HCHsCH o

O^N Oi2CH3a

CH7CH2Q

Although these molecules possess chloroethyl fragments, their activity is not associated with aziridinium ion formation as in the mustards, since the corresponding nitrogen atom is part of a urea structure and so the electron pair on the nitrogen is not available to participate in a cyclisation reaction. Although the mechanism of action has not been firmly established, it is thought that the alkylation of nucleic acids proceeds via generation of a chloroethyl carbonium ion. The alkyl isocyanate fragment also formed is thought to carbamoylate the amino groups of proteins.

Mode of action of nitrosoureas

Although their lipophilic properties render them particularly suitable for treating intracerebral tumours, BiCNU and CCNU are also active in malignant lymphomas and carcinomas of the breast, bronchus and colon. Carcinoma of the GI tract, which is notably intractable to drug treatment, also responds to the nitrosoureas. Unfortunately, the nitrosoureas cause severe bone marrow toxicity which is usually dose-limiting.

The discovery of antitumour activity in the naturally occurring nitrosourea streptozotocin led to the synthesis of the P-chloroethyl analogue, chlorozotocin (9.23) which has significant antileukaemic activity but with reduced bone-marrow toxicity.

This initiated the design of other mono- and disaccharide analogues, whilst association of the 2-chloroethyl-N-nitrosoureidomethyl moiety with an aminopyridine ring has provided other experimental agents active against brain tumours.

0=N (9,33); chJorozcHodn

9.8.1.7 Metal complexes

Cisplatin is often quoted as one of the greatest successes in the development of chemotherapeutic agents. It has pronounced activity in testicular and ovarian cancers, however, as with many other clinically-useful drugs, it was discovered by serendipity rather than by design. In the 1960s, Rosenberg and co-workers observed that passing an alternating electric current through platinum electrodes in an electric cell containing E. coli led to arrest of cell division. The cause of the cytostatic effect was eventually traced to platinum complexes formed electrolytically at concentrations of only 10 parts per million in the presence of ammonium salts and light. cis-Diaminedichloroplatinum (9.24, cisplatin) was identified as one of the most active complexes, and the cis isomer was shown to possess significant antitumour activity. Cisplatin was introduced clinically in the UK in 1979.

Cisplatin has been shown to form intrastrand cross-links in the major groove of DNA with preferential interaction between (guanine N7)-(guanine N7), (guanine N7)-(adenine N7) (in both cases with the bases adjacent to one another) and (guanine N7)-(guanine N7) (with one base inbetween the alkylated guanines). Based on gel electrophoresis and NMR studies, these intrastrand cross-links are known to kink the DNA at adduct sites, a phenomenon that can now be observed directly by Atomic Force Microscopy. In some cisplatin-resistant cell lines the adducts are rapidly repaired, and it is thought that the DNA repair enzymes recognise the distortion around the adduct site. Interestingly, the configurational isomer, trans-platin has significantly less antitumour activity.

Clinical trials of cisplatin in combination with vinblastine have produced complete remission in 59% of patients with testicular cancer, and 30% remission in ovarian carcinoma. Other tumours which respond to cisplatin include squamous cell carcinoma of the head and neck, bladder carcinoma and refractory choriocarcinoma. Cisplatin suffers from dose-limiting adverse effects including leukopenia, extreme nausea, and

renal dysfunction at higher doses. Another disadvantage is the high cost of the drug due to the platinum content.

Carboplatin (9.25) is an analogue of cisplatin incorporating a cyclobutyl-substituted dilactone ring. It is better tolerated than cisplatin in terms of GI toxicity, nephrotoxicity and neurotoxicity. However, myelosuppression is more pronounced than with cisplatin. A new cisplatin analogue (JM 216) that can be administered orally is presently in clinical trials.

