A

Rtpcjl

Guanine,

Mode of action of the aliphatic nitrogen mustards

Theoretically, this type of alkylation resembles a SN2 process since the ratecontrolling step is the bimolecular reaction between the cyclic aziridinium ion and the nucleophile which involves simultaneous bond formation and breakage. The preceding step, involving iminium ion formation, is a fast unimolecular process. In practice it is difficult to draw sharp distinctions between the contributions of SN1- and SN2-type mechanisms.

The aromatic nitrogen mustards were introduced in the 1950s and are milder alkylating agents. The aromatic ring acts as an electron-sink, withdrawing electrons from the nitrogen atom and thus discouraging aziridinium ion formation. These analogues are sufficiently deactivated that they can reach their DNA target sites before being degraded by reaction with other nucleophiles. This means that the aromatic mustards can be taken orally which is a significant advantage. During early Structure-Activity Relationship (SAR) studies it was found that direct attachment of a carboxyl group to the aromatic ring improved solubility but reduced activity due to the additional electron-withdrawing effect. The carboxyl group was thus electronically insulated from the aromatic ring by a number of methylene groups, three proving optimal, giving rise to chlorambucil (9.10) which is one of the slowest acting and least toxic nitrogen mustards, effective in chronic lymphocytic leukaemia, malignant lymphomas and carcinoma of the breast and ovary. Like mustine, it is often administered in conjunction with other drugs such as vinblastine and procarbazine which increase remission rates.

It has been proposed that the central nitrogen atom of an aromatic mustard is not sufficiently basic to form a cyclic aziridinium ion since the nitrogen electron pair is delocalised by interaction with the n electrons of the aromatic ring. Alkylation is therefore thought to proceed via an SN1 mechanism, with normal carbonium ion formation (resulting from chloride ion ejection) providing the rate-determining step.

M 7-GKianmc-DN A

Mode of action of aromatic mustards

Melphalan (9.9, phenylalanine mustard) was synthesised with a view to introducing a degree of selectivity based on the concept that attachment of an amino acid residue (phenylalanine) to the nitrogen mustard might facilitate selective uptake by tumour cells in which rapid protein synthesis occurs. Since melphalan prepared from D-phenylalanine is much less active than material prepared from the L-form, it has been postulated that melphalan may be conveyed into cells by the L-phenylalanine active transport mechanism. Although it not clear whether this is the case, melphalan is widely used in multiple myeloma, breast and ovarian carcinoma and in the rare condition of macroglobulinaemia.

A further attempt to produce more-selective mustards was based on the concept that some tumours are thought to possess high concentrations of phosporamidases. Cyclophosphamide (9.11) is the most successful mustard to result from this work and it has now been in use for over 20 years. The design of this mustard prodrug was based on the concept that the P=O group should decrease the availability of the nitrogen lone pair in an analogous manner to the phenyl ring of the aromatic mustards, thus deactivating the molecule to nucleophilic attack. It was postulated that the P=O group would be removed by phosphoramidases, thus releasing the nitrogen lone pair and restoring the electrophilicity of the molecule. Although shown to be a clinically-useful drug, it was later demonstrated that activation is not due to enzyme-catalysed hydrolysis of the P=O group but to oxidation by liver microsomal enzymes. After 4-hydroxylation, the molecule fragments

(9.11); cyclophosphamide (Endoxaca®)

(9.12); ifosfairiitie

(9.11); cyclophosphamide (Endoxaca®)

(9.12); ifosfairiitie

to give the highly electrophilic acrolein and phosphoramide mustard, although the production of normustine [HN(CH2CH2Cl)2] has also been observed.

Bifr-oiiidalkKi

Bifr-oiiidalkKi

Acrolein

Ptasphoramide musunJ (Reacts with MESNA)

Mechanism of action ofcyclophosphamide

Cylophosphamide has a wide spectrum of activity ranging from malignant lymphomas and lymphoblastic leukaemia to carcinomas of the bronchus, breast, ovary and various sarcomas. It can cause myelosuppression and haemorrhagic cystitis of the bladder which is thought to result from the excretion of electrophilic acrolein in the urine. This problem has been partly overcome by the co-administration of 2-mercaptoethanesulphonate (9.13, MESNA) which acts as a "sacrificial" nucleophile, forming a non-reactive water soluble adduct that is eliminated safely in the urine.

