Industrial natural product chemistry for drug discovery and development

Contents

• 1 Introduction

• 2 Optimization of natural products by total synthesis
  • 2.1 Inhibitors of sodium–glucose co-transporters
  • 2.2 Fingolimod
  • 2.3 Eribulin
  • 2.4 Tetracyclines

• 3 Optimization of natural products by structural modifications
  • 3.1 Glycopeptides
  • 3.2 Echinocandins
  • 3.3 GLP-1 agonists
  • 3.4 Rapamycin and rapalogs

• 4 Immunoconjugation of natural products

• 5 Synthetic supply of unmodified natural products
  • 5.1 Ziconotide
  • 5.2 Trabectedin
  • 5.3 Artemisinin

• 6 Concluding remarks

• References

---

Abstract

Covering: up to March 2013

In addition to their prominent role in basic biological and chemical research, natural products are a rich source of commercial products for the pharmaceutical and other industries. Industrial natural product chemistry is of fundamental importance for successful product development, as the vast majority (ca. 80%) of commercial drugs derived from natural products require synthetic efforts, either to enable economical access to bulk material, and/or to optimize drug properties through structural modifications. This review aims to illustrate issues on the pathway from lead to product, and how they have been successfully addressed by modern natural product chemistry. It is focused on natural products of current relevance that are, or are intended to be, used as pharmaceuticals.

---

Armin Bauer

Armin Bauer studied chemistry at the Technical University of Braunschweig, Germany, and at the University of Bordeaux III, France. He obtained his PhD in organic chemistry in 1999 under the supervision of Prof. D. Schinzer, contributing to the total synthesis of the epothilones. Since 1999 he has been working for Sanofi and predecessor organizations starting as a laboratory head in medicinal chemistry. From 2006 to 2010 he was responsible for the isolation group within the R&D Natural Products Science section, leading discovery projects with a focus on downstream processing and natural products derivatization and total synthesis. Currently he is heading a medicinal chemistry section within Sanofi R&D Lead Generation Chemistry. He has been working in the therapeutic areas of metabolic disorders, thrombosis, osteoarthritis and infectious diseases.

Mark Brönstrup

Mark Brönstrup received a Diploma in Chemistry (supervisor: R.W. Hoffmann) and obtained his Ph.D. in organic chemistry from H. Schwarz at the TU Berlin in 1999. He joined Aventis in 2000 as a lab head for mass spectrometry and spent a research sabbatical on phosphoproteomics with S.P. Gygi at Harvard Medical School in 2003. Between 2005 and 2010, he was leading the Natural Products Science section at Sanofi Aventis in Frankfurt, Germany, which was responsible for drug discovery from natural sources. Between 2010 and 2013, he dealt with translational research as head of sections for Biomarkers & Diagnostics in Diabetes and for Biomarkers, Bioimaging & Biological Assays. At the end of 2013, he became head of the Chemical Biology Department at the Helmholtz Centre for Infection Research in Braunschweig, Germany, coupled with a W3 professorship at the Leibniz University of Hannover.

---

1 Introduction

Natural products represent an excellent source for drug discovery.¹ The careful study of their biological activities and the underlying molecular mechanisms has led to the identification of unprecedented modes of action, pathways and targets that turned out to be of high relevance for the treatment of human diseases in numerous cases.^2,3,4 Furthermore, natural products can either be directly used as drugs to treat human disease, or at least serve as a valuable starting point (‘lead’) for drug discovery programs.^1,5 Among the multiple challenges that have to be mastered along the path from a lead to a viable drug, two require specific attention for natural products with their often complex structures:

(i) How can structural variations be performed in a time- and cost-efficient manner to modulate and enhance their biological activities and to optimize pharmacodynamic, pharmacokinetic and safety (ADMET‡) properties of the lead structure?

(ii) How can a large-scale supply of drug substance in GMP (Good Manufacturing Practices)-quality be realized?

In the current review, we would like to present recent highlights from natural product research and development that illustrate how the lead optimization and/or compound supply challenge has been successfully solved through modern natural product chemistry. For a comprehensive overview on natural products in clinical development, we would like to refer to recent review articles.^6,7

To tackle the above-mentioned challenges, a growing arsenal of methods has been developed in organic chemistry, biochemistry, molecular biology, molecular genetics, and combinations thereof. Innovations in synthetic methodology have always been closely mixed with the total synthesis of natural products; they induced several paradigm shifts in natural product chemistry, as outlined in a recent review.⁸ In fact, an effective synthetic access to a natural product lead continues to be the critical determining factor for the successful accomplishment of medicinal chemistry optimization programs.⁹

Understanding and exploiting the biosynthesis pathways of natural products has an increasing impact on accelerating their discovery and development process.¹⁰ Turn-around times for strain improvement may be considerably reduced by rational approaches based on knowledge of the genetic regulation of secondary metabolite production and of quantitative fluxes of precursors. Progress in genetic engineering techniques like precursor-directed biosynthesis, combinatorial biosynthesis and mutasynthesis approaches^11,12,13,14 are the foundations of “biosynthetic medicinal chemistry”,^15,16 opening up a structural space that is often orthogonal to classical natural product derivatisation or total synthesis approaches.

The extended range of options has led to the intriguing situation that there is no single recipe for success. Instead, we observe revisions of long-prevailing standards for product realization:

– Plant metabolites have been provided as drug substances through horticulture, followed by extraction and purification processes for centuries, with various inherent drawbacks. The example of artemisinin production (chapter 5.3) demonstrates that heterologous expression in genetically engineered standard hosts, followed by semisynthesis, has now become a low-cost, high quality alternative.

– The multistep total synthesis of polyketides or peptides, mostly seen as a purely academic discipline, has become a viable option for industrial production, in particular for drugs with treatment regimens that are limited in time or require low dosages. This is exemplified by eribulin, the GLP-1 analogs and ziconotide (chapters 2.3, 3.3, and 5.1, respectively).

– For several generations, fermentation/semisynthesis was the only practical method to overcome bacterial resistance against the ‘mature’ tetracycline class of antibiotics (chapter 2.4). A recently developed, highly convergent total synthesis enabled a broad structure–activity-relationship (SAR) study of structural parts that have not been amenable to semisynthesis, and led to the identification of a novel candidate that is profiled in Phase II clinical trials.

– The rapamycin scaffold (chapter 3.4) exhibits such unique biological properties that improved, next generation congeners were generated through subtle structural modifications by semisynthesis or genetic engineering. In stark contrast, the optimization of sodium–glucose co-transporters inhibitors (chapter 2.1) or an S1P1 antagonist (chapter 2.2) through medicinal chemistry led to pronounced structural changes, rendering the original natural product lead behind the final compounds being hardly discernible.

– Drug researchers traditionally had to decide whether to address a given biological target either with a biological macromolecule (e.g. a monoclonal antibody, or an oligonucleotide) or a small molecule (e.g. a natural product or a synthetic compound). Recently, it has been proven in a clinical setting that both formats can be combined in a single molecule, a so-called immunoconjugate (chapter 4), through the covalent linkage of natural product effectors to a monoclonal antibody, while retaining key advantages of each format. This approach will be illustrated by ado-trastuzumab emtansine and brentuximab vedotin.

– Is there still space for the classical semisynthetic modification of natural product scaffolds obtained by fermentation to discover drugs? Definitely yes. In most cases, the approach remains the most efficient to explore an SAR in the short timescales of drug discovery, and it is also the most cost-effective, sustainable and reliable supply of complex natural product scaffolds for drug development and production.¹⁷ Its versatility is demonstrated by the successful development of next generation glycopeptides, echinocandins, and rapamycins (chapters 3.1, 3.2 and 3.4, respectively).

Thus, today's natural product chemists have a rich and growing arsenal of methods at hand. In the following chapters, successful applications of this arsenal to optimize natural products by total synthesis (chapter 2), semisynthesis (chapter 3), and immunoconjugation (chapter 4), or to produce them in unmodified forms (chapter 5) will be presented.

2 Optimization of natural products by total synthesis

2.1 Inhibitors of sodium–glucose co-transporters

Sodium–glucose co-transporters (SGLTs) play a key role in the regulation of glucose homeostasis in the body. The physiologically most important representatives of this transporter family are SGLT1 and SGLT2. SGLT1 is expressed in a wider range of tissues, with the highest levels in the small intestine, skeletal muscle and heart, and its main known function is the absorption of galactose and glucose from the intestine. In contrast, SGLT2 is almost exclusively present in the kidney, mediating reabsorption of glucose back into the plasma from the glomerular filtrate.¹⁸ In patients with diabetes this process contributes to hyperglycemia, since the disease is associated with an increased capacity for reabsorption of glucose.¹⁹ Lowering glucose levels by selectively blocking SGLT2 and thus inducing glucosuria was therefore recognized as a valuable therapeutic concept for the management of type 2 diabetes.²⁰

The history of the development of SGLT inhibitors began with phlorizin 1, a dihydrochalcone glycoside that was first isolated in 1835 from the root bark of the apple tree.²¹1 played a key role in (i) uncovering the function of renal glucose re-absorption, (ii) the discovery of the SGLTs and (iii) as a structural starting point for lead optimization.

In the late 19th century, it was observed that 1 induced glucosuria after oral administration, and it was soon recognized that diabetes mellitus symptoms like persistent excretion of glucose and polyuria were mimicked by chronic administration of this compound to dogs.²² As early as 1899, 1 was used for the first time experimentally in a diabetes patient to lower serum glucose levels.^18,23

Subsequently, 1 served as a valuable tool compound for a better understanding of kidney function²⁴ and for uncovering the link between hyperglycemia and insulin resistance.²⁵ Studies on the mechanism of action on the cellular level started in the 1950s. They revealed that 1 blocked sugar transport in both the small intestine and the kidney,²⁶ and finally led to the identification of the sodium–glucose transporters (SGLTs) responsible for the renal re-absorption of glucose.²⁷ The two isoforms SGLT1 and SGLT2 have been cloned from several species,²⁸ and 1 turned out to be a non-selective inhibitor of both human isoforms.²⁹ Since blocking of SGLT1 was thought to be associated with side effects, in particular through affecting gastrointestinal function, selective inhibition of SGLT2 was considered as an important prerequisite for the development of a safe drug. Further issues of 1 included a low metabolic stability due to rapid hydrolysis by glucosidases, resulting in poor pharmacokinetic profiles (in particular, low oral bioavailability and short plasma half lives), and toxic effects associated with the liberated aglycon phloretin 2.

First reports on improved SGLT2 inhibitors based on 1 were reported in the late 1990s by researchers at Tanabe.³⁰ As a result, the O-glycoside analog T-1095 3 emerged as one of the first clinical development candidates.³¹ The toxicity problems were addressed by removing or modifying hydroxy groups in the aglycon part based on earlier reports on structure–activity relationships of 1 and 2.³² In order to prevent hydrolysis by β-glucosidases in the gastrointestinal tract, the glucose moiety was modified as a methyl carbonate prodrug. Although 3 showed promising effects in several diabetes models, its selectivity vs. SGLT1 was moderate (only 4-fold). Its development was discontinued in phase II.³³ Subsequently, several companies developed O-glycoside SGLT2 inhibitors, and many of them entered clinical trials (like sergliflozin etabonate 4 and remogliflozin 5).^34,35 However, the development of all “first-generation” compounds has been abandoned, likely due to the intrinsic metabolic lability of the O-glycoside bond, especially in humans, resulting in insufficient plasma half lives.³⁵ The fact that the O-glycoside bond is more stable in rodents may have contributed to the (too) late recognition of this liability.

This issue could be solved with the C-glycoside-based “second-generation” SGLT2 inhibitors. Initial attempts at replacing the O-glycoside bond by a methylene bridge led to a drastic loss of activity. In contrast, the direct C-aryl glucosides, initially discovered at BMS by coincidence as unanticipated side products of O-glycosylation reactions, turned out to be potent and selective SGLT2 inhibitors after systematic variation of the aryl substitution pattern.³⁶ Dapagliflozin 6, co-developed by BMS and AstraZeneca, represents the frontrunner of this approach,³⁷ quickly followed by other C-glycosides^34,38 like canagliflozin 7,³⁹ empagliflozin 8,⁴⁰ ipragliflozin 9⁴¹ and tofogliflozin 10.⁴² All of them have advanced to phase III clinical trials. It turned out that this new class of oral drugs was generally well tolerated and revealed beneficial effects, such as reductions in fasting and postprandial glucose and HbA1c levels.⁴³ In addition, favorable add-on effects like body weight reduction or blood pressure reduction were observed.⁴⁴ The European Medicines Agency (EMEA) recommended the approval of Forxiga™ (dapagliflozin) 6 for the treatment of type 2 diabetes in April 2012, whereas the FDA has requested further studies assessing the risk–benefit profile before taking a final decision on approval in the US. In March, 2013, canagliflozin 7 (trade name: Invokana™) was approved for the US market to treat adults with type 2 diabetes. The development of SGLT-inhibitors is a success story for medicinal chemistry, but arguably even more for natural product research. Phlorizin has not only been the lead structure for all advanced compounds to date, but its application as a tool compound was essential for disclosing an intriguing area of human biology and medicine.

