**4.3. Cell Cycle Non Specific Agents (CCNSA)**

#### *4.3.1. Alkylating agents*

The first real breakthrough in the search for chemotherapeutics was the chemical synthesis of nitrogen mustards [4,119]. Sulphur mustard was synthesised much earlier, in 1822, but its harmful effects were not known until 1860. It was first used as chemical warfare weapon agent during the latter part of the First World War but its therapeutic activity against squamous cell carcinoma was discovered by accident. In fact most of the first so-called true synthetic chemotherapeutics, were discovered by serendipity, the special term *Serendipity* for accidental discoveries was introduced by Horace Walpole (1717-1797) in the 18th c. Nitrogen mustard, an analogue of the highly toxic sulphur mustard gas, was introduced in 1942 as the first alkylating agent and a true chemotherapeutic. Alfred G. Gilman, Louis S. Goodman and Thomas Dougherty, examined the potential therapeutic effects of nitrogen mustard in rabbits and mice bearing a transplanted lymphoid tumour, while Gustaf E. Lindskog (1903-2002), a thoracic surgeon, administered it to patients with non-Hodgkin's lymphoma. Many cases of cancer regression succeed intensive screening of related alkylating compounds and discovery of busulphan by L.A. Elson, G.M. Timmis, and David A. G. Galton (1922-2006) in 1951, Chlor‐ ambucil by James Everatt in 1953, melphalan by Frank Bergel and John Stock in 1954, Cyclo‐ phosphamide by Herbert Arnold, Friedrich Bourseaux and Norbert Brock in 1956, Lomustine and Carmustine by John A. Montgomery, George S. McCaleb, Thomas P. Johnston in 1966. While many different classes of alkylating agents (nitrogen mustards, nitrosoureas, alkyl sulphonates, triazines, and ethylenimines) are known, the chemical mechanism of their action is common and based on three different mechanisms all of which achieve the same end result - disruption of DNA function and apoptosis. The first mechanism of DNA alkylation results in its fragmentation by repair enzymes to prevent DNA synthesis and RNA transcription from the affected DNA. The second mechanism is the formation of intrastrand or interstrand crosslinks by an alkylating agent, which prevents DNA from being separated for synthesis or transcription. The third mechanism of action is the induction of mispairing of the nucleotides, which leads to mutations, even permanent ones.

increase in the pH of tumour and reduces the formation of spontaneous metastases in mouse models of metastatic breast cancer [115,116]. Another interesting case is selenite known since ancient times but recently revealed as a promising anticancer agent capable of inducing

As yet none of the new natural venom, toxin or minerals derived anticancer agents have reached the status of the clinical drug, but a number of agents are still in study or in preclinical

The first steps toward chemical synthesis of drugs were undertaken by iatrochemistry, a branch of chemistry and medicine concerned with seeking chemical solutions to diseases and ailments. Paracelsus pioneered the use of chemicals and minerals in medicine. He introduced alcohol, arsenic, copper, lead and silver salts into medicine, and developed rules for drug administration and dosages of drugs. Paracelsus also devised methods of extracting the arcanum (active ingredient) from plant materials. For this reason he is considered to be the father of phytochemistry and pharmacognosy. Ehrlich, the father of chemotherapy, developed the animal model to screen a series of chemicals for their potential activity against diseases, which had a major influence on the direction of cancer drug development. He also studied the usability of aniline dyes and the first primitive alkylating agents in cancer treatment. The first overall cancer treatment programme was the work of another pioneer of modern chemother‐ apy - George Clowes (1915-1988). He developed the first transplantable carcinogen-induced tumour systems in mice, which allowed the standardization of models for cancer drug testing. These early model systems including Sarcoma 37 (S37), Sarcoma 180 (S180), Walker 256, and Ehrlich's ascites tumour have been used for several decades [118]. In 1935 Murray Shear developed the most organized program for cancer drug screening. About 3,000 compounds including natural ones, were screened with S37 as a model system. The reason for the failure of this first systematic attempt to search for anticancer drugs - only two drugs have been subjected to clinical trials, but finally dropped because of unacceptable toxicity - was the lack of knowledge on how to test cytotoxic effects in humans. An extension of the number of tumour systems available for studies by the Yoshida's ascites sarcoma model and a murine leukaemia induced by a carcinogen, Leukaemia 1210, described by Lloyd Law allowed fast progress.

