*4.4.1. Antifolates*

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 discovered synthetic anticancer drug i.e. first true chemotherapeutic. Starting from the 1950s, Methotrexate has replaced the more toxic aminopterin and is still in widespread clinical use. In general folate antagonists mechanism of action is linked with competing with folates for uptake into cells and inhibition of the formation of folate co-enzymes or reactions that are mediated by them. Only one of those mechanisms is clinically important, it is the prevention of formation of tetrahydrofolate by inhibition of the enzymes: dihydrofolate reductase (DHFR) or thymidylate synthase (TS). Methotrexate inhibits only DHFR, while a new-generation antifolate Pemetrexed developed by Edward C. Taylor in 1992 inhibits DHFR, TS and also transformylases (GAR and AICAR), but primarily acts as a TS inhibitor. Thus both act similarly by hindering enzymes needed for de novo synthesis of the thymidine and purine nucleotides but show different spectrum of activity. Methotrexate is effective mainly in the treatment of leukaemia, lymphoma and choriocarcinoma but also cancers of breast, head and neck, colorectal, osteosarcoma and bladder, while Pemetrexed is approved for the treatment of mesothelioma and non–small cell lung cancer, active in solid tumours treatment, especially those drug resistant. The risk connected with the use of Methotrexate classified as group 3 agent in IARC classification is smaller than that of alkylating agents [3].

selected by Dennis A. Carson as the most potent enzyme adenosine deaminase (ADA) inhibitor from many candidate congeners. Pentostatin, which also inhibits ADA and similarly to 2CDA is active in hairy cell leukaemia and chronic lymphocytic leukaemia was synthesised by Hollis D.H. Showalter and David C. Baker in 1983. Recently, three new purine antimetabolites nerlabine, clofarabine, and forodesine have been found highly promising. Although these compounds belong to purine antimetabolites and reveal activity against specific types of leukaemia, they differ in metabolic properties and mechanism of action. As long ago as in 1964 Elmer Reist and Leon Goodman synthesized 9-β-Darabinofuranosyl guanine (ara-G), which despite of its antitumor properties in in-vitro canine leukaemia models evaluated by Elion & Kurtzberg was rejected because of inadequate solubility. In 1988, nelarabine, the 6-methoxy derivative of Ara-G, which is 10-fold more soluble than Ara-G, was synthesized by Thomas A. Krenitsky. In 2012, 48 years after synthesis of Ara-G, Nelarabine entered phase II of clinical studies with indication to T-cell acute lymphoblastic leukaemia or T-cell lymphoblastic lymphoma treatment. Clofarabine, a hybrid of Fludarabine and Cladribine, was synthesised by Mongomery in 1992. It also recently entered phase II of clinical studies with indication as antileukemic agent active in acute lymphoblastic leukaemia as well as in myeloid disorders in paediatrics. The third intensively studied purine antimetabolite is forodesine, synthesised by Peter C. Tyler and Vern L. Schramm in 1998. Forodesine, which is not incorporated into DNA and has unexplored mechanism of action is effective for the treatment of relapsed B-cell chronic

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In the 1950s Robert Duschinsky synthesised the first pyrimidine analogue, 5-fluorouracil (5- FU). His discovery was based on the observation of the role of greater uptake of uracil in rat hepatoma metabolism, thus it was the first known case of "targeted" studies. 5-FU introduced into the clinic in 1957 by Charles Heidelberger has broad-spectrum activity against nonhematologic cancers, thus is now widely applied for treatment of many kinds of solid tumours of breast, head and neck, adrenal, pancreatic, gastric, colon, rectal, oesophageal, liver and G-U (bladder, penile, vulva, prostate). Even nowadays, 5-FU apart of its analogue Floxuridine, remains a fundamental drug in the treatment of colorectal cancer. Discovery of 5-FU was not only the first example of targeted studies but also the first targeted therapy, which later attracted much attention in current cancer drug development. However, the target in this case was understood not as a molecular target but as a biochemical pathway. In 1950 two spongonucleosides (spongouridine and spongothymidine) were isolated by Werner Bergman and Robert Feeney from a Caribbean sponge *Cryptotethya crypta*. It inspired Richard Walwick, Walden Roberts, and Charles Dekker to synthesise Cytarabine and Vidarabine in 1959. In 1964, John Evans tested activity of Cytarabine using in-vitro murine S180 model while four years later Rose Ruth Ellison introduced it into clinic for the B-cell leukaemia treatment. Cytarabine is effective in acute non-lymphocytic, lymphocytic, myelogenous, chronic myelocytic leukae‐ mias, as well as leptominingeal carcinomatosis and non-Hodgkin's lymphoma, Other pyri‐ midine antagonists include Capecitabine, which is an oral 5-FU pro-drug adjuvant in colon and breast metastasis therapy, Gemcitabine which is a prodrug of Cytarabine, effective in pancreatic, metastatic breast, bladder, ovarian and non-small cell lung cancers and Decitabine, used in myeloplastic syndrome. The antimetabolites of purines and pyrimidine compounds