2.2 Fingolimod

Multiple sclerosis is an autoimmune disease in which autoreactive B and T cells and autoantibodies aggressively target myelin antigens in the central nervous system, inducing myelin damage, neuroaxonal injury, astrogliosis, inflammation and finally neurodegeneration. Fingolimod 11 (Gilenya™) was the first oral disease-modifying agent approved in 2010 for the treatment of relapsing-remitting multiple sclerosis.⁴⁵11 is a synthetic compound obtained after drastic structural modifications of the fungal secondary metabolite myriocin 12 (ISP-1). 12 was identified as the active agent from culture broths of the fungus Isaria sinclairii by means of a phenotypic screen for immunosuppressive activity that monitored the inhibition of murine T cell proliferation in vitro. In this mouse allogenic mixed lymphocyte reaction (MLR) assay, 12 was 5- to 10-fold more potent than cyclosporin A.⁴⁶In vivo activity could be demonstrated in a rat model of allograft skin rejection after intraperitoneal administration. Further development of the natural product 12, however, was hampered by its low solubility and toxic effects in rats at comparably low doses.⁴⁷ Structure–activity relationships of 12 and the closely related mycestericins showed that structural simplifications, such as reducing the double bond and removing the keto and 4-hydroxy functions, had no impact on activity. The removal of stereogenic centers resulted in ISP-I-28 13 with a hydroxymethyl group instead of the carboxylic acid function of 12. Remarkably, this analogue was found to be more active in vivo and less toxic.⁴⁸ Further improvement of the activity and safety profile was achieved by reducing the linear alkyl chain length from C₁₈ to C₁₄, realized in the simplified analogue ISP-I-55 14.⁴⁹ The number of rotatable bonds was reduced through the introduction of a 1,4-phenyl moiety into the linear alkyl chain. A systematic shift of the phenyl ring along the alkyl chain finally led to the optimum compound 11 with a further potency improvement, which turned out to be 100 times more efficacious than cyclosporin A in an autoimmune encephalomyelitis model of multiple sclerosis.⁵⁰

These remarkable effects triggered various investigations on the mechanism of action of 11. The immunosuppressive effect of 11 could be ascribed to a strong decrease of the number of peripheral blood lymphocytes (T and B cells). The analysis of the lymphocyte distribution in blood, lymph and several secondary lymphoid organs (SLO) revealed that sequestration of lymphocytes into the SLOs was enhanced, while egress into peripheral blood and lymph was decreased,⁵¹ thereby preventing immune cells from reaching inflammatory tissue.⁵² As 11 displayed a close structural homology with sphingosine, further studies were focused on putative interactions with intracellular sphingolipid metabolism.⁵³ They demonstrated that 11 was converted to fingolimod phosphate 15 by sphingosine kinases (Scheme 1). The target of 15 was identified as a new class of transmembrane, G protein-coupled receptors (GPCRs) termed sphingosine 1-phosphate 16 (S1P) receptors.⁵⁴ This disclosure led to intensified research into the whole field of lysophospholipid receptors as a pharmacological relevant target class.⁵⁵15 modulated the function of S1P₁, leading to internalization of the receptor and down-regulation at the level of gene expression.⁵⁶ By this process, the S1P-induced signal that lymphocytes required to egress from the lymph system into inflammatory tissue was quenched. As a result, the attack of myelin antigens in the CNS by autoreactive lymphocytes, a characteristic event in progressing multiple sclerosis, was ultimately reduced.⁵⁷ The discovery of fingolimod impressively illustrates the value of natural products for uncovering intriguing biological mechanisms. Myriocin was in addition the lead structure, notably with features that are remote from lead- or drug-like molecules according to textbook rules. We speculate that it would not survive the majority of today's filtering processes applied in the active-to-lead phase. A reduction of structural complexity of natural products, e.g. through the removal of stereogenic centers, is mostly associated with the loss or drop of biological activity. In contrast, a drastic simplification has been possible along the optimization of 12 to 11, making the final product hardly discernible as one derived from nature.

Scheme 1 The phosphorylation of fingolimod.

2.3 Eribulin

The discovery, development and launch of the new anti-cancer drug eribulin 17 (Halaven™) impressively demonstrates how far the limits of natural product total synthesis have been pushed academically and industrially. 17 represents a simplified, truncated analog of halichondrin B 18, a large polyether macrolide discovered in 1986 by Uemura and coworkers in the sea sponge Halichondria okadai along with homohalichondrin B and norhalichondrin B.⁵⁸ The same organism also produces the phosphatase inhibitor okadaic acid. Later, 18 and its congeners were also isolated from other, unrelated sponges like Phakellia carteri,⁵⁹Lissodendoryx sp.⁶⁰ and Axinella spp.⁶⁰18 was identified as the most potent of these closely related compounds, showing growth inhibition on a panel of various cancer cell lines (including the NCI 60-cell line panel) at nanomolar concentrations.^58,61,62

In further studies, 18 has been shown to interact with tubulin to create non-productive tubulin aggregates, resulting in the suppression of microtubule assembly without disruption of existing tubulin architecture. This interaction led to arrest in the G₂-M phase of the cell cycle and subsequently to apoptosis. Further investigations revealed that 18 bound to the vinblastine site of tubulin in a non-competitive fashion and had no effect on colchicin binding.^62,63 This unprecedented mechanism of action strongly contributes to the therapeutic value of the halichondrin family of antimitotics: activity is still displayed on cancer cells resistant to other antimicrotubule agents like taxanes.⁶⁴ Compound supply was the major hurdle for clinical development since the beginning. The initial amount to start development activities was calculated to be around 10 g of halichondrin B 18 and the future commercial demand of drug substance was estimated to be 1–5 kg year⁻¹.⁶⁵ Due to the rare abundance of organisms producing 18 and the extremely low yield of isolated compound, collection from the wild was soon ruled out. The supply issue for the start of preclinical studies was partially solved by aquaculture of Lyssodendorix, which led to the provision of 310 mg of 18 from one ton of sponge.⁶⁶ Nevertheless it soon became obvious that the only viable alternative would be total synthesis. The first successful syntheses of both halichondrin B 18 and norhalichondrin were reported in 1992 by the Kishi group at Harvard.⁶⁷ However, this route and others developed subsequently⁶⁸ could not be regarded as cost-effective.

The highly convergent and modular synthesis approach was used by the Kishi group and medicinal chemists at Eisai in order to produce simpler analogs and to identify the minimum pharmacophore of 18. The testing of intermediates from the total synthesis efforts revealed that growth inhibitory activity on DLD-1 cells could be traced back to the right half macrolactone fragment 19.⁶⁵

However, 19 was devoid of activity in human tumor xenograft models – in contrast to the parent natural product. Based on the hypothesis that the right half intermediate 19 only induced a reversible mitotic block under the dosing schemes of the in vivo experiments, several regions of the polyether moiety were systematically modified, and the resulting analogs were tested in a sophisticated flow cytometric assay in order to characterize their ability to induce a complete mitotic block after drug washout.⁶⁹ Subsequent studies guided by this assay revealed that simple furan or pyran fragments could replace the western fused pyran moiety. Finally, further functional group exploration and the replacement of the ester linkage in the macrolactone ring by a ketone led to the identification of eribulin 17 (E7389),⁷⁰ an analog that displayed the desired potency on a panel of cancer cell lines without reversing mitotic block. Due to its remarkable biological profile, 17 entered development at Eisai. Although the molecular architecture of 17 is significantly simpler than that of 18, the compound still contains 19 stereogenic centers and can be regarded as the most structurally complex drug substance on the market to date which is prepared by total synthesis.

During preclinical development, the major obstacles that had to be overcome in the context of this unprecedented complex structure were the cost of goods and synthetic feasibility. Continuous scientific contributions from the Kishi group and considerable efforts at Eisai chemical development finally led to a cost-effective, scalable synthesis of 17 in 62 steps,⁷¹ which opened the way for the first clinical phase I trials in collaboration with the NCI. The positive outcome of a phase III study⁷² in patients with metastatic breast cancer previously treated with established drugs in a setting where most treatment schemes fail finally led to the approval of 17 as Halaven™ in 2010 in the US and 2011 in Europe.⁷³

2.4 Tetracyclines

Infectious diseases are one of the leading causes of death worldwide. The tetracyclines have been a cornerstone for the treatment of Gram-positive and Gram-negative bacterial infections with a widespread use in clinical practice for more than 60 years. Their structure is characterized by a linear arrangement of four fused, 6-membered carbocycles, conventionally labeled A through D. The isolation of the first representative chlortetracycline 20 was reported in 1948.⁷⁴20 was introduced into clinical practice as Aureomycin™, quickly followed by several other closely related natural analogs like tetracycline 21 (Achromycin™) and oxytetracycline 22 (Terramycin™) isolated from different Streptomyces strains (Fig. 1). These so-called “first-generation” tetracyclines comprise either unmodified natural products or derivatives obtained by simple synthetic modifications, like the catalytic hydrogenation of 20 to 21.⁷⁵

Fig. 1 First-generation tetracyclines and the ABCD ring system.

Further semisynthetic modifications led to the “second-generation” analogs doxycycline 23 (Vibramycin™) and minocycline 24 (Minocin™) launched in the 1960s and 1970s, which have played a dominant role in clinical practice until today. The discovery, development and application of the first- and second generation tetracyclines has been extensively reviewed.⁷⁶ In order to overcome resistance problems created by the widespread use of tetracyclines in human and veterinary medicine, a third generation is currently being developed. These efforts resulted so far in the launch of tigecycline 25 (Tygacil™) as the first representative of glycyl-modified tetracyclines in 2005.⁷⁷

The high complexity and functional density of the tetracyclines has attracted organic chemists for a long time. The first racemic total synthesis of a tetracyline was published by the Woodward group in 1962⁷⁸ and served as a basis for the synthetic strategies of other approaches realized in the following decades,⁷⁹ in particular by the Muxfeldt group:⁸⁰ the construction of the ring system is performed in a linear manner in the D → A direction. However, the inefficiency of this approach has hampered the application of total synthesis for providing clinical candidates (with one notable exception, the analog (±)-6-thiatetracycline 26⁸¹), leaving semisynthesis as the practical method of choice to generate modified analogs with improved antimicrobial and pharmacokinetic profiles – until recently.

The situation changed with a report of a new, highly convergent approach to the tetracyclines by the Myers group in 2005.⁸² As outlined in Scheme 2, their strategy is based on the parallel preparation of D ring and AB ring fragments (27 and 28, respectively), which are assembled to the protected intermediate 29 at a very late stage of the synthesis with simultaneous formation of the C ring of the tetracyline system 30.⁸³ This approach is approximately 10 steps shorter than previously reported syntheses, rendering it much more efficient and practical for the preparation of sufficient amounts of compounds for biological characterization. After further refinement,⁸⁴ the coupling of building blocks 28 and 27 turned out to be extremely flexible and led to the synthesis of more than 3000 new tetracyclines.⁸⁵

Scheme 2 Myers' convergent approach to the tetracyclines.

Many of those derivatives would have been extremely difficult or impossible to prepare by semisynthesis approaches, in particular heterocyclic D ring analogs or compounds with additional cycles condensed to the D ring. Modifications at this particular region are regarded to be particularly valuable, since X-ray studies with tetracyclines bound to the target – the 30S subunit of the bacterial ribosome – suggested the D ring to be the most promising site for structural variations.⁸⁶ Several of the new analogs displayed an outstanding profile, breaking through resistance vs. tetracyclines and other antibiotics.

Systematic variation of this area has led to the identification of eravacycline 31 (TP-434), the first representative of a new sub-class of tetracylines that are fluoro-substituted at the D-ring, the so-called fluorocyclines.⁸⁷ Eravacycline 31 showed a promising broad-spectrum antibiotic profile and was claimed to overcome common tetracycline resistance mechanisms.⁸⁸ In addition, an oral administration route is principally feasible; its efficacy is currently being investigated. The compound has successfully completed a phase II clinical trial in patients with complicated intra-abdominal infections. The efficiency of the synthesis seems to pave the way for a cost-effective scale-up for the drug substance requirements of antibiotic therapy.

The eravacycline case demonstrates how a novel synthetic route enabled the discovery of new, promising drug candidates in a seemingly mature, well-exploited area.

3 Optimization of natural products by structural modifications

3.1 Glycopeptides

The growing incidence of bacterial infections caused by multidrug-resistant, Gram-positive bacteria represents a serious medical challenge both in the hospital and community environment.^89,90 The glycopeptide vancomycin 32 has been the treatment of choice for such infections for a long time, but emerging resistance against this drug is becoming more and more of an issue. More than 25 years ago, vancomycin-resistant enterococci (VRE) were first discovered, followed by vancomycin intermediate susceptibility (VISA) and recently, vancomycin-resistant Staphylococcus aureus (VRSA) strains.⁹¹

The molecular basis of vancomycin resistance is now well understood. Inhibition of bacterial cell wall synthesis is mediated by tight binding of the antibiotic to the integral precursor peptidoglycan peptide terminus D-Ala-D-Ala. Remodeling of this cell wall precursor to D-Ala-D-Lac is the underlying mechanism for the two most prominent types of resistance, VanA and VanB.⁹²

Glycopeptides like 32 and teicoplanin 33 display a fascinating and complex molecular architecture. It consists of a heptapeptide backbone that is bent to a concave shape through biphenyl and diphenylether cross-links of aromatic amino acids residues. In the case of 32 it took nearly three decades from the first isolation in 1956^93,94 until the complete disclosure of its structure⁹⁵ and total syntheses were not accomplished until 1998.⁹⁶ With a total synthesis at hand, it became possible to break the VanA and VanB resistance through a rational re-design of the vancomycin peptide backbone. Repulsive interactions between the oxygen atom of the (D-Ala-)D-Lac moiety of resistant bacteria and the carbonyl group of the central, substituted phenylglycine amino acid were eliminated through a ‘surgical’ replacement of the carbonyl by a methylene or an imine group, thereby restoring the affinity towards the target.⁹⁷

However, all derivatives in advanced drug development have been generated by semisynthetic modification of the natural products. For obvious practical reasons, the root cause of resistance, i.e. the binding pocket of the peptidoglycan peptide terminus, was not modified, but the periphery was decorated with hydrophobic and hydrophilic moieties. Surprisingly, the new derivatives successfully restored sensitivity of resistant bacteria, as discovered by scientists at Lilly in the 1990s with a series of N-alkyl derivatives of vancomycin.⁹⁸ A fine-tuning of ADME properties of derivatives based on the closely related scaffolds of 32, chloroeremomycin and A40926, a member of the teicoplanin family, led to the development of telavancin 34,⁹⁹ oritavancin 35¹⁰⁰ and dalbavancin 36, respectively, as the most advanced representatives of the second-generation glycopeptides (Fig. 2).⁹⁴

Fig. 2 Structures of lipoglycopeptides. Semisynthetic modifications are highlighted in red.

