**4. Chemotherapeutical agents**

#### **4.1. Drugs of natural origin**

In the second half of the 20th century, one more type of cancer therapy was added to surgery, irradiation and hormonotherapy, which was chemotherapy. Nowadays this term primarily refers to the treatment of cancer with an antineoplastic drug or a combination of drugs, but when it was introduced in 1909 by Paul Ehrlich (1854-1915) it had a broader meaning as it referred as well to antibacterial chemotherapy and treatment of autoimmune diseases, in general use of chemicals to treat disease. Chemotherapy, generally assumed as the youngest method of cancer treatment, is in fact rooted in ancient times. Although cutting out the cancer changed tissue was early found as the main treatment, it was not always effective. Thus various substances of natural origin were applied as complementary medications. Many even ancient cultures had proposed theories explaining the cause of cancer. These theories influenced the search for medicaments. For example Egyptians believed that natural substances similar in look or function to human organs can be used to treat ailments in those organs, thus the use of mixtures of pigs eyes or ears was popular.

belongs to three most serious nuclear accidents ever recorded, although it was revealed only in 1976 [58]. The scarce epidemiological studies suggest very different numbers of cancer deaths among residents associated to radiation exposure. In 1979 the cooling system of Three Mile Is‐ land nuclear power plant near Harrisburg, Pennsylvania failed and the reactor core was parti‐ ally melted. Radiation from the reactor contributed to the premature deaths and cancers in local residents, but the disaster was relatively small [59]. There is still vivid discussion about the carcinogenic effects of nuclear power plant explosion in 1986 in Chernobyl located 80 miles from Kiev, Ukraine, which was the greatest source of long-lived radioactive plutonium and short-lived radioactive caesium (137Cs), iodines (particularly 131I) and strontium (90Sr). The ma‐ jor health effect of Chernobyl was an elevated thyroid-cancer incidence due to iodine absorp‐ tion by the thyroid gland in adolescents and children some of whom were not yet born at the time of the accident, and drastic increase in leukaemia cases caused by distribution the stronti‐ um incorrectly recognized by the body as calcium throughout the bone structure [60]. Radioac‐ tive isotopes of barium, caesium, iodine and tellurium were detected in a radiation plume released by damaged nuclear reactors at the nuclear plant in Fukushima, Japan in 2011. Fu‐ kushima Daiichi disaster was the most serious accident in global scale. As the prolonged expo‐ sure to radiation in the air, ground and food can result in leukaemia and other cancers thus about 160,000 people were evacuated from the region surrounding the plant. According to the‐ oretical 3-D global atmospheric models this nuclear disaster may cause as many as 2,500 cases of cancer, mostly in Japan. Only recently, in the 1990s, much-less energetic UV radiation has been also recognized as carcinogen causing not only genetic mutations but also melanoma or non-melanoma cancers. In 2011 WHO/IARC classified radiofrequency electromagnetic fields as possibly carcinogenic to humans (Group 2B), on the basis of an increased risk of glioma, a malignant type of brain cancer, associated with wireless phone use [3]. Nowadays we are aware that exposure to radiation can be incidental like in Hiroshima, Nagasaki, Chernobyl, Fu‐ kushima [1,61] or systematic due to repeated doses of radiation like UV during sun-bathing or MW during phone-cell use. Anyway the most common radiation induced cancers are basal cell carcinoma and squamous carcinoma of the skin, leukaemia and thyroid cancer. The first two can arise from excessive exposure to UV radiation, while the other are mainly result of ionis‐ ing radiation e.g. γ, X-Ray [1]. The controlled use of ionising radiation in medicine and indus‐ try and annual limits of doses for each individual [62] has reduced the risk connected with ionising radiation but the awareness of UV or MW related risk is still low. Common feature of cancers induced by physical factor is late onset and long period of risk persisting. Most of the

physical carcinogens are classified as group 1 or 2 agents in IARC classification [3].

In the second half of the 20th century, one more type of cancer therapy was added to surgery, irradiation and hormonotherapy, which was chemotherapy. Nowadays this term primarily refers to the treatment of cancer with an antineoplastic drug or a combination of drugs, but when it was introduced in 1909 by Paul Ehrlich (1854-1915) it had a broader meaning as it

