Progress Toward the Development of Noscapine and Derivatives as

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Progress Toward the Development of Noscapine and Derivatives as Anti-cancer Agents Aaron DeBono, Ben Capuano, and Peter J. Scammells J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501180v • Publication Date (Web): 26 Mar 2015 Downloaded from http://pubs.acs.org on April 14, 2015

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Progress Toward the Development of Noscapine and Derivatives as Anti-cancer Agents

Aaron DeBono, Ben Capuano* and Peter J. Scammells*

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria, 3052 Australia.

ABSTRACT: Many nitrogen-moiety containing alkaloids derived from plant origins are bioactive and play a significant role in human health and emerging medicine.

Noscapine, a

phthalideisoquinoline alkaloid derived from Papaver somniferum, has been used as a cough suppressant since the mid 1950s, illustrating a good safety profile. Noscapine has since been discovered to arrest cells at mitosis, albeit with moderately weak activity. Immunofluorescence staining of microtubules after 24 hours of noscapine exposure at 20 µM elucidated chromosomal abnormalities and the inability of chromosomes to complete congression to the equatorial plane for proper mitotic separation.1 A number of noscapine analogs possessing various modifications have been described within the literature and have shown significantly improved anti-prolific profiles for a large variety of cancer cell lines. Several semi-synthetic antimitotic alkaloids are emerging as possible candidates as novel anticancer therapies.

This perspective discusses the advancing

understanding of noscapine and related analogs in the fight against malignant disease.

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1. INTRODUCTION Cancer is a life threatening disease. There were 14.1 million new cases of cancer worldwide in 2012. In the same year 8.2 million deaths were cancer related with an estimated 32.6 million people living with cancer (within 5 years of diagnosis) worldwide. Approximately 57% (8 million) of new cancer cases, 65% (5.3 million) of cancer deaths and 48% (15.6 million) of 5-year prevalent cancer cases occurred in less developed regions.2 Australia and New Zealand have two of the highest incidences of cancer in the developed world followed closely by North America.2 Cancer is the leading cause of death in Australia – 1 in 2 men and 1 in 3 women will be diagnosed with cancer by the age of 85. It is estimated more that 43,700 people died from cancer in 2011, which accounts for 3 in every 10 Australian deaths. In 1996, cancer was declared a National Health Priority Area by Australian Health Ministers.3 Surgery is the most common means of removing accessible tumors however many cancer types remain surgically inaccessible; hence the requirement for new treatment approaches. Even though a plethora of cancer treatments exist for various ailment types, problems exist in current treatment regimens, and for many cancer types there is still no known cure or treatment. As a result chemotherapeutic potential in human cancer merits a thorough evaluation. Cancer is a class of disease, which can be caused by a number of environmental and lifestyle factors. Cancer has been linked to smoking, alcohol consumption, diet, obesity and physical inactivity, chronic infections (viruses, bacteria, parasites), genetic susceptibility, occupational exposures, sunlight, radiation, medical and iatrogenic factors, environmental pollution and reproductive factors.3

Cancer is usually caused when a number of mutagenic cells display

uncontrolled growth and invade healthy tissue. This phenomenon results in the destruction of adjacent tissue and the cells then can cause metastasis via the lymph and/or blood.

These

mutagenic cells are generally able to evade the body’s apoptotic response because of their direct effect on the cell-cycle and apoptotic mechanisms.1


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2. ANTI-MITOTIC AGENTS 2.1 Background. In recent years many new targets have been studied for their role in more effective and specific cancer treatment. The class of drugs receiving considerable attention target microtubules and their major constituent subunit, tubulin.

Microtubules (MTs) are tubular

structures that are key components of the cytoskeletal structure. These structures are rods of approximately 25 nm in diameter, which are polymers of α- and β-tubulin dimers.4 MTs are crucial for both the formation and the destruction of the mitotic spindle, which allows the correct separation of duplicated chromosomes during cellular division.5

Along with microtubule

importance in cellular division, they are also extremely important in facilitating cell movement, cellular locomotion and the intracellular transport of organelles, along with maintaining the structural integrity of both the endoplasmic reticulum and mitochondria. Microtubule formation consists of two stages, the first stage known as nucleation, requires tubulin, GTP and Mg2+ at internal human body temperature. This first step is the rate-determining step and the following step, known as elongation, occurs more frequently. During this step tubulin heterodimers attach to each other in an energetically less favorable oligomeric nucleus for further polymerization.

The

formation of microtubules within cells generally occurs near the nucleus at the microtubule organizing centers.6 The minus end with exposed α-tubulin are anchored at the organizing center, whilst the plus ends with exposed β-tubulin are disposed to the cell periphery.7 The α- and βheterodimers share a non-covalent association and as a result oscillate between a state of polymerization and depolymerization.8 The presence of guanosine triphosphate (GTP) dictates two behaviors shown by microtubules; treadmilling and dynamic instability. Tubulin dimers bind GTP reversibly at a site within the β-subunit. GTP then becomes hydrolyzed to guanosine diphosphate (GDP) and orthophosphate (Pi) during polymerization.9 The hydrolysis of GTP is reversible, which creates unusual non-equilibrium dynamics. The consequence of this is that the microtubule ends can switch rapidly between growing and shortening states. This property is referred to as dynamic instability10 and is crucial for the proper function of MTs to carry out their cellular functions. The



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two ends of the microtubule are non-equivalent; the β-tubulin (plus end) is more kinetically favorable than the α-tubulin (minus end). Both ends have the ability to grow or shorten however changes in length at the plus end are more pronounced than those at the minus end. When net growth is encountered at the plus end and net shortening occurs at the minus end simultaneously, this phenomenon is referred to as the microtubule treadmilling.11 Many important cellular processes such as mitosis are dependent on microtubules. Mitosis is the process during cellular reproduction in which the replicated genetic material in the form of chromosomes is partitioned equally across the cytoplasm to form two new cells known as ‘daughter’ cells. When a cell enters mitosis, the cytoskeletal microtubule network is dismantled and a bipolar mitotic spindle-shaped array of microtubules assemble outward of the microtubule organizing center known as the centrosome. The chromosomes (as chromatids) then become attached to the microtubule spindle where they are equally separated and complete congression to the equatorial plane (spindle pole). If minor alterations to tubulin dynamics exist, this can prevent the onset of anaphase and chromosomal segregation into daughter cells, resulting in the exit of the cell from the cell cycle and imminent apoptotic death.12 When a cell transitions from interphase (resting phase) to mitosis, there is a dramatic increase in microtubule growth and shortening dynamics. It can be assumed that these rapid dynamic changes are critical for proper mitosis. A cell can assemble a normal or nearly normal bipolar mitotic spindle in the presence of an antimitotic agent.9 The assembly of the spindle therefore is not critically dependent upon the dynamics of its microtubules as is spindle function after construction.9 When a cell progresses from metaphase to anaphase, important checkpoints exist to minimize the likelihood of incorrect genetic transcription being passed on. As a result drugs that suppress spindle microtubule dynamics may exert their antiproliferative and cytotoxic effects at the cell-cycle checkpoints. 2.2 Natural Products as Anti-Mitotic Agents. Natural products have proven to be the most reliable single source of new and effective anticancer agents. Newman et al. have shown that 63% of anticancer agents introduced in the last two decades are either natural products or their heritage


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can be traced back to a natural product source.13 Natural products have not only yielded new and effective drugs, but have also provided insight into possible mechanisms of action for naturally occurring compounds on the mitotic death of malignant cells. Natural products are effective antimitotic agents due to the strong correlation between their properties and those of drugs. The unique built in chirality that natural products possess are generally suited to bind to complex proteins and biological receptors.13

2.3 Tubulin-targeting Agents. The effectiveness of MT targeting drugs for the treatment of various neoplastic diseases has been validated by the successful use of a number of naturally occurring and semi-synthetic alkaloids. These compounds include a number of vinca alkaloids and taxanes used in the treatment of a wide variety of human cancers. The US FDA has approved a number of these compounds including the taxanes; paclitaxel and taxostere as chemotherapeutics for the treatment of breast and ovarian cancer, whilst the vinca alkaloids; vincristine, vinblastine and vinorelbine have been approved for the treatment of various hematological pathologies. Colchicine and podophyllotoxin are also two unrelated compounds from plant origins that have shown promise as potent anti-mitotic agents. The above microtubule targeting agents can be broadly classified into two categories. The first group of compounds consists of drugs, which inhibit tubulin polymerization and as a result promote microtubule disassembly.

The drugs

represented within this class include the vinca alkaloids, colchicine and combrestatin. The second class of compounds consists of drugs, which promote tubulin polymerization and cause microtubule over assembly. The compounds represented within this class include the taxanes, laulimalides and discodermolides. For many of these anti-mitotic compounds the precise mechanism of inhibition and the nature of their binding-site interactions are largely unknown. The tertiary structure of the α/β-tubulin heterodimer was first reported by Nogales et al. using electron crystallography at a resolution of 3.7 Å.14 A number of direct and indirect binding site approaches have been used to elucidate possible drug binding sites on tubulin.

These approaches have identified three key



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binding domains on tubulin: 1) the colchicine site15, 2) the vinca alkaloid site16 and 3) the taxane binding domain.17 2.4 Tubulin Polymerization Inhibitors - Vinca Domain. Vinca alkaloids are a group of natural compounds derived from Catharanthus roseus (periwinkle plant), discovered in the extracts of the leaves of the plant at the University of West Ontario in 1958,18 and independently at Lilly Research Laboratories.19 The naturally occurring vinca alkaloids, vincristine and vinblastine and semi-synthetic counterparts; vindesine, vinorelbine and vinflunine are antimitotic drugs, successfully used in the treatment of cancer.20-22 At relatively low concentrations vinca alkaloids have the ability to inhibit the assembly of tubulin into microtubules.9 The mechanism of action is thought to involve the polymerization of spindle microtubules and induction of paracrystalline tubulin-vinca alkaloid arrays.23

The

administration of these compounds at low concentrations exerts subtle effects on MTs, which inhibit their dynamic behavior at concentrations below those required to significantly inhibit polymerization, along with affecting the dynamicity (the total rate of measureable tubulin exchange at microtubule ends attributable to growth and shortening) of MTs.23,24 This results in rapid and reversible binding of a small number of vinblastine molecules to a class of high affinity binding sites located at one or both microtubule ends referred to as kinetic capping.25 Vinblastine (1) binds at the interface between α/β-tubulin heterodimers.16 The structure of 1 bound to the tubulincolchicine:RB3-SLD complex ((TC)2R) was determined at 4.1 Å resolution. Within the (TC)2R complex, one vinblastine binding site exists at the interface between two tubulin heterodimers organized in a head to tail arrangement with approximately 80% of the surface area of 1 buried within the complex which is orientated in a way that the catharanthine and vindoline moieties each interact with the tubulin heterodimers.16 The mechanism of action is suggested to be a result of the crosslinking of two tubulin molecules by vinblastine through the interaction with an α-subunit of one tubulin molecule and the β-subunit of another.26 Vinblastine (1) is able to make several


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interactions with microtubules, which are dependent upon the drug concentration employed, suggesting more than one possible mechanism exists.27

Figure 1. Chemical structure of vinblastine (1) highlighting the vindoline (red) and catharanine (blue) moities.

2.5 Tubulin Polymerization Inhibitors - Colchicine Domain. Colchicine (2) (Fig. 2) is derived from Colchicum autumnale (meadow saffron) and was the first tubulin-destabilizing agent to be discovered. The therapeutic value of 2 for the treatment of cancer however is limited by its poor therapeutic index. Although 2 is not used as a chemotherapeutic agent, there is substantial interest to develop colchicine binding site agents.

Like the vinca alkaloids, 2 also stabilizes

microtubule ends, but by a different mechanism to that of 1. Colchicine (2) first binds to soluble tubulin to give small numbers of tubulin-colchicine complexes, which are then incorporated within tubulin at the microtubule ends affecting the extent and rate of microtubule growth and shortening.9 The percentage of time the cell remains in a state of no net growth is dramatically increased. The mechanism by which 2 may induce cell apoptosis is through the activation of caspase-3.28 Colchicine (2) becomes fluorescent when bound to tubulin providing a convenient method for measuring the binding of 2 on tubulin.29 The colchicine-tubulin complex has an excitation and emission maxima at 362 and 453 nm, respectively.30 Colchicine (2) binding to tubulin exhibits pseudo-irreversible kinetics; it displays a fast step whereby the rapidly formed transient preequilibrium complex does not become incorporated detectably at microtubule ends and does not ACS Paragon Plus Environment


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inhibit tubulin exchange, followed by a slow step where low numbers of final-state TC complexes strongly inhibits tubulin addition.31 It is this slow step that promotes the fluorescent characteristics of the tropolone moiety of 2.32 The effect of 2 on microtubules is dependent on drug concentration. Colchicine (2) inhibits microtubule polymerization in vitro at sub-stoichiometric amounts, well below the concentration of tubulin free in solution.33 Uppuluri et al. using photo-labeling (at > 325 nm) with unmodified [3H]colchicine illustrated that the label is localized almost exclusively within β-tubulin.32,34

Figure 2. Chemical structure of colchicine (2) illustrating the fluorescent tropolone moiety (red).

2.6 Taxanes - Taxol Domain. The National Cancer Institute (NCI) in the USA discovered Paclitaxel (3) as part of a broad screening program for natural products in the 1960s. In 1963, Wall et al. isolated a crude extract from the bark of Taxus brevifolia (Pacific Yew) and in 1971, reported the structural identification of 3 as the active constituent in the bark extract.35 Unlike the previously described classes of anti-microtubule agents, 3 was found to act by promoting the formation of unusually stable microtubules resulting in the disruption of the microtubule network required for proper mitosis and cellular proliferation. Paclitaxel (3) has a complex structure consisting of a 14membered taxane ring system connected to an oxetan ring and an amide side chain.

Early

development of this drug was thwarted because of the low percentage of 3 isolated from the bark of the Pacific yew (0.1 g/kg).36 Preclinical data for 3 was encouraging, however the development of the drug as a clinical agent was hampered by the inability to find a formulation for this highly insoluble compound. In vitro, 3 enhances the rate, extent and nucleation phase of microtubule


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polymerization and the stabilization of microtubules.37 The examination of HeLa cells incubated with 3 elucidated not only normal microtubules, but also bundles of microtubules.37 Schiff and Horwitz found 3 to induce the polymerization of microtubule protein in the absence of factors that are normally essential for microtubule assembly, such as exogenous GTP or microtubule-associated proteins (MAPs).38

Paclitaxel (3) is able to increase the rate, yield and nucleation phase of

microtubule assembly when the ratio between 3 and the tubulin dimer is one.38 A competitive binding study of 3 using [3H]colchicine found 3 not to be a competitive inhibitor of colchicine binding to tubulin.38 Paclitaxel, GTP, MAPs and heat were four components examined by Hamel et al. who illustrated rapid polymerization occurred if any three of these components were present. Microtubules were always formed but in the absence of MAPs, sheets of protofilaments predominated.39 A competitive binding study conducted by Rao et al. using the photoaffinity analog [3H]3′-(p-azidobenzamido)paclitaxel illustrated 3 to compete for the same binding site implicating a very similar binding position within the N-terminal 31 amino acids of the β-tubulin subunit.40 A later model proposed 3 and other microtubule-stabilizing agents (MSAs) to bind between helices H6 and H7 of β-tubulin at an outer putative binding site, which uses its high flexibility to swing the ligand from the pore into the internal luminal binding site.41

Figure 3. Chemical structure of paclitaxel (3) illustrating the oxetan moiety (red), amide side chain (blue) and taxane moiety (green).



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2.7 Combretastatins.

Combretastatins are a class of stilbenoid phenols isolated from

Combretum caffrum.42 Extracts from the stem and bark wood of this tree were found to contain potentially useful activities against P388 lymphocytic leukemia. Combretastatin A-4 (4) is the most potent naturally occurring combretastatin, which is currently being evaluated in phase I clinical trials for the treatment of solid tumors.43 Because of its poor water solubility 4 is prepared as its more soluble phosphate pro-drug CA-4 phosphate (CA-4P).44 The real therapeutic value of 4 is yet to be ascertained with patients in clinical trials experiencing cardiovascular side effects including the presence of tachycardia, bradycardia and hypertension.45 In vitro cell cycle analysis of cells treated with 4 suggests cells are arrested in G2/M a few hours following administration at concentrations close to the IC50 values for cytotoxicity.46 Given the potent cytotoxicity of 4 against a variety of human cancer cell lines, including those that show multidrug resistance, 4 has been envisaged as a very attractive lead compound.47,48 The indirect binding analysis of 4 by Lin et al. illustrated the binding of radiolabelled [3H]colchicine to tubulin was negligible in the presence of 4 therefore indirectly identifying the competitive nature of both ligands.49 The binding of 4 on tubulin did not displace 1 from its binding site, which is consistent with 4 binding at the colchicine site.50

Figure 4. Chemical structure of combretastatin A-4 (4) illustrating the stilbenoid moiety (red).

2.8 Podophyllotoxin. Podophyllotoxin (5) is a naturally occurring cyclolignan isolated from Podophyllin.

In spite of the potential use of 5 as a medicinal drug, its clinical trials were

discontinued due to its systemic toxicity.51 Over the last two decades extensive modifications to the core have been implemented, which have led to the synthesis of a number of pharmacologically


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active drugs which are clinically used.52 Ravelli et al. determined the structure of ternary tubulinpodophyllotoxin:RB3-SLD complex at 4.2 Å and found that 5 binds to tubulin at the same site as 2.15

Podophyllotoxin (5) binds to β-tubulin at its interface with α-tubulin, with the

trimethoxyphenyl nucleus hidden within the β-subunit. The last 20 years has seen an emergence of semi-synthetic derivatives of 5; etoposide, teniposide and etopophos (structures undisclosed), which are widely used as anti-cancer drugs and show good clinical efficacy against several cancer types including testicular cancer, small cell lung cancer, lymphoma, leukemia and Kaposi sarcoma. Although these compounds are analogs of the parent compound 5, they possess an entirely different mechanism of action as topoisomerase II inhibitors and possess no affinity for tubulin. 52,53

Figure 5. Chemical structure of podophyllotoxin (5).