Telavancin 34 (Vibativ™) was launched in 2009,¹⁰¹ and oritavancin 35 and dalbavancin 36 are currently undergoing phase III clinical trials.¹⁰² Due to the hydrophobic side chains that characterize the new glycopeptides, they were placed into a separate sub-class called “lipoglycopeptides”. The structural modifications introduced two favorable properties: 1) a membrane anchoring of the lipid chain positioned the glycopeptide close to its target, and it led to disruption of membrane potential, increased permeability and finally, cell lysis. This additional mode of action also rendered the lipoglycopeptides rapidly bactericidal, whereas vancomycin displayed a bacteriostatic profile. 2) The side chains enhanced the homodimerization of the lipoglycopeptides. Due to multivalency, the homodimers exhibited a much tighter binding to the peptidoglycan, which translated to largely improved in vitro activities.¹⁰³ In addition to enhanced activities against VRE or VRSA, 34–36 also exhibit strongly (10–100-fold) improved activities against S. pneumonia compared to vancomycin.¹⁰⁴ The expansion of the indication areas to nosocomial pneumonia is under current review of regulatory authorities in the EU and the US.

The increased hydrophobicity also led to higher protein binding compared to vancomycin, resulting in longer in vivo half-lives of the drug and thus longer dosing intervals (of up to one weekly injection in the case of 36) in patients.^105,106

In summary, semisynthetic modifications carefully tuned the compound's physicochemical properties. This led to an additional cellular mode of action and in consequence, restored efficacy against resistant pathogens and improved pharmacokinetic properties were obtained as clinical benefits.

3.2 Echinocandins

The echinocandins represent the newest class of drugs approved for the treatment of invasive fungal infections, most notably treatment-refractory invasive aspergillosis and invasive candidiasis. Currently there are three echinocandins available for clinical use. Caspofungin 37 (Cancidas™) was approved in 2001 by the FDA, followed by micafungin 38 (Mycamine™) in 2005 and anidulafungin 39 (Eraxis™) in 2006 (Fig. 3).¹⁰⁷

Fig. 3 Structures of echinocandin antifungals. Semisynthetic modifications are highlighted in red.

There are several key features that make them a valuable addition to the existing treatment options for fungal infections: compared with azoles and polyenes, echinocandins display an enlarged spectrum for Candida spp. beyond C. albicans, an improved renal and hepatic safety profile and reduced cytochrome-mediated drug–drug interactions. These favourable profiles can be partly assigned to the fact that the echinocandins – in contrast to other antifungals – act on a target that is not present in mammals, since they inhibit β-(1,3)-D-glucan synthase, an essential enzyme complex in the formation of the fungal cell wall.¹⁰⁸

The development of all echinocandins launched today can be traced back to a few closely related representatives of a class of large lipopeptides identified from fungal sources in the 1970s and 1980s. The echinocandins are amphiphilic cyclic hexapeptides comprised of rather hydrophilic amino acids and a fatty acid side chain. Echinocandin B 40, a fermentation product of Aspergillus nidulans and the starting point for the development of 39, was discovered in 1974 as the first representative of the class.¹⁰⁹40 showed potent activity against Candida albicans and Candida tropicalis, and in vivo activity was demonstrated in an animal model of candidiasis. On the other hand, low solubility, poor oral bioavailibility and potential hemolytic side effects had to be overcome before clinical development.¹¹⁰ It soon became evident that the lipophilic side chain was an important structural feature that modulated both activity and toxicity and offered potential for the fine-tuning of pharmacokinetic properties of this substance class. The development of a biotransformation process for the deacylation of 40 by Actinoplanes utahensis turned out to be key for access to a broad variety of side-chain modified echinocandins.¹¹¹ As shown in Scheme 3, removal of the linoleoyl unit from 40 led to a cyclic hexapeptide core (deacyl-echinocandin B 41) devoid of antifungal activity. The first new derivative to enter clinical trials, cilofungin 42, was selected by Lilly from several analogs obtained by the re-acylation of this core.

Scheme 3 Synthesis of cilofungin from echinocandin B.

Structure–activity relationship studies revealed that the acyl moiety had to be a fatty acid chain of at least 12 carbon atoms to restore anti-fungal activity. Shorter and/or more polar acyl motifs yielded inactive compounds. These observations led to the assumption that a lipophilic moiety of sufficient length is necessary for attachment to the fungal membrane, preceding the interaction with the glucan synthase as the molecular target; a similar structural requirement and mechanism was discussed above in the context of increased activity of lipoglycopeptides in comparison to vancomycin.¹¹² Although clinical studies with cilofungin 42 gave at first encouraging results for some types of Candida infections, the product had to be discontinued: cilofungin still displayed the low water solubility of its parent echinocandin B; finally, the formulation that had to be used for a parenteral administration caused adverse effects.^111,113

Despite this first setback, inhibition of glucan synthesis remained an attractive approach for antifungals, and further activities in the field were spurred by the increased medical need for antifungal agents in the context of the growing number of immunocompromised patients, in particular those with secondary infections associated with AIDS. New whole-cell based assays for the detection of glucan synthesis inhibitors were developed and led to the discovery of a series of new antifungal lipopeptides.¹¹⁴ Lilly scientists focused on further exploration of structure–activity relationships around 40 and 42. A comparison of the lipophilicities of the side chain fragments revealed that calculated logP values of >3.5 were required for antifungal activity. Furthermore, side chains with linear, rigid moieties (such as [1,1′;4′,1′′]-terphenyls) were most effective in increasing the antifungal potency, and incorporation of such side chains resulted in compounds with long half-lives.¹¹⁵ The development candidate LY303366 was the outcome of these efforts. LY303366 was effective and well tolerated in clinical studies. In contrast to 41, no major adverse effects were observed, and the successful development culminated with the launch of the compound as anidulafungin 39 (Eraxis™).¹⁰⁸

The starting point for the development of caspofungin 37 (Cancidas™) was a new sub-class of echinocandin-like lipopeptides, named pneumocandins, which were discovered at Merck in the 1980s in a broad screening campaign for cell-wall targeting antifungals. The pneumocandins had a particular attraction since they displayed an enlarged spectrum against several Candida pathogens. Moreover, they were active on Pneumocystis carinii, an opportunistic organism causing life-threatening pneumonia in AIDS patients, and showed no hemolysis in vitro. Pneumocandins were produced by the fungus Zalerion arboricola, with pneumocandin A₀43 being the major secondary metabolite in the original culture, accompanied by pneumocandin B₀44 and D₀45 as the most abundant minor fermentation products.¹¹⁶ These compounds differ from 40 by the fatty acid side chain and one or two amino acid residues.

Initially 43 was selected for further optimization by medicinal chemists to overcome some limitations still associated with the natural product. Improvements in antifungal spectrum, potency, chemical stability and water solubility were required. However, after careful profiling of the minor fermentation products, it turned out that 44 was considered as a more suitable starting point for chemical optimization. The limitations of compound supply as a prerequisite for a large program of semisynthetic modifications was overcome by developing a producer strain with increased titers of 44 (now as the main product) and reduction of fermentation by-products. This producer strain was obtained after isolating further related strains which displayed higher titers of 44, followed by mutant selection and media variations.¹¹⁷ In contrast to the Lilly approach, the optimization strategy of the Merck scientists did not follow a biotransformation-based removal–reacylation strategy for a variation of the lipophilic side chain, but focused on modifications of the peptide core structure, leaving the fatty acid chain untouched. Synthetic modifications turned out to be challenging since the natural product 44 was unstable under non-neutral conditions and displayed limited solubility in a variety of solvents. Nevertheless the semisynthesis efforts resulted in a first development candidate which solved the solubility issue: a phosphate ester prodrug 46 was obtained from 44 through esterification of the phenolic hydroxyl group of the homotyrosine moiety.¹¹⁸ In parallel, additional positions on the nucleus 44 were modified. This work was partly guided by the total synthesis of echinocandin D¹¹⁹ and simplified analogs that helped to figure out the minimum structural requirements for antifungal activity.¹²⁰ Both the modification of the hemiaminal group as well as the reduction of the carboxamide function of the hydroxy glutamine moiety to a primary amine (hydroxy ornithine) turned out to be favourable: The 2-aminoethyl-substitution on the aminal conferred improved stability, solubility and higher activity against Candida and Aspergillus in vivo, while the reduction of the glutamine led to even further improved anti-Candida activity.^121,122 Finally, the replacement of the oxygen in the hemiaminal moiety by nitrogen gave the aza analogue MK-0911, now caspofungin 37, with an improved pharmacokinetic and safety profile. MK-0911 proved to be superior to the pneumocandin B₀ phosphate ester prodrug 46 and was therefore chosen for clinical development.¹¹⁰

Scientists at Fujisawa focused on water solubility as a major selection criterion for optimization starting points in their search for β-(1,3)-D-glucan synthase inhibitors.¹²³ These efforts resulted in the discovery of FR901379 47 from the fungus Coleophoma empetri F-11899.¹²⁴ FR901379 47 had an excellent water solubility of 50 mg mL⁻¹ (compared to 0.008 mg mL⁻¹ for 40) due to the presence of a sulfate group on the homotyrosine moiety. The compound was more potent against Candida in vitro than 40. On the other hand, a pronounced hemolytic activity was observed, which was assigned to the linear aliphatic fatty acid moiety.¹²⁵ Optimization was therefore guided by the knowledge obtained in the context of echinocandins and cilofungin chemistry.¹²⁶ Thus, FR901379 47 was deacylated with Actinoplanes utahensis and re-acylation studies confirmed – as in the case of 42 and its analogs – that rigid, linear aromatic moieties were ideally suited to obtain improved compounds.¹²⁷ Finally, a compound with a side chain closely related to that of 39 was chosen as a development candidate: FK463, now micafungin 38, can be regarded as a close analog of anidulafungin 39, the main differential structural features being the sulfate group on the homotyrosine unit and the replacement of the central phenyl ring of the terphenyl side chain by an isoxazole.¹²⁸

3.3 GLP-1 agonists

The human glucagon-like peptide-1 (GLP-1) 48 is secreted from the intestinal endocrine L-cells in response to food or oral glucose uptake. It functions as an incretin hormone enhancing meal-stimulated insulin secretion.^129,130 In contrast to established insulin-releasing drugs like the sulfonylureas, the insulin release is strongly dependent on enhanced blood glucose levels, thereby minimizing the risk of hypoglycemia.¹³¹ Other clinically proven beneficial effects, like delay of gastric emptying, suppression of glucagon secretion and reduction of body weight, led to the development of GLP-1 agonists as a very attractive approach for the treatment of type 2 diabetes.^132,133

However, the use of unmodified human GLP-1 48 is limited by its rapid degradation through dipeptidyl peptidase IV (DPP-IV), resulting in an extremely short plasma half-life of 2–5 min.¹³⁴ The development of modified GLP-1 agonists resistant to degradation by DPP-IV enabled the translation of the incretin mimic concept into clinical practice.¹³⁵ Two principles were followed (Fig. 4): (i) in the case of exendin-4 50 (exenatide, marketed as Byetta™), an altered amino acid sequence originating from an animal homolog (see below) was a much weaker substrate of DPP IV. 50 was the first GLP-1 agonist on the market, launched in the US in 2005 and in Europe in 2007. Lixisenatide 51 (launched as Lyxumia™ in 2013) followed the same principle, but achieved further prolonging of plasma half-life through the attachment of a hexa-lysine residue at the C-terminus. (ii) Liraglutide 49 (launched as Victoza™ in 2010) was directly derived from the human GLP-1 sequence and modified with a palmitate side chain linked through a γ-L-glutamoyl residue. The fatty acid moiety exhibits a strong, reversible binding to human serum albumin, thereby conferring protection against degradation.¹³⁶

Fig. 4 Structures of GLP-1 48 and the GLP-1 receptor agonists liraglutide 49, exenatide 50 and lixisenatide 51. Histidine is the N-terminal amino acid in all analogs. Amino acid modifications are highlighted in purple. The DPP-4 cleavage site is indicated with red arrows.

The discovery of 50 was initially unrelated to the “incretin concept”. 50 is a linear, 39-amino acid peptide that shares about 50% homology with human 48. It was isolated in 1992¹³⁷ from the salivary gland of the lizard Heloderma suspectum, known as the Gila monster originating in Arizona and New Mexico. The discovery was based on earlier observations that the venom of Heloderma suspectum stimulates adenylyl cyclase activity in guinea pig pancreatic acinar cells.¹³⁸ In a first hypothesis it was suggested that 50 activated a putative exendin receptor to increase pancreatic acinar cAMP.¹³⁷

After human GLP-1 was recognized as a close structural homolog,¹³⁹ it was demonstrated that 50 stimulated insulin secretion in murine insulinoma cells through activation of the GLP-1 receptor, a G_i coupled GPCR that indeed enhances intracellular cAMP levels.¹⁴⁰ These findings triggered clinical investigation of 50 in type 2 diabetes.