**4. Chemotherapeutical agents**

**4.1. Drugs of natural origin**

48 Drug Discovery

Although the products of animal and mineral origin had made an important contribution to drug development, the main source of drugs for millennia have been green plants. The most frequently used included castor oil plant (*Ricinus communis* L.), exploding cucumber (*Ecballium elateritum* L.), belladonna (*Atropa belladonna* L.), myrrh (dried sap from trees *Commiphora Myrcha L.*), incense (dried sap from trees *Boswellia thurifera L.*, *Boswellia frereana Birdw.*, *Boswellia bhawdajiana* Birdw.), stinging nettle (*Urtica dioica* L.), gingers (*Zingiber Boehm.* L.), red clover (*Trifolium pratense* L.) and autumn crocus (*Colchicum autumnale* L.). Although, in nature most of them cause sickness, but in small doses or after chemical modifications, they revealed therapeutic effects. Some of them were rediscovered by modern medicine. For example from *Colchicum autumnale* L. described by Pedanius Dioscorides (40-90 A.D.) in De Materia Medica a toxic alkaloid colchicine was extracted in 1820 by Pierre Joseph Pelletier (1788-1842) and Joseph Bienaimé Caventou (1795–1877). Albert Pierre Dustin (1914-1993), described its antymitotic properties in 1934 [63]. In 2009 it was accepted by Food and Drug Administration (FDA) as a drug for gout and Familial Mediterranean Fever. Another interesting case described by Dioscorides is red viscous sap called the dragon's blood mostly collected from *Dracaena cinnabari* Balf. f or *Croton lechleri* L. and used as a dye and anti-inflammatory, antimicrobial and anticancer folk remedy not only by ancient Greek but also Romans and Arabs. Recently methanolic extract of *Croton lechleri* was shown to exert cytotoxic effects on HeLa (Human epithelial carcinoma cell line) cells and its antitumor effect in HeLa tumour in mice was documented [64]. Two other commonly used antileukemic drugs, vinblastine and vincristine, were extracted in 1950 from the species of Madagascar periwinkle (*Catharanthus roseus* L.), for centuries known as folk remedy, and shortly after approved by FDA. Nowadays vinblastine, which binds tubulin, thereby inhibiting the assembly of microtubules, is an important component of a number of chemotherapy regimens, including ABVD for Hodgkin lymphoma, advanced testicular cancer, breast and lung cancers, and Kaposi's sarcoma [65]. Realgar widely used in Chinese traditional medicine because of its anti-inflammation, antiulcer, anticonvul‐ sion, and anti-schistosomiasis activity was recently found capable to induce cell apoptosis and thus effective in the treatment of hematological malignant diseases [66]. As early as in 1021, Avicenna described the medicinal use of *Taxus baccata* L. (Zarnab) as cardiac remedy in The Canon of Medicine. Various parts of *Taxus brevifolia* Peattie*, Taxus Canadensis* Marshall, *Taxus baccata* L. have been used by several Native American Tribes mainly for the treatment of noncancerous diseases [65] but the use for the treatment of cancer was noted only in the Hindu Ayurvedic medicine. Paclitaxel (Taxol®), used in treatment for breast, ovarian, small and nonsmall cell lung cancer and Kaposi sarcoma, was isolated in 1967 by Monroe E. Wall and Mansukh C. Wani from the bark of the 200-years old Pacific yew (*Taxus brevifolia* Nutt.) tree [67]. Its structure was elucidated in 1971 [67,68]. Its cytostatic mechanism of action (mitosis inhibition) was discovered by Susan B. Horowitz in the late 1970s, but only the discovery of total chemical synthesis of Paclitaxel in 1994 by Robert A. Holton widespread its use [69]. Camptothecin isolated from the Chinese and Tibetan ornamental joy tree Decne (*Camptotheca acuminata* var. rotundifolia B. M. Yang & L. D. Duan)*, Nyssaceae* Arnott family, was discovered in 1966 by Wall and Wani in systematic screening of natural products [70]. Although it is a potent topoisomerase inhibitor, it was dropped in the 1970s from clinical trials because of severe bladder toxicity [71]. But two of its semi-synthetic derivatives - topotecan and irinotecan are used for the treatment of ovarian and small cell lung and colon-rectal cancers, respectively [72,73]. Epipodophyllotoxines also belong to active anti-tumour agents derived from plants. Podophyllotoxin and deoxypodophyllotoxin were obtained from the roots of American mandrake or May apple (*Podophyllum peltatum* L.), Himalayan mayapple (*Podophyllum emodi* Wallich ex Hook. f. & Thomson) and Chinese or Asian Mayapple (*Podophyllum pleianthum* L.), respectively [74], all belongs to *Berberidaceae* Juss. family. *Podophyllum peltatum* and *Podophyl‐ lum emodii* were used by the Native American Tribes for the treatment of cancer including skincancers. Podophyllotoxin was isolated from the rhizome in 1880 by V. Podwyssotski [75]. More cytotoxic 4-deoxypodophyllotoxin was isolated from Cow Parsley *(Anthriscus sylvestris* L.) and Korean pasque flower *(Pulsatilla koreana* Y.Yabe ex Nakai). Although native epipodophyllo‐ toxines are not used but its synthetic analogues - etoposide and teniposide, which belongs to topoisomerase II inhibitors, are effective in the treatment of lymphomas and bronchial and testicular cancers [65,76]. Another example is bruceantin isolated from a tree, *Brucea antidy‐ senterica* Mill from *Simaroubaceae* DC family, used traditionally for the tumour treatment in Ethiopia [77]. Recently it was discovered that bruceantin can be an effective agent for the treatment of hematological malignancies (leukaemia, lymphoma and myeloma). Its activity has been linked with the down-regulation of a key oncoprotein. Omacetaxine mepesuccinat (Homoharringtonine), alkaloid isolated from the Cowtail Pine called Japanese Plum Yew (*Cephalotaxus harringtonia* Koch), is one more example of plant-derived anticancer agent [78,79]. Its racemate (harringtonine mixed with homoharringtonine) which induces apoptosis by inhibition of protein synthesis, particularly Mcl-1 (induced myeloid leukemia cell differentia‐ tion protein), is used for the treatment of chronic leukaemia - acute lymphoblastic leukaemia and chronic myelogenous leukaemia [65]. Elliptinium acetate, a derivative of ellipticine, which was isolated from a Fijian plant *Bleekeria vitensis* A.C. Sm., is used for the treatment of breast cancer [65]. Recently numerous potential anticancer compounds have been isolated from different plants. A few of them are currently in clinical or preclinical trials but most require further investigation. A case of considerable interest is indirubine extracted from Mu Lan (*Indigofera tinctoria* L.) from *Leguminosae* Lindl. family called Indigo plant a main component of traditional Chinese herbal remedy called Dang Gui Long Hui Wan used to treat chronic myelogenous leukaemia. Synthetic agents flavopiridol derived from the indirubins - plant alkaloid rohitukine, which was isolated from *Dysoxylum binectariferum* Hook. f. (*Meliaceae*Juss.) [80] and roscovitine derived from olomucine, which was isolated from *Raphanus sativus* L. (*Brassicaceae* Burnett), are respectively in Phase I and II of clinical trials [65,81] against a broad range of cancers including leukaemia, lymphomas and solid tumours [82]. Both are belongs