2.9 Issues with Current Therapies. The clinical success and treatment of various neoplastic diseases is strongly attributed to the acknowledgment, microtubules and their subunits counterparts; tubulin, are one of the best-validated targets for tumor therapy. The widespread occurrence of multi-drug resistance (MDR) within cancerous cells is a major impediment to the clinical success of many unrelated neoplastic agents. There are two general cases of anticancer drug resistance, those that result from the impaired delivery of drug(s) to the tumor cells and those that arise within the cancerous cell due to genetic and epigenetic alterations, which affect the sensitivity of the drug.54 Current chemotherapeutics suffer from poor aqueous solubility, increased drug metabolism, followed by excretion resulting in lower concentrations of drug within the blood and as a consequence, reduced diffusion of drug from the blood, across cellular membranes and into the ACS Paragon Plus Environment


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tumor mass.55 Recently, the in vitro modeling of novel chemotherapeutics using monolayer cells in cultures has been questioned since some cancer cells, which are sensitive to chemotherapy within monolayer cultures have shown resistance when transplanted within animal models.56 This has emphasized the importance of the tumor vasculature system which may embody the activity of a chemotherapeutic in a model more closely related with natural circumstances.57 These observations indicate environmental factors such as extracellular matrix and tumor geometry may also be involved in drug resistance.54 The overexpression of drug efflux pumps such as P-glycoprotein and multi-drug resistance-associated protein 1 (MRP1) is one of the most extensively studied mechanisms of drug resistance. PGP is a broad-spectrum multidrug efflux pump which contains 12-transmembrane regions and two ATP-binding sites.58 Not all multi-drug resistant cellular lines express PGP and a search of other efflux pumps resulted in the discovery of MRP1, which is similar in structure to PGP but differs in the amino-terminal extension which contains 5 membrane spanning domains attached to a PGP like core.54 These pumps belong to a class of ATP-binding cassette (ABC) transporters which share structural homology and sequence.54 Drugs affected by the classical multi-drug resistance include the taxanes and vinca alkaloids. Humans have two MDR genes with one of these; MDR1 being involved in drug transport.59 The induction of the multidrug transporter PGP located on the cellular plasma membrane and cytochrome P450 3A4 (CYP3A4) is apparent.60 This mechanism can infer drug resistance after exposure of any drug and is not specific for chemotherapeutics. Cancer cells are genetically heterogeneous and although the uncontrolled cell growth in cancer favors clonal expansion, the presence of chemotherapeutics results in natural selection of tumor cells, which are able to survive and proliferate resulting in resistance. These two classes of clinically current anti-microtubule agents share similar complications in vivo after chemotherapeutic administration.

Although they are able to influence tubulin and

microtubule formation albeit in opposing mechanisms, the imposing side effects warrant the chemical exploration of new classes of microtubule inhibitors, which can be administered using


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safer administration methodologies, alleviating or eliminating the many side effects associated with current treatment regimens.

3. NOSCAPINE 3.1 Background. Noscapine (6), formerly known as narcotine (renamed noscapine in 1958 by the A.M.A. Council on Drugs), is a phthalideisoquinoline alkaloid, which represents the second highest alkaloid abundance to morphine within opium. Noscapine (6) was one of the first alkaloids to be isolated from Papaver somniferum. Despite the fact it is found within a plant rich in alkaloid content, 6 bears little resemblance to narcotic alkaloids also present within Papaver somniferum either chemically or pharmacologically.61 The early toxicity studies by Krueger et al. demonstrated 6 to be the least toxic of the alkaloid opiates.62,63 The structures of codeine, morphine, thebaine and similar opiate compounds are based on a phenanthrene tetrahydroisoquinoline derivative.

nucleus, whereas 6 is a

Noscapine (6) contains two stereogenic centers, therefore

consists of four possible stereoisomers.

1'

9'

8'

O

7'

2'

N 6'

O 3'

4'

5'

MeO H

3

4

H O2

5

1

6

MeO

O

7

OMe

Noscapine (6)

Figure 6. Chemical structure of noscapine (6) illustrating the tetrahydroisoquinoline moiety (top) and phthalide moiety (bottom).

Noscapine (6) has been used as a cough suppressant since the mid 1950’s, however the mechanism of its antitussive nature is largely unknown. An animal study by Karlsson et al. described L-(S,R)-noscapine [L-(α)-noscapine] was able to inhibit saturable [3H]noscapine with a Ki of 8.4 ± 1.3 nM compared to the non natural stereoisomers D-(α), D-(β) and L-(β) of 2500 ± 460, ACS Paragon Plus Environment


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170 ± 44 and 9700 ± 2400 nM, respectively. Noscapine (6) was determined to bind at a site independent of the dextromethorphan (structure undisclosed) binding site within guinea pig brain homogenates.64 Binding sites of 6 were not exclusive to one area of the guinea pig brain with binding sites present in all major brain areas, with the thalamus exhibiting the largest density. The binding affinity of dextromethorphan was enhanced in the presence of 6 suggesting a possible allosteric modulation.65 Noscapine (6) was described to bind with high affinity, saturable and stereospecific, with the natural occurring L-α-noscapine showing 300-fold greater affinity to the binding site than its enantiomer D-α-noscapine.64 Noscapine (6) is a drug with low toxicity, which can be administered orally in humans.63 Chronic toxicity studies in animal models have illustrated 6 to possess a large margin of safety, with mice models tolerating doses of up to 3500 mg/kg.63 Noscapine (6) is rapidly absorbed after oral administration giving a maximum plasma concentration of 182 ng/mL after 1 h. The absolute oral bioavailability is 30%; levels of 6 then decline with a mean half-life of 124 min.66 The ability to orally administer 6 is not only associated with low cost and convenient administration methodology, but encumbers the likelihood of hypersensitivity issues that have been encountered with the current clinically administered anti-mitotic agents, which generally possess poor water solubility. These current chemotherapeutics require administration methodologies, which include the use of vehicle agents for intravenous administration, which encompass several undesirable characteristics. The low bioavailability for 6 of 30% suggests a high first-pass metabolism after oral administration. Noscapine (6) is extensively metabolized resulting in the main metabolites; cotarnine (7), hydrocotarnine (8) and meconine (9). Metabolism results in the cleavage of the C-C bond between the tetrahydroisoquinoline and phthalide ring systems.67


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Figure 7. Products of noscapine metabolism.

3.2 Antimitotic Activity. The discovery that 6 has the ability to arrest cells at mitosis was made by Lettre et al.68,69 A natural compound screen initiated by Ye et al., of known antimicrotubule assembly inhibitors; 3, 6 and others, illustrated common structural features that may be influential in microtubule binding. Each of the compounds described above share similar motifs, these compounds contain a hydrophobic trimethoxyphenyl group, the presence of a lactone, tropolone or aromatic ring(s) and a small hydrophilic group such as OH or NH2. A series of naturally derived compounds, which shared similar structural motifs were subsequently tested to determine whether they might be useful novel antimitotic agents. Different concentrations of these natural compounds were incubated in the HeLa cell line. The results confirmed that 6 possesses moderately weak anti-mitotic activity.

After exposure of 6 for 24 hours at 20 µM, the

immunofluorescence staining of microtubules elucidated abnormalities of chromosomes, which resulted in the inability of chromosomes to complete congression to the equatorial plane. After 48 hours of administration, many of the cells showed large fragmented nuclei and fewer cells in mitosis. The activity of 6 was not exclusive to the HeLa cell line; other cultured cell types were trialed with similar results. FACS analysis was used to determine the effect of administration of 6 on the cell cycle. The vehicle control DMSO showed normal cell cycle profile with 45.2% cells in G1 phase, 12.9% cells in S phase and 16.5% cells in G2/M phase. After 24 hours of treatment with 6, the cells showed only 6.4% in G1 phase, 11.1% in S phase and 42.9 % in G2/M.1 This was suggestive of G2 mitotic arrest of HeLa cells. ACS Paragon Plus Environment


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Ye et al. were the first to illustrate that noscapine was able to bind stoichiometrically to tubulin.1 This resulted in the overall alteration of the tubulin dimer conformation and subsequently influenced the proper assembly of microtubules. Immunofluorescence staining of microtubules elucidated abnormalities and resulted in the failure of chromosomes to assemble at the metaphase plate.1 The observation of multiple poles and condensed chromosomes was also apparent and after 48 hours of drug treatment there was a reduction in the number of cells observed to be arrested in mitosis. Many cells exhibited fragmented nuclei with evidence of apoptotic morphologies.1

Figure 8.

Noscapine arrested HeLa cells at M phase.

Immunofluorescence micrographs showing

microtubule arrays for noscapine-treated HeLa cells (A and C) and DNA of control cells (B and D). (Bar = 15 µm). Reproduced from Ye et al.1

The initial goal by Ye et al. was to determine whether 6 had the ability to reduce tumor size implanted into mice. The cell line used was the thymocyte E.G7-OVA because these cells produce large palpable tumors within 3 weeks of being subcutaneously injected into syngeneic mice.1 DNA fragmentation was noted within 8 hours of drug application and the number of noscapine-treated


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cells with apoptotic morphologies increased dramatically with more than 50% of cells displaying apoptotic morphology within 24 hours of exposure to 6.1 3.3 Tubulin Binding. Radiolabelled 6 is currently not available and 6 produces a negligible fluorescence spectrum when excited at 279 nm, therefore the binding data for 6 was determined indirectly. To determine whether 6 interacts with tubulin, the fluorescence of tubulin was observed in the presence and absence of 6.1 The dissociation constant (Kd) for 6 on tubulin was determined to be 1.86 ± 0.34 × 10−6 M and a stoichiometry of 0.95 ± 0.02 noscapine molecules per complex of tubulin subunit.1 Noscapine (6) shares similar structural motifs to 2 and 5. Given the similarity in chemical structure it was postulated that they may share a similar binding site. Ye et al. performed a [3H]colchicine-competition study with 6, however the results illustrated an inability for 6 to compete with 2 for the same binding site, demonstrating 6 may bind to an alternate site. These results were confirmed by the inability of 6 to influence the fluorescent properties of the colchicinetubulin complex.1 It is conceivable that the ability of 6 to promote mitotic arrest may be a result of an ability to affect microtubule dynamics rather than directly affecting tubulin polymerization. This hypothesis was tested by Zhou et al. where the effects on tubulin assembly into microtubules in vitro was determined by measuring changes in turbidity produced by tubulin polymerization after administration of 6. Changes in turbidity were unnoticed after 1 µM noscapine administration. Higher doses of 6 only slightly increased the extent of microtubule assembly.12 The microtubule dynamic instability at steady state after 6 administration was studied and microtubule growth was measured. Normal mitotic growth results in alternating periods of net growth and shortening, but also included an attenuated state when neither growth or shortening was detected.12 The addition of 20 µM 6 resulted in reduced growth and shortening rates and an increase in the percentage of time microtubules spent in an attenuated state.12 Noscapine (6) was shown to exert its effect through the slight suppression of both the growth and shortening of microtubules. Growth and shortening rates where determined quantitatively, the mean growth rate for microtubules in the absence of 6 was found to be 0.92 µM/min, the addition of 20 µM 6 reduced the growth rate to 0.60 µM/min. A



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higher dose of 50 µM reduced the mean growth rate by only 42.4%.12 These doses of 6 slightly affected the net shortening of microtubules by 21.6% and 24.3%, respectively.12 A dramatic change in the percentage of time microtubules spent in an attenuated state after 50 µM of 6 administration was observed. Microtubules spent 12.7% of the time in an attenuated state, a 2-fold increase compared to microtubule growth in the absence of 6.12 To test whether 6 affected the tubulin polymer/monomer ratio in cells, cell extracts containing cytoskeletal (polymeric) and soluble (monomeric) tubulin where incubated with different concentrations of 6 (1, 10, 100 µM). Western blot analysis was used to determine the percentage of polymeric tubulin in cells treated with 6. The percentage of polymeric tubulin in cells was found to be 58.3, 59.2 and 59.2%, respectively. These values were similar to control cells treated with an equivalent amount of DMSO solvent therefore indicated that 6 did not induce any noticeable increase or decrease of tubulin in monomeric or polymeric fractions.12 The slight alteration of microtubule dynamics in vitro by 6 appeared to cause a significant disruption of microtubule-mediated proceedings. Noscapine (6) was also found to impair the tension between kinetochore pairs and the attachment between kinetochores and microtubules.12


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Figure 9. Effects of noscapine on the tubulin polymer/monomer ratio in HeLa cells. A, Coomassie Blue staining of the proteins in cell extracts containing polymeric (P) or monomeric/soluble (S) tubulin. Cells were treated with 1, 10, 100 µM noscapine, 10 µm paclitaxel, 10 µM nocodazole, or the equivalent amount of the solvent Me2SO for 4 h, and cell extracts were then isolated as described (20). B and C, Western blot analysis showing polymeric and monomeric tubulin in the cells described in A. In C, cell extracts were loaded at a 1.25-fold serial dilution starting from 30 µg of protein to ensure measurement in the linear range of detection. D, quantitation by densitometry of the fraction of polymeric tubulin in cells treated under different drug conditions. Although paclitaxel increases tubulin in the polymeric fraction and nocodazole increases tubulin in the soluble fraction, noscapine causes no detectable changes in polymeric or soluble tubulin fractions. Reproduced from Zhou et al.12

Using the sister kinetochore distance as a measure of the tension exerted by kinetochore pairs attached to microtubules,70 immunofluorescent staining of sister centromeres and confocal microscopy was used to resolve the kinetochores of sister chromatids.12 The distance between the sister kinetochores in mitotic cells after 20 µM administration of 6 and in the absence of 6 was determined. It was determined that 20 µM administration of 6 resulted in an average reduction in the distance between sister kinetochores of 30%. These results were comparable with low dose vinblastine-treated cells.12 ACS Paragon Plus Environment


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The crystal structure of racemic L-α-noscapine was determined in 2010 by Seidel-Morgenstern et al.71 They described the dihedral angle between the fused ring systems to be 51.87°. The crystal structure of 6 bound within tubulin however, has not been described to date and therefore the exact pharmacophore is not fully elucidated. Along with a competitive binding approach using preexisting ligands, the ‘blind docking’ approaches have also been used to provide information on plausible docking sites of 6 and related analogs. Competitive binding studies using [3H]colchicinetubulin complex have been complemented by studies using ‘blind docking’. This approach was conducted to develop a reasonable predictive model, which could guide rational design for more effective derivatives of 6. The molecular docking method seemed to be the most fitting tool for gaining such understanding.72 However, issues arose using predictive modeling techniques due to the difficulty in ascertaining the correct binding pose of a ligand and the accurate estimation of the corresponding binding affinity. Building predictive models requires two main stages; the first involves the construction of a binding domain and the second involves the ligand preparation. The X-ray crystallographic structure of the colchicine and tubulin protein complex was used for the molecular docking and rescoring.

The structure was manually inspected and cleaning of the

structure resulted in the retention of the complex containing both the ‘A’ and ‘B’ chains of protein. Hydrogen atoms were automatically added to the model via the Maestro interface and all water molecules were removed.72 The molecular structures of each noscapine derivative in the synthetic strategy were generated using the molecular builder of Maestro and structures minimized in vacuo using Impact. The ligands where then docked into the tubulin receptor using Glide, which uses hierarchical filters to systematically search of positions, orientations, and conformations of the ligand in the receptor binding site. This ‘blind docking’ approach allowed the determination of the probable site of interaction of noscapinoids with tubulin. The colchicine-binding site of tubulin gave the best docking scores for noscapine derived ligands.72 The docking results by Naik et al. indicated the binding site for noscapine ligands either proximal to or overlapping with the colchicine binding site.73 Tuszynski et al. identified different clusters of potential binding pockets


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within the tubulin dimer where 6 could be accommodated. The most probable pocket for the binding of 6 was determined to be located at the intra-dimer interface region of the two subunits of α–tubulin (H1, the T5 loop, H6, the H6-H7 loop) and β–tubulin (the H-S2 loop, H7, the T7 loop, H8, S8-H10 and S9).74 Noscapine (6) was found to be surrounded by a network of interactions that consist of Arg214 establishing two hydrogen bonds via its amine groups with the methoxy-ether fragment of the dimethoxybenzene in noscapine along with the lone pair of electrons of the guanidine nitrogen of Arg214 interacting electrostatically with the electromagnetic field induced by the delocalized electron density of the aromatic ring in the dimethoxybenzene of 6.74 A weaker interaction existed between the aromatic ring of Tyr210 and the methoxy group at the C6 position of 6. The NH2 from Gln176 showed a relatively weak hydrogen bond to the oxygen atom of the 1,3dioxalane in 6. The negatively charged carboxylate group of Asp329 interacted with the positivelycharged protonated amine group in the tetrahydroisoquinoline motif of 6. The latter also makes a hydrogen bond with the side-chain hydroxyl group of Ser178.74

Figure 10. Noscapine docking results. FlexX-predictive binding pose for noscapine (blue) surrounded with the interacting amino acids (grey sticks) from its binding site (A). Overlaid docking results of nitrated noscapine (yellow), brominated noscapine (magenta) and the chlorinated (green) derivatives at the binding site (B) located at the intradomain region of the α-subunit (light grey cartoon) and β-subunit (light blue cartoon). Reproduced from Tuszynski et al. 2011.74



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The computationally-derived noscapine binding pocket of tubulin was discovered to be considerably hydrophobic. The ligand plot generated from the tubulin-colchicine and tubulin-9′-Brnoscapine docked complexes revealed identical amino acids within the binding pocket were interacting with both ligands. The calculated Kd scores for ligand binding potentials for 9′-Brnoscapine (10) and 6 illustrated strong correlation with the experimentally measured dissociation constants.74,75 The binding pocket surrounding 6 was defined as a region consisting of residues located approximately within 4.5 Å distance from noscapine atoms, namely: Gln176, Val177, Ser178, Glu207, Tyr210, Asp211, Arg214 in the α-tubulin and Lys326, Asp329, Leu333, Glu330, Asn349 and Thr353 in the β-tubulin subunit.74 3.4 Localization Patterns of Checkpoint Proteins after Noscapine Administration. The localization pattern of three checkpoint proteins, Mad2, Bub1 and BubR1, which monitor whether chromosomes have aligned properly at the spindle equator were also evaluated.12,76,77 The most drastic change was observed in the protein Mad2, which during prometaphase is localized to the kinetochore region and as the cell progresses into metaphase it is no longer observed at the kinetochores. Interestingly in mitotic cells treated with 6, Mad2 was present at the kinetochores near the spindle poles, but not observed at the chromosomes aligned at the metaphase plate.12


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Figure 11. Localisation pattern of spindle checkpoint protein Mad2 within the vicinity of kinetochores. Cells treated with 20 µM noscapine were compared with control metaphase cells and 6.7 nM vinblastinearrested mitotic cells. Mad2 and DNA were stained. Note mitotic cells arrested by noscapine or vinblastine, Mad2 was recruited to the kinetochores on chromosomes near the spindle poles, but not those aligned at the metaphase plate. Bar, 10 µM. Reproduced from Zhou et al.12

Bub1 and BubR1 however where localized to kinetochores of both groups of chromosomes.12 Immunofluorescence microscopy was then used to quantitatively compare differences of these checkpoint proteins at kinetochores on both aligned and unaligned chromosomes. The fluorescence intensity for Mad2 at kinetochores on unaligned chromosomes was 138-fold higher than that on aligned chromosomes within noscapine-treated cells.



