Small Molecule Modulators of ProteinâProtein Interactions: Selected

Review pubs.acs.org/CR

Small Molecule Modulators of Protein−Protein Interactions: Selected Case Studies Madhu Aeluri,† Srinivas Chamakuri,† Bhanudas Dasari,† Shiva Krishna Reddy Guduru,† Ravikumar Jimmidi,† Srinivas Jogula,† and Prabhat Arya* Dr. Reddy’s Institute of Life Sciences (DRILS), University of Hyderabad Campus Gachibowli, Hyderabad 500046, India 5.6.1. Conformationally Constrained Monovalent SMAC Mimetics 5.6.2. Isostere Design Using [7,5]-Bicyclic Scaffold 5.6.3. Design and Synthesis of Azabicyclooctane Scaffold-Based SMAC Mimetics 5.6.4. Some More Monovalent SMAC Mimetics 5.6.5. Diazabicyclic Scaffold-Based Peptide Mimetics 5.7. Bivalent SMAC Mimetics: Design and Synthesis 5.7.1. Unexpected Discovery of First Bivalent SMAC Mimetic 5.7.2. Bivalent Small Molecules Based on Monovalent SMAC Mimetics 6. Case Study 5: Bcl-2 Protein Family and Synthetic Small Molecules 6.1. BCL-2 Protein: Mitochondrial Permeabilization in Apoptosis 6.2. Peptides and Peptidomimetics 6.3. SAHBA 6.4. Terphenyl (α-Helix Mimicry) 6.5. Natural Products and their Derivatives 6.5.1. Antimycin A3 6.5.2. Chelerythrine 6.5.3. Polyphenolic Compounds 6.6. Synthetic Molecules 6.6.1. HA14-1 6.6.2. BH3Is 6.6.3. YC137 6.6.4. ABT-737 6.7. Bax Activators 6.8. BAX Inhibitors 7. Case Study 6: Small Molecule Modulators of 14-33 PPI 7.1. Stabilizers of 14-3-3 PPI 7.1.1. Fusicoccin A 7.1.2. Pyrrolidone and Epibestatin 7.2. Inhibitors of 14-3-3 PPI 7.2.1. Fobisin 101 7.2.2. Molecular Tweezers 8. Concluding Remarks and Future Outlook Author Information Corresponding Author

CONTENTS 1.. Introduction 1.1. Going in for Protein−Protein Interactions and Pathways 1.2. Protein−Protein Interactions and Small Molecules 2. Case Study 1: Tubulin Polymerization and Natural Product-Derived Small Molecules 3. Case Study 2: p53 and MDM2 Interactions and Small Molecules 3.1. Natural Product Inhibitors of p53−MDM2 Interactions 3.2. β-Hairpin Peptidomimetics 3.3. Terphenyls 3.4. Nutlins 3.5. Benzodiazepines 3.6. Spiro-oxindoles 3.7. Chromenotriazolopyrimidines 3.8. Piperidinones 3.9. Indolo-imidazoles 4. Case Study 3: Modulation of HSP90-Related Protein−Protein Interactions by Natural Products and Related Compounds 4.1. Structure, Conformation, and Functions of HSP 4.2. Hsp90 Inhibitors 5. Case Study 4: Protein−Protein Interactions Centered on the Inhibitors of Apoptosis Proteins (IAPs) and Synthetic Small Molecules 5.1. Overview of Functions of IAPs 5.2. Death Receptor Pathway of Caspase Activation 5.3. Mitochondrial Pathway of Caspase Activation 5.4. Converging Points and Cross-Talk 5.5. Nonpeptidic Small Molecules 5.5.1. TWX Molecules: First Reported Nonpeptide Inhibitor of XIAP (BIR2 domain) 5.5.2. Polyphenylureas: Another Class of Nonpeptide Inhibitors of XIAP (BIR2 domain) 5.6. Monovalent SMAC Mimetics © 2014 American Chemical Society

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4668 Special Issue: Chemical Biology of Protein-Protein Interactions

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Received: July 25, 2013 Published: March 27, 2014 4640

dx.doi.org/10.1021/cr4004049 | Chem. Rev. 2014, 114, 4640−4694

Chemical Reviews

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Figure 1. Representation of a small molecule binding to an enzyme and to the protein−protein interface or through an allosteric site .

Author Contributions Notes Biography Acknowledgments References

away from protein−protein interface) to induce conformational changes to prevent the PPI, and (c) somehow blocking protein complex formation which would then lead to prevention of PPI. In the first approach, structural knowledge of the protein− protein interface is often helpful in guiding the design and synthesis of small molecules. Often the second and third approaches are focused on the functional screen, and following the discovery of active small molecules, serious efforts are then needed to determine the allosteric binding site that is away from the protein−protein interface. Both schools of thought have their merits and demerits! In most cases, given the dynamic nature of protein complexes that are made up of several PPI, the later approach is gaining momentum, although for the past several years the former approach (i.e., based on structural knowledge of the protein−protein interface) was dominating the stage in the PPI arena. Keeping in mind several excellent review articles1c,g covering various aspects of PPI, herein, we provide selected case studies in detail that would serve as a good base knowledge for the chemical and biomedical research communities interested in seeking information in this growing area of research. Although predominantly focused on various chemical approaches involving either natural products or structural knowledge-based design strategies, we hope that this would also be a good read for the nonsynthetic community who are seeking opportunities to enter in this arena.

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1.. INTRODUCTION 1.1. Going in for Protein−Protein Interactions and Pathways

There is growing interest within the pharmaceutical community to undertake biological targets that involve protein−protein1 and, in general, biomacromolecular interactions2 (PPI or BMI) and to develop research programs that aim to enhance our current understanding of highly regulated, dynamic signaling pathways.3 It is also well known that deregulation of these pathways that involve complex PPI leads to initiating several diseases, and our quest to correct their deregulation by small molecules is also growing.4 This rapid rise in the paradigm shift, (i.e., a move from classical enzyme-based modulators to pathways) is also providing new opportunities to establish additional competencies to combat in the chemical biology to drug discovery arena.4b For example, a typical enzyme inhibitionbased approach involves seeking the structural information of the enzyme and then working closely with organic synthesis/ medicinal and computational chemists. As in most cases, the enzymes present well-defined, often deep pockets, which provides a good starting point to develop the medicinal chemistry program. Going in for protein interactions is a different territory (see Figure 1), and there are some fundamentals differences that are important to keep in mind for developing the drug discovery program. Some of these key points are outlined as follows: (i) protein−protein interactions are typically a part of the complex signaling network and often involve multiple connectivity; (ii) by contrast to having deep pockets as with enzymes, in general, PPI works through relatively shallow interacting sites and spans over a large surface area. Also, often these interactions involve multiple weak interactions. Over the years, through several outstanding examples, it has also been shown that PPI are known to have “hot spots” from the so-called “connectivity through a large surface area”; thorough structural knowledge gained in this direction also provides a good starting point to the medicinal chemistry community to develop their research program; (iii) in general, PPI involve serious hydrophobic interactions, and this may lead to having functional small molecules with enhanced hydrophobicity. Modulation of a PPI by a small molecule can be envisioned by various approaches, such as (a) the interaction of the small molecule at the protein−protein, thereby preventing the interaction, (b) a small molecule at the allosteric site (i.e.,

1.2. Protein−Protein Interactions and Small Molecules

Classical approaches to design small molecules for typical enzyme inhibition, in general, lead to providing compounds that are rich in sp2 character and typically lack the features that are the hallmark of most bioactive natural products, i.e., compounds having rich sp3 character, that present 3-dimensional (3-D) scaffolds, and that exhibit a rich display of dense chiral groups.4−6 There are also growing reports in the literature that are indicative of emerging problems (e.g., efficacy, high selectivity, etc.) in the drug discovery arena that are attributed to classical medicinal chemistry-derived compounds.4a,5,7 Moreover, it is also well known that natural products have an excellent track record to function as small molecule modulators of protein−protein interactions and thus serve as an excellent starting point leading to design of functional small molecules.5,6,8 One of the key objectives of the present review is to show case several studies where natural products have served to modulate PPI, and this then leads to building the diversity-based organic synthesis program that aims to populate a chemical space having a set of compounds obtained from the inspiration of natural products. The early days of the high-throughput chemistry arena was more focused on accessing a large number of compounds by utilizing rather simple chemistry. However, this paradigm shift from enzyme inhibition to PPI is also challenging the chemistry 4641


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Figure 2. Schematic representation of α/β-tubulin−microtubule equilibrium, and the structure of microtubules.

TOG, MCAK, MAP4, EB1, dynactin 1 (also known as p150 Glued), RAC1, and FHIT, are known to exist.18 Microtubules are highly dynamic polymers exhibiting two important features: dynamic instability and treadmiling. Dynamic instability is a process wherein the individual microtubule ends the switches between the periods of growth and shortening. The two ends of a microtubule being nonequivalent, one end commonly known as the plus end grows and shortens more rapidly than the other end, i.e., the minus end. Dynamic instability is dependent on four main variables: (i) the rate of microtubule growth, (ii) the rate at which it shortens, (iii) the frequency of transition from the growth or paused state to shortening, which is called a “catastrophe”, and (iv) the frequency of transition from shortening to growth or pause called “rescue”.19 Treadmiling is a result of the differences in the critical subunit concentrations at the opposite ends in which net growth occurs at one microtubule end and balanced net shortening at the opposite end, involving the intrinsic flow of tubulin subunits from the plus end of the microtubule to the minus end. The polymerization dynamics of microtubules are unusual compared with those of most polymers in nature in that they are created by the gain and loss of a short region of tubulin− GTP or tubulin−GDP−inorganic phosphate (Pi) at the two microtubule ends, called a GTP cap. As soon as the tubulin− GTP adds to the microtubule ends, the tubulin-bound GTP is hydrolyzed to tubulin−GDP and Pi dissociating the cap from the microtubule, leaving a microtubule core with tubulin and a stoichiometrically bound GDP in β-tubulin. The tubulin−GDP remains in this nondissociable and nonexchangeable position until the tubulin subunit dissociates from the microtubule. A generally accepted model in the field is that the initial straight conformation of tubulin−GTP when it polymerizes into protofilaments changes to a curved form following GTP hydrolysis, thereby locking the tubulin−GDP into the micro-

community in developing novel methods that allow us to populate the chemical space with compounds having (i) a wide variety of 3-dimensional scaffolds with a few chiral centers, (ii) a few diversity points, (iii) an acceptable pharma-related complexity, and (iv) acceptable to medicinal chemistry to be synthesized in a relatively short period.4b,9,6c,4c,5,6,8c

2. CASE STUDY 1: TUBULIN POLYMERIZATION AND NATURAL PRODUCT-DERIVED SMALL MOLECULES Tubulin is a simple subunit of microtubules and was isolated by Borisy and Taylor as a colchicine-binding agent.10 The term tubulin was given by the Mohri team in 1968, although other names like flactin, spactin, and tektin were also considered initially.11 Purification of tubulin from sperm tails was achieved by Shelanski and Taylor12 and from brain by the Weisenberg team in 1968.13 Microtubules are the key apparatus of the cytoskeleton, which are long, filamentous, tube-shaped protein polymers that are fabricated up α/β-tubulin heterodimers and are present in all eukaryotes.14 Microtubules play an important role in the development and maintenance of cell shape, cell transport, cell motility, cell signaling, and cell division.15 The functional diversity of microtubules is attributed to their interaction with various regulatory proteins like microtubule associated proteins (MAPs) and soluble tubulin. The microtubule surfaces and its ends through an expression of different tubulin isotypes display different functions. Most likely, this different tubulin isotype is achieved through several post-translational modifications of tubulin, i.e., polyglutamylation, polyglycylation, phosphorylation, detyrosination/tyrosination, acetylation, and removal of the penultimate glutamic acid residue of α-tubulins.16,17 Other MAPs include the dynein and kinesin motor proteins, and several other microtubule regulatory proteins, such as survivin, stathmin, 4642


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Figure 3. Examples of bioactive natural products known for microtubule destabilization.

tubule core in a strained conformation.14 The presence of GTP or GDP−Pi cap stabilizes the end, and it further helps in growth of the microtubule, while its loss exposes the relatively unstable core of the microtubule leading to shortening of the ends rapidly.20 It is therefore evident that there are two sets of association and dissociation rate constants governing tubulin addition at each microtubule enda stabilized (capped) set and an unstabilized (uncapped) setand also that modification of any of these rate constants at either end by drugs or regulatory

proteins can show a marked effect on polymerization dynamics and microtubule function.21 Microtubule-targeted drugs are extremely effective against fungal and parasitic infections.22 Parasitic diseases including malaria, leishmaniasis, and schistosomiasis have a devastating impact on human health in tropical and subtropical regions and are also highly significantly related to animal health worldwide. FtsZ, the ancestral homologue of eukaryotic tubulins, is a GTPase that assembles into cytokinetic ring structure absolutely vital for cellular division in prokaryotic cells.23 The essential roles 4643


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Figure 4. Examples of bioactive natural products known for microtubule stabilization.