Since the isolation of 50 from an animal source to ensure a sustainable supply was completely out of the question, alternatives had to be elaborated. While the peptide portion of liraglutide 49 is currently being produced in recombinant yeast,¹³⁶50 is manufactured by solid-phase peptide synthesis (SPPS) employing the 9-fluorenyl-methoxycarbonyl (Fmoc) strategy.¹⁴¹ Despite the intrinsic drawbacks of SPPS (high reagent costs, considerable generation of waste) and the need for reversed-phase chromatography for final purification, the drug substance can be provided in a cost-effective manner: since the dose for this hormone-like drug is as low as 5–10 μg twice daily, 3.7–7.3 mg of active pharmaceutical ingredient is sufficient to treat a patient for a year.¹⁴²

3.4 Rapamycin and rapalogs

Rapamycin 52 represents an outstanding example of how a natural product has fertilized various fields of research, as highlighted in numerous review articles. We will focus here on strategies for the development of improved rapamycin analogs (the “rapalogs”) for different therapeutic areas.

Rapamycin 52 was originally discovered in 1975 as a secondary metabolite from a Streptomyces hygroscopicus strain due to its antifungal activity. The compound was named rapamycin after the origin of its producer strain, which had been isolated from a soil sample collected at Rapa Nui (Easter Island).¹⁴³ The complete structural assignment of 52 was accomplished a few years later based on X-ray crystallography and spectroscopic data.¹⁴⁴52 was one of the first complex polyketide synthetase products whose biosynthesis was elucidated.^145,146 Although 52 showed excellent anti-Candida activity,¹⁴⁷ the compound was not developed as an anti-fungal agent. Based on further biological profiling experiments that revealed its potent immunosuppressive activity,¹⁴⁸52 was at first developed without further structural modifications as the oral immunosuppressant drug sirolimus (Rapamune™, approved for prevention of rejection in organ transplantation in 1999).¹⁴⁹ Considerable efforts have been made to unravel the details of the mode of action of 52 after functional effects of the compound on T cells had been studied in more detail.¹⁵⁰ The molecular target was identified in a gene complementation assay in yeast. Rapamycin-resistant strains were selected, and the gene that restored sensitivity to rapamycin was cloned and named TOR for “target of rapamycin”.¹⁵¹ Shortly thereafter, the mammalian homolog referred to today as mTOR (mammalian target of rapamycin) was identified.¹⁵² It was found that 52 had the remarkable ability to occupy two hydrophobic binding sites at the same time (Fig. 5). It inhibited mTOR through binding initially to the immunophilin FKBP-12 (one of the FK506-binding proteins), and then forming a ternary complex with mTOR.¹⁵³ mTOR is a serine-threonine kinase that plays a central role integrating signals from growth factors, nutrients, stress, hormones and mitogens to regulate survival, proliferation and cell growth. Disruption of mTOR activity led to inhibition of protein translation and blocking of the cell cycle transition from the G₁ to the S phase.¹⁵⁴

Fig. 5 Structures of rapamycin 52 and its analogs currently approved for human use or in clinical trials. The FKBP12 and mTOR binding regions are highlighted.

In addition to its application in immunological indications, 52 was also considered as an anticancer agent due to its antiproliferative effect. Further evaluations were at first discouraged by the finding that for an antiproliferative effect in mouse cancer models, much higher doses than for the prevention of allograft rejection were needed, raising concerns that rapamycin would not be tolerated in humans at the high doses estimated to be required for anti-tumour activity.¹⁵⁵52 gained renewed interest when further studies revealed that some tumour cell lines were highly sensitive to 52 and that the compound displayed a unique pattern of reactivity in the NCI-60 cancer cell line panel.¹⁵⁶ Since 52 was poorly soluble and underwent extensive first-pass metabolism, resulting in potentially variable and low absorption and exposure,¹⁵⁷ and since solubility and stability problems prevented the development of a parenteral formulation,¹⁵⁸ analogs with improved pharmaceutical and pharmacokinetic properties were sought after. This effort has led to the development of several rapalogs, which are now in clinical use for a variety of indications in immunology, oncology and cardiology. To date, 5 distinct total syntheses of 52 have been reported.^159,160 Nonetheless all derivatives that are currently in clinical use or late stage development are based on semisynthetic approaches. The most-explored site of derivatization was the unhindered hydroxyl function on the cyclohexyl moiety. Although the pharmacodynamic effects of the rapalogs seemed to be quite similar, the compounds differed in formulation, application and dosing schemes, thereby altering drug exposure.

One approach led to the highly soluble ester prodrug temsirolimus 53 (Torisel™, approved in 2007), which was designed for intravenous administration in oncology in order to achieve high exposures with low inter-individual variabilites in well-controlled chemotherapy settings.¹⁶¹ Temsirolimus 53 was mainly metabolized to 52; through parenteral administration of 53, higher plasma concentrations of the parent compound 52 were achievable than after direct oral administration of 52. Two other non-prodrug derivatives have been developed for cancer treatment: everolimus 54 (Afinitor™, approved in 2009)¹⁶² for oral administration and ridaforolimus (deforolimus) 55 for both oral and intravenous applications (currently in late stage clinical trials). In the structure of 54, the hydroxy group at the cyclohexyl moiety of rapamycin is replaced by a 2-hydroxy ethoxy group, while 55 carries a dimethyl-phosphinic acid ester at this position. 54 had a lower plasma protein binding and higher oral bioavailibility compared with 52.¹⁵⁶ Yet another variation yielded zotarolimus 56 (ABT-578), a rapamycin derivative obtained by replacing the hydroxy function by a tetrazole under inversion of stereochemistry. Zotarolimus 56 is used exclusively in drug-eluting stents for the treatment of coronary artery disease. It is characterized by a high lipophilicity and a shorter terminal half life than 52, properties that were particularly desired for the application in stents to obtain a slow release of the drug directly into the wall of the coronary artery. Lipophilicity was considered to be favorable for crossing cell membranes to inhibit neointimal proliferation of the target tissue, and a short half life led to rapid clearance of the drug once released into the blood stream, thereby reducing systemic toxicity.¹⁶³

Since the mTOR pathway is up-regulated in several malignant diseases, and the dysregulation of this pathway also involves upstream targets, several approaches beyond the development of rapalogs have been pursued. In particular, from directly targeting the active site of the kinase function of mTOR and the upstream kinases PI3K and Akt, several small molecule inhibitors – non-rapalogs – have emerged which are currently undergoing clinical trials.^156,164,165

Like other immunophilin ligands, such as cyclosporin A and FK506, 52 may be regarded as a privileged template for the modulation of protein–protein interactions mediated by small molecules.¹⁶⁶ Therefore both semisynthetic and biosynthetic approaches to new rapamycin analogs have been pursued to address other targets and indications beyond those related to the mTOR pathway. In the early 1990s it was reported that 52 and FK506 exhibited neurotrophic and neuroprotective effects.¹⁶⁷ The finding that these effects were partially independent of inhibition of mTOR or calcineurin led to efforts towards uncoupling neuroprotective from immunosuppressive effects. In the case of 52 this was achieved by semisynthetic modifications in the mTOR binding region while leaving the FKBP (immunophilin) binding region untouched. The derivatives WYE-592 57 and ILS-920 58 were obtained after Diels–Alder reaction of the triene moiety of rapamycin 52 with nitrosobenzene and subsequent hydrogenation, respectively. Both compounds displayed significantly reduced immunosuppressive activity and were active in a rodent model of ischemic stroke.^146,168 More recent efforts to access novel biologically active rapalogs have been based on genetic manipulation, precursor-directed biosynthesis and mutasynthesis.^{14,16,146,169,170}

4 Immunoconjugation of natural products

Natural products have been – and continue to be – a rich source of cytotoxic reagents with high medical utility in anti-cancer therapy.¹ But a large number of natural product leads in that indication are not applied in medical practice, as the cytotoxic effects are also exerted towards healthy, non-tumor cells, thereby inducing systemic toxicities. The balance between tumor defeat and non-tolerable systemic toxicity is often subtle, leading to a therapeutic window that is too small or even non-existent. At the same time, progress in anticancer-therapy has been significantly fueled by the successful development of monoclonal antibodies (mAbs) directed against targets that are specifically expressed in tumor cells. Monoclonal antibodies exert high affinity and high specificity against their targets and are generally well-tolerated, in particular since the introduction of fully humanized mAbs. On the other hand, their efficiency in eradicating tumor cells has often not been sufficient for a lasting clinical response.

In the past three decades, a bold strategy has been pursued to combine the strengths of the two formats summarized above: The covalent coupling of a targeting monoclonal antibody to a natural product effector was expected to combine the specificity and tolerance of mAbs with the cell-killing efficiency of natural products in a single molecule, a so-called immunoconjugate or antibody–drug conjugate (ADC). After binding of the antibody to its cancer cell surface antigen, the ADC is internalized into the cell. Inside the cell, the linker between antibody and effector is cleaved through a chemical or enzymatic mechanism (see below), thereby releasing the ‘free’ natural product for cell killing.

The approach offers a great opportunity to turn extremely active natural products (with potential new modes of action) into useful drugs that might have been otherwise excluded from development due to high toxicity and safety issues. In fact, it is now well recognized that suitable toxic payloads for antibodies should have a potency in the range of 10–100 pM on a cellular level, which is 2–3 orders of magnitude higher than optimum potencies for conventional chemotherapeutic agents.¹⁷¹ While any class of compounds can serve as an effector in principle, almost exclusively natural products have been applied to fulfil this strict design criterion. As immunoconjugates can be regarded as highly sophisticated drug delivery systems, a careful optimization of all components – the monoclonal antibody, the cytotoxic payload and the chemical linker – is crucial for therapeutic efficacy.¹⁷²

Gemtuzumab ozogamicin 60 (Mylotarg™) was the first antibody–drug conjugate to be approved. It is based on the highly cytotoxic agent calicheamicin γ₁^I59, which is linked to an antibody directed against the CD33 antigen present on leukemic myeloblasts.¹⁷³ The acid-labile hydrazone linker is cleaved in the lysosome, thereby releasing the calicheamicin moiety in 60 that acts through a unique mechanism: DNA is targeted through interaction of the sugar moiety with selected base pairs in the minor groove. Upon reductive activation of the disulfide moiety a thiolate is formed, which then attacks the enone function of the enediyne moiety, thus triggering a Bergman cyclisation which generates a diradical. Reaction of this diradical with the DNA backbone leads to double strand cuts, ultimately resulting in cell death.¹⁷⁴

Despite positive clinical results that led to the accelerated approval of gemtuzumab ozogamicin 60 in 2000, the drug was withdrawn in 2010 due to concerns about safety and clinical benefit that were raised after subsequent phase III trials. Another calicheamicin-based immunoconjugate, inotuzumab ozogamicin targeting the CD22 antigen expressed on malignant B cells, is still in late stage clinical development for acute lymphoblastic leukaemia.¹⁷¹ An altered biodistribution of CD33⁺ antigen expressing cells leading to reduced efficacy was discussed as one reason for the failure of 60: High loads of the CD33 antigen in acute myelogenous leukemia (AML) circulating in the blood stream resulted in an increased presence of bound immunoconjugate in the plasma compartment, leading to reduced levels of 60 in CD33⁺ cells in the bone marrow.^172,175

The case of 60 demonstrates that for the development of safe and effective immunoconjugates, many parameters influencing pharmacokinetic and pharmacodynamics must be controlled. It was not until 2011 that the next antibody–drug conjugate was launched.¹⁷⁶

Brentuximab vedotin 63 (Adcetris™) was approved for the treatment of Hodgkin and systemic anaplastic large cell lymphoma. It is based on a fully synthetic analog of dolastatin 10 61 linked to an anti-CD30 antibody. 61 was first reported in 1987 after its extremely challenging isolation from the sea hare Dolabella auricularia.¹⁷⁷ It belonged to a family of very potent antineoplastic pentapeptid-like structures acting as tubulin polymerization inhibitors. Only two years later a convergent total synthesis of dolastatin 10 61 was published,¹⁷⁸ which resolved any resupply issues and paved the way to combinatorial routes that allowed for the synthesis of thousands of analogs, the auristatins. This flexible synthesis route enabled a systematic fine-tuning of physicochemical properties, stability and potency of new derivatives aimed at conjugation to antibodies. A simplified dolastain 10 analog, monomethyl auristatin E (MMAE) 62 was the result of this work. MMAE 62 is characterized by a built-in site for stable linker attachment (a secondary amine) and displayed high potency, solubility and stability under physiological conditions. Brentuximab vedotin 63 was produced by conjugating MMAE 62 to the chimeric anti-CD30 monoclonal antibody cAC10 through a protease-cleavable linker containing a valine-citrulline dipeptide motif.¹⁷⁹ This dipeptide sequence was found to be stable in plasma, but easily cleaved by cathepsin B, a lysosomal protease encountered after binding to the CD30 antigen and internalization by clathrin-mediated endocytosis. After proteolytic cleavage, 62 is released into the cytosol and inhibits microtubule polymerization, leading to cell cycle arrest at the G₂-M transition phase and ultimately to apoptosis in CD30-expressing lymphoma cells.¹⁸⁰

Very recently, the first antibody drug conjugate acting on solid tumors successfully completed clinical development: ado-trastuzumab emtansine 64 (Kadcyla™) was approved in February 2013 by the FDA for use as a single agent for the treatment of patients with human epidermal growth factor receptor 2 (HER-2) positive metastatic breast cancer that had undergone prior therapy. 64 consists of the maytansinoid DM-1 68 attached to a recombinant humanized monoclonal HER2-binding antibody, trastuzumab (Herceptin™).^181,182 The naked antibody is well established in the treatment of HER2-overexpressing breast cancer.¹⁸³