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3.5 Tubulin Binding Domain of Noscapine. The loss of tension within noscapine-treated cells is not only attributed to the suppression of microtubule dynamics, but also results in lower amounts of microtubules attached per kinetochore. This resulted in a further reduction of tension on the kinetochores of chromosomes, which failed to align on the pro-metaphase plate. When slight variations exist in microtubule dynamics in vitro, this often results in severe disruption of microtubule-mediated events. Mitosis is the most dramatic cellular process. It is an irreversible event in which proper segregation of chromosomes during anaphase must prevail.

Mitotic

checkpoints exist to monitor this process and ensure both the proper attachment of microtubules and the maintenance of tension between the kinetochores of sister chromatids. Signal cascades exist to delay the onset of anaphase until the kinetochores of each chromosome pair are correctly attached the spindle microtubules and appropriate tension exists.78

The tubulin-binding site of 6 was

postulated by Naik et al. Colchicine is non-fluorescent in aqueous media but becomes fluorescent after binding to tubulin (excitation λ at 350 nm and emission λ at 430 nm) 2 provides the basis of a useful quantitative standard for competitive binding studies within the colchicine-tubulin binding site.29,30 As previously described the binding of 2 is a complex biphasic reaction. Colchicine (2) first binds to soluble tubulin to give small numbers of tubulin-colchicine complexes which is a low affinity site (phase I), then 2 settles into an irreversible site where it is then incorporated with tubulin at the microtubule ends (phase II).9 It is this slow step that promotes the fluorescent characteristics of the tropolone moiety of 2.32 Competitive inhibition of the colchicine-tubulin binding site by chemically similar ligands is determined through the inhibition of 2 binding on tubulin pre-incubated with test ligands.79-81 3.6 Blood Brain Barrier Penetration on the Treatment of Gliomas. In 2004, Landen et al. published the ability for 6 to inhibit the proliferation of rat C6 glioma cells in vitro. Patients diagnosed with gliobastoma (WHO grade IV) have a median survival rate of 9-12 months after diagnosis.82 Despite surgical resection, radiation therapy and/or chemotherapy, more than 90% of tumors recur within 2 cm of the primary tumor site. These tumors are highly invasive. Intrinsic


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chemo-resistance and/or poor penetrance across the blood brain barrier are common occurrences for current chemotherapeutics. These factors, have resulted in difficulties in the treatment of gliomas.83 To determine whether 6 is able to penetrate the blood brain barrier, a layer of cultured brain microvascular endothelial cells separating a donor and receiver chamber were utilized.

The

penetrance of 6 was compared with known permeates (morphine and met-enkephalin) and nonpermeable ([14C]sucrose) molecules. Noscapine (6) was deposited within the donor chamber at a concentration of 500 µM, and aliquots were removed from the receiver chamber at 15, 30, 60, 90, 120 min. The concentration of 6 was then determined by HPLC analysis. Noscapine (6) was found to cross the simulated blood brain barrier with a permeability coefficient of 21.7 × 10−4 cm/min. This rate was found to be 31.8% more efficient than morphine; an opiate known to possess lipophilic character.84 This model however did not account for drug metabolism that occurs in vivo which reduces the concentration of active drug. However, the model did provide evidence that 6 can efficiently cross the blood-brain barrier compared with other agents.

Landen et al. then

demonstrated the ability of 6 to cross the blood-brain barrier in vivo. Homogenized centrifuged whole brains of animals were treated with 6, and the concentration of 6 was determined by the HPLC of supernatants. The average concentration obtained from brain homogenates of noscapinetreated animals was 18.2 ± 3.7 µM. The examination of rat C6 glioma cells injected into immunedeficient mice illustrated that after 21 days of noscapine administration tumor volumes had reduced by 60% compared to the vehicle treated group.84 Hematological toxicity was absent as determined by complete blood count and no significant toxicity could be detected by histopathology in sites of rapidly dividing tissues such as spleen, duodenum and the liver. It is widely accepted that current microtubule therapy often results in peripheral neuropathy, however administration of 6 showed no evidence of neuropathy in both peripheral motor and sensory nerves.84 Glioblastoma multiforme (GBM); another form of glial cell carcinoma is currently treated with Temozolomide (TMZ).85 GBM is the most common and malignant of all gliomas. The median survival time for GBM patients with the currently therapy, consisting of surgery, radiation and



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chemotherapy is approximately 14.6 months.86 Temozolomide is a DNA alkylating agent which is able to cause DNA damage during cell replication. Treatment of GBM with TMZ has been shown to be effective in delaying tumor progression however its activity is only temporary and only leads to a prolongation of survival, GBM eventually recurs and when it does this is often accompanied by resistance to further TMZ treatment.87 The prevalence of resistance is a major impediment into the treatment of glioblastoma multiforme and therefore identifying new drugs, which can decrease and inhibit the growth of TMZ-resistant tumors is paramount.88 Noscapine (6) has already been shown to inhibit cell proliferation in glioma cells.89 Results from Jhaveri et al. have shown 6 has the ability to decrease the number of colonies in the drug-sensitive glioma cell lines, with an IC50 of 70 µM for LN229, 20 µM for A172 and 40 µM for U251, respectively. This data was consolidated with the prevalent sensitivity of TMZ-resistant populations to 6. It is believed that the invasive nature of glioma cancer after TMZ treatment is a product of the cells ability to migrate. The application of a non-cytotoxic dose of 6 (20 µM) significantly decreased the migration of these glioma cells by >2-fold in both hypoxic and normoxic conditions.88 The activity of 6 within an in vivo xenograft model using U251 TMZ-resistant glioma cells implanted intra-cranially into athymic/nude mice resulted in the increased survival of affected mice compared to vehicle control, showing no significant difference between 6 and 6 plus TMZ treated animals. TMZ alone however showed no significant effect on the survival of the mice with TMZ-resistant tumors compared to vehicle-treated control animals.88


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Figure 12. Effects of drugs on colony formation and cell cycle in glioma cell lines. Three different TMZsensitive and TMZ-resistant glioma cell lines (TMZr) were treated with TMZ (A) or noscapine (B) at increasing concentrations. After 48 h, culture medium was replaced with fresh medium containing no drugs and cells were incubated for another 12-14 days. Colonies were visualized using methylene blue. Individual colonies were counted and IC50 values were determined. U251 TMZ-sensitive (C) and U251 TMZ-resistant glioma cells (D) were treated with medium alone (left) or noscapine (right) at 30 µM for 24 h and cell cycle was analyzed by flow cytometry. Reproduced from Jhaveri et al. 2011.88

3.7 Synergistic Antitumor Activity of Noscapine Co-Administration.

Sachdeva et al.

explored the use of 6 in the treatment of lung cancer in combination with approved anticancer agent Cisplatin. The work by Lobert et al. has previously demonstrated the synergistic antitumor effects in leukemia cells with the combination use of 6 and vincristine within in vitro models.90,91 The combined effects of cisplatin and 6 on cell proliferation were evaluated by an isobolographic analysis method. The confidence interval (CI) values ranged from 0.31 ± 0.5 to 0.57 ± 0.4 for 50% cell kill suggesting synergistic to strong synergistic behavior between 6 (10-50 µM) and Cisplatin against both non-small cell lung cancer cell lines (H460 and A549). Synergism was evaluated through the administration of different concentrations of 6 and the resultant effect on the IC50 of cisplatin.90



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4. DERIVATIVES OF NOSCAPINE

Figure 13. Noscapine scaffold and sites of modification.

4.1 9'-Halonoscapine Analogs. Since the start of the new millennium a number of noscapine derivatives with various modifications to different moieties have been prepared and evaluated as potential anti-cancer agents. This has resulted in the identification of a number of semi-synthetic noscapine analogs with improved in vitro and in vivo activity. One of the first analogs synthesized was the 9′-bromonoscapine (10) (also known as EM011 in patent literature),92 which was subsequently assessed using a competitive binding assay with 2 to determine its binding domain and that of 6 within the tubulin dimer.

Figure 14. 9-Bromonoscapine (10).

Noscapine (6) at concentrations of up to 100 µM showed little inhibition of tubulin binding.93 9’-Bromonoscapine (10) produced a 66 ± 5% reduction in the binding of 2 at 100 µM. The ACS Paragon Plus Environment

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observed activity of 10 compared to parent compound, 6, could be due to the higher affinity of 10 for tubulin. The binding activity of 6 was not observed within this concentration range.75 9’Bromonoscapine (10) was tested at 50 µM and 25 µM, showing a modest inhibition of 2 binding of 40 ± 4% and 23 ± 7%, respectively.93 The competitive binding data for 10 suggest that it’s binding site is on the tubulin heterodimer near or overlapping the colchicine-binding site. However this data cannot exclude the possibility that 10 may bind to a site distinct and distant of the binding domain of 2 which may negatively influence its binding.93 To date the absolute binding site of 6 and related analogs on tubulin remains unknown. The crystal structure of 6 bound within its tubulin-binding domain is yet to be determined and therefore both competitive inhibition and computational “blind docking” approaches are necessary to provide information on plausible docking sites.

Figure 15. Treatment with compound 10 did not cause any pathological abnormalities in normal tissue architecture. (a) Hematoxylin and eosin staining of paraffin-embedded 5 µM sections of the brain, lung, liver, kidney, thymus, spleen, heart, and sciatic nerve. Bar = 20 µM. Reproduced from Li et al.94

The antitumor activity of 6 and 10 on human non-small cell lung cancer cells has been thoroughly investigated. In vitro cytotoxicity of 6 in H460 cells treated with increasing doses of 6



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(10-160 µM) for 72 hours was assessed. Apoptosis in H460 was evaluated using the TUNEL assay after the cells were treated for 72 h with 30 and 40 µM doses of 6, respectively. In vivo studies were also performed using female athymic Nu/nu mice, xenografted with H460 tumors. After 4 days 6 was administered orally at doses of 300, 450 and 550 mg/kg/day for an additional 24 days.95 As a control, xenografted tumors were also treated with Docetaxel (10 mg/kg). Noscapine (6) was shown to inhibit the growth of H460 cells with an IC50 value of 34.7 ± 2.5 µM. In situ DNA Nick end Labeling (TUNEL) was used to determine apoptosis of treated cells after 72 h of treatment with 6.

DAPI staining revealed chromatin condensation characteristic of apoptosis.95

The

administration of 6 at doses of 30 and 40 µM caused apoptosis, which was evident by nuclear condensation in the treated cells. Tumor regression was noted at the cessation of the study period (28 days) of 49, 65, 86 and 93% in xenografted tumor volumes at noscapine doses of 300, 450 and 550 mg/kg/day and docetaxel respectively.95 Aneja et al. investigated the ability for the 10 to activate a survivin-dependent apoptotic program within human non-small cell lung cancer cells. It was demonstrated that 10 inhibited the proliferation of a variety of human lung cancer cells with an IC50 range between 4-50 µM. 9’-Bromonoscapine (10) was able to cause the down-regulation of survivin, which is an important member of the inhibitor of apoptosis (IAP) family of proteins.96 Twelve different lung cancer cell lines were treated with gradient concentrations of 10 and the extent of cell proliferation was measured by the SRB assay.97 Of the 12 lung cancer cell lines assessed, the A549 human epithelial cell line was chosen to demonstrate the cellular and molecular mechanisms of 10. The A549 cellular line is the widely-accepted standardized experimental model for biological properties of alveolar epithlelial type II cells.97 9’-Bromonoscapine (10) was shown to cause a decline in survivin levels as early as 24 h with significantly low levels seen after 72 h post treatment. The lung cancer cell line H1792 however did not show any change in survivin levels over the time of drug treatment, which possibly suggests that survivin may play a role in the causation of resistance to 10-induced apoptosis. Whilst the knock-down of endogenous survivin expression resulted in the increased sensitivity of A549 cells to 10, the overexpression of survivin


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in A549 protected cells against 10-induced apoptosis.97 Noscapine (6) was also shown to induce apoptosis via the downregulation of survivin in human neuroblastoma cells with wild type of null p53.98 The efficacy of 6 was evaluated through the ability of 6 to inhibit the cellular proliferation of a comprehensive panel of neuroblastoma cells exhibiting variable but well defined genotypes. This panel included the neuroblastoma cell lines SK-SY5Y, SH-EP1, SK-N-MC, SK-N-AS, LA1-55N, NB1643, NB1691, SK-N-SH and IMR32. Each of these different neuroblastoma cell lines was treated with gradient concentrations of 6 and the extent of cell proliferation was measured by the SRB assay. Noscapine (6) was demonstrated to successfully suppress the cellular proliferation of neuroblastoma cells.

Each of the neuroblastoma cell lines used in this study presented well

characterized p53 status, namely wildtype (SK-SY5Y, SH-EP1, NB1643, NB1691), null (LA155N, LA1-5S) and mutant (SK-N-AS).

Noscapine (6) was previously shown to induce p53-

dependent apoptosis in colon cancer cells,99 however the data for neuroblastoma cell lines illustrated that the half-maximal growth inhibitory concentrations of 6 did not correlate the p53 status in neuroblastoma cells. For LA1-5S, which lacks endogenous p53, showed similar sensitivity as SK-SY5Y (wild type p53) or SK-N-AS (mutant p53) cells. Regardless of p53 status the IC50 values of administration of 6 for each neuroblastoma cell line were within the range of 21-100 µM.98 The downregulation of survivin contributed to 6-induced apoptosis was then assessed. Joshi et al. used a plasmid to construct encoding survivin specific siRNA, which could selectively knockdown endogenous survivin in SK-SY5Y and LA1-5S cells.

These nonspecific siRNA-

transfected SK-SY5Y cells were exposed to 6 (50 µmol/L) for 24 h which resulted in a ~3-fold increase in survivin protein compared with DMSO-treated control.

Cells transfected with a

survivin-targeted siRNA caused the level of survivin protein to decrease by >95% in SK-SY5Y cells. It was concluded that induction of survivin is marginally cytoprotective against 6-mediated cell death in the SK-SY5Y and LA1-5S cells.98 9’-Bromonoscapine (10) was also shown to inhibit vinblastine-resistant as well as vinblastinesensitive human T-cell lymphoid tumour xenografts in nude mice, resulting in a marked



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improvement in longevity of the cells.

Although 1 is able to inhibit tumors derived from

vinblastine-sensitive lymphoid cells, treatment with 1 often leads to excessive body weight loss and morbidity of the mice used. The examination of CEM and CEM/VLB100 cells after treatment by 10 resulted in a time-dependent accumulation of these cells in the G2/M phase, which was determined by increasing populations of cells with 4N DNA content. For CEM cells the maximum percentage of G2/M arrest was 45% and 47% for CEM/VLB100 cells at 12 hours of treatment with 10. G2/M cell population then decreased in respect to time whilst the sub-G1 population which has less than 2N DNA content increased reaching a peak at 72 hours of treatment with 10 (~57% for CEM and ~75% for CEM/VLB100). These results suggest cells treated with 10 promote G2/M phase arrest prior to apoptosis.100 Aneja et al. also explored whether 10-induced apoptosis occurred through the mitochondrial pathway. The mitochondrial pathway involves the loss of mitochondrial membrane integrity and transmembrane potential. The collapse of transmembrane potential can be monitored by a reduction in the uptake of the fluorochrome, DiOC6. The results demonstrated 10 caused a substantial reduction in the uptake of DiOC6 in CEM/VLB100 cells with 49% of cells showing reduced mitochondrial potential at 24 hours and reaching a maximum of 99% at 72 hours. The collapse of the mitochondrial potential causes an uncoupling of the respiratory chain and an efflux of small pro-apoptotic activating the key executioner caspase; caspase-3, which causes cleavage of the inactive pro-enzyme into an active form. The active form can then be monitored using a small peptide substrate, which becomes luminogenic on cleavage. Administration of 10 (10 µM) caused an increase in cleaved caspase-3 and cleaved Poly ADP ribose polymerase at 48 and 72 hours of treatment, indicative of extensive apoptosis. The prolonged treatment with 10 caused significant morphological changes and induced the formation of apoptotic bodies.100 Aneja et al. explored the in vivo potential of 10 by assessing the efficacy of oral treatment of 10 in xenograft nude mice models. Human tumors were subcutaneously implanted and when the tumors where of about 100 mm3 the mice were grouped randomly into control and treatment groups comprising 6 animals in each. The treatment groups received individually therapeutic dosages of 10 (300 mg/kg


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orally) and 1 (10 mg/kg intravenously) along with matched control groups, which received vehicle orally and intravenously. 9’-Bromonoscapine (10) treatment significantly reduced tumor volume in CEM xenografts. Tumor reduction by 80% compared to vehicle control was recorded on day 26 of treatment with 10. The treatment of mice with 10 did not cause any apparent body weight loss. Although 1 showed better results in tumor regression, mice in this group suffered from body weight loss and death.