thus leading to an arrest in the prometaphase/metaphase state that eventually results in an induction of apoptosis. Most microtubule-targeting compounds exploit this mechanism to eliminate cancer cells, as their high rates of proliferation increase vulnerability to mitotic checkpoint-induced apoptosis. Interestingly, a large number of small molecule natural products bind to soluble tubulin and/or directly to tubulin within the microtubules.28 Most of these compounds are antimitotic agents and further inhibit cell proliferation by performing on the polymerization dynamics of spindle microtubules. Microtubule-targeted antimitotic drugs are typically classified into two main groups. One group, referred to as the microtubule-destabilizing agents (see Figure 3), inhibits microtubule polymerization at high concentrations, and several compounds, such as the vinca alkaloids [e.g., vinblastine (F3.1), vindoline (F3.2), vincristine (F3.3), vinorelbine (F3.4), vindesine (F3.5), and vinflunine (F3.6)], cryptophycins (F3.7−3.9), eribulin mesylate (E7389) (F3.10), and halichondrins (F3.11), are used clinically or in clinical investigation for treatment of cancer. The second main group is referred to as the microtubule-stabilizing agents (Figure 4). These agents induce microtubule polymerization, and some of the representative examples are paclitaxel (F4.1), docetaxel (taxotere), epothilones, ixabepilone (BMS-247550) (F4.2−F4.7), discodermolide, eleutherobins, sarcodictyins, laulimalide (F4.8), rhazinalam, certain steroids, and polyisoprenyl benzophenones.29 The genus vinca alkaloids, a category of antimitotic compounds derived from the periwinkle plant, Catharanthus roseus,30 act by binding to the β-subunit near the GTP-binding site of tubulin (i.e., known as vinca domain) and arrest the mitosis at prometaphase.31 Many other novel chemotherapeutic drugs also bind to this domain. Vinblastine and vincristine are first-generation vinca alkaloids32 that have undergone significant clinical development. Through binding close to the hydrolyzable GTP site they alter the dimeric conformation and therefore inhibit tubulin-dependent GTP hydrolysis and also GDP−GTP exchange. At low concentrations, vinca compounds bind to the plus ends of microtubules that reduces the dynamics and further result to mitotic arrest.33 Vindesine, vinorelbine, and vinflunine are the semisynthetic vinca alkaloids, among which vinflunine is

of FtsZ in prokaryotic cell division, which includes (i) widespread conservation in the bacterial kingdom, (ii) its absence in the mitochondria of higher eukaryotes, (iii) an evolutionary distance from tubulin, and (iv) the known biochemical activity and knowledge of an atomic-level structure, made FtsZ an attractive target to develop agents that may cause selective toxicity to bacterial pathogens.24 Microtubules and their dynamics are the targets of a chemically varied group of antimitotic medication (with numerous tubulin-binding sites) that are used with huge success within the treatment of cancer.15 The antifungal drug griseofulvin inhibits cellular division powerfully in fungal cells and weakly in mammalian cells by touching the mitotic spindle microtubule (MT) function. Collectively, it blocks the cell-cycle progression at G2/M and induces cell death in human tumor cell lines.25 In addition to numerous physiological processes, microtubules and their inherent dynamic behaviors are crucial to productive cell division.26 During mitosis, the microtubule dynamicity roughly increases by 20−100-fold to change the spacial organization and also a rapid remodeling of the interphase arrays to assemble the elegant mitotic machinery within a matter of minutes. The nucleation rate of microtubules at the centrosomes collectively increases by 7-fold throughout mitosis. The fast dynamics of spindle microtubules is important for the capture of chromosomes throughout the prometaphase, because the spindle fibers are compelled to explore the cytoplasmic space to create productive attachments to the kinetochores.27 This is accomplished through an alternating phases of a speedy growth to long distances (usually around 5−10 mm) followed by the whole shortening. After all the chromosomes establish connections at their kinetochore region with the microtubules springing from each opposite poles, they are all aligned on the metaphase plate during a process known as congression. Only after this precise alignment, mitosis proceeds past the metaphase−anaphase check point into an anaphase. Thereafter, the sister chromosomes are synchronously separated and pulled to the alternative ends of the dividing cell. The extraordinarily fast dynamics of microtubules plays an important role within the tangled movement of chromosomes. Even one misaligned or absent chromosome from the metaphase plate will stall mitosis and forestall the cell from progressing beyond the checkpoint, 4644


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Figure 5. Cocrystal structure of epothilone A with binding to β-tubulin. The surface of epothilone A is depicted using the following color coding: C, green; O, red; N, blue; S, yellow. (A) Surface model: epothilone A is shown with hydrogen bonds (yellow dashes) to associated centers of β-tubulin protein. (B) Ribbon model: Hydrogen bonding (yellow dashes) around epothilone A in β-tubulin. Protein secondary structure for helices is shown in red, sheets in aqua, and loops in gray (PDB code 1TVK). All molecular modeling images in this review are taken through the Maestro v9.6 software package (Schroedinger LLC).

Scheme 1. One of the Proposed Retrosynthetic Analyses of Cryptophycin-24

Scheme 2. Synthesis of Cryptophycin-24

found to exhibit a stronger efficacy when compared with the parent vinblastine.34 Cryptophycins (F3.7−3.9, Figure 3) are isolated from the terrestrial cyanobacterium, a Nostoc species,35which shows strong destabilizing properties by blocking hydrolysis of GTP.36 They are not the substrate of P-glycoprotein (Pgp) that favors specific antitumor activity against multiple cancers.37

At lower concentrations, cryptophycins are shown to interrupt the microtubule dynamic instability. They have two forms of binding sites, a high-affinity binding site and a few low-affinity binding sites.38 It interferes with the vinblastine-binding site on tubulin in a very noncompetitive manner.39 The Moore team40 and many other groups41 have succeeded in the synthesis of cryptophycins. Herein, we outline the efforts by the George team 4645


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Scheme 3. Brief Outline of Eribulin (E7389) Synthesis from the Kishi Team

to develop the synthesis of cryptophycins.42 As shown in the retrosynthesis plan, the researchers achieved the crytophycin macrolactone core by a novel macrolactonization approach that utilizes a reactive acyl-β-lactam intermediate (1.4) which incorporates the β-amino acid moiety (Scheme 1). This modular approach permits multiple alterations throughout the structure that can be achieved by modifications in the amino acyl β-lactam and the hydroxyacid. The acyl-β-lactam macrolactonization strategy provides an efficient entry into the macrocyclic core of this promising family of antitumor macrolides. The key synthesis steps are shown in Scheme 2. Halichondrins are microtubule-destabilizing anticancer agents of marine origin.43 Halichondrin B (Figure 3, F3.11) is derived from the marine sponge known as Halichondria okadai by the Uemura and Hirata groups,44 and it inhibits the binding of vinblastine to tubulin in a very noncompetitive fashion. The Kishi research team at the Chemistry and Chemical Biology Department at Harvard University worked with the National Cancer Institute (NCI) and Eisai Research Institute (ERI) to evaluate the in vitro and in vivo antitumor activities of synthetic halichondrins including several intermediates. This extensive study led the discovery of halichondrin B to have antitumor activity. With these significant data, a huge drug discovery effort was then undertaken by ERI that resulted in further identification of eribulin mesylate (E7389, F3.10),45 which is a synthetic intermediate derivative of halichondrin. It was found to be active in patients with metastatic breast cancer relapsing after anthracyclines and taxanes. In a randomized phase III clinical trial when patients received a single agent of eribulin mesylate a substantial improvement in overall survival was achieved when compared to patients treated consistently with the physician’s selection.46 It is now well established that eribulin works by binding to microtubule polymerization without affecting depolymerization and with the extra sequestration of tubulin into nonfunctional aggregates.47 Eribulin stimulates permanent mitotic obstruct by inhibiting mitotic spindle formation that could lead to a cell-cycle arrest in the G2/M phase and consequent apoptosis. Moreover, eribulin binds to a restricted range of high-affinity sites at the plus ends of the microtubules, and there is some evidence against its binding to interdimer interfaces in pre-existing polymers.47,48 The Kishi team and many other groups succeeded in the synthesis of halichondrin B fragments 49 and E7389 by developing the catalytically

asymmetric Cr-mediated coupling reactions.50 A brief outline of the plans leading to synthesis of E7389 is shown in Scheme 3. The catalytic asymmetric Ni/Cr- and Co/Cr-mediated couplings are used for bond formation at the indicated sites. Along with above microtubule destabilizers, Phomopsin A from Phomopsis leptostomiformis43 and ustiloxins from Ustilaginoidea virens,51 Dolastatins (structures not shown) are natural peptides extracted from a species of sea hare, genus Dolabella auricularia.36,39a and inhibit tubulin polymerization.36,52 During the 1990s, paclitaxel and its semisynthetic analog docetaxel (for the taxol structure see Figure 4, 4.1) were added to the chemo-preventive arsenal to be used as anticancer therapeutics. Paclitaxel was extracted from the bark of the yew tree in 1967 by the Wall and Wani teams,53 and it underwent slow development until late 1979, when the Schiff and Horwitz teams made the surprising discovery that, not like the vinca alkaloids, paclitaxel stimulated microtubule polymerization.54 Even then its development for clinical use was obstructed by the limited supplies of the natural compound until procedures for its semisynthesis made its production feasible.55 By 1995 it was approved for clinical use, and it is now widely used to treat breast and ovarian cancer, Kaposi’s sarcoma, and nonsmall-cell lung cancer. Like vinca alkaloids its principal side effects are neurotoxicity and myelo-suppression.52d The taxanes bind poorly to soluble tubulin itself but instead bind directly with a high affinity to tubulin along the length of the microtubule. The binding site for paclitaxel is within the β-subunit, and its location, which is on the inner surface of the microtubule, is also known with precision because determination of the crystal structure of tubulin was carried out with tubulin complexed with paclitaxel.56 The binding of paclitaxel to its site on the within microtubule surface stabilizes the microtubule and increases microtubule polymerization, presumptively by inducing a conformational change in the tubulin that, by an unknown mechanism, increases its affinity for neighboring tubulin molecules.20b Clinical success of the taxanes led to a search for alternative drugs that has the potential to reinforce microtubule polymerization. These efforts were eminent in providing many promising compounds, including discodermolide, epothilones, eleutherobin, sarcodictyins, and laulimalide. Some of these compounds compliment with paclitaxel for binding to microtubules and are regarded to bind at or near the taxane site (discodermolide, 4646


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Scheme 4. Olefin Metathesis-Based Approach to Epothilones A and C by the Nicoloau Team

Scheme 5. Nicolaou’s Olefin Metathesis Strategy to Epothilones A and C

Scheme 6. Synthesis of Ixabepilone (BMS-247550)

the substrate drugs out of tumor cells.62 With the recent publication of the crystallographic structure of the epothilone A/ tubulin complex, the Nettles team was able to solve the puzzle of the bioactive conformation of epothilone A.63 The Danishefsky team at Sloan Kettering was first to accomplish total syntheses of epothilones A and B. Nicolaou’s olefin metathesis strategy to obtain epothilones A and C is highlighted in Schemes 4 and 5.64 Ixabepilone (BMS-247550) (Figure 4, F4.4), a novel synthetic lactam analog of epothilone B, showed better clinical outcomes in metastatic disease notably in triple-negative breast cancer and was further approved by the FDA.65 It is shown to have 2.5-fold higher cytotoxicity when compared with paclitaxel and is equally effective in taxane-resistant tumors.66 Preclinically, ixabepilone showed reduced susceptibility to multiple tumor-resistance mechanisms, including efflux transporters, such as the multidrug-resistance-associated protein 1 and P-glycoprotein, which are involved in the acquired and intrinsic tumor resistance.67 Ixabepilone showed clinical activity in multiple solid tumors including advanced breast, prostate, lung, pancreatic, renal cell, and ovarian cancers.68 Ixabepilone synthesis is shown in Scheme

epothilones, eleutherobins, and sarcodictyins), but others, such as laulimalide, seem to bind to unique sites on microtubules.57 Epothilones (see Figure 4) belong to the macrolide drug family, which act as microtubule stabilizers. Naturally isolated from myxobacterium Sorangium cellulosum, epothilones A and B are known as a novel class of antimicrotubule drugs discovered by the Höfle and Reichenbach teams at the Gesellschaft fur Biotechnologische Forschung (GBF) in Germany.58 Both epothilone A and B seem to compete with paclitaxel for its binding site; however, their unique molecular framework binds at a site that is close to the taxane-binding site. Epothilones exhibit the microtubule-stabilizing activities almost like paclitaxel; however, they somewhat function through the distinct mechanisms.59 Due to their bacterial origin, these drugs have the advantage of easy production. In addition, they are not susceptible to Pgp-mediated drug efflux, so they are useful in treatment of taxane-resistant tumors.60 One of the mechanisms to clarify tumor resistance is the inherent expression of multidrug-resistance proteins like P-glycoprotein (Pgp), which is an ABC (ATP-binding cassette) transporter.61 Expression of these transporters act as drug efflux pumps, causing diffusion of 4647


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Scheme 7. Retrosynthetic Analysis of Laulimalide by the Trost Team

Scheme 8. Synthesis of Laulimalide

6.69 Highlighted in this novel strategy is a regio- and stereoselective π-allyl palladium nucleophilic substitution reaction that provides a pivotal ring-opened intermediate with the nitrogen substituent at C15 of the epothilone core. Chemoselective reduction of an azide intermediate followed by macrolactamization of the resulting amino acid afforded the desired lactam, ixabepilone (BMS-247550). Laulimalides are also known as fijianolides (see Figure 4) and antimitotic agents with complex structures. They are extracted from marine sponges and binds to a unique site on α-tubulin to exhibit microtubule-stabilizing effects similar to paclitaxel.70 Unlike paclitaxel, laulimalide retains activity in P-glycoprotein (PgP)-overexpressing multidrug-resistant (MDR) cells.8e Recently, it was reported that laulimalide binds to a different site on the tubulin polymer than most other microtubule stabilizers,71 and it shows activity against cell lines containing β-tubulin mutations which are shown to promote resistance to paclitaxel and the epothilones.57 The Littlefield team reported the total synthesis of laulimalide capable of producing gram quantities,72

which enabled in-depth evaluation. Many other groups, including those of Wender,73 Mulzer,74 and Trost,75 also succeeded in total synthesis laulimalide and its analogs. Shown in Scheme 7 is Trost’s retrosynthetic analysis of laulimalide76 that involves two key reactions, i.e., Still−Gennari olefination and Ru-catalyzed Alder−ene reaction. The Trost group also achieved macrocyclization with an intramolecular alkene−alkyne coupling utilizing the Ru catalyst approach as shown in Scheme 8. Peloruside A is also extracted from marine sponges, and it has structural similarity with epothilones but binds to the laulimalidebinding site in α-tubulin.77 The diversity within the structure but similarity in the binding site and the vice versa is indeed an intriguing aspect of these drugs. However, a unique binding site but similar polymerization effects of those drugs present an opportunity for synergism with taxanes, which could lead to a combination of drugs with improved antiproliferative activity.78 Discodermolide and dictyostatin (see Figure 6) are naturally derived from marine sponges, in which the microtubule toxins act as a crucial defense mechanism.79 Like paclitaxel, they also 4648


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function p53 gene.91 MDM2 is a 491 amino acid protein, and its 3D structure is available with respect to domain, solved by both X-ray crystallography as well as NMR spectroscopy. MDM2 contains several conserved domains including an N-terminal p53 interaction domain, the structure of which has been solved using X-ray crystallography. MDM2 also contains an acidic domain, and phosphorylation of residues within this domain appears to be important for regulation of MDM2 function. In addition, this region contains nuclear export and import signals that are essential for proper nuclear-cytoplasmic trafficking of MDM2. Another domain is the zinc finger domain, the function of which is poorly understood, and MDM2 also contains a C-terminal RING domain. p53 stimulates expression of MDM2, which in turn negatively acts on p53 in several ways (see Figure 7). MDM2 is a p53-specific ubiquitin E3 ligase92 and thus promotes proteasomal degradation of p53.