The toxic payload DM-1 68, a semisynthetic derivative of the maytansinoid ansamitocin P-3 66, was attached through a non-reducible thioether linker.^181,182 The eponymous first representative of this highly cytotoxic class of compounds, maytansine 65, was isolated from the Ethiopian shrub Maytenus serrata by means of bioassay-guided fractionation. Other congeners have not only been isolated from plants, but also from an Actinomycete and from mosses.¹⁸⁴65 displayed a very potent activity on several carcinoma and leukemic cell lines. It bound directly to tubulin in a mode similar to the Vinca alkaloids and thus interfered with the formation of the mitotic apparatus at cell division.¹⁸⁵ As also observed for other tubulin-binding anti-cancer agents, this interaction led to arrest in the G₂-M phase of the cell cycle and subsequently to apoptosis.¹⁸⁶ Early clinical trials with 65 were initiated in the 1970s. However, dose-limiting toxicity and lack of response in the majority of patients enrolled in phase II trials led to the discontinuation of 65 as a single agent for anti-cancer therapy. From the early 1990s on, the maytansinoids were re-investigated for their use as toxic payloads for antibodies. 66 was chosen as the starting material for semisynthesis, since it was readily available from fermentation. DM1 68 and other related maytansinoids suitable for conjugation (such as DM4 69) were synthesized from maytansinol 67, which is obtained by reductive cleavage of 66 at the acyloxy function at C3 with LiAlH(OMe)₃.¹⁸⁷ Re-esterification of maytansinol with several carboxylic acids mediated by DCC/ZnCl₂ led to new derivatives displaying cytotoxic activity in the 10–90 pM range, which corresponded to an ideal potency for the use as toxic payloads in antibody conjugates.¹⁸⁸

DM1 68 and DM4 69 were combined with various antibodies and linker types, resulting in several new investigational immunoconjugates that have entered clinical trials. The successful development of 65 as the first approved frontrunner of the maytansinoid immunoconjugates may be due to a relatively stable linker and its “dual” mode of action: 65 contains a non-reducible thioether linker, which was cleaved relatively slowly by proteases in the endosome of tumor cells after internalization, resulting in a longer-lasting active metabolite. The linker was highly stable in the extracellular environment and exhibited a relatively low clearance of the immunoconjugate (in contrast to conjugates with reducible disulfide linkers). In addition to the delivery of the toxic payload, ado-trastuzumab emtansine 65 still displayed the properties of the naked antibody, namely inhibition of HER-2 signaling, the therapeutic principle of trastuzumab (Herceptin™), and induced antibody-dependent cellular toxicity.^181,189,212

In the past 3 years, impressive clinical results provided the proof-of-concept for the immunoconjugate approach. They triggered intense R&D activities that has already led to more than 30 clinical development candidates as of May 2013.¹⁹⁰ The fact that most of them employ maytansinoids or 62 demonstrates that the establishment of a single natural product-based effector platform can enable the harvesting of multiple clinical drug candidates. It also suggests that the exploration of additional effector principles is a highly rewarding field for future natural product research.

5 Synthetic supply of unmodified natural products

In fortunate cases, the pharmaceutical profile (in terms of efficacy vs. side effects) of an isolated natural product is so favorable that it can be used as a drug without further structural modification. Roughly 20% of natural product-based drugs fall into this category.¹ Even without a need for lead optimization, the daunting challenge to assure a large-scale supply of (GMP-quality) drug substance remains to be solved by natural product biologists and chemists. For natural products with complex structures, the fermentation of microbial metabolites, or the horticulture of plant metabolites, followed by pure compound isolation, has been the method of choice. In the following paragraphs, we present successfully realized alternatives to this ‘mainstream’ – although not at all trivial – approach.

The first two marketed marine natural product drugs ziconotide (Prialt™, Elan Pharmaceuticals) and trabectedin (Yondelis™, PharmaMar)¹⁹¹ did not have a sustainable biological production source; consequently, chemists have developed a viable access, by total- and semisynthesis, to the products (chapters 5.1 and 5.2, respectively). In order to become independent from the fluctuations of horticulture for the supply of the antimalarial product artemisinin, a combination of molecular biology, creating a novel producer strain for a central drug intermediate, with organic synthesis has been realized (chapter 5.3).

5.1 Ziconotide

Ziconotide is the synthetic form of a 25-amino acid peptide that was originally isolated in 1979 as ω-conotoxin MVIIA 70 (Fig. 6) in the venom of the cone snail, Conus magnus.¹⁹² Toxic peptides (conotoxins) produced by the venomous fish-hunting cone snails (Conus spp.) can be divided into several classes that attack various critical functions of the neuromuscular system of the prey. All conotoxins share several common features: they are relatively small (13 to 29 amino acids), strongly basic peptides, which are highly cross-linked by disulfide bonds.¹⁹³

Fig. 6 The amino acid sequence of ω-conotoxin MVIIA 70.

As the animal sources provided only minute quantities of peptides, total synthesis approaches for the conotoxins were required to ensure a sustainable supply for profiling. A first solid-phase peptide synthesis of 70 was accomplished in 1987 on p-methylbenzhydrylamine resin using the Merrifield protocol. Protection of side-chain amino acid functions was realized by standard methods for the Boc/Bzl strategy, in the case of the cysteine residues as p-methoxy benzyl (Mob) or N-acetyl aminomethyl (Acm) derivatives. After building the ω-conotoxin MVIIA sequence on the resin, the completely deprotected linear peptide with the 6 Cys residues in reduced sulfhydryl form was obtained following cleavage with hydrogen fluoride-based cocktails. Formation of the disulfide bond cross-linking in the correct connectivity was achieved by simple air oxidation at neutral pH in aqueous solution to give correctly folded 70. The yields of the folding step could be improved by the use of additives, such as cysteine and DTT.

In further studies, N-type voltage-sensitive calcium channel blocking was identified as the mode of action of 70.¹⁹⁴ Development of the compound as an antinociceptive agent was initiated through its high effectiveness in a rat model of neuropathic pain.¹⁹⁵ In 2005, ziconotide was launched in the USA for the treatment of severe chronic pain. Due to a therapeutic scheme with doses as low as 2.4–21.6 μg day⁻¹,¹⁹⁶ the multistep total synthesis on solid phase remains an economically viable supply of the drug substance.

5.2 Trabectedin

Trabectedin 71 (ecteinascidin-743, ET-743), launched in Europe in 2007, was the first anti-cancer drug from a marine source. Since its discovery and development and the underlying synthesis efforts have recently been outlined in an excellent review article¹⁹⁷ we will only provide a brief summary. Its discovery dates back to 1969 when antitumour activities of an extract of the marine tunicate Ecteinascidia turbinata were first described.¹⁹⁸ However, the minute quantities of isolated ecteinascidins hampered the final identification and structure elucidation of representatives of this compound class until the 1990s.¹⁹⁹ The ecteinascidins showed exceedingly potent activity in several tumour models in mice.²⁰⁰ ET-743 71 was selected for further development due to its relatively high abundance – though the yields from the tunicate were still very low (∼10 ppm) in absolute terms.¹⁹⁹ Total synthesis enabled the delivery of more material for in vitro and in vivo studies: the first synthesis published in 1996 by the Corey group provided 71 in >30 steps (0.75% total yield).²⁰¹ Improvements were achieved by a more efficient and effective preparation of a key intermediate,²⁰² but the adaptation to a large scale synthesis still remained a major challenge.

Although the establishment of aquacultures of E. turbinata for the production of 71 proved to be very challenging and required considerable efforts, this approach has provided the batches required for clinical trials.^203,204 It was nevertheless considered necessary to establish a sustainable, long-term drug supply capable of serving the needs for commercialization of the drug. A breakthrough was finally achieved by the development of a semisynthetic process starting from the readily available fermentation product cyanosafracin B 72, which can be obtained from Pseudomonas fluorescens in multi-kilogram quantities.²⁰⁵ As shown in Scheme 4, 72 represents a well-suited scaffold for the construction of the more complex 71 and its analogs, since its structure is comprised of two of the three fused tetrahydroisoquinoline rings present in the ecteinascidins and displays a favourable arrangement of functional groups. The transformation into 71 can be accomplished in 20 steps with a reported total yield of 1.14%.

Scheme 4 Trabectedin 71 and cyanosafracin B 72

5.3 Artemisinin

Malaria is the most widespread transmissible disease, affecting most tropical and sub-tropical countries of sub-Saharan Africa, South and South-East Asia, and parts of South America. An estimated one third of the world's population lives in malaria-affected areas. There were an estimated 219 million cases of malaria and over 660 000 deaths estimated in 2012.²⁰⁶ The first-line treatment of the most virulent form of malaria caused by Plasmodium falciparum is based on artemisinin 73, a sesquiterpene lactone peroxide originally extracted from the aerial parts of the shrub Artemisia annua, known as qinghao in traditional Chinese medicine.²⁰⁷

Activity of A. annua extracts against Plasmodium growth in a mouse model was discovered in the early 1970's by Chinese scientists through the systematic investigation of more than 640 traditional herb preparations that might have antimalarial activity.²⁰⁸ In 1972, artemisinin 73 (qinghaosu) was isolated²⁰⁹ and structurally characterized²¹⁰ as the active principle.

In China, artemisinin has been used for the treatment of resistant Plasmodium falciparum malaria, but its therapeutic value is limited by its low solubility both in water (around 60 μg mL⁻¹) and in lipidic formulations.²¹¹ Artemisinin itself has a very short half-life in humans and is rapidly converted mainly to the active metabolite dihydroartemisinin 74.²¹² In order to improve the pharmaceutical and pharmacokinetic properties of artemisinin, several semisynthetic derivatives, such as artemether 75, arteether 76 and artesunate 77 were developed. All of them are accessible through 74 obtained by reduction of 73, and all of them are – as 73 – converted back to 74 as the active metabolite in humans.²¹³ As artemisinin and its derivatives are the most effective antimalarials currently available,²¹⁴ the use of 74, 75 or 77 in combination with other antimalarials in so-called “artemisinin-based combination therapies (ACTs)” is recommended by the WHO in their guidelines for the treatment of malaria.²¹⁵ Whereas 75 and 76 have an improved solubility in lipidic formulations compared with 73,²¹⁶77 displays a high water solubility (around 0.6 mg mL⁻¹) due to its carboxylic acid function.²¹² It is indispensable for the successful treatment of severe P. falciparum infections in areas of low malaria transmission where high doses of an artemisinin-derived drug have to be administered intravenously.^213,217

The first total synthesis of artemisinin starting from the chiral pool building block (−)-isopulegone was reported in 1983.²¹⁸ Although several other improved total syntheses were published,²¹⁹ the approach remains too expensive for commercialization. Today artemisinin is still produced from Artemisia annua cultivated in East Asia (mainly China and Vietnam) and to a lesser extent in Africa.²²⁰

The typical yields of artemisinin based on dried plant material are reported to be in the range of 0.8%. Like for other non-regulated commercial crops, the market for artemisinin experiences strong fluctuations: from 2003–2009 the price ranged between US$ 200–1100 per kilogram and the produced quantities have not always met the demand.^213,219,221 Notable improvements of the production in plants have been made by conventional breeding and selection of high artemisinin genotypes; other attempts using plant cell and hairy root cultures had limited success so far.²²²

The biosynthesis of the isoprenoid 73 proceeds – like with almost all sesquiterpenes – via the cyclisation of farnesyl diphosphate 78 (FPP) generated in the mevalonate pathway (Scheme 5). Detailed knowledge of this pathway enabled metabolic engineering approaches in Artemisia annua.²²³ In one study, overexpression of endogenous farnesyl diphosphate synthase led to an increase of the artemisinin yield from 0.65% to 0.9% (based on dried plant material), indicating that the optimization of a single gene might not be sufficient for a multifold increase of yield.²²⁴

Scheme 5 Biosynthetic pathway of artemisinic acid 82 engineered into yeast strain EPY224. Genes from the S. cerevisiae mevalonate pathway directly up-regulated are highlighted in green, those indirectly upregulated in purple. Down-regulation of squalene synthase (ERG9) is highlighted in red. The biochemical pathway from farnesyl pyrophosphate 78 to artemisinic acid 82 cloned from A. annua is denoted with blue arrows. IPP: isopentenyl pyrophosphate. DMAPP (dimethyl allyl pyrophosphate. GPP: geranyl pyrophosphate. ADS: amorphadiene synthetase.