lymphocytic leukaemia.

#### *4.4.2. Antimetabolites*

The discovery of nitrogen mustard and Methotrexate and their success in medical applications was a breakthrough and stimulated the search for the other antimetabolites as well as new classes of cell cycle specific synthetic anticancer drugs [4,56,119]. In 1944, Richard O. Roblin and James W. Clapp synthesised 8-azaguanine (8-AZA), while 5 years later George W. Kidder and Virginia C., reported that 8-AZA was a guanine antagonist in the metabolism of *Tetrahy‐ mena geleii* (S) (*Colpidium campylum* L.) and inhibited the growth of transplanted mammary adenosarcoma in mice. Since 1944, George Herbert Hitchings (1905-1998) and Gertrude Belle Elion (1918-1999) have investigated the role of purines in nucleic acid metabolism and methods to prevent them from being incorporated to DNA synthesis along the metabolic pathway that would lead to interruption of cell reproduction. By the early 1950s, Hitchings & Elion synthe‐ sized more than 100 purine analogues including 2,6-diaminopurine, 6-thioguanine (6-TG), 6 mercaptopurine (6-MP) and Azathioprine (AZA) as a result of their rational approach to drug development. 6-TG, 6-MP and AZA although categorized as growth inhibitory antimetabo‐ lites, exert their functions more like a genotoxic methylating agents, such as alkylating drug Temozolomide, which methylates DNA. 6-MP, one of the early analogues, is widely used not only for acute leukaemias but also in gout and herpes viral infections, and as immunosup‐ pressive agents in the organ transplantations, 6-TG is predominantly used as antileukaemic agent, while AZA as an immunosuppressive. The discovery of a few purine analogues Fludarabine (FLU), Cladribine (2CDA), and Pentostatin (DCF) was a result of further targeted studies performed within a NIH programme. Fludarabine discovered by John Montgomery and Kathleen Hewson in 1968 was the first halogenated ribonucleotide reductase inhibitor, a new-generation pro-drug of the purine class successfully used in treating refractory chronic lymphocytic and chronic B cell leukaemias, non-Hodgkin's lymphoma and T-cell lymphoma. Cladribine, resembling deoxyadenosine and remarkably active in hairy cell leukaemia was synthesised in 1972 by L.F. Christensen, A. Broom, M.J. Robins, and A.J. Bloch and in 1978 selected by Dennis A. Carson as the most potent enzyme adenosine deaminase (ADA) inhibitor from many candidate congeners. Pentostatin, which also inhibits ADA and similarly to 2CDA is active in hairy cell leukaemia and chronic lymphocytic leukaemia was synthesised by Hollis D.H. Showalter and David C. Baker in 1983. Recently, three new purine antimetabolites nerlabine, clofarabine, and forodesine have been found highly promising. Although these compounds belong to purine antimetabolites and reveal activity against specific types of leukaemia, they differ in metabolic properties and mechanism of action. As long ago as in 1964 Elmer Reist and Leon Goodman synthesized 9-β-Darabinofuranosyl guanine (ara-G), which despite of its antitumor properties in in-vitro canine leukaemia models evaluated by Elion & Kurtzberg was rejected because of inadequate solubility. In 1988, nelarabine, the 6-methoxy derivative of Ara-G, which is 10-fold more soluble than Ara-G, was synthesized by Thomas A. Krenitsky. In 2012, 48 years after synthesis of Ara-G, Nelarabine entered phase II of clinical studies with indication to T-cell acute lymphoblastic leukaemia or T-cell lymphoblastic lymphoma treatment. Clofarabine, a hybrid of Fludarabine and Cladribine, was synthesised by Mongomery in 1992. It also recently entered phase II of clinical studies with indication as antileukemic agent active in acute lymphoblastic leukaemia as well as in myeloid disorders in paediatrics. The third intensively studied purine antimetabolite is forodesine, synthesised by Peter C. Tyler and Vern L. Schramm in 1998. Forodesine, which is not incorporated into DNA and has unexplored mechanism of action is effective for the treatment of relapsed B-cell chronic lymphocytic leukaemia.