9.8.1.8 Carbinolamines

The carbinolamine group [-NR-CH(OH)-] is an electrophilic moiety found in a number of synthetic and naturally-occurring compounds known to interact with DNA. Trimelamol (9.26), which contains three carbinolamines, was developed from the clinically-active

hexamethylmelamine and pentamethylmelamine analogues which do not contain carbinolamines themselves but are converted during oxidative metabolism. Trimelamol was designed to have the carbinolamine moieties already in place. Phase-II trials carried out in the early 1990s indicated that trimelamol is active in refractory ovarian cancer, and is less emetic and neurotoxic than pentamethylmelamine. A number of different mechanisms of action have been suggested for trimelamol including DNA alkylation or the possibility that it can act as a nucleic acid antimetabolite. Although the precise mode of action has not yet been proven, trimelamol is known to be a reasonably potent DNA interstrand cross-linking agent although the exact sites of alkylation have not been well-defined. One of the drawbacks of trimelamol is its poor solubility in a number of physiologically compatible solvents making it difficult to formulate. Some analogues of trimelamol that partially overcome this problem have been developed and may enter clinical trials in the near future.

Anthramycin (9.27) is an example of a naturally occurring carbinolamine-containing compound that also interacts with DNA. These compounds, known as the pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), are perfectly shaped to fit the minor groove of DNA where they covalently attach to the N2-position of guanine. The PBDs are sequence-specific, recognising a purine-guanine-purine sequence and bonding to the central guanine. They have been shown to possess antitumour activity in the clinic although they suffer from side-effects including cardiotoxicity and bone-marrow suppression. The cardiotoxicity is thought to be due to the phenolic -OH group that can be converted to quinone species capable of producing free radicals with the potential to damage heart muscle. Other PBDs which lack a C9-phenolic group are not cardiotoxic. One of the most active analogues known as sibiromycin possesses a sugar moiety at C7 which is thought to enhance cellular uptake.

There is interest in developing an anthramycin analogue suitable for clinical trials, and recent synthetic studies have succeeded in joining two PBD molecules together to make novel minor-groove interstrand cross-linking agents that span up to seven base-pairs and recognise a unique purine-GATC-pyrimidine sequence. The design of these new cross-linking agents was based on the concept that new generations of alkylating and cross-linking agents should recognise longer sequences of DNA potentially leading to a higher selectivity for cancer cells coupled with reduced toxicity. These new cross-linking agents are unique in that they appear to cause negligible distortion of the DNA upon binding thus avoiding detection by the DNA-repair enzymes and potentially reducing the possibility of drug resistance.

9.8.1.9 Cyclopropanes

Adozelesin (9.28), carzelesin (9.29) and bizelesin are synthetic analogues of (+)CC-1065, an extremely cytotoxic antibiotic isolated from Streptomyces zelensis in 1974. It was

(9.28); adozdcsin
(9.29); eanielesin

shown to bind in the minor groove of DNA to 5'-Pu-N-T-T-A or 5'-A-A-A-A-A sequences (N=any other base), alkylating the N3-position of adenine (A italicised). As in adozelesin, the DNA-reactive part of (+)CC-1065 was shown to be a cyclopropane moiety attached to the para-position of a dihydroquinone. It was postulated and later established that attack of the cyclopropane ring by an adenine N3 is driven by the energy released in aromatising the dihydoquinone to a phenol.

Unfortunately, (+)CC-1065 is extremely toxic in vivo and has an unusual delayed lethality due to liver toxicity. Adozelesin, U77779 (bizelesin) and U80244 (carzelesin) were synthesised in an attempt to reduce this toxicity, and all three compounds have recently been studied in clinical trials. It is noteworthy that carzelesin is a prodrug; the cyclopropane-dihydroquinone system is generated in situ from the chloromethyl substituent after release of the phenolic -OH moiety through hydrolysis of the carbamate group. Although adozelesin and carzelesin are monoalkylating agents, bizelesin is a minor groove cross-linking agent.

9.8.1.10 Procarbazine

Procarbazine (9.30), N-(1-methylethyl)-4-[(2-methylhydrazino)methyl]benzamide, is a hydrazine derivative that was first synthesised as a mono-amine oxidase inhibitor. It was later shown to have significant activity in lymphomas and carcinoma of the bronchus.

Metabolic N-oxidation occurs to give azoprocarbazine, and it is believed that subsequent rearrangement produces either methyl diazonium or methyl radicals which act as DNA methylating agents towards guanine residues.