In different studies, a microbial production system for the biosynthetic intermediates amorphadiene 79 and artemisinic acid 82, followed by chemical conversion to 73, was explored as an alternative.²²⁵ The technology for this approach was developed in the Keasling laboratory.²²⁶ Initially, it was based on the biosynthesis of 79 in E. coli via heterologous expression of the mevalonate pathway. This pathway was split into two operons, the “upstream” operon MevT that converts Acetyl-CoA to mevalonate, and the “downstream” operon MBIS, transforming mevalonate further to FPP 78. Introduction of plasmids carrying these operons together with a plasmid carrying the gene for amorphadiene synthetase (ADS) into E. coli DH1 resulted in a strain that produced amorpha-4,11-diene 79 at an average titer of 27.4 g L⁻¹ after careful optimization of the fermentation process.^225,227 Ideally, a biotechnological process should comprise most of the biosynthesis pathway to the target product – in this case, artemisinic acid 82 – as the final result of the cascade of enzymatically controlled chemical transformations. However, CYP activities high enough for an efficient oxidation of amorpha-4,11-diene 79 to artemisinic acid 82 could not be realized in E. coli. As a consequence, the heterologous expression system was switched to baker's yeast. As outlined in Scheme 5, the artemisinic acid-producing strain Saccharomyces cerevisae EPY224 was created by combining an optimized endogenous pathway to 78 with the heterologous expression of the A. annua genes amorphadiene synthase (ADS), CYP71AV1 and its redox partner CPR (which oxidized 79 to 82).²²⁸ Key for a successful increase of product titers was to suppress carbon flux through competing pathways. In particular, high levels of 78 for the conversion to 79 were ensured by down-regulation of the gene ERG9 encoding for squalene synthetase, a pathway that led to depletion of 78. After careful optimization, a defined fed-batch process with titers of 25g L⁻¹ of artemisinic acid 82 was obtained that was ready for industrialization.²²⁹ The heterologous expression process, seen as a prime example for a rising discipline coined ‘synthetic biology’, was followed by the downstream chemical conversion of 82 to 73. It can be achieved in 2 steps by photo-oxidation of dihydroartemisinic acid 83 obtained by reduction of 82.²³⁰ A scaleable process was established in a 3-step sequence via the reaction of 84, a mixed anhydride of 83, with singlet oxygen that has been generated by irradiation of the acidic reaction mixture in the presence of catalytic amounts of tetraphenyl porphyrin (Scheme 6 and Fig. 7).^231,232 The sequence gave less by-products than the direct oxidation of 83, thereby completing the fully industrialized process for the production of 73. With 50–60 tons of 73 planned for 2014, the biotechnological and chemical process is supposed to complement the plant-derived processes, thereby minimizing fluctuations in supply and prices for this essential class of anti-malaria drugs.²³³ For orientation: the global demand for artemisinin in 2013 was estimated to be 180–200 tons during the Artemisinin Conference 2013 (January 15–16, Nairobi, Kenya).

Fig. 7 The photooxidation of 84 under production conditions (plant in Garessio, Italy).

Scheme 6 The scalable synthesis of artemisinin 73 from dihydro-artemisinic acid 83via photo-oxidation of mixed anhydrides.

Recently, a continuous-flow process for the conversion of 83 into 73 was published by the Seeberger group,²³⁴ which might further reduce the costs for artemisinin supply, if it can be demonstrated ultimately that the process is amenable to an industrial scale.²³⁵

6 Concluding remarks

While the classical fermentation of microorganisms followed by semisynthesis still plays a major role for successfully advancing natural products leads towards marketed drugs, the emergence of new technologies will accelerate research and development, and hopefully make under-exploited natural products with interesting biological features usable for clinical applications.

As outlined in this review, sophisticated total synthesis has to be considered as a realistic option for many, even highly complex structures. Significant contributions to natural product development are to be expected from genetic engineering and synthetic biology approaches; those can also be favorably combined or tightly entwined with synthetic steps. Novel, sophisticated delivery systems, such as antibody–drug conjugates, overcoming intrinsic toxicity and pharmacokinetic liabilities of valuable natural compounds, have recently provided a convincing clinical proof-of-concept and await a much broader and systematic exploration.

In summary, a broad and growing arsenal of methods is available today to advance natural products from leads to drugs, with no generic recipe for success and consequently no straightforward prediction of project duration and risk. This characteristic is regarded as a clear drawback from the perspective of planning-oriented operations, and at the same time as an attractive, adventurous challenge from an exploratory research perspective.