The slow rate of in vivo hydroxylation of cyclophosphamide in man has led to the synthesis of a 4-hydroperoxy derivative which spontaneously yields the 4-hydroxy metabolite after administration. Ifosfamide (9.12) is an analogue of cyclophosphamide but is not technically a nitrogen mustard due to the translocation of one chloroethyl moiety to another position within the molecule. Unlike cyclophosphamide, it can only be administered intravenously but it has a similar spectrum of activity.

Despite much research and an in-depth understanding of the chemistry of the nitrogen mustards, their precise mechanism of action at the molecular level is still unknown. It is clear that these molecules "staple" the two strands of DNA together via covalent interactions in the major groove, and it can be demonstrated that enzymes such as RNA polymerase are blocked by mustard-DNA adducts. It can also be shown from both electrophoresis-based experiments and molecular modelling that mustard adducts cause distortion of the DNA around the binding site which can be transmitted through a number of base-pairs (the "teleomeric" effect). Therefore, DNA processing may be affected at a point distant from the adduct site. Apart from the "kinetic" explanation for the antitumour activity of the mustards, an alternative view is that their GC-selectivity may be involved. For example, it is known that some of the gene sequences associated with Burkitt's lymphoma are particularly GC-rich, and that this disease is highly responsive to cyclophosphamide. This view has driven the design of mustard analogues such as tallimustine (a nitrogen mustard conjugated to a netropsin analogue) which has enhanced DNA sequence recognition properties. Tallimustine, which is presently in clinical trial, binds in the minor groove and spans five base pairs, recognising a 5'-GAAAT sequence.

In addition to MDR-related resistance, the effect of nitrogen mustards can be significantly reduced by an increase in concentration of glutathione in the cell. The highly nucleophilic glutathione forms adducts with mustards which are then no longer able to react with DNA. Cells also become resistant to nitrogen mustards by carrying out repair operations whereby the mustard adducts are excised and the damaged DNA resynthesized. Repair inhibitors that could be co-administered with mustards to enhance their clinical effectiveness are under active development.

Rather than form aziridinium ions as reactive intermediates, thiotepa (9.14) and related analogues have an aziridine ring already incorporated in their structure. Ring-opening of the aziridines with nucleophiles is slower compared with the fully-charged aziridinium ions of the mustards. However, depending upon the pKa of the aziridine nitrogen, there is likely to be significant protonation at physiological pH, meaning that, in practice, the aziridinium ion may be the reactive species.

Thiotepa itself has been used in the treatment of ovarian and breast carcinoma. It is injected intramuscularly, intravenously or directly into the tumour mass or administered by intrapleural or peritoneal infusion. A substituted benzoquinone ring has also been employed as an "anchor" for the aziridine groups. In the experimental agents AZQ (9.15) and BZQ (9.16), the aziridine moieties are deactivated by the withdrawal of electrons from the nitrogen into the quinone carbonyl groups via the ring. These molecules are employed as bioreductive prodrugs, as reduction of the quinone ring to either the semiquinone or the hydroxyquinone species reverses the electron flow and raises the pKa of the nitrogen. This allows activation of the aziridine rings to the corresponding aziridinium ions via protonation.

Busulphan (9.17, 1,4-di(methanesulphonyloxy)butane) is the best known example of an alkyl dimethanesulphonate with significant antitumour activity.