discovered synthetic anticancer drug i.e. first true chemotherapeutic. Starting from the 1950s, Methotrexate has replaced the more toxic aminopterin and is still in widespread clinical use. In general folate antagonists mechanism of action is linked with competing with folates for uptake into cells and inhibition of the formation of folate co-enzymes or reactions that are mediated by them. Only one of those mechanisms is clinically important, it is the prevention of formation of tetrahydrofolate by inhibition of the enzymes: dihydrofolate reductase (DHFR) or thymidylate synthase (TS). Methotrexate inhibits only DHFR, while a new-generation antifolate Pemetrexed developed by Edward C. Taylor in 1992 inhibits DHFR, TS and also transformylases (GAR and AICAR), but primarily acts as a TS inhibitor. Thus both act similarly by hindering enzymes needed for de novo synthesis of the thymidine and purine nucleotides but show different spectrum of activity. Methotrexate is effective mainly in the treatment of leukaemia, lymphoma and choriocarcinoma but also cancers of breast, head and neck, colorectal, osteosarcoma and bladder, while Pemetrexed is approved for the treatment of mesothelioma and non–small cell lung cancer, active in solid tumours treatment, especially those drug resistant. The risk connected with the use of Methotrexate classified as group 3

The discovery of nitrogen mustard and Methotrexate and their success in medical applications was a breakthrough and stimulated the search for the other antimetabolites as well as new classes of cell cycle specific synthetic anticancer drugs [4,56,119]. In 1944, Richard O. Roblin and James W. Clapp synthesised 8-azaguanine (8-AZA), while 5 years later George W. Kidder and Virginia C., reported that 8-AZA was a guanine antagonist in the metabolism of *Tetrahy‐ mena geleii* (S) (*Colpidium campylum* L.) and inhibited the growth of transplanted mammary adenosarcoma in mice. Since 1944, George Herbert Hitchings (1905-1998) and Gertrude Belle Elion (1918-1999) have investigated the role of purines in nucleic acid metabolism and methods to prevent them from being incorporated to DNA synthesis along the metabolic pathway that would lead to interruption of cell reproduction. By the early 1950s, Hitchings & Elion synthe‐ sized more than 100 purine analogues including 2,6-diaminopurine, 6-thioguanine (6-TG), 6 mercaptopurine (6-MP) and Azathioprine (AZA) as a result of their rational approach to drug development. 6-TG, 6-MP and AZA although categorized as growth inhibitory antimetabo‐ lites, exert their functions more like a genotoxic methylating agents, such as alkylating drug Temozolomide, which methylates DNA. 6-MP, one of the early analogues, is widely used not only for acute leukaemias but also in gout and herpes viral infections, and as immunosup‐ pressive agents in the organ transplantations, 6-TG is predominantly used as antileukaemic agent, while AZA as an immunosuppressive. The discovery of a few purine analogues Fludarabine (FLU), Cladribine (2CDA), and Pentostatin (DCF) was a result of further targeted studies performed within a NIH programme. Fludarabine discovered by John Montgomery and Kathleen Hewson in 1968 was the first halogenated ribonucleotide reductase inhibitor, a new-generation pro-drug of the purine class successfully used in treating refractory chronic lymphocytic and chronic B cell leukaemias, non-Hodgkin's lymphoma and T-cell lymphoma. Cladribine, resembling deoxyadenosine and remarkably active in hairy cell leukaemia was synthesised in 1972 by L.F. Christensen, A. Broom, M.J. Robins, and A.J. Bloch and in 1978

agent in IARC classification is smaller than that of alkylating agents [3].