Figure 6. Dictyostatin and discodermolide hybrid small molecules.

stimulate tubulin assembly, hypernucleation, increased microtubule stability, and decreased depolymerization. Both are structurally similar and have a therapeutic advantage for cells expressing β-tubulin III isoforms.80 Their binding site appears to be distinct from that of paclitaxel; however, they exhibit synergism with paclitaxel.81 The Paterson team at Cambridge achieved the total synthesis of a potent dictyostatin− discodermolide hybrid82 (Figure 6). This hybrid compound is shown to reinforce cell growth inhibitory activity relative to discodermolide in four human cancer cell lines together with the taxol-resistant NCI/ADR-Res cell line.83

Figure 7. Schematic representation of p53 and MDM2 interactions and its impact on disease biology.

In addition to p53, MDM2 E3 ligase has other substrates such as the ribosomal protein L27 that regulates p53 translation.93 Furthermore, it binds to the N-terminal trans-activation domain of p53 and therefore blocks the latter’s transcriptional activity.89 A third mechanism by which HDM2 regulates p53 activity is by promoting the latter’s nuclear export.94 MDM2 also regulates sumoylation,95 neddylation,96 and acetylation97 of p53. The X-ray crystal structure between p53 peptide and MDM298 showed that MDM2 possesses a deep hydrophobic cleft into which the p53 peptide binds as an amphipathic α-helix (Figure 8). It shows that four hydrophobic residues (Phe19, Leu 22, Trp23, and Leu26) of p53 peptide interact with the deep hydrophobic pocket present in MDM2. Along with these hydrophobic interactions there are two more hydrogen bonds: one is between the F19 backbone amide of p53 and the Q72 side chain of MDM2 at the entrance of the cleft, and the other is between p53 indole (W23) NH and the MDM2 L54 backbone carbonyl deep inside the cleft. The surface area buried between p53−MDM2 is around 1498 and 808 Å2 on p53 and 690 Å2 on MDM2. p53−MDM2 interactions were dominated by only three key residues in p53: F19, W23, and L26, which all together buried no more than about 500 Å2 surface area. The specific interactions between p53 and MDM2 protein are well studied and reported in the literature. It is shown that the 106-amino-acid-long N-terminal domain of MDM2 interacts with the N-terminus of the trans-activation domain of p53.99 The precise structural requirements for the MDM2−p53 protein− protein interaction were demonstrated by the crystal structures of the N-terminal domains of human and Xenopus laevis MDM2

3. CASE STUDY 2: P53 AND MDM2 INTERACTIONS AND SMALL MOLECULES The tumor suppressor protein p53 plays a very important role in the regulation of cell cycle, programmed cell death, DNA repair, and senescence.84 p53 gene was discovered in 1979 as a transformation-related protein and cellular protein which accumulates in the nuclei of cancer cells, and it binds tightly to the SV40 large T antigen.85 p53 does not function properly in all human cancer cells. This will happen in different ways: (i) usually through direct loss of function by mutation in the p53 gene in about one-half of whole tumors86 or (ii) indirectly often by amplification or overexpression of the MDM2 gene.87 MDM2 stands for Mouse Double Minute2 whose human homologue is HDM2; both these nomenclatures are used synonymously. The MDM2 protein was discovered in 198788 as the protein product of a gene that was amplified on a double minute chromosome in some transformed mouse cells. In 1992, Levine and colleagues discovered that this protein could bind tightly to p53 and inhibit its activity as a transcription factor.89 Subsequently, they showed that MDM2 binds the transcriptional activation domain of p53 and blocks its ability to regulate target genes and exert antiproliferative effects. On the other hand, p53 activates expression of the MDM2 gene in an autoregulatory feedback loop.90 MDM2 plays a very important role in regulating p53 function as shown by the observation that deletion of MDM2 is lethal in mice during embryogenesis only in animals carrying a 4649


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Figure 8. (A) NMR structure of free MDM2 (PDB code 1Z1M). (B) X-ray crystal structure of p53 peptide bound to MDM2 (PDB code 1YCR).

in binding affinity than peptide 4, confirming the validity of the design. The keen and detailed observation of the X-ray crystal structure of ligand bound to MDM2 showed incomplete occupancy of Trp, suggesting that substitution at the 6 position of indole would improve binding affinity. Substitution of Trp by 6-Cl-Trp leads to peptide 6, which was >1700-fold more potent than the wild-type p53-derived 12-mer peptide.

complexed with the short peptides, derived from the N-terminal domain of p53 (residues 15−29). Two experiments (A yeast two-hybrid screen and immunoprecipitation)99 were initially used to map the two contact regions of p53 and MDM2. It was shown that the binding domain of MDM2 on p53 was localized between 1 and 52 residues, and the binding domain of p53 on MDM2 was localized between 1 and 118 residues. Further, the Picksely team in 1994100 showed that even shorter peptides such as six amino acids (TFSDLW in human p53, TFSGLW in murine p53) derived from the amino terminus of p53 can bind to MDM2. It was further shown that this short, hexapeptide binds with a low binding affinity for HDM2 (IC50 = 700 μM).101 Bottger and coworkers, using a phage display study, identified a novel MDM2binding peptide (peptide 2). This was 28-fold more potent than wild-type p53-derived peptide (peptide 1). Synthesizing the shortened peptide 2 resulted in an 8-mer peptide 3 that could be identified as the smaller sequence retaining micromolar affinity for HDM2 (see Table 1). It was already known from the X-ray

3.1. Natural Product Inhibitors of p53−MDM2 Interactions

Chlorofusin (9.1, Scheme 9) was isolated by Williams and coworkers in 2001 from the fermentation broth of Microdochium caespitosum in the course of an activity-guided screening program involving examination of over 53 000 microbial extracts.103 It was found to inhibit the p53/MDM2 interaction in a DELFIAmodified ELISA assay and having an IC50 value of 4.6 μM but was inactive against the TNFα−TNFα PPI and showed no cytotoxicity against Hep G2 (human hepatocellular carcinoma) cells at 4.0 μM. Using surface plasmon resonance (SPR) spectroscopy104 it was shown to directly bind to the N-terminal domain of MDM2, and fitting of a biphasic model to the data has been interpreted as an initial fast binding event followed by a second slow step.105 Thus, it was shown to be an interesting lead for antineoplastic intervention that acts by a rare disruption of a PPI. The structure of chlorofusin was proposed based on spectroscopic and chemical degradation studies; it consists of a densely functionalized, azaphilone-derived chromophore linked through the terminal amine of ornithine to a 27-membered cyclic peptide composed of nine amino acid residues.103 Two of the cyclic peptide amino acids possess a nonstandard or modified side chain, and four possess the D configuration. These studies provided the cyclic peptide structure and connectivity, although the stereochemical assignment of the two asparagine residues (Asn3 and Asn4) could not be made and were simply established to have opposite stereochemistry (L and D). Similarly, the structure and relative stereochemistry of the unusual azaphilonederived chromophore was also proposed, although an assignment of its absolute stereochemistry was not possible. The relative stereochemistry was assigned using gradient 1D NOE experiments. Total synthesis of chlorofusin was accomplished by two groups, i.e., Boger106 and Yao,107 but the 27-membered cyclic peptide core of chlorofusin was synthesized by several groups.108 In 2003, Boger and co-workers reported the synthesis of two chlorofusin cyclic peptide diastereomers bearing either the LAsn3/DAsn4 or the D-Asn3/L-Asn4 stereochemistry and correlated the former with the spectroscopic properties (1H and 13C NMR) of the natural product.108c Cyclic peptide containing L-Asn3/D-Asn4 has more close data when compared with natural chlorofusin data.

Table 1 entry 1 2 3 4 5 6

sequence 16

21

Ac-Gln -Glu-Thr-Phe-Ser-Asp Leu-Trp-Lys-Leu-Leu-Pro27-NH2 Ac-Met-Pro-Arg-Phe19-Met-Asp-Tyr-Trp-Glu-Gly-Leu 26 -Asn-NH2 Ac-Phe19-Met-Asp-Tyr-Trp-Glu-Gly-Leu26-NH2 Ac-Phe19-Met-Aib-Tyr-Trp-Glu-Ac3c-Leu26-NH2 Ac-Phe19-Met-Aib-Pmp-Trp-Glu-Ac3c-Leu26-NH2 Ac-Phe19-Met-Aib-Pmp-6-Cl-Trp-Glu-Ac3c-Leu26NH2

IC50 (nM) 8673 ± 164 313 ± 10 8949 ± 588 2210 ± 346 314 ± 88 5±1

structure that p53-derived peptide binds into a deep hydrophobic cleft present on MDM2 as an amphipathic α-helix. Before further optimizing peptide 3 Garcia-Echeverria and co-workers102 studied the conformation of peptide 2 in solution. Circular dichroism (CD) and nuclear magnetic resonance (NMR) studies revealed that peptide 2 has an extended or random coil conformation under physiological conditions. On the basis of these studies, the helical structure of peptide 3 was stabilized by introducing α,α-disubstituted amino acid residues α-aminoisobutyric acid (Aib) and 1-amino-cyclopropanecarboxylic acid (Ac3c) in the place of Asp21 and Gly25 resulting in peptide 4. Using the X-ray crystal structure of p53 peptide and with MDM2 a model complex of peptide 2 with MDM2102 was constructed. Examination of this model revealed that the hydroxyl group of the Tyr and the amino group of the Lys94 of MDM2 are close together. Tyr in peptide 4 was replaced by phosphonomethylphenylalanine in order to form a stabilizing salt bridge with the εamino group of Lys.94 The resulting peptide has a 7-fold increase 4650


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Scheme 9. One of the Proposed Retrosynthetic Approaches to Chlorofusin, 9.1

Scheme 10. Synthesis of Intermediate 9.4

Chlorofusin analogs in the absence of chromophore do not inhibit the p53−MDM2 interaction;109 it is likely that the chromophore, which contains novel fused 1-oxa-6-azaspiro[4.5]dec-7-en-10-ol and butyric acid 3-chloro-1-methyl2,6-dioxocyclohex-3-enyl ester systems, could be responsible for recognition of MDM2, possibly at the p53 F19−W23-L26 site, apart from the fact that the chromophore is essential for biological activity and that the stereochemistry of the D-Ade-8 residue is important for the overall conformation.110

The chlorofusin chromophore is derived from a class of compounds referred to as azaphilones, so named for their ability to readily condense with ammonia or primary amines. In light of this, at least two potential routes to chlorofusin could be envisioned:106b,111 (1) condensation of an appropriate azaphilone 9.5 with the full cyclic peptide 9.6 followed by chromophore oxidation and spirocycle formation or (2) construction of a fully functionalized chromophore appended to a smaller peptide fragment 9.3 followed by peptide 9.2 coupling and macrocyclization to afford chlorofusin (Scheme 9). 4651


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Scheme 11. Total Synthesis of Chlorofusin

Recently, Gerwick and co-workers isolated hoiamide D (F9.3, Figure 9) in bioassay-guided fractionation of two cyanobacterial extracts from Papua New Guinea.114 F9.3 is a polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS)derived natural product that features two consecutive thiazolines and a thiazole as well as a modified isoleucine residue. F9.3 displayed inhibitory activity against p53/MDM2 interaction (EC50 = 4.5 μM). It is a very attractive target for anticancer drug development. Total synthesis of hoiamide D has not been reported to date.

The former requires the conduct of an azaphilone chromophore oxidative spirocyclization on a complex, latestage synthetic intermediate with control of the resulting relative and absolute stereochemistry or effective separation and characterization of the resulting diastereomers, whereas the latter requires that the chromophore N,O-spiroketal in the smaller peptide fragment not unravel during the late stages of the ensuing cyclic peptide synthesis. Synthesis of chlorofusin is shown in Schemes 10 and 11. Hexylitaconic acid (F9.1, Figure 9), an inhibitor of the p53− HDM2 interaction, was isolated from a culture of marine-derived

3.2. β-Hairpin Peptidomimetics

Robinson and co-workers successfully synthesized the mimic to the α-helix of p53 with a cyclic β-hairpin to generate inhibitors of the p53/MDM2 interaction. They identified that the distance between the Cα atoms of two residues i and i+2 along one strand of a β-hairpin is very close to the distance between the Cα atoms of Phe19 and Trp23 on one face of the MDM2-bound p53 αhelix. A designed hairpin mimetic could therefore function as a scaffold to hold the side chains of Phe19 and Trp23 (and possibly also Leu26) in the correct relative positions so that each can interact simultaneously with the p53 binding site on MDM2.115 On the basis of this idea mimetic F10.1 (Figure 10) was obtained, in which an eight-residue loop is preorganized into a regular β-hairpin by mounting upon a D-Pro-L-Pro dipeptide template. It is known that D-Pro-L-Pro can function as a template to stabilize β-hairpin loop conformations in cyclic mimetic.116 This showed only a modest inhibitory activity (IC50 = 125 μM) in a biosensor assay. Lead optimization resulted in compound F10.2, with almost 1000-fold higher affinity (IC50 = 140 nM). [15N, 1H]HSQC spectra115 showed that these mimetics interact with HDM2 at the p53 binding site. On the basis of NMR studies it seemed likely that the weak inhibitory activity of F10.1 was due to the absence of a stable β-hairpin solution conformation. Peptide SAR studies revealed that introduction of aromatic side chains (F10.2) stabilized the hairpin conformation and enhanced binding to HDM2. The X-ray crystal structure between HDM2

Figure 9. Natural product inhibitors of p53−MDM2 interactions.

fungus, Arthrinium sp.112 The structure was identified to be (−)-hexylitaconic acid (F9.1 by spectroscopic analysis, but the absolute configuration (R/S) of the active F9.1 was unknown). Inhibition of p53−HDM2 binding was tested by the ELISA method, and F9.1 inhibited binding with an IC50 value of 50 μg/ mL. F9.1 is the second inhibitor isolated from natural resources. Semipervine (F9.2) was identified as an inhibitor of MDM2 from screening of 144 000 natural product extracts by a novel high-throughput electrochemiluminescent screening by Sasiela and co-workers113 F9.2 inhibits MDM2 autoubiquitination and MDM2-mediated p53 degradation and leads to an accumulation of p53 in cells. 4652


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Figure 11. Substituted terphenyl as p53−MDM2 inhibitors and αhelical-based mimicry on a terphenyl scaffold.

isobutyl, 2-naphthylmethylene, isobutyl side-chain sequence showed a Ki value of 182 nm for displacing p53 binding to MDM2. Comparison of F11.1 (Ki = 3.83 μm) and F11.2 (Ki = 297 μm), which have reverse side-chain sequences, showed the importance of the orientation of terphenyl for binding. The terphenyl compounds F11.1 and F11.3 with 2′,6′-dimethyl substituents showed improved affinities, which may be due to the increased rigidity of the terphenyl backbone and the less entropic loss on binding. Fluorescence experiments suggested that terphenyl compounds bind to MDM2, where p53 binds to MDM2. This was further confirmed using [15N,1H]-HSQC spectroscopy,118 which showed that terphenyl F11.4 targets the same surface area in MDM2 where p53 binds. Terphenyl F11.4 was thus discovered as an α-helical mimetic of p53 having a Ki value of 182 nM. Terphenyl F11.4 disrupts the Bcl-2/Bak binding with a Ki value of 15 μM and Bcl-XL/Bak with a Ki value of 2.5 μM, showing an 82- and 14-fold specificity, respectively, for MDM2 relative to Bcl-2 and Bcl-XL.