9.81.2 Aziridines

(9.14); ihioEepii

9.8.1.3 Methanesulphonates

These compounds are known to cross-link DNA; the methanesulphonyloxy moieties act as leaving groups after attack by nucleophilic sites on DNA. From a mechanistic viewpoint, the methanesulphonate groups should participate in an SN2-type alkylation

reaction. The mode of action has been investigated by SAR studies that have revealed that unsaturated analogues of known stereochemistry such as the corresponding butyne and trans-butene derivatives are inactive, whilst the cis-butene derivatives retain activity. It is thought that the activity of the cis-analogue and the more-flexible saturated busulphan depend on their ability to form a cyclic derivative by 1,4-bisalkylation of suitable nucleophilic groups. 1,4-di(7-guanyl)butane has been identified as a product of reaction between busulphan and DNA suggesting that this drug acts as an interstrand cross-linking agent in a similar manner to the nitrogen mustards. However, a study of the structure of urinary metabolites suggests that cysteine residues in certain proteins are also alkylated.

Busulphan causes significantly less nausea and vomiting than other DNA cross-linking agents and is thus more acceptable to patients. It is highly effective in chronic granulocytic leukaemia where it can keep patients almost symptom-free for long periods of time. Unfortunately, it does have a profound toxic effect on granulocytes and megakaryocytes which requires careful monitoring. Nitrogen mustard analogues have also been synthesised in which their P-chloroethyl groups have been replaced by sulphonate esters.

9.8.1.4 Triazeneimidazoles

Dacarbazine (9.18, 5-(3',3'-dimethyl-1-triazenyl)imidazole-4-carboxamide, DTIC) was one of several triazenes originally evaluated as potential inhibitors of purine biosynthesis. Although it was found to have a wide spectrum of activity ranging from malignant lymphomas to melanomas and sarcomas, it was later established that its mechanism of action is not associated with inhibition of purine biosynthesis. Instead it was demonstrated that demethylation occurs in vivo to afford the corresponding 5-amino derivative and a transient methyl diazonium ion which has been shown through radiolabeling experiments to methylate DNA at guanine N7 positions.

Since triazenes are liable to photochemical decomposition, the infusion bottle must be protected from light during intravenous administration. The irritant properties of the compound also preclude contact with skin and mucous membranes. Combinations of DTIC with adriamycin, bleomycin and vinblastine have also been employed.

OGNH,

OGNH,

9.81.5 Imidazotetrazinones

Temozolomide (9.19) is a relatively new alkylating agent that entered Phase II clinical trials in 1996 and will be licensed for use in melanoma and brain tumours. It is similar in its spectrum of activity and cross-resistance profile to the nitrosoureas, and also causes a dose-limiting bone marrow suppression. Temozolomide was developed from the experimental agent mitozolomide (9.20) and, like DTIC, acts as a source of CH3+ carbocations. This agent has a novel mechanism of action; after positioning itself in the major groove, nucleophilic attack of a water molecule on the lactam carbonyl initiates a degradation process that affords an amino imidazole derivative, N2 and CO2 in addition to the CH3+ ion. Formation of these smaller stable molecules presumably acts as a driving force for the degradation process.

It has now been established that the methyl carbocations generated methylate the N7-positions of guanine bases in the major groove. Furthermore, it is known that there is a preference to alkylate guanines occurring in the centre of runs of three or more guanine bases. It is possible that, as with the nitrogen mustards, the selectivity of temozolomide might be due to its ability to target guanine rich gene sequences associated with some tumours.

The nitrosoureas are known to alkylate DNA and cause both interstrand cross-links and monoadducts at a number of different sites. The screening of a large number of nitrosourea analogues established the structural unit for optimal activity as the 2-chloroethyl-N-nitrosoureido group. The most significant property of the nitrosoureas

{9.19): reiïiozoloriiide if, 20); mitozolomide

{9.19): reiïiozoloriiide if, 20); mitozolomide

9.8.1.6 Nitrosoureas is their activity towards cancer cells in the brain and cerebrospinal fluid, the so-called sanctuary sites. This is due to the relatively high lipophilicity of these molecules compared to other agents. Two examples of clinically-useful nitrosoureas are carmustine (9.21, A,W-bis(2-chloroethyl)-N-nitrosourea, BiCNU) which is administered intravenously and lomustine (9.22, N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea, CCNU) which is administered orally.

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