*4.4.2. Antimetabolites*

58 Drug Discovery

In the 1950s Robert Duschinsky synthesised the first pyrimidine analogue, 5-fluorouracil (5- FU). His discovery was based on the observation of the role of greater uptake of uracil in rat hepatoma metabolism, thus it was the first known case of "targeted" studies. 5-FU introduced into the clinic in 1957 by Charles Heidelberger has broad-spectrum activity against nonhematologic cancers, thus is now widely applied for treatment of many kinds of solid tumours of breast, head and neck, adrenal, pancreatic, gastric, colon, rectal, oesophageal, liver and G-U (bladder, penile, vulva, prostate). Even nowadays, 5-FU apart of its analogue Floxuridine, remains a fundamental drug in the treatment of colorectal cancer. Discovery of 5-FU was not only the first example of targeted studies but also the first targeted therapy, which later attracted much attention in current cancer drug development. However, the target in this case was understood not as a molecular target but as a biochemical pathway. In 1950 two spongonucleosides (spongouridine and spongothymidine) were isolated by Werner Bergman and Robert Feeney from a Caribbean sponge *Cryptotethya crypta*. It inspired Richard Walwick, Walden Roberts, and Charles Dekker to synthesise Cytarabine and Vidarabine in 1959. In 1964, John Evans tested activity of Cytarabine using in-vitro murine S180 model while four years later Rose Ruth Ellison introduced it into clinic for the B-cell leukaemia treatment. Cytarabine is effective in acute non-lymphocytic, lymphocytic, myelogenous, chronic myelocytic leukae‐ mias, as well as leptominingeal carcinomatosis and non-Hodgkin's lymphoma, Other pyri‐ midine antagonists include Capecitabine, which is an oral 5-FU pro-drug adjuvant in colon and breast metastasis therapy, Gemcitabine which is a prodrug of Cytarabine, effective in pancreatic, metastatic breast, bladder, ovarian and non-small cell lung cancers and Decitabine, used in myeloplastic syndrome. The antimetabolites of purines and pyrimidine compounds acts on a cancer cell in S phase of its life cycle, Fig.2 and are classified as a group 3 agents in IARC classification.

chemotherapeutics but also resulted in the use of drug combinations. In 1965 Emil Frei and Emil J. Freireich developed the new treatment program for children leukaemia known as "VAMP" (Nethotrexate, 6-MP, Vincristine and Prednisone). The use of multiple drugs: Methotrexate, which disrupt folic-acid uptake, 6-MP which inhibits synthesis of purine, both critical in cell division, Vincristine which interfered with cell division by binding to spindle protein and Prednisone, anti-inflammatory steroid resulted in the remission rate level as high as 60%. Further modifications like "MOMP" (nitrogen mustard, Vincristine, Methotrexate, and Prednisone), "MOPP" (Procarbazine, nitrogen mustard, Vincristine, and Prednisone) and C-MOPP (Procarbazine, Cyclophosphamide, Vincristine, and Prednisone) resulted in the 80% complete remission rate in advanced Hodgkin's disease in the 1970s. On the basis of the above mentioned first programs dedicated exclusively to leukaemias many modifications - e.g. four drug EBVP (Epirubicin, Bleomycin, Vinblastine, Prednisone) and ABVD (Adriamycin, Bleomycin, Vinblastine and Dacarbazine) and six-drug STANFORD-V (Cyclophosphamide/ Mechlorethamine/Ifosfamide, Doxorubicin, Vinblastine, Vincristine, Bleomycin, Etoposide) have been developed. Nowadays, some cancer diseases like Hodgkin's or acute lymphocytic leukaemia are curable in 90% within the modern protocols using aggressive chemotherapy programs. Despite the reasonable position of classical chemotherapeutics in multidrug combined regimens, their capabilities inevitably decrease because of multidrug resistance (a major factor in the failure of many forms of treatment) and secondary effects (adverse or paradoxically carcinogenic). Significant limitation is also the need for multiple chemotherapy

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in long-term, sequential multidrug regimens and in-hospital administration.