Figure 10. β-Hairpin mimics of α-helical p53 peptide as p53−MDM2 inhibitors (SC, side chain), and X-ray crystal structure of F10.2 bound to MDM2 (PDB code 2AX1).

and peptide F10.2 illustrates that the side chain of 6-Cl-Trp is going deeply into the core of HDM2 and brings the chlorine atom into close contact with the aromatic side chain of F86. Mimetic F10.1 was synthesized on 2-chlorotrityl chloride resin as shown in Scheme 12 and further purified by reverse-phase HPLC. Scheme 12. Synthesis of F10.1

3.4. Nutlins

A breakthrough in the design of nonpeptidic small molecule inhibitors of p53−MDM2 was obtained by Vassilev and coworkers119 by the discovery of small molecules, named nutlins. High-throughput screening of a diverse library of synthetic compounds using surface plasmon resonance technology resulted in a cis-imidazoline class of molecules. These compounds were synthesized as a racemic mixture, and two enantiomers could be separated. rac-F12.1 (Nutiln-1), rac-F12.2 (Nutlin-2), F12.3 (Nutlin 3a), and F12.4 (Nutlin 3b) have IC50 values of 0.26, 0.14, 0.09, and 13.6 μM, respectively. The X-ray crystal structure of human MDM2-Nutlin-2 was obtained at a resolution of 2.3 Ǻ . Examination of the crystal structure showed that the nutlins are mimicking the p53 peptide interactions with MDM2,119 i.e., one bromophenyl group occupying in the Trp pocket and the other bromophenyl group in the Leu pocket and the ethyl ether side chain going into the Phe pocket. NMR

3.3. Terphenyls

Hamilton and co-workers showed that terphenyl derivatives could mimic one face of an α-helical peptide117 by substituting the suitable alkyl or aryl substituents on the three ortho positions of the terphenyl scaffold. These side chains are arranged in an analogous way with i, i+4, and i+7 residues of an α-helix (Figure 11). On the basis of this strategy, the researchers reported that a group of terphenyl-based antagonists mimic the α-helical region of the p53 peptide and disrupt the MDM2−p53 interaction. A fluorescence polarization-based binding assay was used to screen a small library of terphenyl compounds to identify inhibitors of the MDM2−p53 interaction.118 The terphenyl F11.4 with an 4653


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Figure 12. Nutlin analogs as p53−MDM2 inhibitors, and X-ray crystal structure of F12.2 bound to MDM2 (PDB code 1RV1).

studies on the binding mode of rac-Nutlin-3 with MDM2 by the Fry team revealed similar results.120 Examining the effect of MDM2 inhibitors at a cellular level of p53, MDM2, and p21 by treating F12.1 in a dose-dependent manner with cells containing wild-type p53 showed an increase in all three proteins. It was also shown that cells containing mutant p53 under the same conditions showed a high basal level of p53 but no detectable MDM2 and p21. It showed that accumulation of p53 was due to a decreased degradation protein rather than activation of p53 gene.119 Inhibition of the p53− MDM2 interaction increases the cellular level of p21, which plays a crucial role in the CDK-dependent cell-cycle arrest. Cell-cycle analysis of the cells containing overexpressed MDM2 by treating with F12.1 revealed an increase in the G1 and G2/M phase and nearly complete depletion of S phase.119,121 The arrested cells did not show any mitotic index, indicating that G2/M fractions consist of G2 phase cells. It is known that genotoxic drugs activate p53 by phosphorylating serine residues near the MDM2 binding domain. Of these, Ser15 is phosphorylated most frequently.122 Analyzing Ser15 phosphorylation in p53 from cells treated with F12.1 and two genotoxic drugs, doxorubicin and etoposide, revealed that all three compounds induced accumulation of p53, but only doxorubicin and etoposide caused phosphorylation of Ser15. These results suggest that a genotoxic mechanism is unlikely to contribute to activation of p53 by F12.1.119 This shows that possible activation of p53 by nutlins is independent of their effect on MDM2. Upon treatment of cancer cells having a wild-type p53 in the presence of pan-caspase inhibitor Cbz-val-Ala-Asp-OMe it was shown that apoptosis can be reduced, thus confirming the involvement of caspase activation. What is interesting to note is that normal human and mouse diploid fibroblasts with a functioning p53 pathway were far less responsive to nutlins than the transformed cell lines. This selectivity was further translated into an apparent therapeutic margin in a xenograft experiment with the human osteosarcoma cell line SJSA-1 that overexpresses HDM2. It was further shown that F12.3 was well tolerated upon oral administration of 200 mg/kg twice a day for 20 days and achieved the plasma levels above in vitro IC90. A 90% inhibition in tumor growth was seen with mice with well-established tumors in comparison to the vehicle-treated control group, and no significant weight loss and any gross abnormalities were observed. F12.3 (Nutlin-3a) was synthesized from key intermediate βamino nitroalkane 13.3 in five steps, which was obtained from an enantioselective aza−Henry reaction125 between imine 13.1 and the nitro compound 13.2 (Scheme 13).

Scheme 13. Synthesis of Nutlin-3 (F12.3)

3.5. Benzodiazepines

A high-throughput screening of a library of benzodiazepinediones that was designed utilizing directed diversity software was screened by the thermofluormicrocalorimetry technology resulting in a new class of p53−MDM2 inhibitors. An affinitybased high-throughput screening assay using fluorescent dyes led to monitoring protein unfolding as a function of temperature to identify small molecules that bind to MDM2,123 and detection of compounds that bind to MDM2 was measured by the resultant increase in thermal stability. The sensitivity of this assay was further verified by the shift in Tm by adding other peptides that are known to bind to MDM2, with higher affinity peptides, generating significant shifts. Several hits were identified using this method, which were further confirmed by an FP-based peptide displacement binding assay to identify inhibitors of the MDM2−p53 interaction. Using this method Grasberger and co-workers screened 338 000 compounds and identified 1216 hits; out of that 116 originated from benzodiazapinediones.124 One of these hits, a racemic benzodiazepinedione methyl ester (F13.1), is shown in Figure 13. Further optimization studies revealed the importance of stereochemistry.123 This provided the benzodiazepinedione carboxylic acid compounds, such as F13.2 and F13.3, with low to submicromolar potency in a conventional p53−HDM2 fluorescence polarization assay. In this competitive binding assay a p53-derived 9mer peptide [N-terminal fluorescein-RFMDYWEGL101] and an HDM2 fragment (residues 17−125) were used. Like other p53−HDM2 inhibitors, the benzodiazepinediones are highly lipophilic (e.g., cLogP for F13.3 is 5.9). Hit compounds such as F13.1 contain carboxylic acid esters. This group is not directly involved in binding (see Figure 13); the solubility could be improved by hydrolysis of the synthesis precursor esters. 4654


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Figure 13. Benzodiazapine analogs as p53−MDM2 inhibitors, and X-ray crystal structure of F13.4 bound to MDM2 (PDB code 1T4E).

Scheme 14. Synthesis of 1,4-Benzodiazepine-2,5-diones

Figure 14. Spiroxindole analogs as p53−MDM2 inhibitors, and X-ray crystal structure of F14.3 analog bound to MDM2 (PDB code 3LBL).

Scheme 15. Structure-Based Strategy To Design a New Class of MDM2 inhibitors

4655


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Scheme 16. Synthesis of Spiro-oxindoles

The X-ray crystal structure between the p53-derived peptide and MDM2 showed that a fourth residue, Leu 22, seems to play an important role in the overall interaction between p53 and MDM2,98 a suggestion that finds support in results from mutation analysis127 and alanine scanning of p53 peptides.100 Accordingly, Wang and co-workers designed new analogs to capture the additional interaction between Leu 22 in p53 and MDM2. Among these new compounds, compound F14.3 (MI63)126c binds to MDM2 with a Ki value of 3 nM. MI-63 lacked the desirable pharmacological properties in mice and was unsuitable for in vivo evaluations. MI-63 was thus further optimized to obtain new analogs such as F14.4 (MI-219).126b MI-219 mimics all four key residues in p53 and binds to MDM2 with a Ki value of 5 nM, whereas Nutlin 3 has a Ki value of 36 nM.126b MI-219 has highly desirable pharmacological properties, such as 55% oral bioavailability in mice.126b MI-219 was greater than 10 000-fold selective for MDM2 compared to MDMX. In addition, both MI63 and MI-219 showed greater than 10 000-fold specificity for MDM2 relative to Bcl-2 and Bcl-xL.126b,c Consistent with the high binding affinity for MDM2 and disruption of the MDM2− p53 complex, MI-63 and MI-219 induced accumulation of p53 in cancer cells with wild-type p53.126b,c The compounds inhibited growth of cancer cell lines expressing wild-type p53 with submicromolar IC50 values and showed a 20−50-fold weaker activity in cancer cell lines lacking wild-type p53, indicating a critical role of p53 in the cellular activity of the inhibitors.126b,c Spiro-oxindoles MI-63 and MI-219 are the first examples of nonpeptidic MDM2 small molecules that are more potent and selective than F12.3. Compound F14.3 was synthesized from 16.4, which was obtained from asymmetric 1,3-dipolar reaction of 16.1, 16.2, and 16.3 (Scheme 16).

The compounds were synthesized using the Ugi fourcomponent condensation reaction (Scheme 14) between an aldehyde 14.1, anthranilic acid 14.2, 1-isocyanocyclohexene 14.3, and an amino acid ester 14.4 followed by acid-catalyzed cyclization.125 Most of the active compounds appear to possess an iodo group at C-7. Since aryl iodides can undergo facile in vivo deiodination, the alternative substituents are desirable. This pathway seems to be possible, and the isopropyl analog F13.5 was only marginally less potent than the corresponding iodo compound F13.4. 3.6. Spiro-oxindoles

Wang and co-workers designed nonpeptidic small molecule spiro-oxindoles as MDM2 inhibitors using a de novo structurebased design strategy (Figure 14).126 X-ray crystal structure analysis showed that four residues (Phe19, Leu26, Trp23, Leu22) present on the p53 peptide were very crucial for a tight interaction with MDM2 and a small but deep hydrophobic cleft in MDM2.98 Since the indole ring of the Trp23 residue of p53 is buried deeply inside the hydrophobic cavity in MDM2 and its NH group forms a hydrogen bond with the backbone carbonyl in MDM2, Trp23 look to be the most critical for binding of p53 to MDM2. Thus, they looked for chemical entities which can mimic Trp23 interaction with MDM2 and found that oxindole can perfectly mimic the side chain of Trp23 for interaction with MDM2. Initially they tried to identify natural products that contain oxindole substructure using a substructure technique, because many anticancer drugs are natural products. This search resulted in a number of natural alkaloids such as spirotryprostatin A and alstonisine, both of which contain a spiro-oxindole substructure.126a Computational modeling studies suggested that although these compounds fit poorly into the MDM2 cleft due to steric hindrance (Scheme 15) the spiro-oxindole-3,3′-pyrrolidine core structure may be used as the starting point for design of a new class of MDM2 inhibitors. The oxindole could mimic the Trp23 side chain in p53 in both hydrogen-bonding formation and hydrophobic interactions with MDM2, and the spiropyrrolidine ring provides a rigid scaffold from which two hydrophobic groups can be projected to mimic the side chain of Phe19 and Leu26. Ding et al. designed candidate compounds using different R1, R2, and R3 groups with different configurations and docked them into the MDM2 binding cleft using the GOLD program. Docking studies predicted that compound F14.1 binds to MDM2 with good affinity by virtue of its ability to mimic p53.126a An FP-based binding assay showed that F14.1 binds to MDM2 with a Ki value of 8.5 μM and provided a starting point for further optimization. Modification of compound F14.1 yielded spiro-oxindole F14.4,126a which binds to MDM2 with a Ki value of 86 nM. Computational docking predicted that compound F14.4 binds to MDM2 by mimicking Phe19, Trp23, and Leu26 residues in p53.

3.7. Chromenotriazolopyrimidines

Allen and co-workers discovered chromenotriazolopyrimidines as MDM2 inhibitors by a high-throughput screening of 1.4 million compound library using a homogeneous time-resolved fluorescence (HTRF)-based technique,128 FRET was quenched in a concentration-dependent manner by chromenotriazolopyrimidine F15.1 (IC50 = 3.88 ± 1.48 μM). Surface plasmon resonance direct binding experiments demonstrated that F15.1 reversibly bound MDM2 (Kd ≈ 11 μM) but not p53, GST, or the chip matrix, indicating that the FRET quenching behavior of F15.1 was specific to MDM2 binding. Subsequent characterization found that F15.1 was a racemic mixture of syn and anti diastereomers [1:1:3:3 F15.1 (a−d)]. Separation of four diastereomers and retesting demonstrated that only the syn(6R,7S) stereoisomer F15.1a was responsible for activity having IC50 = 1.23 (0.82 μM). During this work it was observed that F15.1 was poorly soluble in organic solvents. The predicted absolute stereochemistry of F15.1a was confirmed by cocrystallization with MDM2 as shown in Figure 4656


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Figure 15. Chromenotriazolopyrimidines analogs as p53−MDM2 inhibitors, and X-ray crystal structure of F15.1a bound to MDM2 (PDB code 3JZK).

Scheme 17. Synthesis of Chromenotriazolopyrimidines

Figure 16. Piperidinone analogs as p53−MDM2 inhibitors, and X-ray crystal structure of F16.3 bound to MDM2 (PDB code 4ERE).

1,2,4-triazol-3-amine at 160 °C to give 17.2. Condensation of 4bromobenzaldehyde with 17.2 gives F15.1(a−d), which was then methylated at the N11 position to give F15.2 as a mixture of four isomers (anti/syn).