9.8.2 Intercalating agents

These agents are flat in shape, usually consisting of three or four fused aromatic rings. They work by inserting between the base pairs of DNA perpendicular to the axis of the helix. Once inserted, they are held in place by interactions including hydrogen bonding and van der Waal's forces. Intercalation can be detected in naked DNA by an increase in helix length which can be evaluated as an increase in viscosity using sedimentation values or as a change in mobility of DNA fragments in an electrophoresis gel. Some intercalators with arrays of functional groups at either end of the molecule protruding into both the minor and major grooves are known as "threading agents".

A number of different mechanisms of action have been ascribed to intercalating-type drugs. It can be demonstrated in the laboratory that some intercalators and threading agents block transcription and interfere with other DNA processing enzymes. Many intercalating agents have a preference for GC-rich sequences and, as with alkylating agents, this has been suggested to account for their selectivity. However, some intercalators are known to "trap" complexes between topoisomerase enzymes and DNA leading to strand cleavage. Others are known to chelate metal ions and produce DNA-cleaving free radicals, or to interact with cell membranes. Some examples of the different classes of intercalating agents are given below.

9.8.2.1 Anthracyclines

The anthracyclines (sometimes known as the anthraquinones) are a group of antitumour antibiotics first isolated from Streptomyces peucetius. They are the best known family of intercalating agents, consisting of a planar anthraquinone nucleus attached to an amino sugar. Doxorubicin (9.32, Adriamycin) is one of the most important anticancer drugs available because of its broad spectrum of activity. It plays a significant role in the treatment of solid tumours such as carcinoma of the breast, lung, thyroid and ovary, as well as soft-tissue carcinomas. Daunorubicin (9.31, Daunomycin) is an important agent in the treatment of acute lymphocytic and myelocytic leukaemias. Although doxorubicin and daunorubicin are both natural products, semi-synthetic analogues have also been developed including epirubicin (9.33), idarubicin and ametantrone.

Four mechanisms have been suggested for the mode of action of the anthracyclines, although there is still controversy about the relative importance of each one. Firstly, the planar ring system can be inserted perpendicular to the long axis of the double helix with the amino sugar appearing to confer stability on the adduct through hydrogen bonding

interactions with the sugar phosphate backbone. Intercalation is known to interfere with DNA processing and transcription, and can be regarded in some cases as a point mutation. Secondly, the anthracyclines are known to form complexes with topoisomerase enzymes leading to strand breaks. Thirdly, binding to cell membranes has been observed, and it has been suggested that this could alter membrane fluidity and ion transport, and disturb various biochemical equilibria in the cell. Lastly, generation of semiquinone species can lead to free radical or hydroxyl radical production (in a fashion similar to that proposed for mitomycin C, see Section 9.15.1) leading to DNA and cellular damage. Radical formation may be mediated by chelation of divalent cations such as calcium and ferrous ions by the phenolic and quinone functionalities, and is believed to be responsible for the cardiotoxicity observed with the anthracyclines.

9.8.2.2 Anthracenes

These compounds are based on the anthracene nucleus and have three rings rather than the four present in the anthracyclines. Mitoxantrone (9.34) and bisantrene (9.35) are examples of anthracenes that have been reasonably successful in the clinic. Both agents are used clinically for childhood and adult myelogenous leukaemia, non-Hodgkins lymphoma and breast cancer. One advantage of these drugs is that they have very low cardiotoxicity compared with the anthracyclines. Mitoxantrone binds to GC-rich sequences whereas bisantrene has little base-pair specificity. Like the anthracyclines, there is evidence that DNA is cleaved although the mechanism is not thought to be linked to the generation of reactive oxygen species.

(9J4); miuixiinlmne (Novanlmiu;®) (9J5); bisanLreni;

9.8.2.3 Phenoxazines

Dactinomycin (9.36), a chromopeptide antibiotic, was isolated from Streptomyces parvulus in the 1940s and used initially as a potent bacteriostatic agent although it was found to be far too toxic. The clinically-useful antitumour activity of dactinomycin was not observed until ten years later when it was tried with great success in the treatment of Wilm's tumour (a kidney tumour in children) and a type of uterine cancer.