This work was also extended to assess whether CEM/VLB100 xenografts

overexpressing Pgp/MDR1 phenotype also respond to 10. Treatment of CEM/VLB100 xenografts with 10 showed remarkable regression in tumour volume by about 81% after 32 days of oral treatment. The median animal survival increase by about 3-fold compared to control.100 The examination of normal tissues sections of the duodenum, liver, spleen, kidney, heart, brain, lung and sciatic nerve of tumor bearing mice using hematoxylin and eosin did not show any detectable pathologic abnormalities or metastatic lesions in the organs of these mice. Micro sections of the brain did not reveal any infarcted areas and the cerebral cortex and gray and white matters were normal for groups treated with 10.100 9’-Bromonoscapine (10) was also tested against a panel of prostate cancer cells with varying degrees of metastatic characteristics and variable hormonedependence.101

Hormone-independent PC-3 cells, along with three additional cell types with

varying metastatic potential, i.e. the parental androgen-responsive non-metastatic LNCaP and its lineage-derived, androgen-independent (C4-2) and bone-metastatic (C4-2B). PC-3 cells which lack endogenous androgen-receptor (AR) expression were determined to be increasingly more sensitive to treatment with 10 then LNCaP, C4-2 and C4-2B cells, which differ by the expression of various forms of mutated AR and exhibit hypersensitivity to androgen stimulation.101

Fluorescence-

activated cell-sorting (FACS) was also employed to determine the cell-cycles profiles of all four prostate cancer cell lines. The accumulate of treated cell in G2/M phase at 24 h post-treatment resulted in the emergence of hypodiploid ( 11 > 10 > 13 > 6.109 9′-Iodonoscapine (12) was found to possess modest cytotoxic effects whilst 10, 11 and 13 exhibited potent cytotoxic activity. These analogs however did not show any correlation between cell types and were cell-type dependent.109 These compounds were evaluated at the National Cancer Institute (NCI), through its Development Therapeutics Program (DTP), against their panel of 60 human cancer cell lines. Compound 10 was shown on average to possess a much lower IC50 value in each of the cancer types (including leukemia, non-small lung, colon, CNS, melanoma, renal, ovarian, breast and prostate cancer) compared to 6, although cell variability was evident.109 Each of the 9′-halogenated noscapine analogs were assessed for their ability to induce spindle abnormalities prior to apoptotic death of MCF-7 cells treated with each of the halogenated analog at 25 µM. Whilst untreated MCF-7 cells ACS Paragon Plus Environment


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exhibited normal radial microtubule arrays in normal interphase cells, treated cells exhibited pronounced spindles and condensed chromosomes that did not complete congression to the metaphase plate. Mitotic arrest was indicated as early as 12 h after drug treatment. After 24 h of drug treatment many mitotically arrested cells were visible using confocal microscopy.109

Figure 18. Halogenated noscapine analogs induce spindle abnormalities. Panels show immunofluorescence confocal micrographs of MCF-7 cells treated for 0, 12, 24, 48 and 72 h with 25 µM concentration of all five compounds [Noscapine (1), 9′-fluoronoscapine (13), 9′-chloronoscapine (11), 9′-bromonoscapine (10) and 9′-iodonoscapine (12)]. As expected, mitotic figures are abundant at 24 h while apoptotic figures start to appear at 48 h (scale bar = 30 µM). Reproduced from Aneja et al.109

In a further communication, Aneja et al. investigated the reduction of the lactone moiety using previously described literature, and subsequent fluorination conditions for the synthesis of the cyclic ether 9′-fluorinated noscapine analog (CEFNA) (structure undisclosed).105,110 CEFNA was shown to significantly inhibit the proliferation of human breast adenocarcinoma cells (estrogen- and progesterone-receptor positive, MCF-7 and estrogen- and progesterone-receptor negative, MDAACS Paragon Plus Environment

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MB-231) irrespective of their hormone status showing ~6-fold lower IC50 in MCF-7 cells and ~7fold lower IC50 in MDA-MB-231 compared to noscapine.110 This activity correlated with the presence of numerous fragmented nuclei at 72 h of drug exposure, which was reminiscent of apoptotic bodies. Cells treated with three different doses of CEFNA (5, 10 and 25 µM) were then analyzed using fluorescence-activated cell sorting (FACS) analysis of DNA content. Treatment of MCF-7 with CEFNA at these concentrations resulted in profound perturbations of the cell cycle resulting in a massive accumulation of cells in G2/M phase at 24 h in a dose dependent manner with 47% of the MCF-7 cells in G2/M compared to 24% in the control at 25 µM.110 6.2 Other 9'-Substituted Noscapine Analogs.

Computational chemistry methods by

Manchukonda et al. identified the 9′-position of 6 could possibly accommodate substituents such as amino and azido groups.72 Computational methods predicted that the 9′-NH2-noscapine analog 16 would bind to tubulin at a site overlapping with the colchicine-binding site and may possess improved antitumor activity compared to 6. The synthesis of the 9′-NO2-noscapine analog 14 was first described by Aneja et al. using 6 dissolved in acetonitrile followed by the addition of silver nitrate and trifluoroacetic anhydride giving the 14 in an undisclosed yield.111 These conditions were reportedly able to bypass the harsh oxidising conditions which compromise the stability of the labile C-C chiral bond joining the tetrahydroisoquinoline and phthalide ring systems.

Other

reagents such as ammonium nitrate, sodium nitrate, or silver nitrate in chloroform were described as giving low yields and had longer reaction times.111 Compound 14 was shown to quench tubulin fluorescence in a saturable manner with a Kd of 86 ± 6 µM.111 The anticancer activity of 14 was evident in its ability to produce a significant sub-G1 population. Lymphoma and ovarian cancer cells treated with 14 for 72 h showed extensive terminal apoptotic figures with fragmented DNA pieces and perturbed microtubule arrays when assessed using immunocytochemistry using a tubulin-specific antibody and a DNA-binding dye.111 Following this work, an attempt to synthesise the 16 was attempted through the 9′-azidonoscapine intermediate 15.93,112



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Scheme 2. Synthesis of nitrogen containing 9′′-Substituted Noscapine Derivativesa

a

Reagents and Conditions: (a) where X= H, AgNO3, TFAA, MeCN, 25 °C, 1 h; (b) where X = Br, NaN3,

NaI, DMF, 85 °C, 15 h; (c) SnCl2, PhSH, Et3N, THF, 25 °C, 2 h; (d) where X = Br, L-proline, NaN3, CuI, DMSO, reflux.

The initial synthesis utilized 10, synthesized using pre-existing literature.109 Compound 10 was reacted with sodium azide and sodium iodide in refluxing DMF for 15 h to yield 15, which was reduced using SnCl2 in the presence of thiophenol and triethylamine to yield 16. This gave the desired compound in a respectable yield of 83%. Compound 16 was shown to reduce the intrinsic fluorescence of tubulin in a concentration dependent manner with a respective Kd value of 14 ± 1 µM.72 Compound 16 was shown to be increasingly more active than 6 in inhibiting the proliferation of various human cancer cells when tested against a panel of 60 human cancer cell lines by the National Cancer Institute (NCI).72 The synthesis of 14 and 15 via the procedure described above was later questioned by Manchukonda et al. The described synthesis of 14 using silver nitrate and excess trifluoroacetic anhydride in acetonitrile111 resulted in mostly cleavage of 6 to give 7 and opianic acid (the open ring form of 9) with very little product. ACS Paragon Plus Environment

Attempts to catalytically

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hydrogenate 14 were sluggish leading to a mixture of unidentifiable products.112 Attempts to nitrate 6 using concentrated nitric acid at low temperature (0 °C), resulted in the acid catalyzed hydrolysis of C-C bond between the two heterocyclic rings of 6 resulting in the formation of 7 (92%) and opianic acid (89%) as the major products.112 The use of nitric acid for the hydrolysis of 6 to 7 and opianic acid was previously described in the literature.67

Following these failed attempts,

Manchukonda et al. attempted to generate 15 using another previously described procedure in the literature starting with 10 dissolved in DMF at 80 °C followed by the addition of sodium azide and sodium iodide.113 The reaction did not proceed even after prolonged reaction time (48 h), giving poor conversion to 15 (~3%). Changes to the conditions including different ratios of sodium azide/sodium iodide, temperature, time and solvent were not fruitful.112 Attempts to replicate the synthesis of 14 and 15 in our laboratory using the above procedures did not yield any of the desired products. The work by Markiewicz and co-workers,114 for the synthesis of primary aryl amines through copper-assisted aromatic substitution reaction with sodium azide, using a modified Ullmann-type coupling reaction, resulted in the successful synthesis of 16 from 10. The reaction facilitated the in situ reduction of the aromatic azide in good yield through the use of sodium azide, copper iodide and L-proline. Early attempts by Manchukonda et al. using the conditions described by Markiewicz et al. did not deliver 16. It was hypothesized that the bromo-aromatic ring may be influenced by a strong electron-rich environment, preventing the azide substitution reaction from occurring.112 The reaction proceeded most efficiently using 10 in the presence of copper(I)iodide, L-proline

and sodium azide in anhydrous DMSO under inert atmosphere to give 16 (62%). The

observation of debromination of 10 to 6 was also observed as a by-product of this reaction (15 %). This facile procedure however did not affect the sensitive C−C bond between the tetrahydroisoquinoline and phthalide ring systems. Porcu et al. explored the introduction of alkyl and aryl moieties at the 9′-position using 10 and the application of modern Pd-catalyzed cross coupling reactions (Suzuki reaction). These novel compounds were assessed for their antiproliferative activity relative to 10 against a panel of human



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cancer cell lines. Generally the activities of both 9′-alkyl and 9′-aryl presented higher IC50 values with respect to 10. This new series of 9′-position noscapine analogues were evaluated for potential inhibition of tubulin assembly in comparison with the potent colchicine binding site agent 4 using GTP- and glutamate-dependent polymerization assay, measuring the assembly after 20 min at 30 °C.115 Only a small number of these analogues possessed activity in this assay. All compounds possessed far less activity than 4 which demonstrated an IC50 value of 1.2 µM. Only one noscapine analogue showed any activity at 400 µM which was a 100-fold weaker that the inhibition observed for 4. The compounds did however demonstrate a significant G2/M arrest in a concentration dependent manner in both HeLa and Jurkat cell lines with the novel 9′-position analogs having essentially identical effects to those observed with 10.115 Compounds within this series were also shown to induce a time and concentration-dependent increase in the proportion of cells with depolarized mitochondria, which was in agreement with the induction of proteolytic cleavage of caspase-9 and caspase-3.115 4.3 Noscapine Derivatives with Modifications in the 7-Position. The chemical modification of 6 to synthesize related analogs with increased cellular potency has been a major focus of each research group working in the area. Each group’s pursuit to design and synthesize noscapine analogs with increased potency has led to an enriched understanding of the SAR of noscapinerelated analogs. Anderson et al. probed the core of 6 at the 7-position using the chemistry of Schmidhammer and Klotzer.116 The reaction of 6 with sodium benzyloxide in benzyl alcohol and DMF at 120 °C, resulted in a 1:1 mixture of 7-OBn diastereomers. The reaction resulted in the epimerization of the phthalide stereocenter of 6. The mixture of diastereomers however showed unexpected S-phase mitotic arrest within HEK293 cells.117 This was an unusual observation since previously described noscapine analogs were known to cause G2/M arrest. HPLC purification allowed the separation of each diastereomer which surprisingly showed similar potency to that of the racemic mixture within the cell cycle assay at 50 µM.117 The introduction of the 7-OBn group (18) resulted in unexpected activity.

Anderson et al. attempted to address the problem of


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epimerization using MeMgBr and BnOH in toluene-THF at 110 °C, but instead of the desired benzyloxide substitution, the reaction resulted in a regioselective O-demethylation at the 7-position with little epimerization. This gave the 7-OH analog 17 which was shown to arrest cells in G2/M.117

Scheme 3. Synthesis of 7-Modified Noscapine Derivativesa

a

Reagents and Conditions: (a) MeMgBr, BnOH, toluene-THF, 120 °C; (b) NaH, BnOH, DMF, 120 °C.

The free phenol facilitated alkylation of the 7-position of 6 with various alkyl halides to give products as pure stereoisomers. A number of substituted benzyl halides along with other aromatic isosteres were reacted with the free phenol 17. It was generally observed that these novel O-benzyl substituted derivatives (and isosteres) were S-phase mitotic arresters.117 Compound 17 was also converted to its triflate and Suzuki coupling reactions were used to generate a small number of biaryl compounds, however these compounds did not show significant S-phase arrest in HEK293 at 50 µM.117 Anderson et al. followed up this body of work by looking at the conversion of 17 to the corresponding triflate (7-OTf) and the subsequent amination reaction using palladium chemistry to generate the amino analog (7-NH2) (19).118

Compound 19 was observed to inhibit tubulin

polymerisation in vitro. Analysis within microtubules using Swiss 3T3 cells showed 19 to be more than 2-fold more potent than 6, with no observable effect on non-dividing Swiss 3T3 cells. The 7OTf was also converted to the 7-thiophenol (7-SH) analog (structure undisclosed) which, interestingly enough, shared similar activity to 19, arresting HEK293 cells in G2/M.118 The activity was presumed to be due to the presence of hydrogen bond donors at the 7-position, but this was refuted when conversion of the 7-OTf analog to the 7-H analog (structure undisclosed) also



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demonstrated G2/M arrest. Stereochemistry was found to be not imperative for noscapine-derived S-phase inhibitors compared to analogs which functioned as G2/M inhibitors.117,118 Compound 17 appeared to have an increased half-life and volume of distribution compared to 6 and 19, with all analogs showing readily measureable oral bioavailability ranging from 11% to 22%.118

An

extension of this work by Mishra et al. investigated acylation reactions of 17 whilst a second strategy, investigated the synthesis of a series of carbamate esters of the key intermediate 17.119 It has been reported that carbamate esters are able to mask free phenolic groups in cytotoxic compounds.120 The regioselective O-demethylation at the 7-position to give 17 which was ~100fold more potent than 6, indicating the chemical modification around the phthalide ring has a significant impact on the biological activity of 6.118 Acylation reactions using acetic anhydride and benzoyl chloride delivered two 7-acyl derivatives (structures undisclosed) for in vitro analysis. The 7-acetyl analog was synthesised to observe the influence of the polarizable carbonyl group on activity in contrast to the inert 7-OMe of parent compound noscapine. The 7-benzoyl analog was prepared to compare the effect of carbonyl containing alkyl to aryl functionality.119 The second strategy investigated the generation of a small number of carbamate esters of 17 using a number of commercially available isocyanate reagents (ethyl, phenyl and benzylisocyanate). Generally the IC50 values for each of the novel 7-position substituted noscapine analogs presenting either acetyl or carbamate functionality showed improved activity compared to 6 in cell lines including A549, CEM, MCF-7 and PC3. Of these compounds the 7-acetyl analog was observed on average to be the most-effective against most cell lines used in the study but showed weaker activity within MCF-7. Different sensitivities by each cancer cell line to the test compounds was observed suggesting the incidence of inter-cell line variability.119 It was ostensible that as the bulk of the substituent increased at the 7-position, activity decreased. It can be postulated that an increase in steric bulk at the 7-position inadvertently had a negative effect on IC50 due to steric interaction with the binding domain. These compounds where also tested on normal human fibroblasts at concentrations as high at 100 µM. Successful chemotherapeutics spare normal


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cells whilst inducing apoptosis of cancerous cells. These novel analogs did not affect the viability of normal human fibroblasts. The expression levels of survivin, an anti-apoptotic protein of the inhibitor of apoptosis (IAP) family which blocks apoptosis by inhibiting caspases121 declined upon treatment with these compounds. This feature was also reported for 10.97,119

Figure 19. Benzofuranone ring substituted noscapine analogs exhibit significant inter-line variability. A, B, C, D and E are the plots of percent cell survival versus concentration of noscapine analogs for cancer cells with varying tissue origins i.e. lung (A549), lymphoma (CEM), breast (MCF-7), pancreas (MIA PaCa-2) and prostate (PC3) used for the determination of IC50 values. F is a bar-graph representation of IC50 values of noscapine analogs in various cancer cells used in the study. The values and error bars shown in the graphs represent average and standard deviations, respectively, of three independent experiments (p < 0.05). Reproduced from Mishra et al.119

Bennani et al. investigated the binding site of 6 compared with 17 and the 7-NH2-noscapine analog (19) in an attempt to understand more clearly the binding site of these analogs.

A

microtubule assembly assay with purified tubulin was used to measure noscapine binding. Tubulin ACS Paragon Plus Environment


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is able to assemble into microtubules with a critical concentration of 3.3 µM.122 Bennani et al. used an assay in which 15 µM of purified tubulin was employed with the expectation that in the absence of a microtubule binding ligand, the tubulin will polymerise to form pellets. The administration of the 19 resulted in the reduction of tubulin pellet formation by 50% at a concentration of around 1 mM with a respective IC50 of 1.2 ± 0.3 µM.

Unlike 19 which possesses sub-stoichiometric

inhibition of tubulin assembly, 17 showed weaker inhibition, requiring above stoichiometric amounts to reduce polymer formation.123

Although 6 and 17, did not express changes in

fluorescence upon incubation with tubulin at 10 µM, 19 could be employed to monitor the binding state of the compound and the number of binding sites on tubulin dimers.123 Three binding assays where utilized to determine whether 19 was affected by the oligomerisation state, if its binding site was located within the previously defined intra-dimer binding domain of 2.15 The first assay involved the use of 5 µM of tubulin, which was titrated with increasing amounts of 19, and the amount of bound and free compound was quantified by changes in fluorescent properties of the compound upon tubulin binding. The binding site for this compound within the tubulin dimer was determined to be 0.87 ± 0.03 sites, indicating a single site and 1:1 stoichiometry.123 The second assay utilized 5 µM of the compound, which was titrated with increasing amounts of tubulin to 100 µM.

The amount of bound and free compound was quantified by employing the change of

fluorescent properties of the compound upon binding to tubulin. The final assay measured the influence of Mg2+ on the binding of 19 to tubulin. Compound 19 (5 µM) was titrated with growing amounts of tubulin to 100 µM in the presence of 0, 0.5 and 2 mM MgCl2 and the amount of bound and free drug was quantified by determining the change in fluorescence upon binding to tubulin. The binding site was identical in each of the three assays indicating the binding site is not affected by oligomerisation state.123 Bennani et al. also investigated a competition assay between the 19 (10 µM) and tubulin (10 µM) incubated in the presence of 100 µM of 5, 1 and isohomohalichrondrin, however no changes in fluorescent properties were observed indicating these compounds did not compete for the same site.123

Co-incubation with 50 µM nocodazole (a synthetic


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aminobenzimidazole) resulted in a slow decrease in fluorescence indicating the possibility of binding to similar or overlapping site.123 Saturation transfer difference (STD) experiments were also used to determine if 6 and related analogs are recognized by the α/β-heterodimer. STD spectra clearly resulted in an enhanced proton signal for noscapine. STD spectrum carried out using 19, also showed clear STD effects for ligand protons. STD experiments were utilized to determine if 6 and 19 compete for the same site on the tubulin heterodimer. The addition of 19 to a sample containing 6, did not decrease the STD signal of 6 indicated these compounds do not compete for the same site.123

Figure 20. Chemical structures of the 7-modified noscapine analogs 17 and 19.

4.5 Noscapine Derivatives with Modifications in the 1- and 6'-Positions. The initial step for the synthesis of N-substituted analogs required the N-demethylation of 6, to give N-nornoscapine 42, which could then be further functionalized. Many classical N-demethylation conditions were trialed for the N-demethylation of 6 including the use of various chloroformate reagents and the von Braun reaction, however these were unsuccessful in the synthesis of the secondary amine scaffold.105,124 It is reported that these conditions resulted in cleavage of 6 at the benzylic and tertiary centers adjacent to the amine functionality.105 conditions125

successfully

delivered

the

desired

Non-classical Polonovski reaction

secondary

amine

core

for

further

functionalization.105 The reaction procedure began with the synthesis of the tertiary amine oxide using m-CPBA in chloroform followed by the acidification of tertiary amine oxide with hydrochloric acid to form the tertiary amine oxide as the hydrochloride salt.126 Noscapine-N-oxide hydrochloride was then dissolved in a citric acid buffer solution containing Fe(III)-citrate which gave



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the desired secondary amine core for further derivatisation.105,126 Aggarwal et al. synthesized a small series of N-substituted phenyl ureas and thioureas.105 The bioactivity data for these analogs however has not been reported. Aggarwal et al. also investigated the reduction of the noscapine lactone moiety, in an attempt to improve the in vivo stability of noscapine analogs given the inherent base sensitivity of lactone functionalities in aqueous media. The cyclic ether variants of both 6 and N-nornoscapine (21) were synthesized by the generation of diborane in situ using BF3•Et2O and NaBH4 in THF at low temperature. The cyclic ether N-nornoscapine analog was then functionalized to generate the N-(3chlorophenyl)carboxamide analog (20).105

Figure 21. N-Carbamoyl cyclic ether noscapine derivative (20).