15. By comparison of known structural data of p53−MDM2,98 it was found that F15.1a and p53 both bound MDM2 using binding interactions. The C7 aryl group of F15.1a was occupying Leu26 of p53 while forming a weak π-stacking interaction with the nearby H96 side chain of MDM2. The C6 aryl group of F15.1a overlapped with the Trp23 region of p53, while the D ring of the chromene group interacted with the binding pocket utilized by p53 Phe19. It was thought that optimizing these interactions would lead to more efficient compound. However, it was found that the C6 position of individual diastereomers of F15.1 was racemizing in DMSO at room temperature over a period of hours. It was envisioned that substituting at N11 would prevent racemization at the C6 position. Interestingly, it was found that N-methylated compounds not only prevented racemization but also increased the solubility without effecting MDM2 potency. Conducting SAR studies on this molecule led to a more potent molecule (6R,7S) isomer F15.2 (6R,7S) which had IC50 = 0.39 μM ± 0.02 μM, by comparison F12.3119 had IC50 = 0.12 ± 0.05 μM in the HTRF assay. Synthesis of chromenotriazolopyimidine F15.2 is shown in Scheme 17. The hydroxyl chalcone 17.1 was condensed with 4H-

3.8. Piperidinones

Structural analysis on known p53−MDM2 inhibitors by Rew and co-workers resulted in several new novel scaffolds, and SAR studies on these scaffolds led identification of 1,3,5,6tetrasubstituted piperidinone F16.1129 (Figure 16), whose IC50 in the HTRF assay was 2.42 μM in serum-free buffer. Changing the cis-diaryl configuration into trans-diaryl, inverting the stereochemical configuration of the C3 acetic acid substituent, and resolving/identifying the active enantiomer resulted in a 50− 70-fold increase in potency (F16.2 vs F16.1). Docking studies of compound F16.2 with MDM2129 revealed that the Leu26 (p53) pocket contains the C5 m-Cl phenyl substituent, with the deepest Trp23 (p53) pocket occupied by the C6 p-Cl phenyl group. The cyclopropylmethyl group occupies the Phe19 (p53) pocket, while the carboxylate anion forms an electrostatic interaction with the H96 imidazole side chain of MDM2 (Figure 16). 4657


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Scheme 18. Synthesis of AM-8553

Figure 17. Indolo-imidazole analogs as p53−MDM2 inhibitors, and X-ray crystal structure of F17.5 bound to MDM2 (PDB code 4DIJ).

A significant potency in both biochemical, i.e., HTRF-based neutralization assay measuring inhibition of the interaction between MDM2 and p53, and cell-based (p21 induction and cell proliferation assays in SJSA-1 tumor cells) assays (Table 1) was shown by compound F16.2. Further, structural optimization of this novel piperidinone series led to F16.4,129 which is a potent and selective inhibitor of MDM2. The ability of F16.4 to inhibit cell proliferation and increase p21 induction of HCT116 p53wt vs HCT116 p53−/− tumor cells further confirmed that the activity of this compound is p53 dependent. Further optimization of F16.4 resulted in the discovery of F16.5 (AM-8553) with an improved potency and pharmacokinetic properties.129 The X-ray crystal structure compound F16.3 bound to human MDM2129 was determined at a resolution of 1.8 Å (Figure 16). This effect was found to be consistent with the proposed binding mode of F16.2 as proposed based on computational binding docking studies. As predicted, the ethyl group occupies the Phe19 (p53) binding pocket and the tert-butyl ester directing group is extending away from the protein with the carbonyl oxygen facing out to the solvent. It was also shown that the tert-butyl group makes van der Waals contact with the surface of the MDM2 protein. The X-ray cocrystal structure of F16.3 further confirmed a gauche-like orientation for the trans C5 and C6 aryl groups when bound to MDM2.129

Synthesis of piperidinone F16.5 is shown in Scheme 18. The chiral scaffold 18.1 was synthesized from phenyl acetic acid in 5 steps as a racemic mixture, which was first converted to a cyclic 6membered lactam and then separated using chiral chromatography to give 18.2a and 18.2b. Sequential methylation and allylation of DMB-protected 18.2b gave 18.3 with the desired epimer as a major product. The primary alcohol 18.4 was obtained from 18.3 in 4 steps. Oxidation of primary alcohol 18.4 followed by treating with Grignard reagent gave secondary alcohol 18.5. The desired product F16.5 was obtained from 18.5 in two steps. 3.9. Indolo-imidazoles

Furet and co-workers synthesized the nonpeptidic inhibitors of p53−MDM2 interaction based on a central valine concept.130 On the basis of the structural information known about the p53− MDM298 interaction they looked for a strategy to design nonpeptide ligands of the MDM2 pocket. A good ligand should fill all three subpockets as best as possible. Thus, one should find a way to connect the three pharmacophore pieces using efficient chemical linkers in such a way that does not increase molecular weight to an unreasonable level while making fruitful interactions with the MDM2 pocket. Through the modeling studies it was shown that the planar aromatic ring is in van der Waals contact with the side chain of Val 93 and provides an appropriate 4658


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Figure 18. (A) Crystal structure of HSP90-SBA1, a closed chaperone complex (PDB code 2CG9). (B) Orthogonal view of HSP90 monomer.

HSP90s are present in bacteria and all eukaryotes. Under normal conditions these proteins are present 1−2% of all cellular proteins.132 Expression of HSP90 is increased up to 10-fold when cells are exposed to physiologic stress including heat, heavy metals, hypoxia, and low pH and various toxins and drugs.133 Heat-shock response is similar in all species, from prokaryotes to eukaryotes, and works through a similar mechanism to maintain intercellular processes.134 HSP90, also known as molecular chaperone family proteins, are present in cytosol, nucleoplasm, endoplasmic reticulum (ER), mitochondria, and chloroplasts.135 Hsp90 is an essential molecular chaperone and involved in the folding process of many client proteins. In eukaryotes, cytoplasmic Hsp90 is absolutely essential for cell survival under all growth conditions. In the functional cycle of the Hsp90 system it interacts with many cochaperones and cofactors. The function of Hsp90 is highly complex; in order to understand that complexity, several groups have attempted to map out the interaction network of this chaperone using yeast and mammalian systems. From these experimental studies it is clearly demonstrated that Hsp90 plays an important role in multiple pathways and cellular processes. The interactions of Hsp90 with client proteins are different from one protein to other. In addition, these interactions are mediated by cochaperones and ATP. The conformation of Hsp90 is different from the conformation of client protein-bound Hsp90.136 The Hsp90 and client protein complex of various client proteins are different from each other and involve different sites on the chaperone.137

substitution pattern to access the three subpockets. On the basis of this central valine a few imidazole compounds were identified; among them, compound F17.2 (IC50 = 3.4 μM) showed a clear signal of activity.130 Docking studies of compound F17.2 (Figure 17) in MDM2 cavity revealed that one hydrogen bond between the NH group of Trp23 and the backbone carbonyl of MDM2 residue Leu 54 was missing. This idea led to the chlorophenyl benzylic group of 17.2 fitting in the Trp 53 subpocket replaced by a 6-chloroindolyl moiety. On the basis of this idea they synthesized compounds F17.3 and F17.4 differing by an aliphatic versus an aromatic group to fill the Leu26 subpocket. In F17.3 the 2-methyl-butyl substituent was designed to exactly mimic the p53 Leu26 side chain, whereas with F17.4 there was more complete occupancy of the space available in this subpocket, and they had IC50 values of 0.9 and 0.2 μM in TR-FRET biochemical assay. To improve the physicochemical properties, the solubilizing moiety in position 2 of the indole ring was introduced, a position facing the solvent in the binding model. Then compound F17.5 was synthesized and tested in the TR-FRET assay showing an IC50 value of 0.03 μM.130 The cocrystal structure of F17.5 with MDM2 was obtained. An explanation of the differences in potency observed between F17.3, F17.4, and F17.5 was given. In the crystal structure the chlorophenyl ring of F17.5 occupying the Leu26 subpocket makes an aromatic stacking interaction with the side chain of the MDM2 residue His 96. This interaction was not anticipated in the model because in the crystal structure of the 15-mer p53 peptide−MDM2 complex on which the model was based the side chain of His 96 adopts a different conformation. The aromatic stacking interaction likely contributes to the higher inhibitory activity of F17.4 compared to F17.3.

4.1. Structure, Conformation, and Functions of HSP

The first crystal structure of the Hsp90 N-terminal domain (NTD) was reported by Prodromou and co-workers in 1996.132 It took nearly another decade to determine the full-length Hsp90 structure.138 A significant difficulty in determining the full-length Hsp90 crystal structure was likely due to its remarkable structural flexibility.139 It resides primarily in the cytoplasm. In its biologically active form it exists as a homodimer. Each homodimer is made up of monomers that consist of three main domains and have important functional interactions. The monomer of Hsp90 consists of a 25 kDa N-terminal ATPase domain, 10 kDa C-terminal domain, and 55 kDa middle domain (Figure 18). The N-terminal domain containing the ATP binding site, which plays an important role in conformational changes of

4. CASE STUDY 3: MODULATION OF HSP90-RELATED PROTEIN−PROTEIN INTERACTIONS BY NATURAL PRODUCTS AND RELATED COMPOUNDS Heat-shock protein 90 (Hsp90) belongs to a family of molecular chaperones, which play a key role in the conformational maturation, stability, and function of client protein substrates within the cell.131 It is a remarkably versatile protein that is involved in various cell functions, particularly when cells are under stress; it then interacts with many client proteins. Through these interactions it either facilitates their stabilization and activation or directs them for the proteosomal degradation to maintain the structural and functional balance within the cell. 4659


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any problem in the folding machinery complex or any disturbance (it competitively blocks the ATP binding site with small molecules) the HSP90 complex undergoes proteosomal degradation.143 Hsp90 is involved in various functions, and it plays an important role in signal transduction, epigenetic regulation, and chromatin remodeling. The perturbation of HSP90 leads to client protein degradation and often cell death. When the cell is under stressful conditions, Hsp90 stabilizes its client proteins and protects the cell from cellular stress, such as in cancer cells. Several onco-proteins act as HSP90 client proteins, and the tumor cells require higher Hsp90 activity than normal cells to maintain their malignancy. Because of these multifacial activities of Hsp90 it has emerged as a promising target for anticancer drug development.131 All these biological studies led to a major current activity in development of inhibitors for treatment of cancer.144

HSP90 homodimer, is essential for molecular chaperoning activity.140 The middle domain is important for client protein interactions. It shows a high affinity for cochaperones and other client proteins, and it differentiates between different substrate types.141 The C-terminal domain plays a crucial role in homodimer formation, and this was further confirmed by electron microscopy and antibody binding studies. It also contains one ATP binding site; however, its role is not clear to date.142 There are various models to explain the mechanism of HSP90 ATPase activity, client protein binding, and subsequent release functions. The N-terminal ATP binding site is responsible for the conformational changes of Hsp90. Once ATP binds to the binding site of NTD, this leads to N-terminal dimerization. This allows the Hsp90 N terminal and the middle domains to undergo a conformational change to form a mature complex. This phenomenon is essential to perform the function(s) of client protein folding and stabilization.138b,141c Once the function of Hsp90 complex is completed, hydrolysis of ATP to ADP occurs which is present at the NTD site, and this facilitates release of the matured proteins (Figure 19). During

4.2. Hsp90 Inhibitors

Several Hsp90 inhibitors (Figure 20) are known over a period of more than two decades, and most of them are in clinical trials. Clinical studies have shown that Hsp90 inhibitors are very potent anticancer agents, exhibit less toxicity, and show few side effects. In some cases, side effects are observed, but these are not directly linked to Hsp90 inhibition.145 Geldanamycin (F20.1) (GA) is one of the early Hsp90 inhibitors discovered in 1994;146 after that numerous inhibitors were identified over the period of two decades. Benzoquinoid anasamycins, such as geldanamycin (F20.1) and herbimycin A (F20.2), are two antibiotics that exhibit antitumor effects. Geldanamycin was isolated from Streptomyces hygroscopicus var. geldanus var. nova in 1970.147 Studies have also shown that the Hsp90 client proteins can be destabilized when geldanamycin binds to the ATP-binding site of Hsp90, and it

Figure 19. Schematic of HSP90 conformational changes.

Figure 20. Natural product inhibitors of HSP90. 4660


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hepatotoxicity.152 These modifications at the C-17 position do not have an effect on formation of Hsp90 and geldanamycin complex. Geldanamycin analogs are also know to form similar complexes with Hsp90 as with the GA.153 17-Allylamino geldanamycin (17-AAG, F21.1) and 17-desmethoxy-17-N,Ndimethylaminoethylamino geldanamycin (17-DMAG, F21.2) are the 17-position derivatives of GA and shown to have lower in vivo toxicity than GA. However, most of them exhibit less affinity toward Hsp90 than the parent natural product, GA. 17-AAG has promising anticancer effects in vitro and in vivo, but it has a low solubility in water. Various research group have developed several other analogs of GA by simply changing various positions.152,150,153,154 From all these studies the C-11-modified analogs of GA and 17-AAG were identified with a slightly improved cytotoxicity over 17-AAG against several cancer cell lines (Figure 21).155 Herbimycin (F20.2) also acts as geldanamycin. Herbimycin A (HA) was isolated from the fermentation broth of Streptomyces hygroscopicus strain AM-3672.156 The structure and absolute configuration of HA was obtained by single-crystal X-ray analysis.157 HA was first synthesized by Tatsuta and co-workers in 1991 by a strategy that established three of the seven stereocenters (i.e., C11, C12, and C14) from a D-mannose derivative.158 The Panek team also reported the synthesis of HA.159 A retrosynthesis by this team involved a disconnection at the lactam to obtain the seco acid 21.1 (Scheme 21). Further simplification led to the disconnection of the C4−C5 (Z)-olefin and the C5−C21 aromatic fragment, from which the (E, Z)dienoate could be established by a series of Wittig reactions. The C6−C7 syn-diol was introduced utilizing Brown’s asymmetric allylboration method, and a C-12 stereocenter was generated by a diastereoselective hydroboration approach. Further, a pair of synmethoxy-methyl stereocenters at C10−C11 and C14−C15 was introduced by Panek’s organosilane methodology (Scheme 22).159 Cossy and co-workers achieved the synthesis from a commercially available Roche ester using allylmetals to control the stereogenic centers at C6, C7, C10, C11, and C12 and a ringclosing metathesis approach to obtain the (Z)-double bond of the (E,Z)-dienic moiety (Scheme 22).160 Another group of Hsp90 inhibitors is radicicol (F20.3) and pochonins (F20.5−F20.10). Radicicol (F20.3) is the most potent natural product inhibitor and has an IC50 of 23 nM.154a It causes cell-cycle arrest in the G1 and G2 phase and further inhibits angiogenesis in vivo.161 From crystal structure analysis it is shown that it binds to the ATP binding pocket which is present on the N-terminal domain (Figure 22A).154a When compared to geldanamycin, radicicol shows 50-fold greater affinity toward the binding site of Hsp90 protein. This leads to destabilization of client proteins.162 There is no structural similarity between radicicol and the ATP, but it is known to bind to the ATP binding site of Hsp90 in a competitive manner.154a Radicicol (monorden) and monocillin I (F20.4) are resorcyclic macrolides, and both of them are isolated from Monocillium nordinii. Radicicol (monorden) was first isolated in 1953 from M. nordinii.163 It is independently isolated from Nectria radicicola.164 The absolute stereochemistry of the three chiral centers of radicicol was assigned by X-ray crystallography. It is known to show some other properties like sedative activity and antibiotic activity164a and also to be a tyrosine kinase inhibitor.165 Like radicicol, monocillin 1 also acts as a HSP90 inhibitor. Because of these biological activities, radicicol became a very interesting target for the synthetic community. The first total synthesis was initially developed by the Lett research

inhibits the chaperone activity of the protein. GA competitively binds to the N-terminal ATP binding site of HSP90, and this then prevents ATP binding and disrupts ATP-dependent conformational cyclization.148 Geldanamycin (F20.1) is a 19-membered macrocyclic lactam and related to ansamycin antibiotics, such as rifamycins and ansamitocins (structures are not shown). Biosynthesis of this class of compounds involves assembly of 3-amino-5-hydroxybenzoic acid (AHBA) as a starting unit, followed by sequential addition of the extender units such as acetate, propionate, and glycolate to form the polyketide backbone, which then undergoes further a downstream processing.149 Total synthesis of GA was carried out initially by Andrus and co-workers150 and later by other researchers.150,151 Recently, the Panek team reported an enantioselective synthesis that was achieved in 20 linear steps with an overall 2% yield. Synthesis of the 19membered macrocycle was achieved from an acyclic amide 19.1 (Scheme 19) through an intramolecular aryl amidation reaction. Scheme 19. Retrosynthesis of Geldanamycin (F20.1) as Reported by the Panek Team