At the beginning of the 20th c., Paul Ehrlich postulated the idea of a "Magic Bullet" (Zauber‐ kugel) i.e. drugs that reach directly intended cell-structural targets. To some extent this idea is the driving force behind the development of modern targeted chemotherapeutics. Conven‐ tional chemotherapeutics are cytotoxic, but affect all cells and work in the so-called statistical manner. Because cancer cells multiply faster than normal, the cancerous cells are killed by the drug with a higher ratio than the normal ones, which are not spared. Therefore the principal criterion applied in the modern anticancer drug design is the principle of selective toxicity, which require activity restricted exclusively to the cancer cells. From the point of view of selective activity directed on tumour cells and mechanisms of carcinogenesis, a few different classes of modern drugs can be distinguished: inhibitors of cytokine stimulating cell prolifer‐ ation, dissociation, motility; cytokine receptor blockers; intracellular kinases inhibitors; transcription factors inhibitors; cell cycle inhibitors; cell adhesion inhibitors and proteasome

Short-lived hopes were raised in the 1990s at the discovery of the inhibitors of angiogenesis, which hold back the growth of capillary vessels in cancer. Avastin, humanized monoclonal antibody, discovered by Napoleone Ferrara in 1997 was the first drug from this class approved by FDA in 2004 to use for several types of metastatic cancer, but the approval of the breast cancer indication was revoked in 2011. Two other inhibitors of angiogenesis include Cetuxi‐ mab invented by Joseph Schlessinger, Michael Sela in 1988, approved for by FDA in 2009 to

*4.4.5. Modern chemotherapeutics*

inhibitors.

### *4.4.3. Plant alkaloids*

A true breakthrough in chemotherapeutics came by in the 1950s as the discovery of the activity of plant alkaloids from Madagascar periwinkle plant *Vinca rosea* (*Catharanthus roseus* (L.) G. Don) by Canadian scientists Robert Laing Noble, Charles T. Beer and Gordon Sloboda [56]. The vinca alkaloids extracted from *Vinca rosea* consist of a subset of structurally similar compounds comprising two multiringed units, vindoline and catharanthine. Initially, they were investigated because of putative hypoglycaemic properties, but strong marrow suppres‐ sion observed in rats and significant antileukaemic effects *in vitro* decided about their clinical use shortly after the discovery of their properties, in 1959. Nowadays, vinca alkaloids are produced synthetically and only four major ones - Vinblastine, Vincristine, Vinorelbine and Vindesine - are in the oncological clinical use. Vinblastine is most often applied in Hodgkin's disease, non-Hodgkin's lymphoma, breast cancer, and germ cell tumours, Vincristine is effective against leukaemia and Hodgkin's, Vinorelbine has significant antitumor activity in patients with breast cancer and antiproliferation effects on osteosarcoma, while Vindesine is used in the treatment of leukaemia, lymphoma, melanoma, breast cancer, and lung cancer. All vinca alkaloids have a unique mechanism of action; they bind to the microtubular proteins of the mitotic spindle, which leads to crystallization of the microtubule and mitotic arrest or apoptosis.

Another group of novel cytotoxic agents from plant alkaloids, taxane diterpenes, was discov‐ ered during long-term screening in the 1970s. The main compound in this class, Paclitaxel, was the first taxane introduced into clinical practice for the treatment of recurrent ovarian cancer metastatic, breast cancer, often in combination with Cisplatin. Nowadays, Paclitaxel is totally synthesized, but less popular than vinca because of its poor solubility. Both plant alkaloids vinca and taxanes are mitotic inhibitors M phase specific, Fig. 2, but they act in different ways. Their principal mechanism of action is the disruption of microtubule function, but in contrast to the vinca alkaloids, taxanes do not destroy mitotic spindles. The plant alkaloids are classified as a group 3 agents in IARC classification [3].