to inhibitors of cyclin-dependent kinases (Cdks), key regulatory proteins in the cell cycle. Most recent studies indicate that drugs of the indirubin family may block brain tumour and thus improve survival in glioblastoma. Other synthetic derivatives of indirubins (3'-monooxime and 5-bromo) reveal comparable activity to other Cdk inhibitors and thus are promising for drug development [83]. Unique source of indirubines are gastropod molluscs: *Bolinus branda‐ ris* L. and *Hexaplex trunculus* L. (*Muricidae* L.) used for over 2,500 years to obtain purplish red dye known as "Tyrian Purple". The 6-bromoindirubine treated as impurity to indigo dye and its synthetic derivative show selective inhibition of glycogen synthase kinase-3 (GSK-3) [81]. The discovery of GSK-3 functions resulted in the search for its inhibitors as potential drugs against neurodegenerative diseases, inflammation and cancer. Combretastatin isolated from the bark of the South African "bush willow", tree *Combretum caffrum* (Eckl. & Zeyh.) Kuntze (*Combretaceae* Loefl. familly) [84] belongs to the most cytotoxic phytomolecules isolated so far [85,86] and is promising in the treatment of colon, lung cancers, lymphomas and leukaemias. Combretastatins belongs to stilbenes, which are anti-angiogenic agents, causing vascular shutdown resulting in tumour necrosis. *Combretum* was widely used in African and Hindu medicine for the treatment of a variety of diseases, but *Terminalia* L. flowering plant from the same family *Combretaceae* Loefl., have been used traditionally for cancer treatment. Another promising stilbene is trans-Resveratrol natural phenol produced by several plants (eg. *Vitis vinifera* L., *Vitis labrusca* L., *Vitis rotundifolia* Michx.), when under attack by pathogens (bacteria or fungi like Botrytis cinerea (De Bary) Whetzel). It was extracted from False Helleborine (*Veratrum Album* L.) by Michio Takaoka in 1939. More than 60 years later in 1997, Ming-Hua Jang reported that trans-Resveratrol prevented skin cancer development in mice treated with a carcinogen, which gain attention to its potential anticancer applications. It was shown that trans-Resveratrol acts on all steps of the process of carcinogenesis [87]. It triggered apoptosis in uterine, colon cancer cell line, colon, human breast, prostate, lung cancer and pancreatic cancer cell lines in vitro, but is also able to arrest the cell cycle or to inhibit kinase pathways. The inhibition in the development of oesophageal, intestinal, and breast cancer after oral administration of resveratrol was revealed in studies on animal models. The human clinical trials for cancer have not been reported. A few promising substances betulinic acid, lupeol have been obtained from white part of *Betula species* (*Betulaceae* Gray) bark. The alcohol precursor of betulinic acid - betulin - was isolated as long ago as in 1788 by Tobias Lowitz (1757-1804). Betulinic acid, a pentacyclic triterpene, is a common secondary metabolite of plants, it was isolated also from *Ziziphus zizyphus* L. H. Karst. species, e.g. *Ziziphus Mauriti‐ ana* Lam., *Ziziphus Rugosa* Lam. and *Ziziphus Oenoplia* (L.) Mill. [88,89], while lupeol was found in a variety of plants, including mango *(Mangifera L.)* and acacia visco *(Acacia visite* Griseb.). All of them are potent anti-inflammatory agents and displayed selective cytotoxicity against human melanoma cell lines [90]. A case of considerable interest is birch polypore fungus Chaga (*Inonotus obliquus* Pers. Pill.), which belongs to *Basidiomycetes* R.T. Moore. It forms black perennial woody growth called a conk on birch trees. It is traditionally used in Russia for the treatment of a number of conditions including cancer, gastritis and ulcers. Two phenolic compounds, hispidin and hispolon extracted from Chaga but also from Japan, Chinese and Korean medicinal fungus *Phellinus linteus* (Japanese *meshimakobu*, Chinese *song gen*, Korean *sanghwang*) were reported to be cytotoxic against human cell line HeLa [91], while the poly‐