The molecule consists of a tricyclic phenoxazin-3-one chromophore with two identical pentapeptide lactones attached. The mode of action of dactinomycin appears, to some extent, to be dependent upon its concentration, with either blockade of DNA synthesis occurring or inhibition of DNA-directed RNA synthesis preventing chain elongation. X-Ray crystallography studies have shown that the phenoxazone ring intercalates preferentially between GC base pairs where it can interact with the N2-amino groups. The peptide moieties position themselves in the minor groove and participate in extensive hydrogen bonding and hydrophobic interactions with functional groups in the floor and walls of the groove, thus providing significant stabilisation of the adduct and blockage of RNA polymerase. It also causes singlestrand DNA breaks in a similar manner to adriamycin, either through radical formation or by interaction with topoisomerase. Dactinomycin is used in combination with vincristine in the treatment of Wilm's tumour, and combined with methotrexate in the treatment of gestational choriocarcinoma. It is also used in some testicular sarcomas and in Kaposi's sarcoma, a tumour associated with AIDS patients. Tumour resistance to dactinomycin is believed to be due to both reduced uptake and active transport of the drug out of the tumour cells.

DNA topoisomerases are a family of enzymes responsible for the cleavage, annealing and topological state (e.g. supercoiling) of DNA. There are two types of topoisomerase enzymes, I and II. Topoisomerase I enzymes are capable of removing negative supercoils in DNA without leaving damaging nicks. They break one strand of DNA only, with the free phosphate residue becoming attached to a tyrosine residue on the enzyme. The complex then rotates, relieving the supercoiled tension of the DNA, and the two ends are then resealed. Topoisomerase II enzymes cleave double-stranded DNA, passing a complete duplex strand through the cut, followed by resealing of the original strands. The inhibition of these enzymes is believed to be involved in the mode of action of some intercalators such as the anthracyclines and anthracenes, although there has been controversy over whether the drugs bind to the enzyme prior to complex formation or after the enzyme-DNA complex has formed. Amsacrine (9.38) is an example of an acridine that is thought to work by topoisomerase inhibition. It is used in the therapy of myelogenous leukaemias, advanced ovarian carcinomas and lymphomas.

In addition to the anthracyclines, some new families of agents such as the ellipticines and camptothecins (see below) have been discovered that specifically inhibit topoisomerase.

(9-36), dactinomyrin (Cosmegari Lyovac®)

9.8.3 Topoisomerase inhibitors

9.8.3.1 Ellipticene

Ellipticine (9.37) is an example of a plant alkaloid that exerts its antitumour action through intercalation and inhibition of the topoisomerase II enzyme. In in vitro cytotoxicity studies, ellipticine is particularly active against nasopharyngeal carcinomas.

9.8.3.2 Camptothecin

Camptothecin (9.39) is a plant alkaloid with a unique five-ring system that exerts antitumour activity through inhibition of topoisomerase I.

9.8.3.3 Etoposide

Podophyllotoxin extracted from the American mandrake rhizome is another example of a natural product with anticancer activity. Etoposide (9.44) is a semisynthetic glucoside of epipodophyllotoxin, and is used clinically to treat small cell bronchial carcinoma for which it is claimed to be one of the most effective compounds known. Its mechanism of action involves inhibition of topoisomerase II.

Many other semisynthetic derivatives, such as podophyllic acid ethyl hydrazide, have been prepared and tested against selected tumours. Some of these appear to have a direct effect on DNA, inducing single-strand cleavage. Antitumour activity has been reported in some leukaemias and lymphomas, oat cell carcinoma of the bronchus and in malignant teratomas.

The bleomycins (9.40) are a group of closely related natural products that exert their antitumour activity by binding to DNA in a sequence selective manner followed by strand cleavage. The preparation know as Bleomycin sulphate consists of a mixture of the glycopeptide bases (e.g. A2, A2I, B1-4 etc.) with A2 as the predominant component.

9.8.4 DNA Cleaving Agents

9.8.4.1 The bleomycins

(9.40); General Slnidurc of the Ulcumyuns

The mixture is obtained from Streptomyces verticillus, and the individual molecular weights are in the region of 1300.