Research by our group was directed to the understanding of the SAR of noscapine related analogs. Initially our aim was to establish improved methods for the N-demethylation of 6 with the intent of introducing activity through functionalization of the tetrahydroisoquinoline amine. Preexisting literature precedence for the N-demethylation of 6 using a ferric citrate buffer solution existed, but was tedious and low yielding, and a more efficient and cleaner method was desired.105,126 The N-demethylation of various opiates using Polonovski-type reaction conditions has been an area of interest within our laboratory.127-130 Conditions discovered for the efficacious N-demethylation of opiate-N-oxides (e.g. morphine, thebaine, codeine)127 using FeSO4•7H2O in methanol provided the best conditions for the N-demethylation of noscapine-N-oxide hydrochloride in good yield.131 The pre-existing literature yield of N-nornoscapine (42) using a ferric citrate


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buffer solution was 26%,126 the use of FeSO4•7H2O in methanol at low temperature improved the yield to 78%. The ability to synthesize 42 in respectable yield provided the opportunity to generate a number of 6′-modified analogs. Compound 42 was sequentially reduced using a modified version of the literature procedure to give 21, which would improve the aqueous stability of our 6′analogs.131,132

Compound 21 was treated with a number of alkyl halides, acid chlorides,

chloroformate reagents, isocyanates and isothiocyanates to yield a focused library of novel cyclic ether N-substituted analogs. Synthesis of each compound class was relatively straightforward, high yielding, and proceeded under facile conditions. The purification of a small number of analogs was challenging, with reagent by-products enclosing similar retentions on silica as the desired compounds.

Each of the compound classes (N-alkyl, amide, carbamate, thiourea, urea) were

assessed for their ability to cause mitotic arrest of various cancer cell lines. Our results illustrated a proportion of the 6′-position analogs, were able to cause G2/M arrest, showing anticancer activity within PC3, MCF-7 and Caco-2 cell lines.

At similar concentrations 6 was found to be

ineffective.131 A handful of the compounds showed high potency, with EC50 values ≤10 µM. Substituting the N-methyl group for longer N-alkyl derivatives was detrimental to activity and these analogs seemed to be less stable. Moderate activity was observed for both N-benzoyl and Nphenacyl amides, however N-carbamoyl and N-thiocarbomyl (urea and thiourea) with short saturated alkyl chains showed promising in vitro activity in both PC3 and MCF-7 cell lines and to a lesser extent within Caco-2. Cell line variability was apparent for these active compounds. Strong activity within PC3 and MCF-7 was often accompanied by weaker activity in Caco-2 cell lines. The activities of phenyl and substituted phenyl urea/thiourea systems resulted in severe reductions in activity.131 The cyclic ether N-ethyl urea analog 22 was shown to be the most active compound within the series with EC50 values of 6.7 and 3.58 µM (PC3 and MCF-7, respectively) and became a lead core for further development.



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Scheme 4. Synthesis of 6-Modified Noscapine Derivativesa

a

Reagents and Conditions: (a) RX, Cs2CO3, MeCN, reflux, 18 h; (b) ROCOCl, Et3N, CH2Cl2, rt, 24 h; (c)

RCOCl, Et3N, CH2Cl2, rt, 3 h; (d) RNCO/RNCS, MeCN, rt, 3 h.

Manchukonda et al. followed up this body of work through the rational design and synthesis of a number of noscapinoids through the functionalization of 42, which they referred to as ‘third generation’ α-noscapine analogs.133 The overall aim was to maintain the electronic environment of the isoquinoline nitrogen mostly intact (i.e. keeping the ionization state of the N-methyl group consistent with the parent alkaloid, 6). The compounds synthesized consisted of a small number of substituted aromatics possessing a methylene linker (23a−23g) along with N-propan-2-one and Nmethyl/ethyl ethanoate analogs (23h−23j)

Figure 22. Third generation α-noscapine analogs ACS Paragon Plus Environment

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In silico molecular modeling calculations were conducted for each compound to investigate their binding affinity based on a reasonable predictive model prior to chemical synthesis.133 The compounds were chemically synthesized and assessed for their tubulin binding properties and their effect on cell cycle progression in rapidly dividing cancer cells of different tissue origins; CEM (human lymphoblast cell line), HeLa (human cervix cell line), A549 (human lung adenocarcinoma epithelial cell line) and MCF-7 (human breast epithelial cell line). Molecular structures of each novel noscapine derivative were prepared using molecular builder of Maestro and the structures were energy minimized using macromodel and OPLS 2005 force field with PRCG algorithm. A reasonable predictive model was developed for calculating the binding free energy (∆Gbind) of ligands utilizing the linear interaction energy (LIE) empirical equation. All the designed analogs yielded better docking scores than the parent compound, 6.133 The in vitro results illustrated each of the cancer cell types utilized for their biological evaluation were more susceptible to these novel third generation compounds compared to 6 with lower IC50 values. The IC50 values however do not show any correlation among these analogs and were cell-type dependent.133 The effects of each of these compounds on the percentage of G2/M and sub-G1 cell populations in MCF-7 cancer cells as a function of time using fluorescence activated cell sorting (FACS) analysis was also assessed. The effects of each compound were assessed at 25 µM for 0, 24 and 72 hours of drug treatment. Treatment of MCF-7 cells with the analogs for 24 and 72 hours led to considerable perturbations in the cell cycle progression leading to a massive accumulation of cell populations with 4N DNA indicative of G2/M arrest. Treatment of MCF-7 cancer cell lines with each analog resulted in significant increases in sub-G1 populations having hypodiploid (2N) DNA content, which reflects fragmented DNA, a characteristic of apoptosis.



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Table 1. IC50 values (a drug concentration required to achieve a 50% inhibition of cellular proliferation) of noscapine derivatives N-nornoscapine & 23a-j for various cancer cell types.134 Compound

CEM (µM)

HeLa (µM)

A549 (µM)

MCF-7 (µM)

N-nornoscapine 23a 23b 23c 23d 23e 23f 23g 23h 23i 23j Noscapine

8.9 ± 0.6 8.3 ± 0.4 9.0 ± 0.4 10.0 ± 0.8 7.7 ± 0.3 7.5 ± 0.6 6.7 ± 0.3 11.9 ± 0.8 6.9 ± 0.5 9.5 ± 0.7 13.6 ± 1.3 14.5 ± 2.5

20.9 ± 1.8 19.4 ± 1.4 21.2 ± 2.1 23.7 ± 1.5 17.9 ± 0.9 17.3 ± 0.7 15.3 ± 0.5 28.3 ± 2.1 15.8 ± 0.5 22.3 ± 1.2 32.4 ± 2.4 24.0 ± 2.9

41.6 ± 2.1 37.7 ± 1.8 42.4 ± 2.3 49.0 ± 2.5 33.5 ± 1.7 32.1 ± 1.6 26.9 ± 1.4 61.3 ± 3.1 28.0 ± 1.5 45.4 ± 2.5 72.3 ± 3.5 72.9 ± 4.6

33.6 ± 1.3 31.2 ± 1.5 34.1 ± 1.6 38.2 ± 2.5 28.7 ± 0.8 27.8 ± 1.3 24.5 ± 0.8 45.8 ± 2.2 25.3 ± 1.2 36.0 ± 1.9 52.6 ± 3.8 42.3 ± 2.7

Cancer cells used in the assay namely, CEM: human lymphoblast cell line, HeLa: human cervix cell line, A549: human lung adenocarcinoma epithelial cell line and MCF7: human breast epithelial cell line. Each value represents mean ± S.D. from three different experiments performed in triplicates. The variation in IC50 values are statistically significant among the third generation α-Noscapine derivatives (F = 169.93, P < 0.001) as well as among different cancer cell lines (F = 1530.48, P < 0.001) based on 2-way anova test.

4.6. Water-soluble Noscapine Analogs. The limited water solubility of noscapine and related analogs encumbers its development as an oral antimitotic agent for clinical administration. Henary et al. have recently reported the development of water-soluble noscapine analogs with negatively charged sulfonato and positively charged quaternary ammonium groups using noscapine, 9bromonoscapine 10 and 9-aminonoscapine 16 as scaffolds.134 The insolubility of noscapine analogs in water has emerged to be a major issue for in vivo experimentation,134 which is ascribed to be the result of the substituted isoquinoline and isobenzofuranone ring systems which present hydrophobic structural characteristics. Henary et al. prepared 16, 10 and 17 using existing literature. Compound 16 was reacted with 3-bromopropyltrimethylammonium bromide to give the desired positively charged 9-quaternary ammonium alkylamino derivative 25a. Compound 16 was also reacted with alkyl sultones, 1,4-butane sultone and 1,3-propane sultone to generate the charged alkyl sulfonato groups on the amino moiety (25b and 25c). Compound 17 was also alkylated with the above ACS Paragon Plus Environment

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quaternary ammonium alkyl bromide and sultones (24a-24c). Compound 10 was O-demethylated to facilitate the direct comparison of adding water-solubilizing groups 26a-c (Fig. 23) to identify whether the introduction of charged species could impart aqueous solubility and improved biological efficacy.134

Figure 23. Water-soluble noscapine analogues.

The solvation free energy of each analog was calculated using the Poisson Boltzmann electrostatics. The results highlighted the compounds with negatively charged alkylsulfonato group (24b, 24c, 25b, 25c, 26b and 26c) were the most soluble with the amino-substituted compounds (24a, 25a and 26a) being more soluble then 6 and 10. Each of the compounds was evaluated against tubulin assembly using two systems: MAPs rich tubulin and pure tubulin to establish whether tubulin was interacting with the test ligands. Compounds 24b, 26b and 26c were found to be the most effective in decreasing the polymerization of MAPs-rich tubulin with a percent decrease in MAPs-rich tubulin polymerization at 50 µM concentrations of 74±13%, 68±18% and 61±21% respectively.134 The presence of bromine at the 9′-position in 26b and 26c may contribute to the depolymerizing effect on MAPs-rich tubulin shown by the presence of 4-oxy-butane-1sulfonic acid and 4-oxy-propane-1-sulfonic acid groups. These three compounds; 24b, 26b and 26c along with 6 and 10 were assessed using in silico docking studies to determine how these compounds docked into the active site. The docking results suggested the structure of 6 can be divided into two halves a) the tetrahydroisoquinoline moiety which can insert deep and interact



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closely with the tubulin dimer, whereas b) dimethoxyisobenzofuranone group gets closer to GTP in ligand-tubulin docked complex. Results from this study suggested 26b possessed the strongest affinity for the tubulin dimer, with the bromo-substituted compounds adopting similar binding configurations in the active site, which was determined to be different from the unsubstituted compounds. Compounds with a longer side chain (26b) docked better in the active site, which is possibly a result of allowing the sulfate group to move away from the phosphate group of GTP compared with compounds possessing shorter side chain (26c) 4.7 Multi-functionalised Noscapine Derivatives. Our research group further elaborated the SAR of noscapine analogs by investigating the synthesis and biological evaluation of multifunctionalized derivatives.135 We introduced multiple modifications to 6, combining modifications at the 1-, 7-, 6′ and 9′-positions to determine if any additive SAR existed. The introduction of the N-ethyl urea functionality at the 6′-position of 21 to give compound 22 proved to be our most potent analog of our initial work with EC50 values of 6.70 and 3.58 µM in the PC3 and MCF-7 cell lines, respectively.131 Compound 22 exhibited comparable activity to the best noscapine analogs described within current literature. Introduction of the ethyl carbamoyl moiety was combined with changes to the noscapine core at the 7- and 9′-positions. Modifications at both the 7- and 9′positions have been extensively described within the literature resulting in a number of analogs with superior anti-cancer activity compared to 6 in various cancer cell lines.109-112,117,118,133 Our chemical efforts explored the coupling of 22 with modifications, which included the halogenation and amination of the 9′-position and the regioselective O-demethylation at the 7position. Suzuki coupling conditions were also utilized to install substituted aromatic systems at the 9′-position with the reaction of 30 with a number of substituted phenylboronic acids.135,136 Highly functionalized noscapine analogs with multiple modifications could be synthesized by a plethora of reaction sequences.

Our initial work demonstrated the N-demethylation of 6 and subsequent

lactone reduction of 42 to give the cyclic ether derivative 27 was the critical sequence of steps for multi-step synthesis involving 27. The overall yield across these two steps (i.e. N-demethylation


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and reduction) was found to be 61%.131 The introduction of various halogen atoms at the 9′position ensued through the use of facile conditions. The use of N-halosuccinimides (i.e. N-chloro, N-bromo and N-iodosuccinimides) gave the desired 9′-halogen derivatives albeit under slightly differing solvent conditions and reaction temperatures. Use of N-chlorosuccinimide for the synthesis of 28 was replaced with sulfuryl chloride, due to prolonged reaction times, high temperature required and the formation of multiple by-products, which were difficult to separate using flash chromatography.

The use of sulfuryl chloride at low temperature in chloroform

delivered the desired compound 28 in 54%. N-Bromosuccinimide in acetic acid successfully delivered the 9′-bromo analog 29 in 72% yield whilst the use of N-iodosuccinimide required the use of trifluoroacetic acid to activate the succinimide for aromatic substitution, which gave the desired 9′-iodo analog 30 in 68% yield.135

Attempts to synthesise the fluorinated derivative using

conditions described within the literature were not fruitful.110 For each of the cyclic ether 9′-haloN-nornoscapine analogs (28-30), the 6′-position ethyl urea functionality was installed by reacting each intermediate with ethyl isocyanate. These reactions proceeded efficiently with respective yields ranging from 61-68% (Scheme 5).135



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Scheme 5. Synthesis of Multi-functionalised Noscapine Derivativesa

a

Reagents and Conditions: (a) for X = Cl: SO2Cl2, CHCl3, 0 °C for 28; for X = Br: NBS, AcOH, rt for 29; for

X = I: NIS, TFA, rt. for 30; (b) 29, F2, Amberlyst-A, THF, reflux; (c) EtNCO,MeCN, rt, (d) 33, ArB(OH)2, PdCl2(PPh3)2, THF, 1 M Na2CO3, 100 °C; (e) 32, L-proline, NaN3, CuI, DMSO, reflux.

Suzuki coupling reactions between the 9′-iodinated analog 33 and various phenylboronic acids using coupling conditions similar to those within the literature,136 delivered a small number of substituted aryl moieties at the 9′-position which were assessed for in vitro cellular activity. The Suzuki reactions proceeded in low yields ranging from 7 to 48%, but furnished sufficient quantities of the desired products for pharmacological evaluation.135 Our group also explored the introduction of the 9′-amino moiety. The reaction procedure was derived from the existing literature for 9′position amination of 6.112 Compound 32 was dissolved in anhydrous DMSO and heated at 130 °C for 8 h in the presence of NaN3, NaI, L-proline and CuI giving 41 in a modest yield of 25% (Scheme 5).135 Attempts to synthesize the 9′-fluoro derivative from 41 using classical fluorination conditions (i.e. Sandmeyer and Balz-Schiemann) were unsuccessful. Compounds 31−33 were assessed for in


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vitro cytotoxicity in PC3, MCF-7 and PANC-1 cells. These compounds possessed significantly improved activity compared to both 6 and 22, across each of the cell lines assessed. Cell variability was prevalent with most analogs showing reduced activity within PANC-1 as calculated by the percentage increase in arrested cells with reference to the percentage of cells in G2/M in vehicle control (DMSO). Compounds 31–33 showed higher EC50 values within PANC-1 compared to the EC50 values within PC3 and MCF-7 cell lines.135 Compound 41 on average was 14-fold less active in each of the cell lines tested. The presence of any aryl functionality in the 9'-position (34–40) abolished activity completely.135 The relatively weak antimitotic activity of 6 resulted in the precipitation of 6 from vehicle control at concentrations which may elucidate activity in our assays.131 The 9'-chloro analog 31 proved to be the most active of the halo derived compounds with EC50 values of 1.5, 1.7 and 0.9 µM at PC3, MCF-7 and PANC-1, respectively. Compound 31 was further elaborated by the regioselective O-demethylation at the 7-position. Compound 31 demonstrated significant activity and reduced cell line variability compared with the noscapine analog previously synthesized in our group. This compound was subsequently subjected to regioselective O-demethylation to deliver the 7-OH analog 46. Synthesis began with 42 (Scheme 6) and initially involved chlorination at the 9'-position using N-chlorosuccinimide in trifluoroacetic acid. The reaction proceeded in low yield to give 34% of the desired compound 43, which was subsequently reacted with ethyl isocyanate in acetonitrile at low temperature to give the desired 6' and 9'-position modified compound 44. The lactone was reduced in the final step since it is suggested that the source of regioselectivity for the O-demethylation is due to the stabilization of the resultant phenoxide by the neighbouring carbonyl group.117 The O-demethylation reaction was completed in accordance with the pre-existing literature,117 resulting in the synthesis of 45 in good yield 64%.

The reduction of the lactone moiety was fulfilled using the modified literature

procedure described by our lab131 to give 46, which pertained modifications at the 1-, 6'-, 7- and 9'positions. Existing literature had described each of these modifications to 6 independently resulted



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in improved activity, the combination of 1-, 6'-, 7- and 9'-position modifications on the same scaffold did not correlate with improved activity, but rather a compound with modest activity. Compound 46 demonstrated weaker activity across PC3, MCF-7 and PANC-1 cell lines compared to 31; our most active compound to date. For noscapine related analogs it is often difficult to suggest which compound(s) or compound class are the most active; a number of significantly active compounds in comparison to 6 have been described in the current literature. Each of the research groups working on improving the efficacy of noscapine derivatives assess their compounds using different biological assays, cellular lines and varying concentrations which makes directly comparing the efficacy of derivatives difficult. This is compounded by the cell line variability that noscapine analogs demonstrate across many cancer cell lines, with some cellular lines being significantly more resistant than others. Joshi et al. published the activity of 6 and 10 against the broad National Cancer Institute (NCI) panel of 60 human cancer cell lines organised into subpanels of cancer types.109 This was the most expansive study into the sensitivities of multiple cancer cell lines to 6 and 10 indicating multiple examples of cell line variability.