Anticancer Drug Discovery — From Serendipity to Rational Design

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

51

to inhibitors of cyclin-dependent kinases (Cdks), key regulatory proteins in the cell cycle. Most recent studies indicate that drugs of the indirubin family may block brain tumour and thus improve survival in glioblastoma. Other synthetic derivatives of indirubins (3'-monooxime and 5-bromo) reveal comparable activity to other Cdk inhibitors and thus are promising for drug development [83]. Unique source of indirubines are gastropod molluscs: *Bolinus branda‐ ris* L. and *Hexaplex trunculus* L. (*Muricidae* L.) used for over 2,500 years to obtain purplish red dye known as "Tyrian Purple". The 6-bromoindirubine treated as impurity to indigo dye and its synthetic derivative show selective inhibition of glycogen synthase kinase-3 (GSK-3) [81]. The discovery of GSK-3 functions resulted in the search for its inhibitors as potential drugs against neurodegenerative diseases, inflammation and cancer. Combretastatin isolated from the bark of the South African "bush willow", tree *Combretum caffrum* (Eckl. & Zeyh.) Kuntze (*Combretaceae* Loefl. familly) [84] belongs to the most cytotoxic phytomolecules isolated so far [85,86] and is promising in the treatment of colon, lung cancers, lymphomas and leukaemias. Combretastatins belongs to stilbenes, which are anti-angiogenic agents, causing vascular shutdown resulting in tumour necrosis. *Combretum* was widely used in African and Hindu medicine for the treatment of a variety of diseases, but *Terminalia* L. flowering plant from the same family *Combretaceae* Loefl., have been used traditionally for cancer treatment. Another promising stilbene is trans-Resveratrol natural phenol produced by several plants (eg. *Vitis vinifera* L., *Vitis labrusca* L., *Vitis rotundifolia* Michx.), when under attack by pathogens (bacteria or fungi like Botrytis cinerea (De Bary) Whetzel). It was extracted from False Helleborine (*Veratrum Album* L.) by Michio Takaoka in 1939. More than 60 years later in 1997, Ming-Hua Jang reported that trans-Resveratrol prevented skin cancer development in mice treated with a carcinogen, which gain attention to its potential anticancer applications. It was shown that trans-Resveratrol acts on all steps of the process of carcinogenesis [87]. It triggered apoptosis in uterine, colon cancer cell line, colon, human breast, prostate, lung cancer and pancreatic cancer cell lines in vitro, but is also able to arrest the cell cycle or to inhibit kinase pathways. The inhibition in the development of oesophageal, intestinal, and breast cancer after oral administration of resveratrol was revealed in studies on animal models. The human clinical trials for cancer have not been reported. A few promising substances betulinic acid, lupeol have been obtained from white part of *Betula species* (*Betulaceae* Gray) bark. The alcohol precursor of betulinic acid - betulin - was isolated as long ago as in 1788 by Tobias Lowitz (1757-1804). Betulinic acid, a pentacyclic triterpene, is a common secondary metabolite of plants, it was isolated also from *Ziziphus zizyphus* L. H. Karst. species, e.g. *Ziziphus Mauriti‐ ana* Lam., *Ziziphus Rugosa* Lam. and *Ziziphus Oenoplia* (L.) Mill. [88,89], while lupeol was found in a variety of plants, including mango *(Mangifera L.)* and acacia visco *(Acacia visite* Griseb.). All of them are potent anti-inflammatory agents and displayed selective cytotoxicity against human melanoma cell lines [90]. A case of considerable interest is birch polypore fungus Chaga (*Inonotus obliquus* Pers. Pill.), which belongs to *Basidiomycetes* R.T. Moore. It forms black perennial woody growth called a conk on birch trees. It is traditionally used in Russia for the treatment of a number of conditions including cancer, gastritis and ulcers. Two phenolic compounds, hispidin and hispolon extracted from Chaga but also from Japan, Chinese and Korean medicinal fungus *Phellinus linteus* (Japanese *meshimakobu*, Chinese *song gen*, Korean *sanghwang*) were reported to be cytotoxic against human cell line HeLa [91], while the poly‐