The (E,Z)-diene was installed by reduction of an enyne from alkynylation of precursors 19.2 and 19.3, which were generated from easily accessible aldehyde 19.5 and a chiral silane reagent 19.4. Organosilane 19.4 has unique features as it establishes the C10−C11 syn stereochemistry while simultaneously creating the C8−C9 (E)-trisubstituted olefin. The synthesis is also highlighted by (i) a regio- and stereoselective hydroboration reaction and a Sc(OTf)3/Et3SiHmediated pyran ring-opening reaction, (ii) an enantioselective crotylation to simultaneously install the C8−C9 (E)- trisubstituted olefin and the C10 and C11 stereocenters, and (iii) a chelation-controlled asymmetric metalated acetylide addition followed by an intramolecular copper(I)-mediated aryl amidation reaction to obtain the 19-membered macrolactam (Scheme 20).151 Due to the high hepatotoxicity and poor solubility shown during preclinical studies in animals there is a growing interest in developing geldanamycin analogs to overcome these hurdles.152 The analogs of geldanamycin with alkylamino groups in place of the methoxy moiety at C-17 are shown to be less reactive toward nucleophiles and have excellent biological activity and reduced 4661


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Scheme 20. Synthesis of Geldanamycin (F20.1)

Figure 21. (A) Crystal structure of HSP90 and GDA Complex (PDB code 1YET). (B) Crystal structure of HSP90 and 17DMAG complex (PDB code 1OSF).

group.166 Later, Danishefsky167 and Winssinger and other research groups also reported their total synthesis efforts.164b The Danishefsky group reported the synthesis of radicicol and monocillin I in 2001. Synthesis was achieved by a highly convergent coupling sequence to obtain three key intermediates (23.1, 23.2, and 23.3, Scheme 23). It was highlighted by application of a ring-closing metathesis to the macrolide formation. The researchers achieved the synthesis in a highly convergent manner but failed to remove the two aryl methyl ethers. Further, many experiments were performed by changing the protecting groups to develop a successful route to obtain both natural products. Synthesis starts with esterification of an appropriately substituted benzoic acid with an optically pure secondary alcohol containing all three stereogenic centers of radicicol. The next

reaction is substitution of benzylic chlorine using a dithiane anion in the presence of a sensitive vinyl epoxide. In the last stage, a stereospecific ring-closing metathesis of a diene with the vinyl epoxide gives the 14-membered lactone containing the cis−trans diene. This route also give monocillin I in a single step from the radicicol by an aromatic chlorination reaction (Scheme 24).167b,168 Radicicol shows potent in vitro antiproliferative activity against a wide variety of human tumor cell lines, but it is inactive when tested against in vivo antitumor models.169 This is because radicicol gets rapidly converted into inactive metabolites due to the electrophilicity of an epoxide ring and α,β,γ,δ-unsaturated carbonyl functionality. To overcome this limitation, the Danishefsky and Winssinger team and a few other groups synthesized several analogs of radicicols having different ring 4662


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key hydrogen-bond network with the conserved water molecules coordinated through Asp93 in the region that typically binds the adenine ring of ATP. The carbamate functional group on GDA provides similar interactions with this portion of the binding pocket. The quinone ring of GDA interacts with Asp54, Lys58, Lys112, and other amino acids, while the epoxide on RDC maintains only one hydrogen bond with Lys58. From this information it was proposed that a compound containing the quinone ring of GDA and the resorcinol ring of RDC would provide a new scaffold for development of Hsp90 inhibitors. A hybrid of radicicol and geldanamycin was named radanamycin ester (Figure 24).171 Radamide (F24.2) is a hybrid molecule that is composed of the natural products radicicol and geldanamycin and prepared through an amide linkage connecting the resorcinol moiety of radicicol to the quinone ring of geldanamycin. This hybrid molecule was tested in HER-2 in MCF-7 breast cancer cells. It inhibits ATPase activity along with degradation of the Hsp90 client protein.172 The resorcinol moiety of RDC binds in the same location as the adenine ring of ADP and thus mimics its functions (the hydrogen-bond donor/acceptor properties). The quinone ring of GDA binds toward the exterior of the pocket and participates in hydrogen-bond interactions with the amino acids. The key interactions observed between the quinone and the ATP binding pocket suggest that binding to this region is critical to GDA’s affinity for Hsp90. The cocrystallization studies suggested that a hybrid molecule composed of RDC’s resorcinol ring and GDA’s quinone may provide a molecule with high affinity for Hsp90. Radanamycin amide (radamide, Figure 24) is such a molecule that connects the resorcinol ring of radicicol to the quinone moiety of geldanamycin through an amide linkage. Molecular modeling and docking experiments supported alignment of these two portions into the appropriate locations within the ATP binding site of Hsp90 (Figure 24).172 A new family of resocyclic macrolides was isolated from the fermentation of Pochonia chlamydosporia and is named pochonins A−F (F20.5−F20.10).173 Pochonin A (F20.5) is closely related to radicicol and was shown to be a 90 nm inhibitor of HSP90. Pochonin C (F20.7) is also closely related to radicicol, and it can be easily converted into radicicol.170c Although

Scheme 21. Retrosynthesis of Herbimycin (F20.2)

sizes and replacement of an epoxide with the cyclopropyl group.164b,165,170 A series of radicicol analogs was synthesized and evaluated in different enzymatic assays. These experiments demonstrated that 13-, 15-, and 16-membered lactones have good inhibitory activity. All these macrolactones are known to bind to yeast Hsp90 N-terminal domain similar to radicicol, suggesting that these molecules adopt such a conformation to fit in the ATP binding site. In many cases, this was further confirmed by cocrystallization studies (Figures 22 and 23). Blagg and co-workers synthesized hybrid molecules of geldanamycin and radicicol. Radester (F24.1) is a hybrid compound composed of radicicol resorcinol ring and geldanamycine quinone through an isopropyl ester. The cytotoxicity of radester and the corresponding hydroquinone was determined in MCF-7 breast cancer cells to be 13.9 and 7.1 μM, respectively. The cocrystal structures of Hsp90 with radicicol and GDA show that the resorcinol ring of RDC and the quinone moiety of GDA bind in opposite orientations. The resorcinol moiety provides a Scheme 22. Synthesis of Herbimycin (F20.2)

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Figure 22. (A) Crystal structure of HSP90 and radicicol (F20.3) complex (PDB code 1BGQ). (B) Crystal structure of HSP 90 and compound F23.5 complex (PDB code 2XD6).

Scheme 23. Retrosynthesis of Radicicol (F20.3)

radicicol is the most potent inhibitor (0.2−0.8 mm), it is also cytotoxic, whereas the closely related pochonin C (F20.7) is less active (6 mm) and not toxic at that concentration and then had the highest selectivity index (cytotoxicity 50/IC50) among the resorcyclides.164b The Winssinger research group studied extensively pochonins and various analogs of pochonines.164b,170c,174 This group developed an expedient stereoselective synthesis of pochonin C and further demonstrated conversion of the product into radicicol. As shown in the disconnection approach to pochonin c in Scheme 25, both possible diastereoisomers of the C5 chloro group can stem from the cis- or trans-epoxide moiety 25.2. Given that the ring-closing metathesis of the conjugated diene can lead to four different products (12- versus 14-membered ring and cis/ trans isomers), it was controlled by keeping the α,β-conjugated

Figure 23. Synthetic analogs of radicicol.

olefin masked as a thioether. This can prevent formation of an undesired 12-membered ring. Synthesis starts with esterification of the dihydroxy toluic acid under Mitsunobu or carbodiimide conditions to obtain an intermediate 26.5. Deprotonation of the benzylic position of 26.5 with LDA at −78 °C and further quenching with the Weinreb amide 25.3 yields the cyclization precursor 26.6 (Scheme 26).

Scheme 24. Synthesis of Radicicol (F20.3)

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Figure 25. Novobiocin analog.

further induces degradation of Hsp90 client proteins. Structural modification of this compound led to analogs with 1000-fold greater efficacy in antiproliferative assays against various cancer cell lines.175e Various research groups reported the synthesis of novobiocin analogs in an attempt to improve its poor Hsp90 inhibitory activity.176 Novobiocin is composed of three distinct parts, and further, analogs can be made by their modification. These include (i) the benzamide side chain, (ii) the coumarin core, and (iii) the noviose sugar. The role of each part and the structure−activity relationships of novobiocin have been studied very well. A library of novobiocin analogs published in 2005 reported that F25.1, with a shortened N-acyl side chain, an absent 4-hydroxy substituent, and a missing carbamoyl group on the noviose appendage, induced degradation of Hsp90-dependent client proteins at ∼70-fold lower concentration than novobiocin.177 This study demonstrated that attachment of the noviose appendage to the 7 position and an amide linker at the 3 position of the coumarin ring are essential for Hsp90 inhibition.177 (−)-Gambogic acid (F20.12) is a natural product isolated from the resin of Garcinia hurburyi tree in Southeast Asia. The structure of gambogic acid was elucidated mainly by detailed NMR spectrum analysis and further confirmed by X-ray crystallographic analysis.178 It was identified as a potential Hsp90 inhibitor. Subsequent testing established that gambogic acid inhibited the cell proliferation thus brought about

Figure 24. Hybrid molecules of GDA and radicicol.

Scheme 25. Retrosynthesis of Pochonin C (F20.7)

In addition, a few more natural products are known to inhibit the HSP90, and these are novobiocin (F20.11), (−)-epigallocatechin-3-gallate [(−)-EGCG], Celastrol, Gedunin, and Derrubone, Figure 25. Very recently, it was reported that gambogic acid (F20.12) also inhibits the activity of HSP90. The coumarin antibiotics novobiocin (F20.11), chlorobiocin, and coumermycin A1 were isolated from several Streptomyces strains, and all of them exhibit potent activity against Grampositive bacteria. These compounds bind to type II topoisomerases, including DNA gyrase, and thus inhibit enzymecatalyzed hydrolysis of ATP.175 In addition, novobiocin was reported to bind weakly to the newly discovered Hsp90 Cterminal nucleotide-binding site (∼700 μM in SKBr3 cells) and Scheme 26. Synthesis of Pochonin C (F20.7)

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Figure 26. IAPs in humans.

of host cells; the inhibitor of the apoptosis (IAP) family of proteins is well known for its antiapoptotic function(s).182 IAPs are present at high levels in many types of tumors; by inhibiting the enzymatic activity of caspases they suppress cell death.186 Caspases (cysteine-dependent aspartate-directed proteases) are a family of cysteine proteases that play pivotal roles in apoptosis. Inhibition of caspases is performed by the zinc-binding BIR (baculovirus IAP repeats) domains of the IAP proteins.183 All members of the IAP protein family contain 1−3 BIR domains. BIRs were identified originally as a sequence of approximately 70 amino acids. Different BIR domains may exhibit distinct functions. The second BIR domain of XIAP (XIAP-BIR2) potentially inhibits caspase 3, whereas the third BIR domain of XIAP (XIAP-BIR3) primarily targets the active caspase 9.184 Shown in Figure 26 are eight mammalian IAPs, and these are (i) neuronal apoptosis inhibitory protein (BIRC1), (ii) cellular IAP1 (c-IAP1 or BIRC2), (iii) cellular IAP2 (c-IAP2 or BIRC3), (iv) X chromosome-linked IAP (XIAP or BIRC4), (v) survivin (BIRC5), (vi) ubiquitin-conjugating BIR domain enzyme apollon (BIRC6), (vii) melanoma IAP (ML-IAP or BIRC7), and (viii) IAP-like protein 2 (ILP2 or BIRC8).185

degradation of Hsp90 client proteins in cultured cells. Gambogic acid also disrupted the interaction of Hsp90, Hsp70, and Cdc37 with the heme-regulated eIF2R kinase (HRI, an Hsp90dependent client) and blocked maturation of HRI in vitro. Surface plasmon resonance spectroscopy indicated that it is bound to the N-terminal domain of Hsp90 with a low micromolar Kd in a manner that was not competitive with the Hsp90 inhibitor geldanamycin. Molecular docking experiments supported further that gambogic acid binds to Hsp90 at a site distinct from Hsp90’s ATP binding pocket. The data obtained further established gambogic acid as a novel Hsp90 inhibitor, and it also provided evidence of a new site that can be targeted for development of improved Hsp90 inhibitors.179 Sansalvamide A-amide (San A-amide) (F20.13), which was isolated from a marine fungus, Fusarium spp, exhibits antitumor activity against multiple cancer cell lines. It influences a subset of cancer-related pathways involving heat-shock protein 90 (Hsp90). San A-amide is an allosteric inhibitor of Hsp90, and it binds at the N-middle domain of Hsp90. It disrupts the Cterminal interacting proteins without disturbing the N-terminal domain client proteins. Because of this specific character, San Aamide became a very good target in the development of cancer therapeutics.180 San A-amide is over 100-fold more potent at inhibiting C-terminal client proteins than novobiocin. The interactions of San A-amide, Hsp90 cochaperone, and Hsp90client protein are in in vivo studies.180 The complexity of the bioactive natural products and their instability in the biological systems promoted a widespread search for synthetic inhibitors of Hsp90. To overcome these problems, various synthetic groups reported several novel Hsp90 inhibitors that were identified through high-throughput screening.181

