1.2 Plants As Source of Anti-Cancer Agents

Chapter 1

1.1Introduction

Natural products (NPs) extracted from speckled life formsof plants, animals, marine organisms or micro-organisms are the evolutionary shaped molecules with profoundmedicinalsignificance.The biosynthetic engine of nature produces myriadNPsin almost incredible chemical diversity with distinct biological properties. These NPs are by and large stereochemically complex with diverse functional groups that explicitlyinteract with biological targets thus make them valuable as health products or structural templates for drug discovery. Rightly said by Aristotle that “Nature does nothing without purpose or uselessly” the world of plants, and indeed all natural sources, represents a virtually untapped pool of novel drugs awaiting imaginative and progressive organisation. Inearly 1900s, when the synthetic chemistry was atinfancystage, more than80% drugs/medicines were generally obtained from plants. Over the past centuries the countries like USA,China, India, Egypt, and Greece etc., emerged with different plant based traditional medicine systems.The continuous growth in the knowledge of plant, animal, and microbial species lends support to constantdiscovery ofnovel secondary metabolites from these sources.Even today 80% of the world’s population mainly uses traditional medicines developed from plant-based compounds for health care and therapeutic purposes (World Health Organisation, 2008). The significant contribution of these plant-derived drugs to medicine even in present era of science and technology is also evident by theimmense therapeutic potential of Morphine, Quinine, Digitalis, Atropine, Reserpine, Vincristine, Vinblastine etc.

The exploration ofnatural product sources associated with anti-cancer and other biological properties are studied for several principal reasons. NPs present unmatched chemical diversity and structural complexity.Several useful drugs viz., Quinine, Morphine andPenicillin used against malaria, dulling pain and infectious diseases respectively were all nature based. Further they increase our understanding of the genetics and biosynthesis of natural products and possibly lead to the discovery and better understanding of the disease process and pathways underlying. Apart from this natural products can go straight from hit to a drug in comparison to synthetic drugs.Thus there is a continued interest in the investigation of NPs extracted fromliving organismsto search for bio-active compounds[1-4].Natural productbased modified drugs are also accumulating steadily in the market.In this backdrop the present work was planned to achieve the following objectives; 1) structural modification of various natural products 2) subsequent biological evaluation of these molecules for development of anti-cancer and anti-microbial lead molecules.

1.2 Plants as source of anti-cancer agents

Cancer is the most feared disease second only to heart disease as a leading cause of death in most of the developed countries including United States. Cancer can affect people at all ages, even foetus, but the risk for the more common varieties tends to increase with age[5]. Cancer causes about 13% of all deaths. Although surgery and radiation therapy are key weapons to fight against cancer, chemotherapy also plays an important role and is the only essential and possible approach for disseminated cancers. Cancer chemotherapy is aimed at using selective and more appropriate drugs that can kill malignant tumour cells or render them benign without effecting normal cells. Cancer chemotherapy was in practice since 1940 when nitrogen mustard and folic acid antagonist drugs were used. Since then,cancer drug development has gone upinto a multi-billion dollar industry.As a result of this ongoing research, a number of clinically useful and market-approved natural product based drugs are available.Today, this strategy remains an essential route to new pharmaceuticals. Towards the end of 19th century and till date US approved a number of plant-derived compounds as anticancer drugs.

The historical isolation of twoalkaloids Vinblastine1 and Vincristine 2 from the Madagascar periwinkle, Catharanthus roseus G. Don (Apocynaceae) introduced a new era of the use of plant material as anticancer agents. These two agents first advanced into clinical use for the treatment of cancerby inhibiting mitotic cell division[6]. They irreversibly bind to tubulin, thereby blocking cell multiplication and eventually causing cell death thereby showpotential activity against lymphocytic leukaemia.A series of semi-synthetic analogues of these two important drug molecules have been developed in due course of time to increase the therapeutic index. The two semi synthetic analogs such asNavelbineor Vinorelbine (VRLB)3 and Vindesine (VDS),were synthesised which showed potential activity against leukaemia’s, lymphomas, advanced testicular cancer, breast cancer, lung cancer and Kaposi’s sarcoma when treated in combination with other chemotherapeutic drugs.