#### *4.4.4. Combination regimens*

In 2005, conventional chemotherapeutics still made the majority of the Top 20 Cancer Thera‐ peutics. Their popularity was dictated not only by wide spectrum of activity and also long history of use in oncology but also their key role in the multidrug treatment programs. In the early 1960s, single alkylating agents were basic for all cancer treatment programmes. Although the remissions were observed for example up to 25% in advanced Hodgkin's disease but they were still incomplete and temporary. Increasing resistance of the cancer cells to classical drugs and numerous side effects forced new strategies. After Jacob Furth and Morton Kahn discovery that a single leukemic cell was sufficient to cause the death of an animal, Howard E. Skipper formulated "Cell Kill" hypothesis, according to which a given dose of drug is able to kill only a constant fraction of tumour cells. This hypothesis favoured search for more aggressive chemotherapeutics but also resulted in the use of drug combinations. In 1965 Emil Frei and Emil J. Freireich developed the new treatment program for children leukaemia known as "VAMP" (Nethotrexate, 6-MP, Vincristine and Prednisone). The use of multiple drugs: Methotrexate, which disrupt folic-acid uptake, 6-MP which inhibits synthesis of purine, both critical in cell division, Vincristine which interfered with cell division by binding to spindle protein and Prednisone, anti-inflammatory steroid resulted in the remission rate level as high as 60%. Further modifications like "MOMP" (nitrogen mustard, Vincristine, Methotrexate, and Prednisone), "MOPP" (Procarbazine, nitrogen mustard, Vincristine, and Prednisone) and C-MOPP (Procarbazine, Cyclophosphamide, Vincristine, and Prednisone) resulted in the 80% complete remission rate in advanced Hodgkin's disease in the 1970s. On the basis of the above mentioned first programs dedicated exclusively to leukaemias many modifications - e.g. four drug EBVP (Epirubicin, Bleomycin, Vinblastine, Prednisone) and ABVD (Adriamycin, Bleomycin, Vinblastine and Dacarbazine) and six-drug STANFORD-V (Cyclophosphamide/ Mechlorethamine/Ifosfamide, Doxorubicin, Vinblastine, Vincristine, Bleomycin, Etoposide) have been developed. Nowadays, some cancer diseases like Hodgkin's or acute lymphocytic leukaemia are curable in 90% within the modern protocols using aggressive chemotherapy programs. Despite the reasonable position of classical chemotherapeutics in multidrug combined regimens, their capabilities inevitably decrease because of multidrug resistance (a major factor in the failure of many forms of treatment) and secondary effects (adverse or paradoxically carcinogenic). Significant limitation is also the need for multiple chemotherapy in long-term, sequential multidrug regimens and in-hospital administration.

#### *4.4.5. Modern chemotherapeutics*

acts on a cancer cell in S phase of its life cycle, Fig.2 and are classified as a group 3 agents in

A true breakthrough in chemotherapeutics came by in the 1950s as the discovery of the activity of plant alkaloids from Madagascar periwinkle plant *Vinca rosea* (*Catharanthus roseus* (L.) G. Don) by Canadian scientists Robert Laing Noble, Charles T. Beer and Gordon Sloboda [56]. The vinca alkaloids extracted from *Vinca rosea* consist of a subset of structurally similar compounds comprising two multiringed units, vindoline and catharanthine. Initially, they were investigated because of putative hypoglycaemic properties, but strong marrow suppres‐ sion observed in rats and significant antileukaemic effects *in vitro* decided about their clinical use shortly after the discovery of their properties, in 1959. Nowadays, vinca alkaloids are produced synthetically and only four major ones - Vinblastine, Vincristine, Vinorelbine and Vindesine - are in the oncological clinical use. Vinblastine is most often applied in Hodgkin's disease, non-Hodgkin's lymphoma, breast cancer, and germ cell tumours, Vincristine is effective against leukaemia and Hodgkin's, Vinorelbine has significant antitumor activity in patients with breast cancer and antiproliferation effects on osteosarcoma, while Vindesine is used in the treatment of leukaemia, lymphoma, melanoma, breast cancer, and lung cancer. All vinca alkaloids have a unique mechanism of action; they bind to the microtubular proteins of the mitotic spindle, which leads to crystallization of the microtubule and mitotic arrest or

Another group of novel cytotoxic agents from plant alkaloids, taxane diterpenes, was discov‐ ered during long-term screening in the 1970s. The main compound in this class, Paclitaxel, was the first taxane introduced into clinical practice for the treatment of recurrent ovarian cancer metastatic, breast cancer, often in combination with Cisplatin. Nowadays, Paclitaxel is totally synthesized, but less popular than vinca because of its poor solubility. Both plant alkaloids vinca and taxanes are mitotic inhibitors M phase specific, Fig. 2, but they act in different ways. Their principal mechanism of action is the disruption of microtubule function, but in contrast to the vinca alkaloids, taxanes do not destroy mitotic spindles. The plant alkaloids are classified

