NH3

Cl

H3N

NH2(CH2)6NH2

Pt

NH3

H3N NH2(CH2)6NH2

Pt

NH3

H3N Cl

4+

4 NO3-

Initially dinuclear Pt compounds, e.g., [ClPt(NH3)2NH2(CH2)nNH2Pt(NH3)2Cl]2+, were studied and found to be active. They were able to chelate two guanines at N7 forming a hair-pin structure with B-DNA. However these compounds turned out to be too toxic for clinical trials.

However, a trinuclear compound, BBR3464, is now in phase II clinical trials for melanoma, lung cancer and pancreatic cancers. It is very active against cis-platin resistant cell lines and in addition is 10x more potent than cis-platin. BBR3464 forms long-range interstrand and intrastrand crosslinks with B-DNA (up to 6 base pairs apart) leading to significant unwinding of the duplex.

7.14 Synthesis of Pt anti-tumour agents

Variation of the amine groups in cis-Pt(II) complexes

Cis-Pt complexes with two different amine groups can be prepared from cis-platin using the following procedure:

Pt

H3N

H3N

Cl

Cl

Et4NClPt

H3N

Cl

Cl

Cl

-

Pt

H3N

Cl

I

Cl

-

NaIPt

H3N

RH2N

I

Cl

RH2NPt

H3N

RH2N

Cl

Cl

1. AgNO3

2. HCl

BBR3464

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Variation of the leaving groups in cis-Pt(II) complexes (X = di-acid)

Pt(IV) complexes

PtR3N Cl

R3N Cl Cl2Pt

R3N Cl

R3N ClCl

Cl

PtR3N Cl

R3N Cl H2O2Pt

R3N Cl

R3N ClOH

OH

(CH3OCO)2O PtR3N Cl

R3N Cl

OC(O)CH3

OC(O)CH3