Camptothecin (CPT)4, is a quinoline alkaloid isolated fromCamptotheca acuminata. It is a potential anticancer agent showing topoisomerase-I inhibitoractivity, and cause cell death by DNA damage [7].It showed poor solubility andsevere toxicity. To overcome these limitations a panel of analogues of CPT were synthesized. Some of the reputed and most promising analogues liketopotecan5, irinotecan6, (CPT-11), 9-aminocamptothecin (9-AC), lurtotecan andrubitecanworked well by inhibitingDNA topoisomerase-I which plays a majorrole in various DNA functions likereplication and transcription[8].However,CPT itself is too insoluble to be used as a drug but its modified analogs, namely, topotecan 5and irinotecan6 have been developed as effective drugs.

Phodophyllotoxin 7isanother important anti-cancer compound obtained from Podophyllum peltatum in 1944 [9].It was initially used therapeutically as a purgative and in the treatment of venereal warts [10].An extensive research was initiated on this molecule particularly in its chemical synthesis and bio-evaluation. Later in 1974, this molecule has come up with a promising anticancer activity by binding irreversibly to tubulin [11].Etoposide 8 and Teniposide 9are the two important modified analogs of Phodophyllotoxin out of a range of analogues synthesized. These analogues showed cell death activity by inhibition of topoisomerase II, thus preventing the cleavage of the enzyme-DNA complex and arresting the cell growthand therefore useful in the treatment of various cancers[12,13].

The discovery of paclitaxel (Taxol, 10) from the bark of the Pacific Yew, Taxus brevifolia Nutt (Taxaceae) is another evidence of the success in natural product drug discovery.Anextract ofT. brevifolia was discovered to possessan excellent anticancer property in 1963, and its active component paclitaxel (Taxol 10) was isolated and characterized only few years latter[14,15]. It was reported to bindirreversibly withβ-tubulin, thus promoting microtubule stabilization[16]. This tubulin-microtubule equilibrium is essential for cell multiplication, and its stabilization causes programmed cell death [17].Paclitaxel was the first compound to be discovered to promote microtubule formation. Since then it has been used in the treatment of several types of cancer particularly for ovarian and breast cancers as well as non-small cell lung tumours [18]. The structure ofPaclitaxelis highly complex and it was very difficult to have ever been produced synthetically prior to its discovery. Hence combinatorial chemistry would have ever led to the discovery of paclitaxel. However, the structural complexity of molecule made it a good candidate for combinatorial modifications to produce a panel of analogues [19].An extensive and targeted research was started on this molecule by many groups around the globe for both semi- and total synthesis in view of its complex structure, unique activity and low bioavailability.

There is a long list of bioactive compounds available in the literature that has beenisolated from plant sources. Out of them a good share is currently in clinical trials or preclinical trials or undergoing further investigation e.g.flavonoids(12, 13, 14, 15),sesquiterpenoid lactones(16, 17, 18, 19) and many others.

1.3Sesquiterpeniods in cancer chemotherapy

Sesquiterpene lactones (SLs) constitute a large and diverse group of biologically active plant chemicals that have been identified in several plant families such as Asteraceae, Canthaceae, Anacardiaceae, Apiaceae, Euphorbiaceae, Lauraceae, Magnoliaceae, Menispermaceae, Rutaceae, Winteraceae and Hepaticeae etc [20]. With over 3000 different structures these compounds are reported to be present in greatest numbers in the family Asteraceae[21]. Sesquiterpene lactones are diverse and unique class of natural products of plant terpenoids. They are important constituent of essential oils, which are formed from head-to-tail condensation of three isoprene units and subsequent cyclization and oxidative transformation to produce cis-or trans-fused lactones. These secondary compounds are primarily classified on the basis of their carbocyclic skeletons into pseudoguainolides, guaianalides, germanocranolides, eudesmanolides, heliangolides and hyptocretenolides etc.,(Fig-6).The suffix "olide" refers to the lactone function, a germanacranoride which is related to the ten-membered carbocyclic sesquiterpene, germacrone. However, SLs exhibit variety of other skeletal arrangements. An individual plant species generally produces one skeletal type of SLs concentrated primarily in the leaves and flower heads. These compounds exhibit a wide range of biological activities. An important feature of SLs is the presence of a γ-lactone ring (closed towards either C-6 or C-8) containing in many cases, andα-methylene group. Among other modifications, the incorporation of hydroxyls or esterified hydroxyls and epoxide ring are common. A few SLs occur in glycoside form and some contain halogen or sulphur atoms [22]. Majority of SLs are associated with cytotoxic activity (κB and P388 leukaemia in vitro) and activity against in vivo P388 leukaemia.