In 2005, conventional chemotherapeutics still made the majority of the Top 20 Cancer Thera‐ peutics. Their popularity was dictated not only by wide spectrum of activity and also long history of use in oncology but also their key role in the multidrug treatment programs. In the early 1960s, single alkylating agents were basic for all cancer treatment programmes. Although the remissions were observed for example up to 25% in advanced Hodgkin's disease but they were still incomplete and temporary. Increasing resistance of the cancer cells to classical drugs and numerous side effects forced new strategies. After Jacob Furth and Morton Kahn discovery that a single leukemic cell was sufficient to cause the death of an animal, Howard E. Skipper formulated "Cell Kill" hypothesis, according to which a given dose of drug is able to kill only a constant fraction of tumour cells. This hypothesis favoured search for more aggressive

IARC classification.

60 Drug Discovery

*4.4.3. Plant alkaloids*

apoptosis.

as a group 3 agents in IARC classification [3].

*4.4.4. Combination regimens*

At the beginning of the 20th c., Paul Ehrlich postulated the idea of a "Magic Bullet" (Zauber‐ kugel) i.e. drugs that reach directly intended cell-structural targets. To some extent this idea is the driving force behind the development of modern targeted chemotherapeutics. Conven‐ tional chemotherapeutics are cytotoxic, but affect all cells and work in the so-called statistical manner. Because cancer cells multiply faster than normal, the cancerous cells are killed by the drug with a higher ratio than the normal ones, which are not spared. Therefore the principal criterion applied in the modern anticancer drug design is the principle of selective toxicity, which require activity restricted exclusively to the cancer cells. From the point of view of selective activity directed on tumour cells and mechanisms of carcinogenesis, a few different classes of modern drugs can be distinguished: inhibitors of cytokine stimulating cell prolifer‐ ation, dissociation, motility; cytokine receptor blockers; intracellular kinases inhibitors; transcription factors inhibitors; cell cycle inhibitors; cell adhesion inhibitors and proteasome inhibitors.

Short-lived hopes were raised in the 1990s at the discovery of the inhibitors of angiogenesis, which hold back the growth of capillary vessels in cancer. Avastin, humanized monoclonal antibody, discovered by Napoleone Ferrara in 1997 was the first drug from this class approved by FDA in 2004 to use for several types of metastatic cancer, but the approval of the breast cancer indication was revoked in 2011. Two other inhibitors of angiogenesis include Cetuxi‐ mab invented by Joseph Schlessinger, Michael Sela in 1988, approved for by FDA in 2009 to use in colorectal cancer therapy and Sunitinib discovered by Joseph Schlessinger and Axel Ullrich approved by FDA in 2006 to use for renal cell carcinoma and gastrointestinal stromal tumour. Recent progress in genetic sequencing has led to the discovery of Vemurafenib by Fritz Hoffmann, approved by FDA in 2011. It targets the B-Raf gene that signals the growth of new blood cells in melanoma tumours, which are extremely difficult to treat.

General Chemotherapy Drugs (the alkylating agents, anti-neoplastics, anti-metabolites), (2) Steroids, (3) Bisphosphonates, (4) Hormone therapies and (5) Biological therapies/Immuno‐ therapy. This modern classification reflects the fact that anticancer drugs evolved from classical chemotherapeutics discovered mostly by serendipity to drugs acting directly against abnormal proteins in cancer cells designed by rational drug design. All of them have remarkable influence on the growth of cancer cells and on the mechanisms whereby cells replicate,

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Current research are so multidirectional that it is impossible to discuss all of them in a short chapter. New directions in this field include the search for the improved pharmaceutical forms, new analogues of currently used drugs, new chemical compounds (natural or synthetic) of anticancer activity, selective anticancer agents (acting on the basis of pathophysiological mechanisms), the search for drugs among old-known drugs currently used for other indica‐ tions than cancer, search for the methods of precise delivering the anticancer drugs to cancer tissue and stroma or to stimulate the immune system to generate anti-tumor immune responses