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Scheme 6. Synthesis of Multi-functionalised Noscapine Derivativesa

a

Reagents and Conditions: (a) NCS, TFAA, reflux; (b) EtNCO, MeCN, -5 °C; (c) MeMgBr, BnOH, toluene,

120 °C; (d) BF3.Et2O, NaBH4, rt.

4.8 Susceptibility of Noscapine Derivatives to Multi-Drug Resistance. The frequent and unwanted, observation for cytotoxic agents is the presence or emergence of drug resistance, which is generally a result of the over-expression of multidrug efflux pumps, which confers a broad resistance to many unrelated classes of drugs. Compounds, which are substrates of the drug efflux pumps ABCB1 and ABCG2 experience considerable reductions in their cytotoxic potential in drug resistant cell lines. The cytotoxic drugs 2 and mitoxantrone are substrates for ABCB1 and ABCG2 respectively.137,138 The presence of ABCB1 in NCI-AdrRES cell line reduced the cytotoxicity of 2 by 260-fold from 0.36 ± 0.01 nM in the parental cell line to 96 ± 15 nM. The presence of ABCG2 in the MCF-7FLV1000 cell line reduced the potency of mitoxantrone by 17-fold from 26 ± 13 nM in MCF-7 cell to 435 ± 123 nM. Given the major problem for existing antimitotic compounds is the emergence of resistance, which is a function of the active efflux of drug out of a cell by ATPbinding cassette transporters, our research group assessed whether our active lead compounds were



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substrates for cellular efflux by drug efflux pumps ABCB1 and ABCG2. The cytotoxic potency of each of the reduced 9'-halo-6'-ethylaminocarbonyl-noscapine analogs (31–34) along with the nonhalogenated reduced 6'-ethylaminocarbonylnoscapine analog (22) was assessed in both normal MCF-7 cell line which did not express either multidrug efflux pump and the drug resistant MCF7FLV1000 cell line.135 To date limited data is described regarding the efflux status of noscapine related analogs to drug resistant cell lines.

Our studies suggested there was no statistically

significant difference in the ability of 22, 31-34 to produce cell cytotoxicity between the resistant and sensitive cells. Compound 31 illustrated very marginal deviation in its efficacy between control cells (MCF-7) and the resistant cell lines MCF-7FLV1000 and NCI-AdrRES. This trait was also common for the other 9'-halogen analogs. The lack of variance between the resistant and sensitive cell lines to these noscapine related analogs indicates these compounds are unlikely to be substrates for transport by either ABCB1 or ABCG2.135 4.9 Related Tetrahydroisquinoline Derivatives with Antimitotic Activity. Recently the concept of biology-oriented synthesis (BIOS) has been described.139 Bioactive small molecules are excellent tools and probes for the analysis of complex biological networks and systems endowed with robust and redundant functionality.139 The limitation contained within the evolution of both small molecules and protein binding sites means the structural motifs of proteins and natural products is limited, these scaffolds which are characteristic of natural product classes can be regarded as “privileged”, with compound classes derived from or inspired by natural product classes regarded as biologically relevant and pre-validated.140,141

Along with this validation,

properties such as structural complexity and drug likeness render these small molecule compound classes as valuable probes for medicinal chemistry and chemical biology investigations. BIOS employs biological relevance as a key evolutionary pre-selection criteria of natural product scaffolds. This facilitates inspiration for the design and synthesis of focused compound collections with biological activity related to but not necessarily identical to the activity of the guiding compound classes.139,141,142 Using the logic of BIOS Zimmermann et al. explored the synthesis and


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biological evaluation of a noscapine-inspired collection of tetrahydroisoquinolines. The synthesis of

tetrahydroisoquinolines

commenced

dihydroisoquinolin-1(2H)-ones,

obtained

with through

the

readily

accessible

Bischler-Napieralski

substituted

cyclisation

of

3,4the

corresponding carbamates. The cyclic lactam was subsequently deprotonated and reacted with bocanhydride giving the corresponding N-Boc lactams.142,143 These compounds were alkynylated in an efficient one-pot procedure, which included coupling of the alkynyl boranes with lactams. The alkynyl lactam’s were subsequently deprotected and copper(I)-catalysed azide-alkyne cycloaddition of the terminal alkynes with various azides, or Sonogashira coupling to give the target noscapineinspired compounds. To expand the collection further, the natural product cotarnine was used in a reaction sequence comprising both nucleophilic addition and subsequent N-methylation to yield both tertiary amines and quaternary ammonium salts. Given 6 contains two chiral centers with specific stereochemistry, enantioselective methods were developed for the alkyne addition to generate compounds with identical stereochemistry of 6 at the tetrahydroisoquinoline stereocenter.142 Copper-catalysed additions of alkynes to the naturally occurring iminium salt cotarnine in the presence of chiral phosphine ligand QUINAP/CuBr system reported by Schreiber et al.144 gave the highest enantiomeric excess.142 The compounds were tested to determine whether they possessed biological activity resembling those of the guiding alkaloid. Each of the synthetic analogs was screened at 30 µM to determine the phenotypic changes associated with microtubule cytoskeleton and mitotic arrest after compound administration. The treatment of BSC-1 and HeLa cells for 24 h, with several of these tetrahydroisoquinolines resulted in accumulation of round cells with condensed DNA, indicative of mitotic arrest.142 The compounds identified as positive hits displayed the substitution patterns of the noscapine tetrahydroisoquinoline core showing enhanced activity for the R enantiomer in comparison with the S-enantiomer. Three of the most active compounds contained either a phenylacetylene either unsubstituted or equipped with one or two meta-methoxy groups reminiscent of both the podophyllotoxin and colchicine core.142 Modifications of this structure, such as saturation of the triple bond, introduction of aliphatic alkyne



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residues, bulky meta substituents, quaternization of the nitrogen, or the introduction of a methoxy group at ortho, para or both meta-positions, led to a decrease in activity.142 Immunofluorescent staining of HeLa and BSC-1 cells showed the active compound’s ability to disturb the microtubule, without compromising the cytoskeleton.

Chromosome congression defects along with the

formation of multipolar mitotic spindles was apparent. Compound 48 illustrated higher mitotic induction than 6, and higher antiproliferative activity.

The cancer cell lines treated with 48

remained arrested for several hours before undergoing mitotic cell death, determined using timelapse experiments. To determine whether 48 binds to either the colchicine or the vinca site on tubulin, the displacement of 2 and a fluorescently labelled BODIPY FL vinblastine was measured, however 48 could not displace either from their binding domain implying the compound in question binds at a site distinct of these two domains.142

Scheme 7. Synthesis of Related Tetrahydroisoquinolines with Antimitotic Activitya

a

Reagents and Conditions: (a) iPrMgCl, 3-methoxyphenylacetylene, THF, 0 oC.

It was apparent from the work conducted by Zimmerman et al. that compounds demonstrating the stereochemistry and/or substitution pattern of 6 possess significantly better activity then those of the alternate isomer. Rigidity of the scaffold was demonstrated to be imperative to activity with the saturation of the triple bond resulting in a significant decrease in activity. Whilst the introduction of larger bulky substituents on the phenylacetylene portion resulted in reduced activity which was a similar outcome described in previous literature.119,142


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7. SUMMARY AND PERSPECTIVES Several research groups from around the world continue to actively investigate noscapine and synthesize novel noscapine-derived analogs and some comprehensive reviews have been published.6,105,109-112,117,118,131,135,145,146 Many of these research groups have evaluated noscapine and noscapine-derived analogs using an array of techniques including molecular modelling approaches and cell-based assays. The use of competitive tubulin binding studies using [3H]colchicine have shown noscapine and other noscapine-related analogs lack the ability to directly compete with colchicine for the same binding site, illustrating they may bind to an alternate site.1 The treatment of various cancer cell lines with noscapine demonstrated noscapine possessed the ability to reduce growth and shortening rates of microtubules and increased the percentage of time microtubules remained in an attenuated state which was a 1.9-fold increase compared to microtubule growth in the absence of noscapine.12 It was later shown by Naik et al. that administration of the 10 within a competitive binding assay was able to reduce the binding of colchicine by 66 ± 5% when administered at 100 µM, with reduced activity at lower doses.73 This observation suggested the binding site of 10 is at a site on the tubulin heterodimer near or overlapping the colchicine-binding site on tubulin but the data could not exclude the possibility that 10 may bind to a site distinct and distant from the colchicine-binding domain, which may negatively influence the binding of colchicine. To date the crystal structure of noscapine and/or a noscapine-related analog bound within its tubulin binding domain does not exist within the literature and thus an array of analytical tools and observations can only postulate the plausible binding domain of these compounds based on the competitive nature of these ligands with other tubulin binding compounds of known binding sites. Research groups interested in investigating noscapine as a novel chemotherapeutic have focused their attention on the modification of the noscapine core to generate new novel compounds. The noscapine core has been modified in the 1-, 7-, 6′ and 9′-positions to give a large number of chemically diverse analogs. These modifications have led to a number of analogs which possess improved anticancer activities and reduced cell line variability compared with parent compound;



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noscapine.109,110,117,118,131 Recently our group has shown the introduction of multiple modifications in the 1-, 7-, 6′ and 9′-positions on noscapine could lead to analogs with improved activity and reduced cell line variability.135 It is often difficult to directly compare noscapine analogs of each research group due to the variety of assays in which the compounds are assessed; cancer cell lines used and the concentration of compounds used in biological assays. To date, the most expansive analysis of noscapine analogs as anti-proliferative compounds was conducted by Aneja et al. on compound 10, which entered the clinical phase. The anti-proliferative activity of 10 was assessed by the National Cancer Institute (NCI) against their panel of 60 human cancer cell lines which represented the cancer classes of leukemia, non-small cell lung cancer, central nervous system (CNS), melanoma, renal, ovarian, breast and prostate cancer lines. It was shown from this broad cell based testing approach that 10 was consistently more active than noscapine in inhibiting the proliferation of the tested cancer cell lines. The overexpression of drug efflux pumps such as Pglycoprotein and multi-drug resistance-associated protein 1 (MRP1) result in reduced in vivo activity of the current clinical chemotherapeutics for the treatment of cancer. These mechanisms can infer drug resistance after exposure to any drug and is not specific for chemotherapeutics. It is widely accepted that current chemotherapeutics of the drug classes taxanes and vinca alkaloids amongst others are affected by the classical multi-drug resistance phenotype resulting in lower intracellular drug concentrations and increased drug efflux.54 Importantly we were able to illustrate that a number of our most active noscapine analogs to date, did not show any statistically significant difference in the ability to produce cell cytotoxicity between the resistant and sensitive cells (MCF7 and MCF-7FLV1000 respectively).135 Recently the concept of biology-oriented synthesis (BIOS) has been adopted by Zimmermann et al. in the synthesis and biological evaluation of noscapine-inspired tetrahydroisoquinolines (THIQs), which have shown cytotoxic abilities and also demonstrated the importance of retaining the natural chirality of noscapine.142 In summary a number of noscapine and noscapine like scaffolds have resulted in an improved understanding of the SAR of noscapine related analogs. It is


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imperative going forward that we are able to elucidate the binding domain(s) of noscapine and/or related analogs on the tubulin heterodimer to facilitate the targeted synthesis of potent noscapinoids.

AUTHOR INFORMATION Corresponding Authors *PJS: Phone: +61 9903 9542. E-mail: [email protected]; BC: Phone: +961 9903 9556. E-mail: [email protected]

Notes The authors declare no competing financial interest.

Biographies Aaron DeBono is currently completing a Ph.D. degree in the medicinal chemistry in the Monash Institute of Pharmaceutical Sciences at Monash University under the supervision of Professor Peter J. Scammells and Dr Ben Capuano. He has a strong interest in the development of noscapine derivatives as anti-microtubule agents in various cancer cell types. His dissertation has focused on identifying novel noscapine analogues possessing multiple modifications to the noscapine core as improved antimitotic agents against numerous in vitro cancer cell lines. Ben Capuano completed a PhD at Monash University in 2000 under the supervision of Dr Edward J. Lloyd and Dr Ian T. Crosby.

Much of his research has been centered on the atypical

antipsychotic clozapine, which has culminated in the synthesis and biological evaluation of the first clozapine-based homobivalent ligand with nanomolar affinity for the dopamine D2 receptor. He has published extensive medicinal chemistry-related papers in the area of schizophrenia research. Dr Capuano is currently a Senior Lecturer at Monash University and continues to undertake medicinal chemistry research in the area of small-molecule ligands targeting G protein-coupled receptors



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implicated in psychological and neurological disorders, and investigate new avenues centered on analogues of the opium poppy-derived alkaloid noscapine as novel anticancer agents. Peter J. Scammells completed a PhD at Griffith University under the supervision of Prof. Ronald J. Quinn in 1991. After a postdoctoral fellowship at the University of South Florida and an Alexander von Humboldt Fellowship at the Technische Universität Darmstadt, he commenced his independent academic career at Deakin University in 1993. He was appointed as Professor of Medicinal Chemistry at Monash University in 2001 and is currently the Medicinal Chemistry Theme Leader at the Monash Institute of Pharmaceutical Sciences (MIPS). Prof. Scammells’ research interests include the medicinal chemistry of G protein-coupled receptor ligands and the development of noscapine derivatives as potential anti-cancer agents.

ABBREVIATIONS USED 1A9, Human Ovarian Carcinoma cell line; 1A9/PTX10, Paclitaxel Resistant Human Ovarian Carcinoma cell line; 1A9/PTX22, Paclitaxel Resistant Human Ovarian Carcinoma cell line; A172, Human Glioblastoma Cell line; A2780, Human Ovarian Carcinoma cell line; A2780/AD10, Human Ovarian Carcinoma cell line; A549, Adenocarcinomic Human Alveolar Basal Epithelial Cells; ABCB1, ATP-binding cassette, sub-family B; ABCG2, ATP-binding cassette sub-family G; A.M.A., American Medical Association; BALB/c, Bagg Albino (inbred research mouse strain); BIOS, biology orientated synthesis; BSC-1, Kidnet Epithelial Cells of Monkey Origin; BODIPY FL, Boron-dipyrromethene fluorescent label; Bub1, Budding Uninhibited by Benzimidazoles 1; BubR1, Budding Uninhibited by Benzimidazole-related 1; CA-4P, combretastatin A-4 phosphate; Caco-2 – human colon carcinoma cells; Ca Ski, Human Caucasian cervical epidermoid carcinoma cell line; CBSIs, Colchicine binding site agents; CD complex, Colchicine Dimer complex; CEFNA, cyclic

ether

fluorinated

noscapine

analogue;

CEM,

Human

T

cell;

CI, confidence interval; CNS, Central Nervous System; DAPI, 4’,6-diamidino-2-phenylindole blue; DMF, Dimethylformamide; DMSO, dimethylsulfoxide; DNA, deoxyribonucleic acid; DTP, Development Therapeutics Program; DU 145, Human Prostate cancer cell line; E.G7-OVA, Mouse lymphoma cell line EL4; FACS, fluorescene-activated cell sorting analysis; GBM, gliobastoma multiforme; GDP, guanosine diphosphate; GTP, guanosine triphosphate; H460, Hypotriploid Human Cell line; HEK293, Human Embryonic Kidney 293 cell line; HeLa, Henrietta lacks (cervical cancer cell line); HPLC, High Performance Liquid Chromatography; IAP, inhibitor of ACS Paragon Plus Environment

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apoptosis; IC50, Inhibitor concentration causing 50% inhibition of cell replication in a cell culture system; IMR32, Human Neuroblastoma cell line; Kd, dissociation constant; LA1-55N, Human Neuroblastoma cell line; LIE, linear interaction energy; LN229, Front parieto-occipital gliobastoma cells; Mad2, Mitotic Arrest Deficient 2; MAPS, Microtubule Associated Proteins; MCF-7, Michigan foundation breast cancer cell line; MCF-7FLV1000, Human Breast cancer cell line; MDAMB-231, Human Breast Adenocarcinoma cell line; MIA PaCa-2, Human Pancreatic Carcinoma cell line; MRP1, multi-drug resistance associated protein 1; MSA’s, Microtubule-stabilising agents; MTP, Microtubule Protein; MTs, microtubules; NB1643, Human Neuroblastoma cell line; NB1691, Human Neuroblastoma cell line; NCI, National Cancer Institute; NCI-AdrRES, Multidrug-resistant ovarian cancer cell line; NP, natural product; OPLS 2005, Optimized Potentials for Liquid Simulations; PANC-1, Human Non-endocrine Pancreatic cell line; PC3, prostate cancer cell line; PGP, phosphoglycoprotein; Pi, orthophosphate; PRCG, Polak-Ribiere Conjugare Gradient; RB3SLD, RB3 protein Stathmin Like Domain; SAR, Structure Activity Relationship; SH-EP1, Human Epithelial cell line; SigC, Clonal granulosa cell line; siRNA, Small Interfering RNA; SK-N-AS, Human Neuroblastoma cell line; SK-N-MC, Human Neuroepithelioma cell line; SK-N-SH, Human Neuroblastoma cell line; SK-OV-3, Hypodiploid human cell line; SK-SY5Y, Human Neuroblastoma cell line; STD, saturation transfer difference; Swiss 3T3, Human Fibroblast; TC complex, Tubulin Colchicine complex; THIQ, tetrahydroisoquinoline; TMZ, temozolomide; TMZr, temozolomide resistant; TUNEL, Terminal Deoxynucleotidyl transferase dUTP nick end labeling; USFDA, United States Food and Drug Administration; WHO, world health organization; U251, Human Glioblastoma Cell line.

REFERENCES (1)

Ye, K.; Ke, Y.; Keshava, N.; Shanks, J.; Kapp, J. A.; Tekmal, R. R.; Petros, J.; Joshi, H. C.

Opium Alkaloid Noscapine Is an Antitumor Agent That Arrests Metaphase and Induces Apoptosis in Dividing Cells. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1601–1606. (2)

Ferlay, J.; Shin, H.-R.; Bray, F.; Forman, D.; Mathers, C.; Parkin, D. M. Estimates of

Worldwide Burden of Cancer in 2008: GLOBOCAN 2008. Int. J. Cancer 2010, 127, 2893–2917. (3)

Ho, W.; Shubhada, S.; Mills, L.; Negrello, T.; Hao, M.; Connell, E.; Australian Institute of

Health and Welfare & Australasian Association of Cancer Registries 2012. Cancer in Australia: An Overview 2012. 2012, 74, 1–215.