The first sesquiterpeniodswith potential antitumor activity were vernolepin 20 and vernomenin 21(Fig-7).These were isolated from Vernonia hymenolepis by Kupchan and colleagues in 1968[23]as tumour inhibitors against KB cellsand Walker intramuscular carcinosarcoma at appreciable doses.

The discovery of Vernolepin and its antitumor properties was the impetus for a decade of intensive searching for cytotoxic and anti-cancer active sesquiterpenoid lactones during the 1970s. A large number of active agents were isolated from plants, primarily from Asteraceae. A majority of the hundreds of compounds evaluated were cytotoxic, and a small number have shown activity in-vivo against P-388 leukaemia and other tumour systems.Someother antitumor sesquiterpenoid lactones include the following compounds(Fig-8) with different skeletal types [24].

1.3.1Anticancer sesquiterpenoid lactones in clinical trials

The SL drugs presently in clinical trials are parthenolide 16 from Tanacetum parthenium and artemisinin 17 from Artemisia annua L and Thaipsigargin 19 from Thiapsia (Apiaceae), and a panel of their synthetic derivatives. Artemisinin derived drugs are promising for laryngeal carcinomas, uveal melanomas and pituitary macrodenomas as indicated by clinical evidences. Some of these drugs and are in phase I-II trials against lupus nephritis and breast, colorectal and non small cell lung cancers(Table-1).Thaipsigargin derived drugs are undergoing phase-I clinical trials for breast, kidney and prostate cancer treatment. The orally bioavialable parthenolide analogue, dimethyl amino-parthenolide, or LC-1 is at present in phase I against acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) and other blood and lymph node cancer.

Table-1: List of some sesquiterpeniods in cancer clinical trials

1.3.2Mechanism of action of sesquiterpenoid lactones

Effective cancer treatment is through elevationof tumour load and inhibition of cancer stem cells which are concerned in cancer clinical degeneration and treatment resistance.SLs in cancer clinical trials have properties that enable them to target tumour and cancer stem cells while sparing normal cells [32-34].The selectivity of thiapsigargin,artemisinin and/or parthenolide towards tumour cells are attributed to their ability to target the sarco/endoplasmic reticulum calcium ATPase(SERCA) pump [35],particularly proteases secreted by cancer cells [36], high iron content and cell surface transferrin receptors [37-38], nuclear factor-(NF-κB) signalling [39-40], MDM2 degradation and p53activation [41], angiogenesis [42], metastasis [43]and epigenetic mechanism [44-45] as shown in (Fig-10).

Fig 10:Mechanism of action of SLs (taken from Ref: [46])

But most of the anticancer SLs inhibit the NF-κB pathway (Fig-11) e.g Parthenolide and artemisinin are established NF-κB inhibitors and render cancer cells sensitive to chemotherapy. Parthenolide was found to directly modify the NF-κB, p65 subunit or to suppress the activity of upstream IκB kinase complex leading to the stabilization of the NF-κB inhibitors IκBα and IκBβ. The nucleophillic attack by parthenolide occurs through α-methylene-γ lactone ring and epoxide moieties that target specific nucleophiles but not others.Several artemisinin type compounds also inhibit NF-κB activity. Normal cells are usually not sensitive to these SLs because their basal NF-κB activity is often low.

Fig 11:NF-κB cell signalling pathway (taken from) Ref: [47]

Besides anticancer activity SLs show a broad spectrum of other biological properties e.g.,anti-inflammatory [48], anti-bacterial [49], anti-malarial [50], antiviral [51],anti-fungal [52].

1.3.3. Structural-activity relationships (SAR) of sesquiterpene lactones

The biological activity of SLs can be affected by three major chemical properties viz., 1) alkylating centre reactivity 2) side chain and lipophilicity3) molecular geometry and electronic features.