5.1. Overview of Functions of IAPs

cIAP-1 and cIAP-2 inhibit TNF (tumor necrosis factor)mediated apoptosis,187 and X-chromosome-linked IAP (XIAP) is a potent inhibitor of caspases that plays a critical role in resistance to chemotherapeutic agents and other pro-apoptotic stimuli.188 XIAP, cIAP-1, and cIAP-2 have three BIR domains. The BIR3 domain of XIAP binds to an initiator caspase 9, whereas the BIR2 domain of XIAP mediates inhibition of an effector caspase 3 and caspase 7. The BIR2 domain of XIAP and the linker residues N-terminal to XIAP-BIR2 contribute to an inhibition of effector caspases and further blocks both the intrinsic and the extrinsic apoptotic pathways.189 The BIR3 domain of cIAP-1 and cIAP-2 can also bind to caspase 9, but they do not inhibit the activity of this caspase. However, cIAP-1 and cIAP-2 bind to SMAC with high affinities and prevent it from blocking XIAP-mediated inhibition of caspases.190 A melanoma inhibitor of apoptosis (ML-IAP) is up-regulated in a number of melanomas but not expressed in most normal adult tissues,191 and down regulation of ML-IAP by RNA interference leads to induction of apoptosis in tumor cells.192 At present, IAPs follows mainly two pathways for initiation of caspase activation (Figure 27): (a) using the tumor necrosis factor (TNF) family of death receptors, i.e., the death receptor

5. CASE STUDY 4: PROTEIN−PROTEIN INTERACTIONS CENTERED ON THE INHIBITORS OF APOPTOSIS PROTEINS (IAPS) AND SYNTHETIC SMALL MOLECULES Apoptosis, also commonly known as programmed cell death (PCD), is a key regulator of the physiological growth control and regulation of homeostasis in tissues. In cancer cells, various signaling pathways leading to normal cell death are often hampered (or blocked) by an abnormal expression of several antiapoptotic agents. Identified as viral products initially, they were used by baculoviruses for inhibiting the defensive apoptosis 4666


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which in turn then activates procaspase 9. Activation of procaspase 9 cleaves procaspase 3 for its activation.198 The role of the SMAC is to promote caspase activation and apoptosis by inhibiting several IAP proteins, like XIAP, c-IAP1, and c-IAP2, via alanine-valine-proline-isolucine (A1-V2-P3-I4), a highly conserved amino acid sequence which presents on its amino terminal. 5.4. Converging Points and Cross-Talk

Both intrinsic and extrinsic pathways finally converge at the activation of downstream effector caspases such as caspase 3. It is known that the activated caspase 3 leads to further cleavage of several crucial intracellular proteins, and this then causes cell death. These two pathways that converge at some stage allow the cooperation to enhance apoptosis through BID (BH3interacting domain death agonist), which is a member of the BH3-only family of pro-apoptotic proteins and is cleaved and activated by caspase 8. After cleavage, BID then migrates to the mitochondrial membrane. This further causes permeabilization of the mitochondrial membrane for the cytochrome c release and initiation of the intrinsic pathway of caspase activation.199 In this section we are going to discuss the chemical biology of XIAPs and other related proteins and how they are inhibited using nonpeptidic and peptidic (SMAC mimetics) small molecules. The X-linked inhibitor of apoptosis protein (XIAP) is a prototypical member of IAP proteins and a central regulator of apoptosis. This has been shown to bind and inhibit directly three members of caspases, the two effectors, caspase 3 and caspase 7, and an initiator, caspase 9 (Figure 27). XIAP has three BIR (baculovirus IAP repeat) domains, and the third BIR domain, commonly known as BIR3, selectively targets caspase 9, whereas the BIR2 domain along with the linker preceding BIR2 inhibit both caspase 3 and caspase 7.183a,185a The apoptosissuppressing functions of XIAPs are well known in the literature, and generally, it is found at high concentrations in many human tumor cell lines.200 Due to these reasons, a variety of anticancer drugs face serious resistance that is attributed to XIAPs. Further, it is also known that SMAC/DIABLO are released from mitochondria into the cytosol in response to apoptotic stimuli.201 It targets both the BIR2 and the BIR3 domains in XIAP by forming an elongated dimer. Structural and biological studies have shown that SMAC binds to BIR3 domain in XIAP using its N-terminus four amino acid residues, AVPI202 (Figure 28). Through these interactions it competes with the binding of caspase 9 with the XIAP. X-ray crystallographic studies have shown further that the linker preceding BIR2 in XIAP binds to caspase 3/7. Further, the modeling studies also demonstrate that SMAC protein binds to XIAP BIR2 through its AVPI motif and

Figure 27. Apoptosis: extrinsic and intrinsic pathways.

pathway (extrinsic pathway) and (b) using the mitochondrial pathway with cytochrome c (intrinsic).193 IAPs also influence a third minor pathway in which granzyme B directly activates caspase 3. 5.2. Death Receptor Pathway of Caspase Activation

The death receptor pathway (see Figure 27) for caspase activation is initiated with the TNF family of cytokine receptors (TNFR), which includes DR4 (Trail-R1), TNF-R1 (CD120a), and FAS (CD95). The death receptors are activated by a ligand binding to an extracellular domain of the receptor and then recruit the death domain protein Fadd/Mort-1. This leads to binding of FAS-associated death domain with caspase 8,194 thus initiating the death-inducing signaling complex (DISC). Dimerization of caspase 8 leads to its activation and is then released from the DISC into cytosol.195 At this place it then cleaves and activates the downstream effector caspases.196 5.3. Mitochondrial Pathway of Caspase Activation

The intrinsic pathway for caspase activation is dependent on release of cytochrome c and the second mitochondria-derived activator of caspase (SMAC) also known as DIABLO (direct IAP binding protein with low pI) from the mitochondria. The cytochrome c release is caused by a variety of pro-apoptotic stimuli.197 Following this, it then binds and activates Apaf-1,

Figure 28. Structure of the XIAP BIR3 domain/SMAC peptide complex (PDB ID 1g73). (A) Close-up of the binding site. (B) Surface representation. (C) Alignment of SMAC AVPI with those from the Drosophila proteins Hid, Grim, and Reaper. 4667


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prevents binding of XIAP to caspase 3/7.189c,203 On the basis of this, in the past few years intense research efforts have been devoted to the design and development of nonpeptidic and peptidic (SMAC mimetics) small molecules.

compete with the SMAC peptides for their interactions with XIAP provides good evidence that they are functioning through a different mechanism than the conventional small molecule SMAC mimetics for the BIR3 interactions.

5.5. Nonpeptidic Small Molecules

5.6. Monovalent SMAC Mimetics

5.5.1. TWX Molecules: First Reported Nonpeptide Inhibitor of XIAP (BIR2 domain). TWX molecules, obtained and studied by Schultz and Deveraux research teams204 following the standard solid-phase organic transformations, consist of two aromatic/nonaromatic dinitrogen linkers and two sp2 carbonrich (mostly aromatic) moieties spaced alternatively (Figure 29A). Initially, a library containing 160 000 molecules was built

5.6.1. Conformationally Constrained Monovalent SMAC Mimetics. In 2004, Wang and co-workers designed SMAC AVPI mimetics206 based on the crystal structures of SMAC in complex with XIAP BIR3. The cocrystal structure shows that the hydrophobic side chain of Ile4 in SMAC AVPI peptide 27.1 (Scheme 27) binds to a hydrophobic pocket in XIAP BIR3, and also the carbonyl group of the Ile4 residue did not have any specific interactions with the protein. Scheme 27. Design To Access Conformationally Constrained SMAC Mimetics

Figure 29. (A) Structures of TWX 006 and TWX 024. (B) General structure of polyphenylureas.

The synthetic design of peptidomimetics was taken in such a way that the Ile4 residue was replaced with a benzylamine group, and it was further determined that 27.2 binds to XIAP BIR3 protein twice as potent as SMAC AVPI. The researchers further constructed a two diastereoisomeric six-membered bicyclic lactam rings by cyclizing the isopropyl group of Val2 and the five-membered ring of Pro3 that gave compounds 27.3 and 27.4 (Scheme 27), which mimic the binding conformation of 27.2. Compounds 27.3 and 27.4 have not shown promising binding affinities like compound 27.2 toward mimicking SMAC/XIAP BIR3 interactions. They further modified the six-membered ring to a seven-membered ring by inserting the additional carbon moiety to obtain compound 27.5, which was determined to have a Ki value of 150 nM. Hence, seven-membered bicyclic compound 27.5 was found to be 30 times more potent than compound 27.3. The additional structure-based design and modifications of 27.5 to further improve its interactions with XIAP BIR3 led to compounds 27.6 and 27.7 (Scheme 27). It was further determined that 27.6 and 27.7 have Ki values of 60 and 25 nM, respectively. Chemical synthesis of these compounds is shown in Scheme 28.206a Briefly, the key intermediates 28.5a and 28.6a were prepared from the methyl ester of L-glutamic acid (28.1a).207 Phenylfluorenation of the amine group gives compound 28.2a. Homo-Claisen condensation of 28.2a gives β-keto ester 28.3a, hydrolysis and decarboxylation of β-keto ester 28.3a in NaOH gives 2,8-diamino azelaic acid, followed by esterification gives

in 1536-well format and identified several moderately active molecules. On the basis of the structural characteristics of the successful molecules, a more focused library was attempted using different small fragments attached by two linkers (Figure 29A) that led to finding small molecules that can prevent the XIAP− caspase 3 interactions with IC50 values up to 10 (TWX 006) and 25 μM (TWX 024) (Figure 29A). These compounds bind to XIAP to mask its functions in cancer cells. 5.5.2. Polyphenylureas: Another Class of Nonpeptide Inhibitors of XIAP (BIR2 domain). Reed and co-workers screened a library of nearly one million compounds against the IAP family of protein, XIAP. They identified some nonpeptidic small molecules having polyphenylureas (F29.3) as antagonists of XIAP (Figure 29B).205 The most active compound induces apoptosis of a broad range of tumor and leukemia cells in vitro as single agents while having comparatively little toxicity in normal cells. It was further shown that the polyphenylurea-based compounds overcome the inhibitory effects of XIAP on caspases 3 and 7 but not caspase 9 in vitro. These compounds bind to the BIR2 domain of the XIAP site, which is known to be sufficient for binding and inhibiting caspase 3. The interactions of these compounds differ from the endogenous XIAP antagonist SMAC, which is known to bind through BIR3, and this then leads to caspase 9 suppression. The fact that these compounds do not 4668


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Scheme 28. Synthesis of Conformationally Constrained SMAC Mimetics

28.4a. Indolizidinone methyl esters 28.5a and 28.6a were synthesized using Pd/C in hydrogen condition proceeded by deprotection of phenylfluorenyl groups, intramolecular imine formation, protonation, and hydrogen addition to the iminium ion intermediate. Methyl esters 28.5a and 28.6a were hydrolyzed and followed by condensation with benzylamine to obtain two amides. Removal of the Boc protecting groups in these two amides, condensation with N-tert-butoxycarbonyl- L-alanine, followed by further removal of the Boc protective groups in these two compounds then afforded 27.3 and 27.4. Another class of potent seven-membered bicyclic conformationally constrained SMAC mimetics 27.6 and 27.7206b was prepared from L-pyroglutamic acid. The key intermediates (28.7b and 28.8b) were prepared according to the literature procedure,208 and further, these intermediates were converted into 27.5, 27.6, and 27.7 using an amino acid coupling strategy. 5.6.2. Isostere Design Using [7,5]-Bicyclic Scaffold. The Deshayes team in 2006 reported SMAC mimetics using a [7,5]bicyclic moiety as a central scaffold (Scheme 29).209 The researchers carried out docking studies with different scaffolds at the P2 and P3 positions of SMAC AVPI peptide. It revealed that the 7-membered ring was more flexible and might attain a conformation suitable for binding. They further substituted the proline ring in the SMAC AVPI tetrapeptide with γ-thioproline, which is shown to be not losing any activity. Finally, this was combined with the caprolactam ring and γ-thioproline ring to obtain a new peptidomimetic of the P2 and P3 positions of AVPI tetrapeptide. In the synthesis section the key intermediate 29.5a/b was obtained by condensing compound 29.3a/b with 29.4 (see Scheme 29). 29.3a and 29.3b were then synthesized in 4 steps from cysteine and penicillamine derivatives, respectively. 29.4

Scheme 29. Synthesis of Isosteres

was also synthesized from diphenylmethylene glycine ethyl ester. Ester hydrolysis followed by condensation of 29.5a/b gives a [7,5]-bicyclic lactam. Deprotection of the Boc protecting group in acidic conditions led to the 1:1 diastereomeric mixture, which 4669


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Scheme 30. Synthesis of Azabicyclooctane Scaffold-Based SMAC Mimetics

upon separation gave compounds 29.6−29.9. All of these compounds were evaluated against BIR domains of XIAP, cIAP1, cIAP2, and ML-IAP, and 29.6 showed binding to ML-IAP-BIR and cIAP1-BIR3 domains at 50 nM concentration. 5.6.3. Design and Synthesis of Azabicyclooctane Scaffold-Based SMAC Mimetics. Cohen and co-workers in 2009 designed and synthesized IAP antagonist 30.9 based on the azabicyclooctane scaffold (Scheme 30).210 This compound binds to the XIAP BIR3 domain, the BIR3 domain of c-IAP1, and the BIR domain of ML-IAP with Ki values of 140, 33, and 38 nM, respectively. They are shown to promote degradation of c-IAP1, activate caspases, and lead to decreased viability of breast cancer cells. Synthesis of the azabicyclooctane scaffold was started with amine 30.1 (Scheme 30), which was then converted into the bicyclic amino iodide 30.2 by iodocyclization using I2. This crude mixture was reacted with NaN3, which led to displacement of the iodide by azide to obtain the diastereomeric azides 30.3 and 30.4. They separated both diastereomers by column chromatography, and further hydrogenation under mild conditions provided the primary amines 30.5 and 30.6. Then primary amine 30.6 was protected with Teoc to give 30.7; removal of the benzyl group under Pd(OH)2 condition allowed them to obtain cyclic secondary amine 30.8. This amine was then converted to 30.9 using simple amino acid coupling reactions and, finally, Teoc removal followed by amidation with diphenylacetylchloride. 5.6.4. Some More Monovalent SMAC Mimetics. Vucic and colleagues in 2009 reported monovalent c-IAP-selective antagonists.211 IAP antagonists were initially designed to have broad specificity and target different IAP proteins. However, structural and biological studies revealed the roles of BIR domains of individual IAP proteins; with this information scientists started designing IAP antagonists that are specific for a particular IAP protein or a group of IAP proteins. Such c-IAPselective antagonists induce cancer cell apoptosis, promote degradation of c-IAP1 and c-IAP2, and activate canonical and noncanonical NF-κB signaling pathways. They developed a compound CS3 (F30.1, Figure 30), which binds selectively (>2000-fold) to c-IAP1 BIR3 over XIAP BIR3 with a high affinity (Ki = 16 nM). Although it has good selectivity, CS3 was a much weaker inducer of cell death in short-term and long-term viability assays in comparison with a chemically similar pan-selective IAP antagonist, PS1. Therefore, although the single-agent proapoptotic activity and activation of NF-κB signaling pathways