The first real breakthrough in the search for chemotherapeutics was the chemical synthesis of nitrogen mustards [4,119]. Sulphur mustard was synthesised much earlier, in 1822, but its harmful effects were not known until 1860. It was first used as chemical warfare weapon agent during the latter part of the First World War but its therapeutic activity against squamous cell carcinoma was discovered by accident. In fact most of the first so-called true synthetic chemotherapeutics, were discovered by serendipity, the special term *Serendipity* for accidental discoveries was introduced by Horace Walpole (1717-1797) in the 18th c. Nitrogen mustard, an

apoptosis in malignant mesothelioma and sarcoma cells [117].

**4.3. Cell Cycle Non Specific Agents (CCNSA)**

*4.3.1. Alkylating agents*

development.

54 Drug Discovery

**4.2. Synthetic drugs**

Alkylating agent acts on a cancer cell in every phase of its life cycle, Fig. 2, thus can be used in the treatment of a wide range of cancers from various solid tumours to leukaemia. However strong adverse effect is their ability to induce secondary cancers, which is reflected by their classification as definite carcinogens by IARC [3].

**Figure 2.** Cell replication occurs in the cell cycle (G0, G1, S, G2 and M). The cell cycle nonspecific agents (alkylating agents, platinum compounds, cytotoxic antibiotics) are able to kill a cell during any phase of the cycle, while cell cycle specific (antimetabolites, antifoliates, planta alkaloids, some cytotoxic antiniotics line bleomycin) are only able to kill only during a specific phase.

#### *4.3.2. Cytotoxic antibiotics*

A number of cytotoxic antibiotics that have been derived from natural sources such as grampositive bacteria in soil and water, belonging to genus *Streptomyces* (phylum *Actinobacteria*) [4,119]. They produce secondary metabolites, many of which have been successfully isolated and used as antifungals, antibiotics and anticancer drugs. The large-scale screening of fermentation products by the pharmaceutical industry which resulted in the discovery of antibiotics to treat wound infections is one more example of finding anticancer drugs by serendipity. Although penicillin, which was the basic compound for the above mentioned studies has no antitumor properties itself, but the chromo oligopeptide actinomycin D, isolated from *Streptomyces antibioticus* by Selman Abraham Waksman (1888-1973) and Boyd Woodruff in the 1940s as a result of search for drugs to treat tuberculosis, has significant antitumor properties and was applied in the 1950/1960s in paediatric oncology. This antibiotic was approved by the U.S. Food and Drug Administration (FDA) in 1964. In 1950, the search for anticancer compounds from soil-based microbes in the area of Castel del Monte, Italy, resulted in the discovery of an antibiotic - Daunorubicin (red pigment) - independently by Aurelio di Marco, Arpad Grein and Celestino Spalla from bacterium *Streptomyces peucetius* and by M. Dubost from *Streptomyces caeruleorubidus.* It was found to be active against murine tumours (Yoshida sarcoma). Although clinical trials which began in the 1960s suggested its significant activity against acute leukaemia and lymphoma, but shortly after, in 1967, it was recognized that daunorubicin had significant cardiac toxicity. In general, many antibiotics produced by *Streptomyces* are too toxic for use as antibiotics in humans, but their activity towards specific cells lines makes them useful in chemotherapy. The search for more effective antitumor antibiotics over 2,000 analogues of slightly modified structures yielded in a series of com‐ pounds, some of which are in common use till today. In 1969, Federico Arcamone developed a derivative of Doxorubicin which in the same year was tested against animal tumours by di Marco. Daunroubicin and Doxorubicin belongs to inhibitors of the topoisomerase II, one of two enzymes that regulate overwinding/underwinding of DNA. Inhibition of the topoiso‐ merase II block cleavage of both strands of the DNA which ultimately leads to cell death. An important antibiotic of a wide spectrum of anticancer activity is Mitomycin C isolated from *Streptomyces caespitosu* in 1955 and *Streptomyces lavendulae*in 1958 and clinically used since the first successful trials against childhood leukaemia reported by Charlotte Tan in 1965. Mito‐ mycin C belongs to bifunctional alkylating agents, whose biological activity mode is DNA alkylation and cross-linking. It has a broad activity against a range of tumours. In 1966 Hamao Umezawa discovered an important unique antibiotic in this group - bleomycin - a glycopeptide showing anticancer activity, while screening a culture filtrates of *Streptomyces verticullus.* Bleomycin is used to treat many types of cancer, including testicular cancer, non Hodgkin's lymphoma, Hodgkin's lymphoma and cancers of the head and neck. Anticancer antibiotics, apart from Bleomycin, act on a cancer cell in every phase of its life cycle and prevent cell divisions, but Bleomycin is considered as cell cycle agent specifically working in G2 and M phase, Fig. 2. However, the risk connected with the use of cytotoxic antibiotics classified as group 2 or 3 agent in IARC classification is smaller than that related to alkylating agents [3].