1.3.3a Alkylating centre reactivity

It is commonly believed that the bioactivity of SLs is mediated by alkylation of nucleophiles through theirβ orγ-unsaturated carbonyl structures, such as α-methylene-γ-lactones or α,β-unsaturated cyclopentenones. These structural elements react with nucleophiles especially the cystiene sulfhydryl groups by Michael-type addition. Therefore, it is widely accepted that thiol groups such as cystiene residues in proteins, as well as the free intracellular GSH, serve as the major targets of SLs. In essence, the interaction between SLs and protein thiol groups or GSH leads to reduction of enzyme activity or causes the disruption of GSH metabolism and vitally important intracellular redox homeostasis. The relationship between chemical structure and bioactivity of SLs has been studied in several systems, especially with regard to cytotoxicity. It is believed that the exo methylene group on the lactone is essential for cytotoxicity as structural modifications such as saturation or addition to the methylene group resulted in the loss of cytotoxicity and tumour inhibition. However, it has also been shown that the factor responsible for the cytotoxicity of SLs might be the presence of the O=C-C=CH2 system, regardless of lactone or cyclopentenone. It was latter demonstrated that the presence of additional alkylating groups greatly enhanced the cytotoxicity of SLs. Furthermore, it was established that the α-methylene-γ-lactones and α, β-unsaturated cyclopentenone ring (or α-epoxy cyclopentenone) present in SLs is essential for their in vivo anti-tumour activity. Further it has been confirmed by various published reports that the spectrum of biological activities displayed by SLs is due to presence of either α-methylene-γ-lactones or α, β-unsaturated cyclopentenone ring.

1.3.3bSide chain and lipophilicity

In general, higher lipophilicity can facilitate penetration through cell membrane, thereby increasing the SLs cytotoxicity in vitro butsteric hindrances set up a threshold limit.Moreover higher lipophilicityis often associated with lower drug bioavailability in vivo. In bi-functional helanalin33 and maxicanin I34analogues, the increasedlipophilicity due tolipophilic chain ester and liphophilic conjugated ester group at C-6, enhanced cytotoxicity aganist Ehrilish ascites both in vitro and in vivo[53]. However there was a size optimum of lipophilic ester groups beyond which SLs toxicity decreased. In contrast to this, within the mono-functional 11α-13-dihydrohelanalin35, cytotoxicity was directly proportional to the size of the ester side chain at C-6. Although, larger groups can increase lipophilicity, these moieties, beyond a size limit, cause steric hindrance on to the exocyclic methylene group, preventing it from approaching its target.

The number and position of H-bond acceptors do influence SLs cytotoxicity. Non covalent interaction, such as hydrogen bond formation between oxygen atoms in SLs and amino acid residues adjacent to the target protein, can precede alkylation and increase SLs bioactivity. In addition chemical environment around the target sulfhydryl groups which are SLsMichael addition sites is important for bioactivity.

1.3.3c Molecular geometry and electronic features

Conformational flexibility effects SLs bioactivity to a very great extent. Flexible bi-functional helanalins with 7,8- cis fused lactone ring, were more toxic than rigid maxicanin I derivative with 7,8-trans-fused lactone ring [54]. Within 2,3-dihydrohelanalin36,37derivatives flexibility accounted for five fold differences in cytotoxicity between two compounds having identical structures but one bearing carbonyl group, instead of hydroxyl group at C-4.

Perusal of literature has witnessed that stereochemistry of SLs plays an important role in defining their anti-tumorigenic properties. Studies of structurally related pseudoguanolides showed that β-OH isomer (parthenin 30) at C-1 are active than α-OH equivalent(hymenin 35) [55].In summary, the differences in activity among individual SLs may be explained by differences in the number of alkylating elements, lipophilicity, molecular geometry, and thechemical environment of the target sulfhydryl group.

1.4 Natural products as anti-infective agents

Today, infectious diseases are the second major cause of deathworldwide and third leading cause of death in economically advanced countries [56].Bacterial pathogens are responsible for several seriousdiseases. Resistant strains to antibiotics in clinical useposegreat threat to mankind. The ability of bacteria todeceive any kind of conventional therapy has become apparent andpathogens resistant to one or more antibiotics are emerging and spreadingworldwide [57].The discovery of vancomycin resistant S. aureus (VRSA) and multiresistantS. aureus has evoked worldwide response. Thus,novel antibacterial drugs with broader spectrum of activity are urgently needed.There is long list of herbs known to be used for many infectious diseases such as Acacia, Garlic, Turmeric, Neem, Ginger, Clove, Plum and Pomegranate etc. The extracts from most of these herbs have been screened in quest for potential and safer antibacterial agents [58-63].The majority of antibacterial agents that are in use today find their origin in natural products or their semi-synthetic variants. More than 75% of new chemical entities that entered in the market between 1984 and 2004 were based on natural product lead structures [64].