Page 72 of 87

72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(4)

Kavallaris, M. Microtubules and Resistance to Tubulin-Binding Agents. Nat. Rev. Cancer

2010, 10, 194–204. (5)

Wittmann, T.; Hyman, A.; Desai, A. The Spindle: a Dynamic Assembly of Microtubules

and Motors. Nat. Cell Biol. 2001, 3, E28–E34. (6)

Singh, H.; Singh, P.; Kumari, K.; Chandra, A.; Dass, S. K.; Chandra, R. A Review on

Noscapine, and Its Impact on Heme Metabolism. Curr. Drug Metab. 2013, 14, 351–360. (7)

Zhou, J.; Shu, H.-B.; Joshi, H. C. Regulation of Tubulin Synthesis and Cell Cycle

Progression in Mammalian Cells by ?-Tubulin-Mediated Microtubule Nucleation. J. Cell. Biochem. 2002, 84, 472–483. (8)

Campos, S. M.; Dizon, D. Antimitotic Inhibitors. Hematol. Oncol. Clin. North Am. 2012,

607–628. (9)

Wilson, L.; Jordan, M. A. Microtubule Dynamics: Taking Aim at a Moving Target. Chem.

Biol. 1995, 2, 569–573. (10) Zhou, J.; Giannakakou, P. Targeting Microtubules for Cancer Chemotherapy. Curr. Med. Chem.: Anti-cancer Agents 2005, 5, 65–71. (11) Margolis, R. L.; Wilson, L. Opposite End Assembly and Disassembly of Microtubules at Steady State in Vitro. Cell 1978, 13, 1–8. (12) Zhou, J.; Panda, D.; Landen, J. W.; Wilson, L.; Joshi, H. C. Minor Alteration of Microtubule Dynamics Causes Loss of Tension Across Kinetochore Pairs and Activates the Spindle Checkpoint. J. Biol. Chem. 2002, 277, 17200–17208. (13) Cragg, G. M.; Grothaus, P. G.; Newman, D. J. Impact of Natural Products on Developing New Anti-Cancer Agents. Chem. Rev. 2009, 109, 3012–3043. (14) Nogales, E.; Wolf, S. G.; Downing, K. H. Structure of the Αβ Tubulin Dimer by Electron Crystallography. Nature 1998, 391, 199–203. (15) Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.; Knossow, M. Insight Into Tubulin Regulation From a Complex with Colchicine and a Stathmin-Like Domain.


Page 73 of 87


73 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nature 2004, 428, 198–202. (16) Gigant, B.; Wang, C.; Ravelli, R. B. G.; Roussi, F.; Steinmetz, M. O.; Curmi, P. A.; Sobel, A.; Knossow, M. Structural Basis for the Regulation of Tubulin by Vinblastine. Nature 2005, 435, 519–522. (17) Burkhart, C. A.; Berman, J. W.; Swindell, C. S.; Horwitz, S. B. Relationship Between the Structure of Taxol and Other Taxanes on Induction of Tumor Necrosis Factor-Α Gene Expression and Cytotoxicity. Cancer Res. 1994. 54, 5779–5782. (18) Noble, R. L.; Beer, C. T.; Cutts, J. H. Role of Chance Observations in Chemotherapy: Vinca Rosea. Ann. N. Y. Acad. Sci. 2006, 76, 882–894. (19) Svoboda, G. H.; Neuss, N.; Gorman, M. Alkaloids of Vinca Rosea Linn. (Catharanthus Roseus G. Don.) v. Preparation and Characterization of Alkaloids. J. Pharm. Sci. 2006, 48, 659– 666. (20) Bunn, P. A.; Kelly, K. New Chemotherapeutic Agents Prolong Survival and Improve Quality of Life in Non-Small Cell Lung Cancer: a Review of the Literature and Future Directions. Clin. Cancer. Res. 1998, 4, 1087–1100. (21) Crown, J. Optimising Treatment Outcomes: a Review of Current Management Strategies in First-Line Chemotherapy of Metastatic Breast Cancer. Eur. J. Cancer 1997, 33 Suppl 7, S15– S19. (22) Johnson, S. A.; Harper, P.; Hortobagyi, G. N.; Pouillart, P. Vinorelbine: an Overview. Cancer Treat. Rev. 1996, 22, 127–142. (23) Ngan, V. K.; Bellman, K.; Panda, D.; Hill, B. T.; Jordan, M. A.; Wilson, L. Novel Actions of the Antitumor Drugs Vinflunine and Vinorelbine on Microtubules. Cancer Res. 2000, 60, 5054– 5051. (24) Dhamodharan, R.; Jordan, M. A.; Thrower, D.; Wilson, L.; Wadsworth, P. Vinblastine Suppresses Dynamics of Individual Microtubules in Living Interphase Cells. Mol. Biol. Chem, 1995, 6, 1215–1229.



Page 74 of 87

74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(25) Wilson, L.; Jordan, M. A.; Morse, A.; Margolis, R. L. Interaction of Vinblastine with Steady-State Microtubules in Vitro. J. Mol. Biol. 1982, 159, 125–149. (26) Haskins, K. M.; Donoso, J. A.; Himes, R. H. Spirals and Paracrystals Induced by Vinca Alkaloids: Evidence That Microtubule-Associated Proteins Act as Polycations. J. Cell Sci. 1981, 47, 237–247. (27) Jordan, M. A.; Margolis, R. L.; Himes, R. H.; Wilson, L. Identification of a Distinct Class of Vinblastine Binding Sites on Microtubules. J. Mol. Biol. 1986, 187, 61–73. (28) Chen, X.-M.; Liu, J.; Wang, T.; Shang, J. Colchicine-Induced Apoptosis in Human Normal Liver L-02 Cells by Mitochondrial Mediated Pathways. Toxicol. In Vitro. 2012, 26, 649–655. (29) Arai, T.; Okuyama, T. Purification and Properties of Colchicine-Binding Protein from the Bovine Brain. Seikagaku. 1973, 45, 19–29. (30) Bhattacharyya, B.; Wolff, J. Promotion of Fluorescence Upon Binding of Colchicine to Tubulin. Proc. Natl. Acad. Sci. 1974, 71, 2627–2631. (31) Skoufias, D. A.; Wilson, L. Mechanism of Inhibition of Microtubule Polymerization by Colchicine: Inhibitory Potencies of Unliganded Colchicine and Tubulin-Colchicine Complexes. Biochemistry 1992, 31, 738–746. (32) Uppuluri, S.; Knipling, L.; Sackett, D. L.; Wolff, J. Localization of the Colchicine-Binding Site of Tubulin. Proc. Natl. Acad. Sci. 1993, 90, 11598–11602. (33) Margolis, R. L.; Wilson, L. Addition of Colchicine-Tubulin Complex to Microtubule Ends: the Mechanism of Substoichiometric Colchicine Poisoning. Proc. Natl. Acad. Sci. 1977, 74, 3466– 3470. (34) Wolff, J.; Knipling, L.; Cahnmann, H. J.; Palumbo, G. Direct Photoaffinity Labeling of Tubulin with Colchicine. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2820–2824. (35) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P. Plant Antitumor Agents. VI. Isolation and Structure of Taxol, a Novel Antileukemic and Antitumor Agent From Taxus Brevifolia. J. Am. Chem. Sci. 1971, 93, 2325–2327.


Page 75 of 87


75 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(36) Wall, M. E.; Wani, M. C. Camptothecin and Taxol: Discovery to Clinic—Thirteenth Bruce F. Cain Memorial Award Lecture. Cancer Res. 1995, 55, 753–760. (37) Schiff, P. B.; Fant, J.; Horwitz, S. B. Promotion of Microtubule Assembly in Vitro by Taxol. Nature 1979, 277, 665–667. (38) Schiff, P. B.; Horwitz, S. B. Taxol Assembles Tubulin in the Absence of Exogenous Guanosine 5'-Triphosphate or Microtubule-Associated Proteins. Biochemistry 1981, 20, 3247–3252. (39) Hamel, E.; del Campo, A. A.; Lowe, M. C.; Lin, C. M. Interactions of Taxol, MicrotubuleAssociated Proteins, and Guanine Nucleotides in Tubulin Polymerization. J. Biol. Chem. 1981, 256, 11887–11894. (40) Rao, S.; Krauss, N. E.; Heerding, J. M.; Swindell, C. S.; Ringel, I.; Orr, G. A.; Horwitz, S. B. 3'-(P-Azidobenzamido)Taxol Photolabels the N-Terminal 31 Amino Acids of Beta-Tubulin. J. Biol. Chem. 1994, 269, 3132–3134. (41) Snyder, J. P. The Microtubule-Pore Gatekeeper. Nature Chem. Biol. 2007, 3, 81–82. (42) Pettit, G. R.; Cragg, G. M.; Herald, D. L. Isolation and Structure of Combretastatin. Can. J. Chem. 1982, 60, 1374–1376. (43) Rustin, G. J. S. Phase I Clinical Trial of Weekly Combretastatin A4 Phosphate: Clinical and Pharmacokinetic Results. J. Clin. Oncol. 2003, 21, 2815–2822. (44) Young, S. L.; Chaplin, D. J. Combretastatin A4 Phosphate: Background and Current Clinical Status, Expert Opinion on Investigational Drugs, Informa Healthcare. Expert Opin. Investig. Drugs. 2004, 13, 1171–1182. (45) Cooney, M. M.; Radivoyevitch, T.; Dowlati, A.; Overmoyer, B.; Levitan, N.; Robertson, K.; Levine, S. L.; DeCaro, K.; Buchter, C.; Taylor, A.; Stambler, B. S.; Remick, S. C. Cardiovascular Safety Profile of Combretastatin A4 Phosphate in a Single-Dose Phase I Study in Patients with Advanced Cancer. Clin. Cancer Res. 2004, 10, 96–100. (46) Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. Medicinal Chemistry of Combretastatin A4: Present and Future Directions. J. Med. Chem. 2006, 49, 3033–



Page 76 of 87

76 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3044. (47) McGown, A. T.; Fox, B. W. Differential Cytotoxicity of Combretastatins A1 and A4 in Two Daunorubicin-Resistant P388 Cell Lines. Cancer Chemother. Pharmacol. 1990, 26, 79–81. (48) Bellina, F.; Cauteruccio, S.; Monti, S.; Rossi, R. Novel Imidazole-Based Combretastatin a4 Analogues: Evaluation of Their in Vitro Antitumor Activity and Molecular Modeling Study of Their Binding to the Colchicine Site of Tubulin. Bioorg. Med. Chem Lett. 2006, 16, 5757–5762. (49) Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Antimitotic Natural Products Combretastatin a-4 and Combretastatin a-2: Studies on the Mechanism of Their Inhibition of the Binding of Colchicine to Tubulin. Biochemistry 1989, 28, 6984–6991. (50) McGown, A. T.; Fox, B. W. Structural and Biochemical Comparison of the Anti-Mitotic Agents Colchicine, Combretastatin A4 and Amphethinile. Anticancer Drug Des. 1989, 3, 249–254. (51) Gordaliza, M.; Garcıa, P. A.; del Corral, J. M. Podophyllotoxin: Distribution, Sources, Applications and New Cytotoxic Derivatives. Toxicon 2004, 44, 441–459. (52) Labruère, R.; Gautier, B.; Testud, M.; Seguin, J.; Lenoir, C.; Desbene-Finck, S.; Helissey, P.; Chabot, G. G.; Vidal, M.; Giorgi-Renault, S. Design, Synthesis, and Biological Evaluation of the First Podophyllotoxin Analogues as Potential Vascular-Disrupting Agents. ChemMedChem. 2010, 5, 2016–2025. (53) Bohlin, L.; Rosen, B. Podophyllotoxin Derivatives: Drug Discovery and Development. Drug Discov. Today 1996, 1, 343–351. (54) Gottesman, M. M.; Fojo, T.; Bates, S. E. Multidrug Resistance in Cancer: Role of AtpDependent Transporters. Nat. Rev. Cancer 2002, 2, 48–58. (55) Jain, R. K. Delivery of Molecular Medicine to Solid Tumors: Lessons From in Vivo Imaging of Gene Expression and Function. J. Control. Release 2001, 74, 7–25. (56) Green, S. K.; Frankel, A.; Kerbel, R. S. Adhesion-Dependent Multicellular Drug Resistance. Anticancer Drug Des. 1999, 14, 153–168. (57) Jain, R. K. Normalizing Tumor Vasculature with Anti-Angiogenic Therapy: a New


Page 77 of 87


77 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Paradigm for Combination Therapy. Nature Med. 2001, 7, 987–989. (58) Chen, C.; Chin, J. E.; Ueda, K.; Clark, D. P.; Pastan, I. Internal Duplication and Homology with Bacterial Transport Proteins in the Mdr1 (P-Glycoprotein) Gene From Multidrug-Resistant Human Cells. Cell 1986, 47, 381–389. (59) Chin, K. V.; Pastan, I.; Gottesman, M. M. Function and Regulation of the Human Multidrug Resistance Gene. Adv. Cancer Res. 1993, 156–180. (60) Schuetz, E. G.; Beck, W. T.; Schuetz, J. D. Modulators and Substrates of P-Glycoprotein and Cytochrome P4503A Coordinately Up-Regulate These Proteins in Human Colon Carcinoma Cells. Mol. Pharmacol. 1996, 49, 311–318. (61) Segal, M. S.; Goldstein, M. M.; Attinger, E. O. The Use of Noscapine (Narcotine) as an Antitussive Agent. Chest 1957, 32, 305–309. (62) Krueger, H. M.; Eddy, N. B.; Sumwalt, M.; Martin, H. The Pharmacology of the Opium Alkaloids. No. 165. US Government Printing Office. 1941. (63) Winter, C. A.; Flataker, L. Toxicity Studies on Noscapine. Toxicol. Appl. Pharmacol. 1961, 3, 96–106. (64) Karlsson, M. O.; Dahlström, B.; Neil, A. Characterization of High-Affinity Binding Sites for the Antitussive [3H]noscapine in Guinea Pig Brain Tissue. Eur. J. Pharmacol. 1988, 145, 195– 203. (65) Craviso, G. L.; Musacchio, J. M. High-Affinity Dextromethorphan Binding Sites in Guinea Pig Brain. II. Competition Experiments. Mol. Pharmacol. 1983, 23, 629–640. (66) Dahlström, B.; Mellstrand, T.; Löfdahl, C. G. Pharmakokinetic Properties of Noscapine Springer. Eur. J. Clin. Pharmacol. 1982, 22, 535–539. (67) Ashour, A.; Hegazy, M. A. M.; Moustafa, A. A.; Kelani, K. O.; Fattah, L. E. A. Validated Stability-Indicating TLC Method for the Determination of Noscapine. Drug Test. Analysis 2009, 1, 327–338. (68) Lettre, H.; Albrecht, M. Narcotin, Ein Mitosegift. Naturwissenschaften 1942, 12, 184–185.



Page 78 of 87

78 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(69) Lettre, H. Synergists and Antagonists of Mitotic Poisons. Ann. N. Y. Acad. Sci. 1954, 58, 1264–1275. (70) Nicklas, R. B. How Cells Get the Right Chromosomes. Science 1997, 275, 632–637. (71) Langermann, J. V.; Lorenz, H.; Boehm, O.; Flemming, A.; Bernsdorf, A.; Kockerling, M.; Schinzer Z.; Seidel-Morgenstern, A. (3 R*,5′ S*)-6,7-Dimethoxy-3-(4′-Methoxy-6′-Methyl5′,6′,7′,8′-Tetrahydro-1,3-Dioxolo[4,5- G]Isoquinolin-5′-Yl)Isobenzofuran-1(3 H)-One (Racemic Α-Noscapine). Acta Crystallogr E Struct Rep Online 2010, 66, o570–o570. (72) Naik, P. K.; Chatterji, B. P.; Vangapandu, S. N.; Aneja, R.; Chandra, R.; Kanteveri, S.; Joshi, H. C. Rational Design, Synthesis and Biological Evaluations of Amino-Noscapine: a High Affinity Tubulin-Binding Noscapinoid. J. Comput. Aided Mol. Des. 2011, 25, 443–454. (73) Naik, P. K.; Santoshi, S.; Rai, A.; Joshi, H. C. Molecular Modelling and Competition Binding Study of Br-Noscapine and Colchicine Provide Insight Into Noscapinoid–Tubulin Binding Site. J. Mol. Graphics Modell. 2011, 29, 947–955. (74) Alisaraie, L.; Tuszynski, J. A. Determination of Noscapine’s Localization and Interaction with the Tubulin-Α/Β Heterodimer. Chem. Biol. Drug Des. 2011, 78, 535–546. (75) Zhou, J.; Gupta, K.; Aggarwal, S.; Aneja, R.; Chandra, R.; Panda, D.; Joshi, H. C. Brominated Derivatives of Noscapine Are Potent Microtubule-Interfering Agents That Perturb Mitosis and Inhibit Cell Proliferation. Mol. Pharmacol. 2003, 63, 799–807. (76) Jablonski, S. A.; Chan, G.; Cooke, C. A.; Earnshaw, W. C. The hBUB1 and hBUBR1 Kinases Sequentially Assemble Onto Kinetochores During Prophase with hBUBR1 Concentrating at the Kinetochore Plates in Mitosis. Chromosoma 1998, 107, 386–396. (77) Shin, H.-J.; Baek, K.-H.; Jeon, A.-H.; Park, M.-T.; Lee, S.-J.; Kang, C.-M.; Lee, H.-S.; Yoo, S.-H.; Chung, D.-H.; Sung, Y.-C.; McKeon, F.; Lee, C.-W. Dual Roles of Human BubR1, a Mitotic Checkpoint Kinase, in the Monitoring of Chromosomal Instability. Cancer Cell 2003, 4, 483–497. (78) Biggins, S.; Murray, A. W. Sister Chromatid Cohesion in Mitosis. Curr. Opin. Cell Biol.