Mansukh C. Wani from the bark of the 200-years old Pacific yew (*Taxus brevifolia* Nutt.) tree [67]. Its structure was elucidated in 1971 [67,68]. Its cytostatic mechanism of action (mitosis inhibition) was discovered by Susan B. Horowitz in the late 1970s, but only the discovery of total chemical synthesis of Paclitaxel in 1994 by Robert A. Holton widespread its use [69]. Camptothecin isolated from the Chinese and Tibetan ornamental joy tree Decne (*Camptotheca acuminata* var. rotundifolia B. M. Yang & L. D. Duan)*, Nyssaceae* Arnott family, was discovered in 1966 by Wall and Wani in systematic screening of natural products [70]. Although it is a potent topoisomerase inhibitor, it was dropped in the 1970s from clinical trials because of severe bladder toxicity [71]. But two of its semi-synthetic derivatives - topotecan and irinotecan are used for the treatment of ovarian and small cell lung and colon-rectal cancers, respectively [72,73]. Epipodophyllotoxines also belong to active anti-tumour agents derived from plants. Podophyllotoxin and deoxypodophyllotoxin were obtained from the roots of American mandrake or May apple (*Podophyllum peltatum* L.), Himalayan mayapple (*Podophyllum emodi* Wallich ex Hook. f. & Thomson) and Chinese or Asian Mayapple (*Podophyllum pleianthum* L.), respectively [74], all belongs to *Berberidaceae* Juss. family. *Podophyllum peltatum* and *Podophyl‐ lum emodii* were used by the Native American Tribes for the treatment of cancer including skincancers. Podophyllotoxin was isolated from the rhizome in 1880 by V. Podwyssotski [75]. More cytotoxic 4-deoxypodophyllotoxin was isolated from Cow Parsley *(Anthriscus sylvestris* L.) and Korean pasque flower *(Pulsatilla koreana* Y.Yabe ex Nakai). Although native epipodophyllo‐ toxines are not used but its synthetic analogues - etoposide and teniposide, which belongs to topoisomerase II inhibitors, are effective in the treatment of lymphomas and bronchial and testicular cancers [65,76]. Another example is bruceantin isolated from a tree, *Brucea antidy‐ senterica* Mill from *Simaroubaceae* DC family, used traditionally for the tumour treatment in Ethiopia [77]. Recently it was discovered that bruceantin can be an effective agent for the treatment of hematological malignancies (leukaemia, lymphoma and myeloma). Its activity has been linked with the down-regulation of a key oncoprotein. Omacetaxine mepesuccinat (Homoharringtonine), alkaloid isolated from the Cowtail Pine called Japanese Plum Yew (*Cephalotaxus harringtonia* Koch), is one more example of plant-derived anticancer agent [78,79]. Its racemate (harringtonine mixed with homoharringtonine) which induces apoptosis by inhibition of protein synthesis, particularly Mcl-1 (induced myeloid leukemia cell differentia‐ tion protein), is used for the treatment of chronic leukaemia - acute lymphoblastic leukaemia and chronic myelogenous leukaemia [65]. Elliptinium acetate, a derivative of ellipticine, which was isolated from a Fijian plant *Bleekeria vitensis* A.C. Sm., is used for the treatment of breast cancer [65]. Recently numerous potential anticancer compounds have been isolated from different plants. A few of them are currently in clinical or preclinical trials but most require further investigation. A case of considerable interest is indirubine extracted from Mu Lan (*Indigofera tinctoria* L.) from *Leguminosae* Lindl. family called Indigo plant a main component of traditional Chinese herbal remedy called Dang Gui Long Hui Wan used to treat chronic myelogenous leukaemia. Synthetic agents flavopiridol derived from the indirubins - plant alkaloid rohitukine, which was isolated from *Dysoxylum binectariferum* Hook. f. (*Meliaceae*Juss.) [80] and roscovitine derived from olomucine, which was isolated from *Raphanus sativus* L. (*Brassicaceae* Burnett), are respectively in Phase I and II of clinical trials [65,81] against a broad range of cancers including leukaemia, lymphomas and solid tumours [82]. Both are belongs

50 Drug Discovery

saccharides ß (1→3)-D glucopyrans and ß (1→6)-D-glucosyl, found also in ornamental plant *Pteris ensiformis* Burm., originating from tropical Africa, Asia and Pacific region, have prom‐ ising anticancer activity against a number of different cell lines.

In general, over 120 currently prescribed drugs including anticancer ones being the basis of modern chemotherapy were first extracted from plants. About 60% of the anticancer drugs available prior to 1983 were of natural origin [65]. As much as 40% of anticancer drugs developed from 1940 to 2002 had natural or natural-product origins, while another 8% were natural-product mimics. Although nowadays about 300,000 different plant species are known but less than 5,000 have been studied for their potential drug usefulness. Since 1989, the National Cancer Institute (NCI) has screened up to 10,000 potential anticancer agents per year including minerals, exotic plants from tropical rain forests and animal venoms and toxins.