The bleomycins have attracted interest because they tend to accumulate in squamous cells and are therefore suitable for inclusion in regimens to treat tumours of this cell type in the head, neck and genitalia. Bleomycin sulphate has also been used in Hodgkin's disease and testicular carcinomas. It may be given intravenously, intramuscularly or subcutaneously. Unlike most anticancer drugs, it is only slightly myelosuppressive, and dose-limiting toxicity is confined to the skin, mucosa and lungs. Enzymes in most other tissues rapidly deactivate the bleomycins, probably as a result of deamination or peptidase activity. However, the bleomycins cause erythema, pain and hypertrophic changes in the skin in areas where there is a lot of keratin. As a result, ulceration in these areas and pigmentation of the nails may occur. In addition, pulmonary fibrosis occurs in 5%-l5% of patients. A carboxylic acid derivative, bleomycin acid, has been prepared and the substitution of various amino moieties in the molecule has enabled the synthesis of over 100 analogues. For example, Pepleomycin is a derivative with less tendency to cause pulmonary fibrosis. Despite the size and complexity of the molecule, particularly with regard to the number of chiral centres, the first total synthesis of bleomycin was reported in 1982.

Within the bleomycin molecule there are three distinct regions which are believed to contribute towards its mechanism of action. First, the heterocyclic bithiazole moiety (top right as drawn) is thought to intercalate with DNA. Electrostatic attraction of the

highly basic sulphonium ion ^2» R = NHtCH^ SMej)t0 ^g phosphate residues in DNA stabilise the adduct. Once bound, the second domain (top left) which consists of a P-hydroxyhistidine, a P-aminoalanine and a pyrimidine forms an iron (II) complex which interacts with oxygen to generate free radicals leading to single and double-

strand breaks. Currently, it is not clear whether the activation of this complex is self-initiating or the result of enzyme catalysis. The third region of bleomycin (bottom left) is glycopeptidic in nature and, while having no direct antitumour activity of its own, may contribute to either drug uptake by tumour cells or provide additional stabilising hydrogen bonding interactions with DNA or associated histone proteins.

9.8.4.2 The Enediynes

A new class of DNA-cleaving agent known as the enediynes is in the development stage. These compounds are based on natural products such as esperamycin and calicheamycin whose structures contain two triple bonds in close proximity. These molecules interact with DNA and then undergo a unique thiol-mediated cyclisation (the Bergman cyclisation) during which the triple bonds rearrange to form an aromatic ring. This process causes a proton to be abstracted from a sugar in the DNA backbone leading to strand cleavage. The basis for any tumour cell selectivity with the enediynes is not entirely clear, and it is likely that they will be developed mainly as "warheads" to be attached to other sequence-selective DNA-targeting agents.

9.9 ANTITUBULIN AGENTS

9.9.1 Vinca Alkaloids

Plants have traditionally been an invaluable source of medicinal compounds, including anticancer drugs. Following a screening program for agents with potential hypoglycaemic properties, two alkaloids, vinblastine and vincristine, which occur as minor constituents of the Madagascar periwinkle (Vinca rosea), were isolated and shown to reduce white blood cell count. These agents have subsequently played an important role in cancer chemotherapy.

The two Vinca alkaloids have complex but similar chemical structures. Vincristine (9.42) is more widely used than vinblastine (9.41) but the plant produces the latter in approximately 100-fold greater quantity. Fortunately, vinblastine may be converted to vincristine by a simple chemical step involving oxidation of the methyl to a formyl group. Furthermore, since these Vinca alkaloids have proved so useful in therapy, efforts

fl / OOOfcfc aip fl / OOOfcfc ft Rt aip

(9.41); vinblastine (Velbe®) Me OMe COMe {9.42); vincristine (Oncovin©) CHO OMc COMe (9.43); vindcsine (Eidcsine©) Me NH, H

have been directed towards the design of new analogues, and the synthesis of vinblastine itself was reported in 1979. Research has also been carried out into cell culture techniques and the use of immobilised plant enzymes as a means to produce the alkaloids more efficiently. Compounds representing the two halves of the structure of the dimeric Vinca molecules occur in much higher proportions in the plant extract, and the possibility of linking these at the appropriate positions and with the correct stereochemistry, has also become feasible.