*4.3.3. Platinum compounds*

**4.4. Cell Cycle Specific Agents (CCSA)**

*4.4.1. Antifolates*

Cisplatin was synthesized in 1845 but its potential as an antitumour agent was not recognized until 1965 when its capabilities were discovered by Barney Rosenberg, Loretta van Camp and Thomas Krigas. The inhibition of growth caused by platinum complex of ammonia and chloride (Peyrone's salt i.e. Cis-platinum) was discovered by serendipity during the studies of the influence of electric current on bacterial growth. The positive result during the studies of murine tumours in vivo confirmed its antitumor activity and prompted the studies of other compounds from this class [4,56,119]. It was introduced into clinical practice one decade later in the 1970s [120]. By 1978 about 1,000 platinum complexes had been screened, but only seven were selected for detailed pharmacological evaluation on rats and only two - Carboplatin and Oxaliplatin - were non-toxic at effective antitumour dose. Although Cisplatin belongs to three most commonly used chemotherapeutics, progress made to improve its use since its discovery is in fact limited. The mechanism of action of Cisplatin and other platinum compounds resemble that of the alkylating agents. They interact covalently with DNA and form intrastrand (within the same DNA molecule; >90%) or interstrand (between two different DNA molecules; <5%), cross links between adjacent guanine molecules [121]. The formation of DNA adducts results in an inhibition of DNA synthesis and transcription. Platinum compounds act on a cancer cell in every phase of its life cycle, Fig.2. Their use is widespread and includes the treatment of bladder and colorectal cancer, upper gastrointestinal disease, germ cell tumours, head and neck malignancies, lung and ovarian cancer. Their ability to induce secondary

Anticancer Drug Discovery — From Serendipity to Rational Design

http://dx.doi.org/10.5772/52507

57

cancers reflected by their classification as definite carcinogens by IARC is high [3].

Antimetabolites (folic acid, pyrimidine or purine analogues), which structurally resemble naturally occurring molecules necessary for DNA and RNA synthesis and either inhibit enzymes needed for nucleic acid production or induce apoptosis during the S phase of cell growth, Fig. 2, were among the first effective chemotherapeutics discovered. In the early 1940s, Sidney Faber (1903–1973) studied the effect of folic acid (pteroylglutamic acid; Vitamin B9) first isolated from spinach leaves. In 1945, Rudolf Leuchtenberger reported that folic acid inhibited tumour growth in mice, while Richard Lewisohn reported complete regression of spontaneous breast in mice observed with folic acid. Farber, Robert D. Heinle, and Arnold D. Welch tested folic acid in leukaemia and concluded spuriously that deficiency of Vitamin B9 accelerates leukaemia cell growth. Efforts to treat leukaemia resulted in pharmacological folic acid analogues with effects antagonistic to those of Vitamin B9. Shortly after Sidney Farber and Harriet Kilte developed a series of foliate antagonists including highly active aminopterin (4-amino-pteroylglutamic acid). Its analogue 4-amino-4-deoxy-10-*N*-methyl-pteroylglutamic acid, known nowadays as Methotrexate, was discovered by Yellapragada Subbarao (1895-1948) and successfully applied by Sidney Farber in 1947 to induce remissions in children with leukaemia. In 1958 Min Chiu Li, reported fully effective treatment of a very rare tumour of the placenta, choriocarcinoma with Methotrexate, which was the first-ever intentionally