Figure 30. Monovalent SMAC mimetics CS3, GDC-0152, ML101, and its derivative (F30.2b).

as well as cytokine production rely on the antagonism of c-IAP proteins, both c-IAP and XIAP should be targeted simultaneously for efficient stimulation of cell death. Flygare, Fairbrother, and co-workers in 2012 designed and synthesized a series of compounds and screened against different IAPs.212 From this screening they discovered GDC-0152 (F30.2) (Figure 30), which binds to the BIR3 domain of XIAP with an affinity of 28 nM and further blocks the PPI that involve IAPs. GDC-0152 also binds to the BIR domain of ML-IAP and the BIR3 domains of cIAP1 and cIAP2 with Ki values of 14, 17, and 43 nM, respectively, and promotes cell death by activating caspases. Cosford’s research group in 2011 developed monovalent SMAC mimetics which bind to the BIR2 domain of the antiapoptotic protein XIAP.213 They synthesized different sets of compounds with the modification at Val2 position and replacement of Ile4 with diphenylmethyl amine. Also, they obtained compounds in which Ile4 was replaced with different heterocyclic compounds and further evaluated them for selectivity with BIR2 over BIR3. Finally, they came up with the compound quinolin-5-amine derivative ML101 (F30.3a), which was less potent (BIR2 Ki = 1.91 μM; BIR3 Ki = 12.71 μM) but showed selectivity for BIR2 versus BIR3 (7:1). Further, the indole moiety on nitrogen (C-terminal position) provided a compound F30.3b that was 7-fold selective for BIR2 over BIR3 and retained potency (BIR2 Ki = 0.74 μM). 5.6.5. Diazabicyclic Scaffold-Based Peptide Mimetics. Wang and co-workers in 2011 discovered monovalent, 4670


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conformationally constrained SMAC mimetic containing a diazabicyclic scaffold SM-406/AT-406214 which were found to be orally active IAP antagonist using the structure-based design (Scheme 31). This compound binds to XIAP, cIAP2, and cIAP1 Scheme 31. Design and Synthesis of SM-406/AT-406

Figure 31. Recently developed monovalent SMAC mimetics.

of XIAP. Among all of them F31.1 showed the best potency (Ki = 9.64 μM). It also binds ML-IAP with a binding affinity of 2 nM. Ishikawa and his team designed an octahydropyrrolo[1,2a]pyrazine scaffold (Figure 31B) as a novel proline mimetic to synthesize a novel class of SMAC mimetics (F31.2) with different moieties at R1, R2, R3, and R4. From these compounds they found one compound217 with binding affinities of 1.3 and 200 nM toward cIAP1 and XIAP, respectively. 5.7. Bivalent SMAC Mimetics: Design and Synthesis

SMAC is known to form a homodimer and then promotes apoptosis by direct interactions with XIAP and cIAP1 and cIAP2. It is also well documented that its homodimeric form binds to BIR2 and BIR3 domains of XIAP using two AVPI binding motifs, and this then does not allow XIAP to interact with caspase 9. Xray crystal structures of SMAC/DIABLO with the BIR3 domain of XIAP218 led to design two different types of SMAC mimetics, and these are (i) monovalent SMAC mimetics based on one AVPI tetrapeptide mimetic and (ii) bivalent SMAC mimetics containing two AVPI mimics tethered with a linker. It is also known that the AVPI peptide sequence of SMAC binds to both BIR2 and BIR3 domains, although binding with BIR 3 is relatively stronger compared to BIR2. It was perceived that the bivalent SMAC mimetics having two AVPI binding motifs may mimic the mode of action through targeting both the BIR2 and the BIR3 domains of XIAP, and this may lead to much higher affinities for the interactions. In general, the bivalent agents that can exhibit two-point bindings are considered more stable than single-site affinities. 5.7.1. Unexpected Discovery of First Bivalent SMAC Mimetic. The Harran and Wang team in 2004 reported the first bivalent SMAC mimetic,219 which initiated a new era of making bivalent compounds. They wanted to combine compounds 32.1 and 32.2. In this study, they found an unexpected product in a copper-mediated reaction. Eventually the compound was characterized as C2-symmetric diyne 32.3/32.4, which came from oxidative homodimerization of 32.2 known as a Glaser coupling (Scheme 32). 32.4 and monomer 32.2 showed comparable binding affinity toward XIAP BIR3 domain. When the XIAP full-length protein was used, compounds 32.3/33.4 showed much higher affinity than the SMAC protein with an estimated Kd value of 0.3 nM. 5.7.2. Bivalent Small Molecules Based on Monovalent SMAC Mimetics. The Wang group in 2007 designed of a new class of bivalent small molecules based upon conformationally

proteins with Ki values of 66.4, 5.1, and 1.9 nM, respectively. SM406 effectively inhibits XIAP BIR3 protein in a cell-free functional assay and, moreover, induces degradation of cellular cIAP1 protein. They had shown that SM-406 has all the desirable properties as a drug candidate for clinical development, which is currently in phase Ia clinical trial for treatment of human cancer. Synthesis of intermediate 31.5 was started with L-pyroglutamic acid (31.2) and further converted to 31.3 according to literature procedures215 (see Schemes 31) The primary alcohol was protected with TBS followed by debenzylation to give amine 31.4, and this secondary amine was then coupled with BocDap(Z)-OH to obtain 31.5. This intermediate was further converted to 31.6 by TBS deprotection, Dess−Martin periodinane oxidation, followed by Cbz removal. The key intermediate 31.6 was converted to 31.1 (SM-406/AT-406) in 6 steps by simple amino acid coupling reactions. Since the past decade, many groups have focused on designing new moieties to mimic SMAC interactions with XIAP. In their design strategies, they prepared SMAC mimetics in more than 10 steps; another drawback is the linear synthesis; therefore, lead optimization is a challenge in these cases. Keeping these limitations in mind, the Casford team in 2013 designed a scaffold that could mimic SMAC and was synthesized rapidly using the Ugi four-component reaction (Ugi 4CR)216 (Figure 31A). In this two-step reaction sequence the researchers built six bonds and two stereocenters (with a high degree of stereocontrol). Using this methodology they synthesized a series of compounds with different scaffolds, and further evaluated their inhibitory activity against IAPs. These compounds showed low to moderate levels of inhibitory activity toward the BIR2 domain 4671


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Scheme 32. Unexpected Discovery of the First Bivalent SMAC Mimetic

Scheme 33. (A) Bivalent Derivative with a Linker; (B) Another Bivalent Derivative That Is Linked from a Different Site

constrained monovalent SMAC mimetics.220 Compound 33.1 binds to the BIR3 domain of XIAP protein with an IC50 value of 91 nM, and this binding affinity is 13 times higher than that of the SMAC AVPI tetrapeptide. Also, compound 33.1 inhibits cell growth and induces apoptosis in cancer cells. This is also a good indication that compound 33.1 is a good cell permeable. It was further concluded with these results that compound 33.1 shows desirable qualities for design of bivalent SMAC mimetics to target concurrently the BIR2 and BIR3 domains. To design bivalent mimetics, the researchers identified suitable sites in compound 33.1 for chemically tethering two molecules. The modeling studies further suggested that the pro-(S) phenyl group was suitable as an anchoring site for tethering two molecules of 33.1. It was also suggested that the pro-(S) phenyl group in 33.1 can be replaced by other aromatic rings with comparable binding affinity. To test this idea, compound 33.2 in which the pro-(S) phenyl group is replaced by a substituted [1,2,3]-triazole ring was synthesized using a click approach, and the resulted compound showed a binding affinity with an IC50 value of 57 nM. To obtain further next-generation compounds, SM-164 (33.3), a bivalent SMAC mimetic having a tethering of two monovalent SMAC mimetics (compound 33.2) through a flexible linker, which provides a distance of approximately 15 Å between the two triazole rings, was planned. Compound SM-164 (33.3) concurrently binds to BIR2 and BIR3 domains in the same XIAP protein and achieves an extremely high binding affinity to XIAP. This bivalent SMAC mimetic SM-164 (33.3) is 271 times more potent than the monovalent compound (33.1) and >7000 times more potent than the SMAC AVPI peptide. The Wang research group further developed bivalent nonpeptidic SMAC mimetics221 based on their previous monovalent SMAC mimetic that was having an azabicyclic core (Scheme 33). This team is continuously developing monovalent as well as bivalent mimetics using different linkers having various lengths and hydrophobic groups,220b and one of the designs led to a new bivalent SMAC mimetic (33.4) based upon the core structure of monovalent SMAC mimetic SM 406 (31.1).214 These compounds were evaluated, and it was found that the bivalent SMAC mimetics having a diazabicyclic moiety are highly potent antagonists of XIAP, cIAP1, and cIAP2, efficiently inducing degradation of cIAP1 and cIAP2 in cancer

cells at concentrations as low as 1 nM in MDA-MB-231 and SKOV-3 cancer cells Varfolomeev and Vucic co-workers in 2007222 designed a compound BV6 (F32.1) in which the monomeric form of a SMAC mimetic is tethered by a 7-numbered carbon chain. BV6 binds to the BIR domains of IAPs and leads to apoptosis by proteosomal degradation of cIAPs. The researchers also discovered that cIAP proteins are important regulators of NFkB signaling and the ubiquitin ligases for the crucial kinase in the noncanonical nuclear factor kappa-light-chain-enhance of activated B cells pathway (NFkB), NIK. Further, they also showed that the IAP antagonist-induced cell death is dependent on the TNF signaling. Barker and co-workers in 2008223 discovered that AEG40730 (F32.2), which is a dimeric ATPF mimetic, can activate caspase 8 and then induces cell death through binding to the BIR domain of XIAP. The region of SMAC that binds the XIAP BIR3 domain is an N-terminal site with four residues (AVPI) and is structurally similar to the amino tetrapeptide in caspase 9 (ATPF) (Figure 28c). This was taken as the structural template to develop XIAP antagonists. Extensive studies in this direction led to access several cell-permeable peptides, peptidomimetics, and nonpeptidic small molecules that are based on this sequence and disrupt XIAP−caspase interactions and further promote apoptosis in cancer cells. This led to a novel compound AEG40730 as an IAP antagonist which binds to BIR domains with a nanomolar affinity. Shown in Figure 32 is another class of bivalent SMAC mimetics TL32711 (F32.3), the most potent bivalent IAP antagonist.

6. CASE STUDY 5: BCL-2 PROTEIN FAMILY AND SYNTHETIC SMALL MOLECULES Apoptosis (i.e., programmed cell death) is a fundamental process in organ development, which is characterized by many biological 4672


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Figure 32. Structures of bivalent SMAC mimetics BV6, AEG40730, and TL32711.

Figure 33. Three subfamilies of Bcl-2-related proteins according to the conserved domains.

and morphological changes.224 There are diseases with insufficient apoptosis such as cancer and autoimmune diseases or excessive apoptosis such as Alzheimer’s and Parkinson’s diseases. Apoptosis proceeds via one of the two major pathways, termed extrinsic and intrinsic.225 The extrinsic pathway is triggered by ligation of cell-surface death receptors that drive formation of a death-initiating signaling complex (DISC). Activation of the multidomain pro-apoptotic Bcl-2 (B-cell lymphocyte/leukemia-2) family proteins Bax or Bak by a subgroup of Bcl-2 homology (BH)-3 only family members play an important role in the intrinsic pathway (Figure 33). The Bcl-2 family of proteins plays a critical intracellular checkpoint in the intrinsic pathway of apoptosis. The bcl-2 gene was first isolated as a proto-oncogene at the breakpoint of a t(14,18) chromosomal translocation associated with follicular Bcell lymphoma.226 Expression of Bcl-2 proved not to promote cell proliferation, like other oncogenes, and instead blocks cell death following multiple physiological and pathological stimuli.227,228 These proteins are localized to the mitochondria, perinuclear membrane, and smooth endoplasmic reticulum.229,230 The plasma membrane blebbing, nuclear condensation, volume contraction, and endonucleolytic cleavage of DNA termed apoptosis231 was blocked by Bcl-2, which unexpectedly localized to the mitochondrion, nominating this intracellular organelle for a prominent role in apoptosis.230 Currently, more than 20 Bcl-2 family members have been identified, and they have either anti- or pro-apoptotic functions.232 Bcl-2 proteins are characterized by the presence of up to four relatively short sequence motifs, which are 280). The dominant component of serum deactivation was identified as domain III of human serum albumin (HSA).296 The NMR solution structures of the inhibitors bound to both Bcl-XL and HSA domain III indicated two potential optimization sites for separation of affinities. Modifications at both sites resulted in compounds with improved Bcl-XL binding and further greatly increased activity in the presence of human serum, A-385358. F39.1 binds to Bcl-XL with a Ki of 0.8 nM and Bcl-2 with a Ki of 67 nm. In a cellular assay compound 2 reversed the protection afforded by Bcl-XL overexpression against cytokine deprivation in FL5.12 cells with an EC50 of 0.47 μM and a little effect on the viability of the human nonsmall cell lung cancer cell line A549.297,298 A-385358 shows moderate single-agent activity recorded on tumor cell lines, cytotoxicity compromised in the presence of serum, and synergizes with multiple cytotoxic agents in vitro and with paclitaxel in vivo.298 Further studies led to the discovery of ABT737 (F39.2), a molecule with a high affinity for Bcl-2 and Bcl-XL (IC50 values of

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