7 References

1. D. J. Newman and G. M. Cragg, J. Nat. Prod., 2012, 75, 311–335.
2. J. J. LaClair, Nat. Prod. Rep., 2010, 27, 969–995.
3. E. K. Schmitt, C. M. Moore, P. Krastel and F. Petersen, Curr. Opin. Chem. Biol., 2011, 15, 497–504.
4. N. Dixon, L. S. Wong, T. H. Geerlings and J. Micklefield, Nat. Prod. Rep., 2007, 24, 1288–1310.
5. F. E. Koehn and G. T. Carter, Nat. Rev. Drug Discovery, 2005, 4, 206–220.
6. M. S. Butler and M. A. Cooper, J. Antibiot., 2011, 64, 413–425.
7. M. S. Butler, Nat. Prod. Rep., 2008, 25, 475–516.
8. R. W. Hoffmann, Angew. Chem., Int. Ed., 2013, 52, 123–130.
9. G. T. Carter, Nat. Prod. Rep., 2011, 28, 1783–1789.
10. (a) H. B. Bode and R. Müller, Angew. Chem., Int. Ed., 2005, 44, 6828–6846; (b) C. Hertweck, Angew. Chem., Int. Ed., 2009, 48, 4688–4716.
11. K. Witting and R. D. Süssmuth, Curr. Drug Targets, 2011, 12, 1547–1559.
12. J. Kennedy, Nat. Prod. Rep., 2008, 25, 25–34.
13. A. Kirschning and F. Hahn, Angew. Chem., Int. Ed., 2012, 51, 4012–4022.
14. A. Kirschning, F. Taft and T. Knobloch, Org. Biomol. Chem., 2007, 5, 3245–3259.
15. M.-Q. Zhang, S. Gaisser, M. Nur-E-Alam, L. S. Sheehan, W. A. Voudsen, N. Gaitatzis, G. Peck, N. J. Coates, S. J. Moss, M. Radzom, T. A. Foster, R. M. Sheridan, M. A. Gregory, S. M. Roe, C. Prodromou, L. Pearl, S. M. Boyd, B. Wilkinson and C. J. Martin, J. Med. Chem., 2008, 51, 5494–5497.
16. F. E. Koehn, Med. Chem. Commun., 2012, 3, 854–865.
17. F. von Nussbaum, M. Brands, B. Hinzen, S. Weigand and D. Haebich, Angew. Chem., Int. Ed., 2006, 45, 5072–5129.
18. M. Pfister, J. M. Whaley, L. Zhang and J. F. List, Clin. Pharmacol. Ther., 2011, 89, 621–625.
19. S. J. Farber, E. Y. Berger and D. P. Earle, J. Clin. Invest., 1951, 30, 125–129.
20. Review: E. C. Chao and R. R. Henry, Nat. Rev. Drug Discovery, 2010, 9, 551–559.
21. Review: J. R. L. Ehrenkranz, N. G. Lewis, C. R. Kahn and J. Roth, Diabetes/Metab. Res. Rev., 2005, 21, 31–38.
22. P. G. Stiles and G. Lusk, Am. J. Physiol., 1903, 10, 61–79.
23. C. Archard and V. Delamare, Soc. Med. Des. Hôpiteaux, 1899, 379–393.
24. H. Chassis, N. Jolliffe and H. Smith, J. Clin. Invest., 1933, 12, 1083–1090.
25. L. Rossetti, D. Smith, G. I. Shulman, D. Papachristou and R. A. DeFronzo, J. Clin. Invest., 1987, 79, 1510–1515.
26. F. C. Alvaro and R. K. Crane, Biochim. Biophys. Acta, 1962, 56, 170–172.
27. H. D. Vick, D. F. Deidrich and K. Baumann, Am. J. Physiol., 1973, 224, 552–557.
28. (a) K. Amsler and J. Cook, Am. J. Physiol., Cell Physiol., 1982, 242, C94–C101; (b) M. A. Hediger, M. J. Coady, T. S. Ikeda and E. M. Wright, Nature, 1987, 330, 379–381; (c) W.-S. Lee, Y. Kanai, R. G. Wells and M. A. Hediger, J. Biol. Chem., 1994, 269, 12032–12039; (d) G. You, W.-S. Lee, E. J. G. Barros, Y. Kanai, T.-L. Huo, S. Khawaja, R. G. Wells, S. K. Nigam and M. A. Hediger, J. Biol. Chem., 1995, 270, 29365–29371; (e) T. Ohta, K.-J. Isselbacher and D. B. Rhoads, Mol. Cell. Biol., 1990, 10, 6491–6499; (f) M. A. Hediger, E. Turk and E. M. Wright, Proc. Natl. Acad. Sci. U. S. A., 1989, 86, 5748–5752; (g) Y. Kanai, W.-S. Lee, G. You, D. Brown and M. A. Hediger, J. Clin. Invest., 1994, 93, 397–404; (h) R. G. Wells, A. M. Pajor, Y. Kanai, E. Turk, E. M. Wright and M. A. Hediger, Am. J. Physiol., 1992, 263, F459–F465.
29. C. S. Hummel, C. Lu, J. Liu, C. Ghezzi, B. A. Hirayama, D. D. F. Loo, V. Kepe, J. R. Barrio and E. M. Wright, Am. J. Physiol.: Cell Physiol., 2012, 302, C373–C382.
30. (a) K. Tsujihara, M. Hongu, K. Saito, M. Inamasu, K. Arakawa, A. Oku and M. Matsumotu, Chem. Pharm. Bull., 1996, 44, 1174–1180; (b) M. Hongu, T. Tanaka, N. Funami, K. Saito, K. Arakawa, M. Matsumotu and K. Tsujihara, Chem. Pharm. Bull., 1998, 46, 22–33; (c) M. Hongu, N. Funami, Y. Takahashi, K. Saito, K. Arakawa, M. Matsumotu, H. Yamakita and K. Tsujihara, Chem. Pharm. Bull., 1998, 46, 1545–1555.
31. K. Tsujihara, M. Hongu, K. Saito, H. Kawanishi, K. Kuriyama, M. Matsumotu, A. Oku, K. Ueta, M. Tsuda and A. Saito, J. Med. Chem., 1999, 42, 5311–5324.
32. (a) D. F. Diedrich, Biochim. Biophys. Acta, 1963, 71, 688–700; (b) J. Hase, K. Kobayashi and R. Kobayashi, Chem. Pharm. Bull., 1973, 21, 1076–1079.
33. T. C. Hardman and S. W. Dubrey, Diabetes Ther., 2011, 2, 133–145.
34. Reviews: (a) E. C. Chao and R. R. Henry, Nat. Rev. Drug Discovery, 2010, 9, 551–559; W. N. Washburn, Expert Opin. Ther. Pat., 2012, 22, 483–494; (b) M. A. Abdul-Ghani, L. Norton and R. A. DeFronzo, Curr. Diabetes Rep., 2012, 12, 230–238.
35. M. Isaji, Kidney Int., 2011, 79, S14–S19.
36. (a) B. A. Ellsworth, W. Meng, M. Patel, R. N. Girotra, G. Wu, P. M. Sher, D. L. Hagan, M. T. Obermeier, W. G. Humphreys, J. G. Robertson, A. Wang, S. Han, T. L. Waldron, N. M. Morgan, J. M. Whaley and W. N. Washburn, Bioorg. Med. Chem. Lett., 2008, 18, 4770–4773; (b) W. N. Washburn, J. Med. Chem., 2009, 52, 1785–1794.
37. W. Meng, B. A. Ellsworth, A. A. Nirschl, P. J. McCann, M. Patel, R. N. Girotra, G. Wu, P. M. Sher, E. P. Morrison, S. A. Biller, R. Zahler, P. P. Deshpande, A. Pullockaran, D. L. Hagan, N. Morgan, J. R. Taylor, M. T. Obermeier, W. G. Humphreys, A. Khanna, L. Discenza, J. G. Robertson, A. Wang, S. Han, J. R. Wetterau, E. B. Janovitz, O. P. Flint, J. M. Whaley and W. N. Washburn, J. Med. Chem., 2008, 51, 1145–1149.
38. Review: J. Liu and T. Lee, Annu. Rep. Med. Chem., 2011, 46, 103–115.
39. S. Nomura, S. Sakamaki, M. Hongu, E. Kawanishi, Y. Koga, T. Sakamoto, Y. Yamamoto, K. Ueta, H. Kimata, K. Nakayama and M. Tsuda-Tsukimoto, J. Med. Chem., 2010, 53, 6355–6360.
40. (a) G. Luippold, T. Klein, M. Mark and R. Grempler, Diabetes, Obes. Metab., 2012, 14, 601–607; (b) I. Aires and J. Calado, Curr. Opin. Invest. Drugs, 2010, 11, 1182–1190.
41. (a) M. Imamura, K. Nakanishi, T. Suzuki, K. Ikegai, R. Shiraki, T. Ogiyama, T. Murakami, E. Kurosaki, A. Noda, Y. Kobayashi, M. Yokota, T. Koide, K. Kosakai, Y. Ohkura, M. Takeuchi, H. Tomiyama and M. Ohta, Bioorg. Med. Chem., 2012, 20, 3263–3279; (b) A. Tahara, E. Kurosaki, M. Yokono, D. Yamajuku, R. Kihara, Y. Hayashizaki, T. Takasu, M. Imamura, L. Qun, H. Tomiyama, Y. Kobayashi, A. Noda, M. Sasamata and M. Shibasaki, Naunyn-Schmiedebergs Arch. Pharmacol., 2012, 385, 423–436; (c) S. A. Veltkamp, T. Kadokura, W. J. J. Krauwinkel and R. A. Smulders, Clin. Drug Investig., 2011, 31, 839–851; (d) S. L. Schwartz, B. Akinlade, S. Klasen, D. Kowalski, W. Zhang and W. Wilpshaar, Diabetes Technol. Ther., 2011, 13, 1219–1227.
42. (a) Y. Ohtake, T. Sato, T. Kobayashi, M. Nishimoto, N. Taka, K. Takano, K. Yamamoto, M. Ohmori, M. Yamaguchi, K. Takami, S.-Y. Yeu, K.-H. Ahn, H. Matsuoka, K. Morikawa, M. Suzuki, H. Hagita, K. Ozawa, K. Yamaguchi, M. Kato and S. Ikeda, J. Med. Chem., 2012, 55, 7828–7840; (b) M. Suzuki, K. Honda, M. Fukazawa, K. Ozawa, H. Hagita, T. Kawai, M. Takeda, T. Yata, M. Kawai, T. Fukuzawa, T. Kobayashi, T. Sato, Y. Kawabe and S. Ikeda, J. Pharmacol. Exp. Ther., 2012, 341, 692–701.
43. (a) P. Cole, M. Vicente and R. Castañer, Drugs Future, 2008, 33, 745–751; N. Katsiki, N. Papanas and D. P. Mikhailidis, Expert Opin. Invest. Drugs, 2010, 19, 1581–1589; (b) R. K. Ghosh, S. M. Ghosh, S. Chawla and S. A. Jasdanwala, J. Clin. Pharmacol., 2012, 52, 457–463; (c) J. E. Gerich and A. Bastien, Expert Rev. Clin. Pharmacol., 2011, 4, 669–683.
44. C. Foote, V. Perkovic and B. Neal, Diabetes Vasc. Dis. Res., 2012, 9, 117–123.
45. D. Pelletier and D. A. Hafler, N. Engl. J. Med., 2012, 366, 339–347.
46. T. Fujita, K. Inoue, S. Yamamoto, T. Ikumoto, S. Sasaki, R. Toyama, K. Shiba, Y. Hoshino and T. Okumoto, J. Antibiot., 1994, 47, 208–215.
47. Review: C. R. Strader, C. J. Pearce and N. H. Oberlies, J. Nat. Prod., 2011, 74, 900–907.
48. T. Fujita, M. Yoneta, R. Hirose, S. Sasaki, K. Inoue, M. Kiuchi, S. Hirase, K. Adachi, M. Arita and K. Chiba, Bioorg. Med. Chem. Lett., 1995, 5, 847–852.
49. T. Fujita, N. Hamamichi, M. Kiuchi, T. Matsuzaki, Y. Kitao, K. Inoue, R. Hirose, M. Yoneta, S. Sasaki and K. Chiba, J. Antibiot., 1996, 49, 846–853.
50. H. Kataoka, K. Sugahara, K. Shimano, K. Teshima, M. Koyama, A. Fukunari and K. Chiba, Cell. Mol. Immunol., 2005, 2, 439–448.
51. K. Chiba, Y. Yanagawa, Y. Masubuchi, H. Kataoka, T. Kawaguchi, M. Ohtsuki and Y. Hoshino, J. Immunol., 1998, 160, 5037–5044.
52. J. Ingwersen, O. Aktas, P. Kuery, B. Kieseier, A. Boyko and H.-P. Hartung, Clin. Immunol., 2012, 142, 15–24.
53. Reviews: (a) V. Brinkmann, A. Billich, T. Baumruker, P. Heining, R. Schmouder, G. Francis, S. Aradhye and P. Burtin, Nat. Rev. Drug Discovery, 2010, 9, 883–897; (b) J. Chun and H.-P. Hartung, Clin. Neuropharmacol., 2010, 33, 91–101; (c) C. W. Lee, J. W. Choi and J. Chun, Arch. Pharmacal Res., 2010, 33, 1567–1574.
54. (a) V. Brinkmann, M. D. Davis, C. E. Heise, R. Albert, S. Cottens, R. Hof, C. Bruns, E. Prieschl, T. Basumruker, P. Hiestand, C. A. Foster, M. Zollinger and K. A. Lynch, J. Biol. Chem., 2002, 277, 21453–21457; (b) S. Mandala, R. Hajdu, J. Bergstrom, E. Quackenbush, J. Xie, J. Milligan, R. Thornton, G. J. Shei, D. Card, C. Keohane, M. Rosenbach, J. Hale, C. L. Lynch, K. Rupprect, W. Parsons and H. Rosen, Science, 2002, 296, 346–349.
55. Review: T. Mutoh, R. Rivera and J. Chun, Br. J. Pharmacol., 2012, 165, 829–844.
56. (a) M. Matloubian, C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, M. L. Allende, R. L. Proia and J. G. Cyster, Nature, 2004, 427, 355–360; (b) F. Mullershausen, F. Zecri, C. Cetin, A. Billich, D. Guerini and K. Seuwen, Nat. Chem. Biol., 2009, 5, 428–434.
57. (a) V. Brinkmann, J. G. Cyster and T. Hla, Am. J. Transplant., 2004, 4, 1019–1025; (b) R. Pappu, S. R. Schwab, I. Cornelissen, J. P. Pereira, J. B. regard, Y. Xu, E. Camerer, Y.-W. Zheng, Y. Huang, J. G. Cyster and S. R. Coughlin, Science, 2007, 316, 295–298; (c) T. H. M. Pham, P. Baluk, Y. Xu, I. Grigorova, A. J. Bankovich, R. Pappu, S. R. Coughlin, D. M. McDonald, S. R. Schwab and J. G. Cyster, J. Exp. Med., 2010, 207, 17–27.
58. (a) D. Uemura, K. Takahashi and T. Yamamoto, J. Am. Chem. Soc., 1985, 107, 4796–4798; (b) Y. Hirata and D. Uemura, Pure Appl. Chem., 1986, 58, 701–710.
59. G. R. Pettit, R. Tan and M. D. Williams, J. Org. Chem., 1993, 58, 2538–2543.
60. M. Litaudon, J. B. Hart, J. W. Blunt, R. J. Lake and M. H. G. Munro, Tetrahedron Lett., 1994, 35, 9435–9438.
61. G. R. Pettit, C. L. Herald, M. R. Boyd, J. E. Leed, C. Dufresne, D. L. Doubek, J. M. Schmidt, R. L. Cerny, J. N. A. Hooper and K. C. Rutzler, J. Med. Chem., 1991, 34, 3339–3340.
62. R. Bai, K. D. Paull, C. L. Heraldy, L. Malspeis, G. R. Pettit and E. Hamel, J. Biol. Chem., 1991, 266, 15882–15889.
63. (a) R. F. Ludueña, M. C. Roach, V. Prasad and G. R. Pettit, Biochem. Pharmacol., 1993, 45, 421–427; (b) D. Dabydeen, J. C. Burnett, R. Bai, P. Verdier-Pinard, S. J. H. Hickford, G. R. Pettit, J. W. Blunt, M. H. G. Munro, R. Gussio and E. Hamel, Mol. Pharmacol., 2006, 70, 1866–1875; (c) M. A. Jordan, K. Kamath, T. Manna, T. Okouneva, H. P. Miller, C. Davis, B. A. Littlefield and L. Wilson, Mol. Cancer Ther., 2005, 4, 1086–95.
64. B. Overmoyer, Clin. Breast Cancer, 2008, 8, S61–S70.
65. Case history: M. J. Yu, W. Zheng, B. M. Seletsky, B. A. Littlefield and Y. Kishi, Annu. Rep. Med. Chem., 2011, 46, 227–241.
66. M. H. G. Munro, J. W. Blunt, E. J. Dumdei, S. J. H. Hickford, R. E. Lill, S. Li, C. N. Battershill and A. R. Duckworth, J. Biotechnol., 1999, 70, 15–25.
67. T. D. Aicher, K. R. Buszek, F. G. Fang, C. J. Forsyth, S. H. Jung, Y. Kishi, M. C. Matelich, P. M. Scola, D. M. Spero and S. K. Yoon, J. Am. Chem. Soc., 1992, 114, 3162–3164.
68. Review: K. L. Jackson, J. A. Henderson and A. J. Philips, Chem. Rev., 2009, 109, 3044–3079.
69. M. J. Towle, K. A. Salvato, B. F. Wels, K. K. Aalfs, W. Zheng, B. M. Seletsky, X. Zhu, B. M. Lewis, Y. Kishi, M. J. Yu and B. A. Littlefield, Cancer Res., 2011, 71, 496–505.
70. (a) B. M. Seletsky, Y. Wang, L. D. Hawkins, M. H. Palme, G. J. Habgood, L. V. DiPietro, M. J. Towle, K. A. Salvato, B. F. Wels, K. K. Aalfs, Y. Kishi, B. A. Littlefield and M. J. Yu, Bioorg. Med. Chem. Lett., 2004, 14, 5547–5550; (b) W. Zheng, B. M. Seletsky, M. H. Palme, P. J. Lydon, L. A. Singer, C. E. Chase, C. E. Lemelin, Y. Shen, H. Davis, L. Tremblay, M. J. Towle, K. A. Salvato, B. F. Wels, K. K. Aalfs, Y. Kishi, B. A. Littlefield and M. J. Yu, Bioorg. Med. Chem. Lett., 2004, 14, 5551–5554.