Page 79 of 87


79 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1998, 10, 769–775. (79) Sarrazin, M.; Briand, C.; Menendez, M.; Laynez, J. Mechanism of Binding of the New Antimitotic Drug MDL 27048 to the Colchicine Site of Tubulin: Equilibrium Studies. Biochemistry 1992, 31, 11125–11132. (80) Chakrabarti, G.; Sengupta, S.; Bhattacharyya, B. Thermodynamics of ColchicinoidTubulin Interactions. J. Biol. Chem. 1996, 271, 2897–2901. (81) Ray, K.; Bhattacharyya, B.; Biswas, B. B. Role of B-Ring of Colchicine in Its Binding to Tubulin. J. Biol. Chem. 1981, 256, 6241–6244. (82) Burger, P. C.; Jouvet, A.; Scheithauer, B. W. The 2007 WHO Classification of Tumours of the Central Nervous System. Acta Neuropathol. 2007, 114, 97–109. (83) Hofer, S.; Herrmann, R. Chemotherapy for Malignant Brain Tumors of Astrocytic and Oligodendroglial Lineage. J. Cancer Res. Clin. Oncol. 2001, 127, 91–95. (84) Landen, J. W.; Hau, V.; Wang, M.; Davis, T.; Ciliax, B.; Wainer, B. H.; Van Meir, E. G.; Glass, J. D.; Joshi, H. C.; Archer, D. R. Noscapine Crosses the Blood-Brain Barrier and Inhibits Glioblastoma Growth. Clin. Cancer Res. 2004, 10, 5187-5201. (85) Ashby, L. S.; Ryken, T. C. Management of Malignant Glioma: Steady Progress with Multimodal Approaches. Neurosurg. Focus 2006, 20, E3 1–13. (86) Stupp, R.; Mason, W. P.; van den Bent, M. J.; Weller, M.; Fisher, B.; Taphoorn, M. J. B.; Belanger, K.; Brandes, A. A.; Marosi, C.; Bogdahn, U.; Curschmann, J.; Janzer, R. C.; Ludwin, S. K.; Gorlia, T.; Allgeier, A.; Lacombe, D.; Cairncross, J. G.; Eisenhauer, E.; Mirimanoff, R. O. Radiotherapy Plus Concomitant and Adjuvant Temozolomide for Glioblastoma. New Engl. J. Med. 2005, 352, 987–996. (87) Lu, C.; Shervington, A. Chemoresistance in Gliomas. Mol. Cell. Biochem. 2008, 312, 71– 80. (88) Jhaveri, N.; Cho, H.; Torres, S.; Wang, W.; Schönthal, A. H. Noscapine Inhibits Tumor Growth in TMZ-Resistant Gliomas. Cancer Lett. 2011, 312, 245–252.



Page 80 of 87

80 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(89) Newcomb, E. W.; Lukyanov, Y.; Schnee, T.; Ali, M. A.; Lan, L.; Zagzag, D. Noscapine Inhibits Hypoxia-Mediated HIF-1alpha Expression Andangiogenesis in Vitro: a Novel Function for an Old Drug. Int. J. Oncol. 2006, 28, 1121–1130. (90) Chougule, M.; Patel, A. R.; Sachdeva, P.; Jackson, T.; Singh, M. Anticancer Activity of Noscapine, an Opioid Alkaloid in Combination with Cisplatin in Human Non-Small Cell Lung Cancer. Lung Cancer 2011, 71, 271–282. (91) Hiser, L.; Herrington, B.; Lobert, S. Effect of Noscapine and Vincristine Combination on Demyelination and Cell Proliferation in Vitro. Leuk. Lymphoma 2008, 49, 1603–1609. (92) Aneja, R.; Joshi, H. C.; Vandapandu. S.; Noscapine Analogs and their Use in Treating Cancers, Including Drug-Resistant Cancers. WO 2008/109609 A1, 2008. (93) Naik, P. K.; Lopus, M.; Aneja, R.; Vangapandu, S. N.; Joshi, H. C. In Silico Inspired Design and Synthesis of a Novel Tubulin-Binding Anti-Cancer Drug: Folate Conjugated Noscapine (Targetin). J. Comput. Aided Mol. Des. 2012, 26, 233–247. (94) Li, S.; Ghaleb, A. M.; He, J.; Bughani, U. Chemoprevention of Familial Adenomatous Polyposis by Bromo-Noscapine (EM011) in the ApcMin/+ Mouse Model. Int. J. Cancer. 2012, 131, 1435–1444. (95) Jackson, T.; Chougule, M. B.; Ichite, N.; Patlolla, R. R.; Singh, M. Antitumor Activity of Noscapine in Human Non-Small Cell Lung Cancer Xenograft Model. Cancer Chemother. Pharmacol. 2008, 63, 117–126. (96) Mita, A. C.; Mita, M. M.; Nawrocki, S. T.; Giles, F. J. Survivin: Key Regulator of Mitosis and Apoptosis and Novel Target for Cancer Therapeutics. Clin. Cancer Res. 2008, 14, 5000–5005. (97) Karna, P.; Sharp, S. M.; Yates, C.; Prakash, S.; Aneja, R. EM011 Activates a SurvivinDependent Apoptotic Program in Human Non-Small Cell Lung Cancer Cells. Mol. Cancer 2009, 8, 93. (98) Li, S.; He, J.; Li, S.; Cao, G.; Tang, S.; Tong, Q.; Joshi, H. C. Noscapine Induced Apoptosis via Downregulation of Survivin in Human Neuroblastoma Cells Having Wild Type or


Page 81 of 87


81 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Null P53. PLoS ONE 2012, 7, e40076. (99) Aneja, R.; Ghaleb, A. M.; Zhou, J.; Yang, V. W.; Joshi, H. C. P53 and P21 Determine the Sensitivity of Noscapine-Induced Apoptosis in Colon Cancer Cells. Cancer Res. 2007, 67, 3862– 3870. (100) Aneja, R.; Zhou, J.; Vangapandu, S. N.; Chandra, R.; Joshi, H. C. Drug-Resistant TLymphoid Tumors Undergo Apoptosis Selectively in Response to an Antimicrotubule Agent, EM011. Blood 2006, 107, 2486–2492. (101) Aneja, R.; Miyagi, T.; Karna, P.; Ezell, T.; Shukla, D.; Vij Gupta, M.; Yates, C.; Chinni, S. R.; Zhau, H.; Chung, L. W. K.; Joshi, H. C. A Novel Microtubule-Modulating Agent Induces Mitochondrially Driven Caspase-Dependent Apoptosis via Mitotic Checkpoint Activation in Human Prostate Cancer Cells. Eur. J. Cancer 2010, 46, 1668–1678. (102) Karna, P.; Rida, P. C. G.; Pannu, V.; Gupta, K. K.; Dalton, W. B.; Joshi, H. C.; Yang, V. W.; Zhou, J.; Aneja, R.; A Novel Microtubule-Modulating Noscapinoid Triggers Apoptosis by Inducing Spindle Multipolarity via Centrosome Amplification and Declustering. Cell Death Differ. 2011, 18, 632–644. (103) Aneja, R.; Kalia, V.; Ahmed, R.; Joshi, H. C. Nonimmunosuppressive Chemotherapy: EM011-Treated Mice Mount Normal T-Cell Responses to an Acute Lymphocytic Choriomeningitis Virus Infection. Mol Cancer. Ther. 2007, 6, 2891–2899. (104) Dey, B. B.; Srinivasan, T. K. Cotarnine series. IV. 5-Bromonarcotine, 5-Bromocotarnine, 5-Bromohydrocotarnine and 5-Bromonarceine and Their Derivatives. J. Indian. Chem. Soc. 1935, 12, 526–536. (105) Aggarwal, S.; Ghosh, N. N.; Aneja, R.; Joshi, H. A Convenient Synthesis of ArylSubstituted N-Carbamoyl/N-Thiocarbamoyl Narcotine and Related Compounds. Helv. Chim. Acta. 2002, 85, 2458–2462. (106) Giannakakou, P.; Sackett, D. L.; Kang, Y.K.; Zhan, Z.; Buters, J. T. M.; Fojo, T.; Poruchynsky, M. S. Paclitaxel-Resistant Human Ovarian Cancer Cells Have Mutant Β-Tubulins



Page 82 of 87

82 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

that Exhibit Impaired Paclitaxel-Driven Polymerization. J. Biol. Chem. 1997, 272, 17118–17125. (107) Pryor, D. E.; O'Brate, A.; Bilcer, G.; Díaz, J. F.; Wang, Y. The Microtubule Stabilizing Agent Laulimalide Does Not Bind in the Taxoid Site, Kills Cells Resistant to Paclitaxel and Epothilones, and May Not Require Its Epoxide Moiety for Activity. Biochemistry 2002, 41, 9109– 9115. (108) Zughaier, S.; Karna, P.; Stephens, D.; Aneja, R. Potent Anti-Inflammatory Activity of Novel Microtubule-Modulating Brominated Noscapine Analogs. PLoS ONE 2010, 5, e9165. (109) Aneja, R.; Vangapandu, S. N.; Lopus, M. Synthesis of Microtubule-Interfering Halogenated Noscapine Analogs That Perturb Mitosis in Cancer Cells Followed by Cell Death. Biochem. Pharmacol. 2006, 72, 415–426. (110) Aneja, R.; Vangapandu, S. N.; Joshi, H. C. Synthesis and Biological Evaluation of a Cyclic Ether Fluorinated Noscapine Analog. Bioorg. Med. Chem. 2006, 14, 8352–8258. (111) Aneja, R.; Vangapandu, S. N.; Lopus, M.; Chandra, R.; Panda, D.; Joshi, H. C. Development of a Novel Nitro-Derivative of Noscapine for the Potential Treatment of DrugResistant Ovarian Cancer and T-Cell Lymphoma. Mol. Pharmacol. 2006, 69, 1801–1809 (112) Manchukonda, N. K.; Sridhar, B.; Naik, P. K.; Joshi, H. C. Copper(I) Mediated Facile Synthesis of Potent Tubulin Polymerization Inhibitor, 9-Amino-Α-Noscapine From Natural ΑNoscapine. Bioorg. Med. Chem. 2012, 22, 2983–2987. (113) Santoshi, S.; Naik, P. K.; Joshi, H. C. Rational Design of Novel Anti-Microtubule Agent (9-Azido-Noscapine) From Quantitative Structure Activity Relationship (QSAR) Evaluation of Noscapinoids. J. Biomol. Screen. 2011, 16, 1047–1058. (114) Markiewicz, J. T.; Wiest, O.; Helquist, P. Synthesis of Primary Aryl Amines Through a Copper-Assisted Aromatic Substitution Reaction with Sodium Azide. J. Org. Chem. 2010, 75, 4887–4890. (115) Porcù, E.; Sipos, A.; Basso, G.; Hamel, E.; Bai, R.; Stempfer, V.; Udvardy, A.; Bényei, A. C.; Schmidhammer, H.; Antus, S.; Viola, G. Novel 9′-Substituted-Noscapines: Synthesis with


Page 83 of 87


83 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Suzuki Cross-Coupling, Structure Elucidation and Biological Evaluation. Eur. J. Med. Chem. 2014, 84, 476–490. (116) Schmidhammer, H.; Klötzer, W. Neue Reaktionen an Phthalidisochinolinalkaloiden. Alkoxytauschreaktionen Und Isomerisierungen an Α- Und Β-Narcotin. Arch. Pharm. 1978, 311, 664–671. (117) Anderson, J. T.; Ting, A. E.; Boozer, S.; Brunden, K. R.; Danzig, J.; Dent, T.; Harrington, J. J.; Murphy, S. M.; Perry, R.; Raber, A.; Rundlett, S. E.; Wang, J.; Wang, N.; Bennani, Y. L. Discovery of S-Phase Arresting Agents Derived From Noscapine. J. Med. Chem. 2005, 48, 2756– 2758. (118) Anderson, J. T.; Ting, A. E.; Boozer, S.; Brunden, K. R.; Crumrine, C.; Danzig, J.; Dent, T.; Faga, L.; Harrington, J. J.; Hodnick, W. F.; Murphy, S. M.; Pawlowski, G.; Perry, R.; Raber, A.; Rundlett, S. E.; Stricker-Krongrad, A.; Wang, J.; Bennani, Y. L. Identification of Novel and Improved Antimitotic Agents Derived From Noscapine. J. Med. Chem. 2005, 48, 7096–7098. (119) Mishra, R. C.; Karna, P.; Gundala, S. R.; Pannu, V.; Stanton, R.; Gupta, K. K.; Robinson, H. M.; Lopus, M.; Wilson, L.; Henary, M. Second Generation Benzofuranone Ring Substituted Noscapine Analogs: Synthesis and Biological Evaluation. Biochem. Pharmacol. 2011, 82, 110-121. (120) Ray, S.; Chaturvedi, D. Application of Organic Carbamates in Drug Design. Part 1: Anticancer Agents - recent reports. Drugs Future 2004, 29, 343. (121) Altieri, D. C. Survivin, Versatile Modulation of Cell Division and Apoptosis in Cancer. Oncogene 2003, 22, 8581–8589. (122) Oosawa, F.; 1922; Asakura, S.; 1927. Thermodynamics of the Polymerization of Protein. Academic Press in London, New York. 1975. (123) Bennani, Y. L.; Gu, W.; Canales, A.; Dı́az, F. J.; Eustace, B. K.; Hoover, R. R.; JiménezBarbero, J.; Nezami, A.; Wang, T. Tubulin Binding, Protein-Bound Conformation in Solution, and Antimitotic Cellular Profiling of Noscapine and Its Derivatives. J. Med. Chem. 2012, 55, 1920– 1925.



Page 84 of 87

84 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(124) Braun, J. V. Die Aufspaltung Cyclischer Basen Durch Bromcyan. Ber. Dtsch. Chem. Ges. 1909, 42, 2035–2057. (125) Grierson, D. The Polonovski Reaction. Organic Reactions 2004, 39, 85–295. (126) Allen, A. C.; Cooper, D. A.; Moore, J. M.; Gloger, M. Illicit Heroin Manufacturing byProducts: Capillary Gas Chromatographic Determination and Structural Elucidation of Narcotineand Norlaudanosine-Related Compounds. Anal. Chem. 1984, 56, 2940–2947.. (127) McCamley, K.; Ripper, J. A.; Singer, R. D. Efficient N-Demethylation of Opiate Alkaloids Using a Modified Nonclassical Polonovski Reaction. J. Org. Chem. 2003, 68, 9847–9850. (128) Dong, Z.; Scammells, P. J. New Methodology for the N-Demethylation of Opiate Alkaloids. J. Org. Chem. 2007, 72, 9881–9885. (129) Kok, G.; Ashton, T. D.; Scammells, P. J. An Improved Process for the N-Demethylation of Opiate Alkaloids Using an Iron(II) Catalyst in Acetate Buffer. Adv. Synth. Catal. 2009, 351, 283– 286. (130) Kok, G.; Scammells, P. Polonovski-Type N-Demethylation of N-Methyl Alkaloids Using Substituted Ferrocene Redox Catalysts. Synthesis 2012, 44, 2587–2594. (131) Debono, A. J.; Xie, J. H.; Ventura, S.; Pouton, C. W.; Capuano, B.; Scammells, P. J. Synthesis and Biological Evaluation of N-Substituted Noscapine Analogues. ChemMedChem 2012, 7, 2122–2133. (132) Aggarwal, S.; Ghosh, N. N.; Aneja, R. Mass Spectral Studies on Aryl-Substituted NCarbamoyl/N-Thiocarbamoyl Narcotine and Related Compounds. Rapid Commun. Mass Spectrom. 2002, 16, 923–928. (133) Manchukonda, N. K.; Naik, P. K.; Santoshi, S.; Lopus, M.; Joseph, S.; Sridhar, B.; Kantevari, S. Rational Design, Synthesis, and Biological Evaluation of Third Generation ΑNoscapine Analogues as Potent Tubulin Binding Anti-Cancer Agents. PLoS ONE 2013, 8, e77970. (134) Henary, M.; Narayana, L.; Ahad, S.; Gundala, S. R.; Mukkavilli, R.; Sharma, V.; Owens, E. A.; Yadav, Y.; Nagaraju, M.; Hamelberg, D.; Tandon, V.; Panda, D.; Aneja, R. Novel Third-


Page 85 of 87


85 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Generation Water-Soluble Noscapine Analogs as Superior Microtubule-Interfering Agents with Enhanced Antiproliferative Activity. Biochem. Pharmacol. 2014, 92, 192–205. (135) Debono, A. J.; Mistry, S. J.; Xie, J.; Muthiah, D.; Phillips, J.; Ventura, S.; Callaghan, R.; Pouton, C. W.; Capuano, B.; Scammells, P. J. The Synthesis and Biological Evaluation of Multifunctionalised Derivatives of Noscapine as Cytotoxic Agents. ChemMedChem 2014, 9, 399– 410. (136) Verma, A. K.; Jha, R. R.; Chaudhary, R. 2-(1-Benzotriazolyl)Pyridine: a Robust Bidentate Ligand for the Palladium-Catalyzed C– C (Suzuki, Heck, Fujiwara–Moritani, Sonogashira), C– N and C– S Coupling Reactions. Adv. Synth. Catal. 2013, 355, 421–438. (137) Woodahl, E. L.; Crouthamel, M. H.; Bui, T.; Shen, D. D.; Ho, R. J. Y. MDR1 (ABCB1) G1199A (Ser400Asn) Polymorphism Alters Transepithelial Permeability and Sensitivity to Anticancer Agents. Cancer Chemother. Pharmacol. 2009, 64, 183–188. (138) Limtrakul, P.; Chearwae, W.; Shukla, S.; Phisalphong, C.; Ambudkar, S. V. Modulation of Function of Three ABC Drug Transporters, P-Glycoprotein (ABCB1), Mitoxantrone Resistance Protein (ABCG2) and Multidrug Resistance Protein 1 (ABCC1) by Tetrahydrocurcumin, a Major Metabolite of Curcumin. Mol. Cell Biochem. 2007, 296, 85–95. (139) Wetzel, S.; Bon, R. S.; Kumar, K.; Waldmann, H. Biology-Oriented Synthesis. Angew. Chem. Int. Ed. 2011, 50, 10800–10826. (140) Kumar, K.; Waldmann, H. Synthesis of Natural Product Inspired Compound Collections. Angew. Chem. Int. Ed. 2009, 48, 3224–3242. (141) Vetter, I. R.; Waldmann, H. From Protein Domains to Drug Candidates—Natural Products as Guiding Principles in the Design and Synthesis of Compound Libraries. Angew. Chem. Int. Ed. 2002, 41, 2878–2890. (142) Zimmermann, T. J.; Roy, S.; Martinez, N. E.; Ziegler, S. Biology-Oriented Synthesis of a Tetrahydroisoquinoline-Based Compound Collection Targeting Microtubule Polymerization. ChemBioChem 2013, 14, 295–300.



Page 86 of 87

86 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(143) Flynn, D. L.; Zelle, R. E.; Grieco, P. A. A Mild Two-Step Method for the Hydrolysis of Lactams and Secondary Amides. J. Org. Chem. 1983, 48, 2424–2426. (144) Taylor, A. M.; Schreiber, S. L. Enantioselective Addition of Terminal Alkynes to Isolated Isoquinoline Iminiums. Org. Lett. 2006, 8, 143–146. (145) Aneja, R.; Mishra, R. C. Synthesis and Characterization of Second Generation Benzofuranone Ring Substituted Noscapine Analogs. US 2014/0121233 A1, 2014. (146) Tripathi, M.; Reddy, P. L.; Rawat, D. S.; Noscapine and its Analogues as Anti-Cancer Agents. J. Chem. Biol. Interfaces. 2014, 4, 1–22.


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Progress Toward the Development of Noscapine and Derivatives as

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