The vinca alkaloids are cell-cycle-specific agents that block mitosis by metaphase arrest. Their cytotoxic effects appear to result from binding to the microtubules. These structures were first characterised in the cytoplasm over 20 years ago and are comprised of two main proteins, the a and p tubulins (Mr c 55 000), which form the microtubule scaffolding upon which many of the dynamic internal processes in living cells, including cell division, depend. Microtubules are long tubular structures of about 25 nm in diameter which form the major component of the mitotic spindle apparatus responsible for the movement of chromosomes during cell division. Binding of the Vinca alkaloids to the tubulins interferes with microtubule assembly causing damage to the mitotic spindle apparatus and preventing chromosomes from travelling out to form daughter cell nuclei. However, the basis for the tumour cell selectivity of these compounds is less clear.

Vinblastine sulphate is included on a weekly basis in several drug regimens for treating Hodgkin's disease, disseminated carcinoma of the breast, choriocarcinoma and testicular carcinoma. Vincristine sulphate, despite its similarity in structure to vinblastine, has a different spectrum of both antitumour activity and side-effects. Several drug combinations include vincristine for the treatment of acute lymphoblastic and myeloblastic leukaemias, Wilm's tumour, rhabdomyosarcoma, neuroblastoma, retinoblastoma, soft tissue sarcomas and disseminated cancer of the breast, testes, ovaries and cervix. Although neurotoxicity can be pronounced, a relatively low bone-marrow toxicity renders it suitable for combination with drugs that cause greater bone-marrow depression.

Vindesine sulphate (9.43) is a semi-synthetic product derived from vinblastine, although its spectrum of activity corresponds more closely to that of vincristine. Efforts towards the synthesis of new analogues are generally directed at reducing the neurotoxicity (peripheral parasthesia and autonomic neuropathy) associated with the vinca alkaloids.

9.9.2 The Taxanes

Paclitaxel (9.45, Taxol®) is a highly complex tetracyclic diterpene found in the bark and needles of the Pacific yew tree Taxus brevifolia. The cytotoxic nature of extracts of Taxus brevifolia was first demonstrated in 1964; pure taxol was isolated in 1966 and its structure published in 1971. However, taxol has only recently appeared in the clinic, over 30 years since its discovery.

The mechanism of action of this agent also involves the microtubules. There is an equilibrium between the microtubules and tubulin dimers, and the assembly and disassembly of dimers is governed by cell requirements. Taxol is thought to promote microtubule assembly, shifting the equilibrium in favour of the polymeric form of tubulin and reducing the critical concentration of the non-polymerised form by stabilising the microtubule complex. There are also some reports of taxol acting as an immunomodulator and activating macrophages to produce interleukin-1 and tumour necrosis factor (see section 9.14).

6Bz

The main indication for taxol is in the treatment of refractory ovarian cancer, although clinical trials are currently underway to assess its therapeutic value in breast cancer. Side effects include myelosuppression, peripheral neuropathy, cardiac conduction defects (arrhythmias), alopecia, muscle pain, nausea and vomiting. The barks from a very large number of yew trees would be required to provide a single course of treatment for an ovarian cancer patient, and the problems associated with producing sufficient quantities of this agent account, in part, for its delay in arriving in the clinic. Fortunately, semi-synthesis is now possible by extracting baccatin (taxol without the ester group) in large amounts from the leaves (a renewable resource) of a related species, Taxus baccata. The ester side chain (shown to the left of the molecule as drawn above) can now be made synthetically and then joined on to baccatin to provide taxol. There is also interest in the total synthesis of taxol, with the first route being reported in the early 1990s. Taxol itself has relatively poor water solubility, and so there is interest in producing analogues with improved solubility profiles. A related compound called taxotere has also generated interest.

9.10 MISCELLANEOUS AGENTS

9.10.1 Hydroxyurea

Hydroxyurea (9.46) is thought to exert its antitumour action in the S-phase of the cell cycle where it inhibits ribonucleotide reductase. This has the effect of depleting the deoxynucleoside triphosphate pool. An advantage of hydroxyurea is that it can be taken orally. It is generally used in combination chemotherapy with other agents in the treatment of melanoma and chronic myelogenous leukaemia.

0 0

Post a comment