Anticancer Drug Discovery — From Serendipity to Rational Design

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Animal venoms and toxins which has been used as therapeutics in ancient Ayurvedic, Unani, Chinese folk medicine as well as in Homeopathy are also screened. Venoms of snakes, scorpions, toads, frogs and their derivatives protein or non-protein toxins, peptides, enzymes are promising and show some potential in cancer treatment. Léon Charles Albert Calmette (1863-1933) a French physician, bacteriologist and immunologist, was the first to describe an antitumor effect of the venom of Indian cobra Naja naja sp. on adenocarcinoma cells. Thereafter many reports have established the anticancer potential of venoms of different species of Elapidae, Viperidae, Crotalidae snakes [102-107] and Hydrophis spimlis sea snake [108,109] and assigned it to phospholipase activity. Scorpions venom has been used by traditional and folk medicine in India, China, Africa and Cuba. Chinese red scorpion (Buthus martensi Karsch) venom and skin extracts, known as Chan Su in China and Senso in Japan, have been used by traditional Chinese medicine for as long as 2000 years also as anti-leukaemia agents. 4',6 diamidino-2-phenylindole extracted from Buthus martensi Karsch induced cell apoptosis in malignant glioma cells in vitro [110], while serine proteinase and hylauronidase have prom‐ ising anticancer activity against a number of different cell lines including breast ones [111]. Bengalin protein isolated from Hindu black scorpion is suspected to have anti-leukemic properties [112]. Chlorotoxin and Charybdotoxin, 36- and 37-amino acid peptides, respectively isolated from the venom of death stalker scorpion (Leiurus quinqestriatus Hebraeus) are promising for the treatment of several types of cancers including glioma and human breast cancer [113,114]. The anticancer effect of the venom of Cuba red scorpions (Rhopalurus junceus) was discovered 20 years ago in Guantanamo, but after 15 years of studies Vidatox drug was announced in 2011. The skin extract from Hindu toads (Bufo melanostictus, Bufo gargarizans Cantor), Chan Su used by Chinese traditional medicine, was discovered to contain a few bufadineolides showing specific activity against human leukemic, liver carcinoma and melanoma cell lines. Species belonging to the families Bufonidae (toads), Lampyridae (fireflies) and Colubridae (snakes) as well as mammalian tissues contain bufadienolides, but the richest source of them are toad species. Although all the bufadienolides showed potent cytotoxicity in vitro, but the evidence of their activity in vivo is limited to human hepatocellular carcinoma

and HeLa human cancer cells in mice and require further investigation.

Some hope rises with the use of minerals as a source of anticancers drugs. Most important example is sodium bicarbonate, NaHCO3, which was originally derived from Nahcolite (thermokalite) carbonate mineral. The ancient Egyptians used natural natron, a mixture of sodium carbonate decahydrate, and sodium bicarbonate as a soap and embalming tool. Recently it has been shown that sodium bicarbonate administered orally causes a selective

Claims for another efficient plant derivative - *Tabebuia* Gomes (*Bignoniaceae* Juss.) used traditionally by the indigenous people in the Amazonian region for the treatment of variable diseases, appeared in the 1960s. Numerous bioactive compounds including naphthaquinones, particularly lapachol and ß-lapachone have been isolated from the stem bark and wood of *Tabebuia impetiginosa* (Mart. Ex DC.) Standl., *Tabebuia rosea* Bertol., and *Tabebuia serratifolia* (Vahl) Nicholson. Lapachol revealed potent in vivo anti-tumour activity, but was dropped out because of unacceptable level of toxicity [92]. ß-lapachone was recently found active against breast cancer, leukaemia, prostate tumour and several multidrug resistant (MDR) cell lines and more promising than lapachol [93]. It is a potent inhibitor of Cdc25 phosphatases enzyme that play a key role in cell cycle progression [83]. Another potent and promising in the field of MDR is pervilleine A, aromatic ester tropane, selectively cytotoxic against oral epidermoid cancer cell line which was isolated from the roots of the Madagascar tree *Erythroxylum pervillei* Baill. from *Erythroxylaceae* Kunth family [94,95]. In the early 1970s another plant originated substance, maytansine was isolated from the Ethiopian plant, *Maytenus serrata* (Hochst. Ex A. Rich.) Wilczek from *Celastraceae* R. Br. family. Although the results of preclinical animal tests were very promising but the lack of efficacy in clinical trials in the early 1980s resulted in dropping it out from further study. However, related compounds, the ansamito‐ cins, isolated from actinomycete *Actinosynnema pretiosum* shed some light on its possible microbial origin [96]. Its synthetic derivative - cytotoxic Mertansine is a component of human‐ ized monoclonal antibodies: Cantuzumab mertansine, Bivatuzumab mertansine, Lorvotuzu‐ mab mertansine and Trastuzumab emtansine effective in colorectal, squamous cell carcinoma, small-cell lung or ovarian cancer and breast cancer, respectively. Another case of considerable interest is thapsigargin isolated from the umbelliferous plant, *Thapsia garganica* L. (*Apiaceae* Lindl.) from Mediterranean island of Ibiza [97]. Thapsigargin, induces apoptosis in prostate cancer cells and synthetic prodrug derived from it called "G-202" is in Phase II clinical trials. Silvestrol isolated from the fruits of *Aglaila sylvestre* Roemer from *Meliaceae* Juss. family [98], exhibit cytotoxicity against lung and breast cancer cell lines [65]. Its synthetic analogue 4′ desmethoxyepisilvestrol is cytotoxic against lung and colon cancer cell lines. Two alkaloids, schischkinnin and montamine isolated recently from the seeds of *Centaurea schischkinii* Tzvelev and *Centaurea montana* L. [99,100] exhibit significant cytotoxicity against HCCLs (human colon cancer cell lines). The essential oil of *Salvia officinalis* L., most popular folk remedy in Middle East known for its antitumor effects, which contains monoterpenes thujone, β-pinene, and 1,8 cineol was shown to be cytotoxic against squamous human cell carcinoma cell line of the oral cavity [101]. There are many other natural substances like extracts of unknown composition from *Colubrina macrocarpa* (Cav.) G. Don., *Hemiangium excelsum* (Kunth) A.C. Sm*, Acacia pennatula* (Schltdl. & Cham.) Benth., *Commiphora opobalsamum* Jacq.*, Astragalus* L.*, Paris polyphylla* Sm.*, Teucrium polium* L.*, Pistacia lentiscus* L. used as anticancer remedies on folk medicine in China, Israel, Plestina, Saudi Arabia etc.