Even nowadays precursors or generic drugs are frequently discovered by serendipity, while their analogues are developed by purely rational design. Often the newly synthesised drug proved effective in quite different than expected applications. For example Aminoglutethi‐ mide was found to be effective in breast cancer treatment instead of being an antiepileptic, Cisplatin, an electrolysis product, was discovered to be cytotoxic or Tamoxifen antiestrogenic activity of *cis*-isomer was discovered as unexpected bonus in the search for drugs to treat mania in bipolar disorder. Sometimes surprisingly a new field of activity is revealed for a long known drug. For example potassium-sparing diuretic Amiloride is effective in glioma; sedative Thalidomide, linked to birth defects, slows the propagation prostate cancer; Tebrophen, antiviral drug, slows the propagation of breast cancer; S-dimethylarsin-gluthathione, an organic form of arsenic, slow solid tumours expansion; anti-epileptic Valproic and Rapamycin, immunosuppressor in organ transplantation are valuable in antitumor therapy; Gossypol, potential male contraceptive overcome resistance to Cisplatin; antimalarial chloroquine may address a critical cell nutrition issue with proliferating cancer cells while insecticide benzoyl‐ phenylurea helps understand the microtubule assembly process important for growth of the pancreatic cancer cell. Anticancer properties have been ascertained to be shown by aspirin used commonly as analgesic, antipyretic and an anti-inflammatory medication, 9-aminoacri‐ dine used as an antiprotozoa and antibacterial agent and Quinacrine commonly used antima‐ larial drug. The use of long know drugs fasten modern process of drug discovery, which involves the identification of candidates, synthesis, characterization, screening, and assays for therapeutic efficacy and proceeds through many stages including discovery, product charac‐ terisation, formulation, pharmacokinetics, preclinical toxicology testing and IND (Investiga‐ tional New Drug) application, bioanalytical testing and clinical trials. The new drugs (new completely or a long known one) often have many adverse effects. Sometimes in the final step the new drug proves toxic, ineffective or even carcinogenic. Many widely applied chemother‐ apeutic anticancer agents (nitrogen mustards, HN2 and HN3, treatamine, Chlorambucil,

transmit, and translate genetic information.

and protect against cancer.

Recently widespread attention has been given to inhibitors of protein kinases, enzymes that catalyze phosphorylation reactions, a principal mechanism of signal transduction governing various cellular processes including growth, division, migration and apoptosis. Imatinib, developed in the late 1990s by Nicholas Lydon, introduced to clinic by Brian Druker and approved to treat chronic myelogenous leukaemia by FDA in 2001 was the first drug of this new class of small active molecules. It inhibits the oncogene BCR-ABL1 and blocks the signals for cell proliferation, controlling tumour growth. Many imatinib analogues: including Nilotnib, Dasatinib, Bosutinib, Ponatinib, Bafetinib were obtained further by rational drug design. Gefitinib invented by ASTRA/ZENECA (approved by FDA in 2003 but withdrawn in 2005) and Erlotinib invented by OSI (approved by FDA in 2004), were the first selective inhibitors of epidermal growth factor receptor among the kinase inhibitors used for treatment of lung cancer.

Monoclonal antibodies that allow the cytotoxin to reach a target required to kill the malignant cells (induce apoptosis), without harming normal cells belong to the unique class of chemo‐ immunotherapeutics. In contrast to small molecule drugs which have a direct impact on their targets, the monoclonal antibodies stimulate the immune system i.e. re-direct targets to the immune system. Most popular includes: Gemtuzumab ozogamicin invented by Wyeth Ayerst and used for the treatment of acute myelogenous leukaemia, but withdrawn from the market in 2010 due to its toxicity, Ibritumomab tiuxetan developed by IDEC Pharmaceuticals used for the treatment of non-Hodgkin's lymphoma, but known to cause serious side effects, Panitu‐ mumab used for the treatment of colon cancer but ineffective, Rituximab developed by IDEC Pharmaceuticals and still used for the treatment of B-cell non-Hodgkin's lymphoma and tositumomab developed by Mark Kaminski and Richard Wahl used for the treatment of non-Hodgkin's lymphoma (mainly follicular lymphoma), currently in the clinical trials.

Although modern targeted therapies provide a new approach to cancer therapy and similarly to conventional ones are able to suppress tumour growth, but they also have drawbacks and limitations, while true cancer treatment is still a challenge for oncology.