In general, over 120 currently prescribed drugs including anticancer ones being the basis of modern chemotherapy were first extracted from plants. About 60% of the anticancer drugs available prior to 1983 were of natural origin [65]. As much as 40% of anticancer drugs developed from 1940 to 2002 had natural or natural-product origins, while another 8% were natural-product mimics. Although nowadays about 300,000 different plant species are known but less than 5,000 have been studied for their potential drug usefulness. Since 1989, the National Cancer Institute (NCI) has screened up to 10,000 potential anticancer agents per year including minerals, exotic plants from tropical rain forests and animal venoms and toxins.

saccharides ß (1→3)-D glucopyrans and ß (1→6)-D-glucosyl, found also in ornamental plant *Pteris ensiformis* Burm., originating from tropical Africa, Asia and Pacific region, have prom‐

Claims for another efficient plant derivative - *Tabebuia* Gomes (*Bignoniaceae* Juss.) used traditionally by the indigenous people in the Amazonian region for the treatment of variable diseases, appeared in the 1960s. Numerous bioactive compounds including naphthaquinones, particularly lapachol and ß-lapachone have been isolated from the stem bark and wood of *Tabebuia impetiginosa* (Mart. Ex DC.) Standl., *Tabebuia rosea* Bertol., and *Tabebuia serratifolia* (Vahl) Nicholson. Lapachol revealed potent in vivo anti-tumour activity, but was dropped out because of unacceptable level of toxicity [92]. ß-lapachone was recently found active against breast cancer, leukaemia, prostate tumour and several multidrug resistant (MDR) cell lines and more promising than lapachol [93]. It is a potent inhibitor of Cdc25 phosphatases enzyme that play a key role in cell cycle progression [83]. Another potent and promising in the field of MDR is pervilleine A, aromatic ester tropane, selectively cytotoxic against oral epidermoid cancer cell line which was isolated from the roots of the Madagascar tree *Erythroxylum pervillei* Baill. from *Erythroxylaceae* Kunth family [94,95]. In the early 1970s another plant originated substance, maytansine was isolated from the Ethiopian plant, *Maytenus serrata* (Hochst. Ex A. Rich.) Wilczek from *Celastraceae* R. Br. family. Although the results of preclinical animal tests were very promising but the lack of efficacy in clinical trials in the early 1980s resulted in dropping it out from further study. However, related compounds, the ansamito‐ cins, isolated from actinomycete *Actinosynnema pretiosum* shed some light on its possible microbial origin [96]. Its synthetic derivative - cytotoxic Mertansine is a component of human‐ ized monoclonal antibodies: Cantuzumab mertansine, Bivatuzumab mertansine, Lorvotuzu‐ mab mertansine and Trastuzumab emtansine effective in colorectal, squamous cell carcinoma, small-cell lung or ovarian cancer and breast cancer, respectively. Another case of considerable interest is thapsigargin isolated from the umbelliferous plant, *Thapsia garganica* L. (*Apiaceae* Lindl.) from Mediterranean island of Ibiza [97]. Thapsigargin, induces apoptosis in prostate cancer cells and synthetic prodrug derived from it called "G-202" is in Phase II clinical trials. Silvestrol isolated from the fruits of *Aglaila sylvestre* Roemer from *Meliaceae* Juss. family [98], exhibit cytotoxicity against lung and breast cancer cell lines [65]. Its synthetic analogue 4′ desmethoxyepisilvestrol is cytotoxic against lung and colon cancer cell lines. Two alkaloids, schischkinnin and montamine isolated recently from the seeds of *Centaurea schischkinii* Tzvelev and *Centaurea montana* L. [99,100] exhibit significant cytotoxicity against HCCLs (human colon cancer cell lines). The essential oil of *Salvia officinalis* L., most popular folk remedy in Middle East known for its antitumor effects, which contains monoterpenes thujone, β-pinene, and 1,8 cineol was shown to be cytotoxic against squamous human cell carcinoma cell line of the oral cavity [101]. There are many other natural substances like extracts of unknown composition from *Colubrina macrocarpa* (Cav.) G. Don., *Hemiangium excelsum* (Kunth) A.C. Sm*, Acacia pennatula* (Schltdl. & Cham.) Benth., *Commiphora opobalsamum* Jacq.*, Astragalus* L.*, Paris polyphylla* Sm.*, Teucrium polium* L.*, Pistacia lentiscus* L. used as anticancer remedies on folk

ising anticancer activity against a number of different cell lines.

52 Drug Discovery

medicine in China, Israel, Plestina, Saudi Arabia etc.

Animal venoms and toxins which has been used as therapeutics in ancient Ayurvedic, Unani, Chinese folk medicine as well as in Homeopathy are also screened. Venoms of snakes, scorpions, toads, frogs and their derivatives protein or non-protein toxins, peptides, enzymes are promising and show some potential in cancer treatment. Léon Charles Albert Calmette (1863-1933) a French physician, bacteriologist and immunologist, was the first to describe an antitumor effect of the venom of Indian cobra Naja naja sp. on adenocarcinoma cells. Thereafter many reports have established the anticancer potential of venoms of different species of Elapidae, Viperidae, Crotalidae snakes [102-107] and Hydrophis spimlis sea snake [108,109] and assigned it to phospholipase activity. Scorpions venom has been used by traditional and folk medicine in India, China, Africa and Cuba. Chinese red scorpion (Buthus martensi Karsch) venom and skin extracts, known as Chan Su in China and Senso in Japan, have been used by traditional Chinese medicine for as long as 2000 years also as anti-leukaemia agents. 4',6 diamidino-2-phenylindole extracted from Buthus martensi Karsch induced cell apoptosis in malignant glioma cells in vitro [110], while serine proteinase and hylauronidase have prom‐ ising anticancer activity against a number of different cell lines including breast ones [111]. Bengalin protein isolated from Hindu black scorpion is suspected to have anti-leukemic properties [112]. Chlorotoxin and Charybdotoxin, 36- and 37-amino acid peptides, respectively isolated from the venom of death stalker scorpion (Leiurus quinqestriatus Hebraeus) are promising for the treatment of several types of cancers including glioma and human breast cancer [113,114]. The anticancer effect of the venom of Cuba red scorpions (Rhopalurus junceus) was discovered 20 years ago in Guantanamo, but after 15 years of studies Vidatox drug was announced in 2011. The skin extract from Hindu toads (Bufo melanostictus, Bufo gargarizans Cantor), Chan Su used by Chinese traditional medicine, was discovered to contain a few bufadineolides showing specific activity against human leukemic, liver carcinoma and melanoma cell lines. Species belonging to the families Bufonidae (toads), Lampyridae (fireflies) and Colubridae (snakes) as well as mammalian tissues contain bufadienolides, but the richest source of them are toad species. Although all the bufadienolides showed potent cytotoxicity in vitro, but the evidence of their activity in vivo is limited to human hepatocellular carcinoma and HeLa human cancer cells in mice and require further investigation.

Some hope rises with the use of minerals as a source of anticancers drugs. Most important example is sodium bicarbonate, NaHCO3, which was originally derived from Nahcolite (thermokalite) carbonate mineral. The ancient Egyptians used natural natron, a mixture of sodium carbonate decahydrate, and sodium bicarbonate as a soap and embalming tool. Recently it has been shown that sodium bicarbonate administered orally causes a selective 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 apoptosis in malignant mesothelioma and sarcoma cells [117].

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,

Anticancer Drug Discovery — From Serendipity to Rational Design

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

55

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

> **CELL CYCLE NON SPECYFIC AGENTS (CCNSA) ALKYLATING AGENTS CYTOTOXIC ANTIBIOTICS PLATINUM COMPOUNDS NATURAL PRODUCTS CELL CYCLE SPECYFIC AGENTS (CCSA)**

> > **S 2%**

**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

**G2** 

 **40% (DNA - synthesis)** ANTIMETABOLITES ANTIFOLIATES

> **39% G (Pre-synthesis) 1**

 **19% (Pre-mitosis)** ANTIBIOTICS - BLEOMYCIN

**M**

**(Mitosis)** PLANT ALKALOIDS

> **G (Resting) 0**

which leads to mutations, even permanent ones.

classification as definite carcinogens by IARC [3].

only during a specific phase.

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 development.