71. (a) D.-S. Kim, C. G. Dong, J. T. Kim, H. Guo, J. Huang, P. S. Tiseni and Y. Kishi, J. Am. Chem. Soc., 2009, 131, 15642–15646; (b) B. Austad, C. E. Chase and F. G. Fang, US Pat. Appl., 2007, 2007/0244187–A1; (c) C. Chase, A. Endo, F. G. Fang and J. Li, US Pat. Appl., 2009, 2009/0198074–A1; (d) K. Inanaga, M. Kuboto, A. Kayana and K. Tigami, US Pat. Appl, 2009, 2009/0203771–A1; (e) Y. Wang, Drugs Future, 2007, 32, 681–698.
72. J. Cortes, J. O'Shaughnessy, D. Loesch, J. L. Blum, L. T. Vahdat, K. Petrakova, P. Chollet, A. Manikasd, V. Diéras, T. Delozier, V. Vladimirov, F. Cardoso, H. Koh, P. Bougnoux, C. E. Dutcus, S. Seegobin, D. Mir, N. Meneses, J. Wanders and C. Twelves, Lancet, 2011, 377, 914–923.
73. P. Aftimos and A. Awada, Adv. Ther., 2011, 28, 973–985.
74. (a) B. M. Duggar, Ann. N. Y. Acad. Sci., 1948, 51, 177–181; (b) R. W. Broschard, A. C. Dornbush, S. Gordon, B. L. Hutchings, A. R. Kohler, G. Krupka, S. Kushner, D. V. Lefemine and C. Pidacks, Science, 1949, 109, 199–200.
75. L. H. Conover, W. T. Moreland, A. R. English, C. R. Stephens and F. J. J. Pilgrim, J. Am. Chem. Soc., 1953, 75, 4622–4623.
76. (a) I. Chopra and M. Roberts, Microbiol. Mol. Biol. Rev., 2001, 65, 232–260; (b) M. L. Nelson and M. Y. Ismail, in Comprehensive Medicinal Chemistry II, ed. J. B. Taylor and D. J. Triggle, Elsevier, Oxford, 2007, vol. 7, pp. 597–628.
77. (a) J. E. Frampton and M. P. Curran, Drugs, 2005, 65, 2623–2635; (b) H. Giamarellou and G. Poulakou, Expert Opin. Drug Metab. Toxicol., 2011, 7, 1459–1470.
78. (a) L. H. Conover, K. Butler, J. D. Johnston, J. J. Korst and R. B. Woodward, J. Am. Chem. Soc., 1962, 84, 3222–3224; (b) J. J. Korst, J. D. Johnston, K. Butler, E. J. Bianco, L. H. Conover and R. B. Woodward, J. Am. Chem. Soc., 1968, 90, 439–457.
79. Reviewed in: K. C. Nicolaou and J. S. Chen, Classics in Total Synthesis III, Wiley-VCH, Weinheim, 2011, pp. 345–375.
80. (a) H. Muxfeldt and W. Rogalski, J. Am. Chem. Soc., 1965, 87, 933–934; (b) H. Muxfeldt, G. Hardtmann, F. Kathawala, E. Vedejs and J. B. Mooberry, J. Am. Chem. Soc., 1968, 90, 6534–6536; (c) H. Muxfeldt, H. Döpp, J. E. Kaufman, J. Schneider, P. E. Hansen, A. Sasaki and T. Geiser, Angew. Chem., 1973, 85, 508–510; (d) H. Muxfeldt, G. Haas, G. Hardtmann, F. Kathawala, J. B. Mooberry and E. Vedejs, J. Am. Chem. Soc., 1979, 101, 689–701.
81. (a) R. Kirchlechner and W. Rogalski, Tetrahedron Lett., 1980, 21, 247–250; (b) M. Bakhtiar and S. Selwyn, J. Antimicrob. Chemother., 1983, 11, 291; (c) I. Chopra, Antimicrob. Agents Chemother., 1994, 38, 637–640.
82. (a) M. G. Charest, C. D. Lerner, J. D. Brubaker, D. R. Siegel and A. G. Myers, Science, 2005, 308, 395–398; (b) M. G. Charest, D. R. Siegel and A. G. Myers, J. Am. Chem. Soc., 2005, 127, 8292–8293.
83. Review: M. D. Burke, Nat. Chem. Biol., 2009, 5, 77–79.
84. (a) C. Sun, Q. Wang, J. D. Brubaker, P. M. Wright, C. D. Lerner, K. Noson, M. Charest, D. R. Siegel, Y.-M. Wang and A. G. Myers, J. Am. Chem. Soc., 2008, 130, 17913–17927; (b) D. A. Kummer, D. Li, A. Dion and A. G. Myers, Chem. Sci., 2011, 2, 1710–1718.
85. P. M. Wright and A. G. Myers, Tetrahedron, 2011, 67, 9853–9869.
86. (a) R. H. Connamacher and H. G. Mandel, Biochem. Biophys. Res. Commun., 1965, 20, 98–103; (b) D. E. Brodersen, W. M. Clemons, A. P. Carter, R.-J. Morgan-Warren, B. T. Wimberly and V. Ramakrishnan, Cell, 2000, 103, 1143–1154; (c) J. Wirmer and E. Westof, Methods Enzymol., 2006, 415, 180–202.
87. (a) X.-Y. Xiao, D. K. Hunt, J. Zhou, R. B. Clark, N. Dunwoody, C. Fyfe, T. H. Grossman, W. J. O'Brien, L. Plamondon, M. Rönn, C. Sun, W.-Y. Zhang and J. A. Sutcliffe, J. Med. Chem., 2012, 55, 597–605; (b) R. B. Clark, D. K. Hunt, M. He, C. Achorn, C.-L. Chen, Y. Deng, C. Fyfe, T. H. Grossman, P. C. Hogan, W. J. O'Brien, L. Plamondon, M. Rönn, J. A. Sutcliffe, Z. Zhu and X.-Y. Xiao, J. Med. Chem., 2012, 55, 606–622.
88. T. H. Grossman, A. L. Starosta, C. Fyfe, W. O'Brien, D. M. Rothstein, A. Mikolajka, D. N. Wilson and J. A. Sutcliffe, Antimicrob. Agents Chemother., 2012, 56, 2559–2564.
89. H. W. Boucher, G. H. Talbot, J. S. Bradley, J. E. Edwards, D. Gilbert, L. B. Rice, M. Scheld, B. Spellberg and J. Bartlett, Clin. Infect. Dis., 2009, 48, 1–12.
90. J. Merlino, M. Leroi, R. Bradbury, D. Veal and C. Harbour, J. Clin. Microbiol., 2000, 38, 2378–2380.
91. (a) A. H. C. Uttley, C. H. Collins, J. Naidoo and R. C. George, Lancet, 1988, 331, 57–58; (b) J. May, K. Shannon, A. King and G. French, J. Antimicrob. Chemother., 1998, 42, 189–197; (c) H. M. Ziglam and R. G. Finch, Clin. Microbiol. Infect., 2001, 7(4), 53–65; (d) D. M. Sievert, M. L. Boulton, G. Stoltman, D. Johnson, M. G. Stobierski, F. P. Downes, P. A. Somsel, J. T. Rudrik, W. Brown, W. Hafeez, T. Lundstrom, E. Flanagan, R. Johnson, J. Mitchell and S. Chang, MMWR Morbity Mortality Weekly Rep., 2002, 51, 565–567; (e) S. Chang, D. M. Sievert, J. C. Hageman, M. L. Boulton, F. C. Tenover, F. P. Downes, S. Shah, J. T. Rudrik, G. R. Pupp, W. J. Brown, D. Cardo and S. K. Fridkin, N. Engl. J. Med., 2003, 348, 1342–1347; (f) L. M. Weigel, D. B. Clewell, S. R. Gill, N. C. Clark, L. K. McDougal, S. E. Flannagan, J. F. Kolonay, J. Shetty, G. E. Killgore and F. C. Tenover, Science, 2003, 302, 1569–1571.
92. T. D. H. Bugg, G. D. Wright, S. Dutka-Malen, M. Arthur, P. Courvalin and C. T. Walsh, Biochemistry, 1991, 30, 10408–10415.
93. Reviews: (a) D. H. Williams and B. Bardsley, Angew. Chem., Int. Ed., 1999, 38, 1172–1193; (b) K. C. Nicolaou, C. N. C. Boddy, S. Bräse and N. Winssinger, Angew. Chem., Int. Ed., 1999, 38, 2096–2152.
94. Review: D. Kahne, C. Leimkuhler, W. Lu and C. Walsh, Chem. Rev., 2005, 105, 425–448.
95. (a) F. J. Marshall, J. Med. Chem., 1965, 8, 18–22; (b) D. H. Williams and J. R. Kalman, J. Am. Chem. Soc., 1977, 99, 2768–2774; (c) G. M. Sheldrick, P. G. Jones, O. Kennard, D. H. Williams and G. A. Smith, Nature, 1978, 271, 223–225; (d) C. M. Harris and T. M. Harris, J. Am. Chem. Soc., 1982, 104, 4293–4295.
96. (a) D. A. Evans, M. R. Wood, B. W. Trotter, T. I. Richardson, J. C. Barrow and J. L. Katz, Angew. Chem., Int. Ed., 1998, 37, 2700–2704; (b) K. C. Nicolaou, H. J. Mitchell, N. F. Jain, N. Winssinger, R. Hughes and T. Bando, Angew. Chem., Int. Ed., 1999, 38, 240–244; (c) D. L. Boger, S. Miyazaki, S. H. Kim, J. H. Wu, O. Loiseleur and S. L. Castle, J. Am. Chem. Soc., 1999, 121, 3226–3227.
97. (a) B. M. Crowley and D. L. Boger, J. Am. Chem. Soc., 2006, 128, 2885–2892; (b) J. Xie, J. G. Pierce, R. C. James, A. Okano and D. L. Boger, J. Am. Chem. Soc., 2011, 133, 13946–13949; (c) J. Xie, A. Okano, J. G. Pierce, R. C. James, S. Stamm, C. M. Crane and D. L. Boger, J. Am. Chem. Soc., 2012, 134, 1284–1297.
98. T. I. Nicas, M. L. Zeckel and D. K. Braun, Trends Microbiol., 1997, 5, 240–249.
99. M. R. Leadbetter, S. M. Adams, B. Bazzini, P. R. Fatheree, D. E. Karr, K. M. Krause, B. M. T. Lam, M. S. Linsell, M. B. Nodwell, J. L. Pace, K. Quast, J.-P. Shaw, E. Soriano, S. G. Trapp, J. D. Villena, T. X. Wu, B. G. Christensen and J. K. Judice, J. Antibiot., 2004, 57, 326–336.
100. N. E. Allen, Anti-Infect. Agents Med. Chem., 2010, 9, 23–47.
101. G. R. Corey, M. E. Stryjewski, W. Weyenberg, U. Yasothan and P. Kirkpatrick, Nat. Rev. Drug Discovery, 2009, 8, 929–930.
102. P.-A. Ashford and S. P. Bew, Chem. Soc. Rev., 2012, 41, 957–978.
103. (a) D. L. Higgins, R. Chang, D. V. Debabov, J. Leung, T. Wu, K. M. Krause, E. Sandvik, J. M. Hubbard, K. Kaniga, D. E. Schmidt, Q. Gao, R. T. Cass, D. E. Karr, B. M. Benton and P. P. Humphrey, Antimicrob. Agents Chemother., 2005, 49, 1127–1134; (b) Y. Song, C. S. Lunde, B. M. Benton and B. J. Wilkinson, Antimicrob. Agents Chemother., 2012, 56, 3157–3164; (c) S. J. Kim, L. Cegelski, D. Stueber, M. Singh, E. Dietrich, K. S. E. Tanaka, T. R. Parr, A. R. Far and J. Schaefer, J. Mol. Biol., 2008, 377, 281–293; (d) M. Jeya, H.-J. Moon, K.-M. Lee, I.-W. Kim and J.-K. Lee, Curr. Pharm. Biotechnol., 2011, 12, 1194–1204.
104. J. L. Pace and J. K. Judice, Curr. Opin. Invest. Drugs, 2005, 6, 216–225.
105. K. A. Lyseng-Williamson and S. K. A. Blick, Drugs, 2009, 69, 2607–2620.
106. Reviews: (a) D. Abbanat, B. Morrow and K. Bush, Curr. Opin. Pharmacol., 2008, 8, 582–592; (b) M. T. Guskey and B. Tsuji, Pharmacotherapy, 2010, 30, 80–94; (c) F. Van Bambeke, Y. Van Laethem, P. Courvalin and P. M. Tulkens, Drugs, 2004, 64, 913–936.
107. Reviews: (a) S. C.-A. Chen, M. A. Slavin and T. C. Sorrell, Drugs, 2011, 71, 11–41; (b) S. L. Holt and R. H. Drew, Am. J. Health-Syst. Pharm., 2011, 68, 1207–1220.
108. D. W. Denning, Lancet, 2003, 362, 1142–1151.
109. F. Benz, F. Knüsel, J. Nüesch, H. Treichler, W. Voser, R. Nyfeler and W. Keller-Schierlein, Helv. Chim. Acta, 1974, 57, 2459–2477.
110. Review: M. B. Kurtz and J. H. Rex, Adv. Protein Chem., 2001, 56, 423–475.
111. L. D. Boeck, D. S. Fukuda, B. J. Abbott and M. Debono, J. Antibiot., 1989, 42, 382–388.
112. (a) M. Debono, B. J. Abbott, J. R. Turner, L. C. Howard, R. S. Gordee, A. S. Hunt, M. Barnhart, R. M. Molloy, K. E. Willard, D. S. Fukuda, T. F. Butler and D. J. Zeckner, Ann. N. Y. Acad. Sci., 1988, 544, 152–167; (b) M. Debono, B. J. Abbott, D. S. Fukuda, M. Barnhart, K. E. Willard, R. M. Molloy, K. H. Michel, J. R. Turner, T. F. Butler and A. H. Hunt, J. Antibiot., 1989, 42, 389–397.
113. (a) T. J. Walsh, J. W. Lee, P. Kelly, J. Bacher, J. Lecciones, V. Thomas, C. Lyman, D. Coleman, R. Gordee and P. A. Pizzo, Antimicrob. Agents Chemother., 1991, 35, 1321–1328; (b) J. G. Sundelof, R. Hajdu, W. J. Cleare, J. Onishi and H. Kropp, Antimicrob. Agents Chemother., 1992, 36, 607–610; (c) N. H. Georgopapadakou and T. J. Walsh, Antimicrob. Agents Chemother., 1996, 40, 279–291.
114. (a) M. L. Coen, C. G. Lerner, J. O. Capobianco and R. C. Goldman, Microbiology, 1994, 140, 2229–2237; (b) D. J. Frost, K. Brandt, J. Capobianco and R. Goldman, Microbiology, 1994, 140, 2239–2246; (c) D. Frost, K. Brandt, C. Estill and R. Goldman, FEMS Microbiol. Lett., 1997, 146, 255–261.
115. M. Debono, W. W. Turner, L. LaGrandeur, F. J. Burkhardt, J. S. Nissen, K. K. Nichols, M. J. Rodriguez, M. J. Zweifel, D. J. Zeckner, R. S. Gordee, J. Tang and T. R. Parr, J. Med. Chem., 1995, 38, 3271–3281.
116. (a) R. E. Schwartz, R. A. Giacobbe, J. A. Bland and R. L. Monaghan, J. Antibiot., 1989, 42, 163–167; (b) C. F. Wichmann, J. M. Liesch and R. E. Schwarz, J. Antibiot., 1989, 42, 168–173; R. A. Fromtling and G. K. Abruzzo, J. Antibiot., 1989, 42, 174–178; (c) S. A. Morris, R. E. Schwartz, D. F. Sesin, P. Masurekar, T. C. Hallada, D. M. Schmatz, K. Bartizal, O. D. Hensens and D. L. Zink, J. Antibiot., 1994, 47, 755–764.
117. (a) R. E. Schwartz, D. F. Sesin, H. Joshua, K. E. Wilson, A. J. Kempf, K. A. Goklen, D. Kuehner, P. Gailliot, C. Gleason, R. White, E. Inamine, G. Bills, P. Salmon and L. Zitano, J. Antibiot., 1992, 45, 1853–1866; (b) P. S. Masurekar, J. M. Fountoulakis, T. C. Hallada, M. S. Sosa and L. Kaplan, J. Antibiot., 1992, 45, 1867–1874.
118. J. M. Balkovec, R. M. Black, M. L. Hammond, J. V. Heck, R. A. Zambias, G. Abruzzo, K. Bartizal, H. Kropp, C. Trainor, R. E. Schwartz, D. C. McFadden, K. H. Nollstadt, L. A. Pittarelli, M. A. Powles and D. M. Schmatz, J. Med. Chem., 1992, 35, 194–198.
119. (a) Y. Ohfune and N. Kurokawa, J. Am. Chem. Soc., 1986, 108, 6041–6043; (b) Y. Ohfune and N. Kurokawa, J. Am. Chem. Soc., 1986, 108, 6043–6045; (c) D. A. Evans and A. E. Weber, J. Am. Chem. Soc., 1987, 109, 7151–7157.
120. R. A. Zambias, M. L. Hammond, J. V. Heck, K. Bartizal, C. Trainor, G. Abruzzo, D. M. Schmatz and K. M. Nollstadt, J. Med. Chem., 1992, 35, 2843–2855.
121. J. M. Balkovec and R. M. Black, Tetrahedron Lett., 1992, 33, 4529–4532.
122. F. A. Bouffard, R. A. Zambias, J. F. Dropinski, J. M. Balkovec, M. L. Hammond, G. K. Abruzzo, K. F. Bartizal, J. A. Marrinan, M. B. Kurtz, D. C. McFadden, K. H. Nollstadt, M. A. Pauls and D. M. Schmatz, J. Med. Chem., 1994, 37, 222–225.
123. D. Barrett, Biochim. Biophys. Acta, Mol. Basis Dis., 2002, 1587, 224–233.
124. (a) T. Iwamoto, A. Fujie, K. Sakamoto, Y. Tsurumi, N. Shigematsu, M. Yamashita, S. Hashimoto, M. Okuhara and M. Kohsaka, J. Antibiot., 1994, 47, 1084–1091; (b) T. Iwamoto, A. Fujie, K. Nitta, S. Hashimoto, M. Okuhara and M. Kohsaka, J. Antibiot., 1994, 47, 1092–1097.
125. A. Fujie, Pure Appl. Chem., 2007, 79, 603–614.
126. M. Debono and R. S. Gordee, Annu. Rev. Microbiol., 1994, 48, 471–497.
127. (a) A. Fujie, T. Iwamoto, B. Sato, H. Muramatsu, C. Kasahara, T. Furuta, Y. Hori, M. Hino and S. Hashimoto, Bioorg. Med. Chem. Lett., 2001, 11, 399–402; (b) M. Tomishima, H. Ohki, A. Yamada, K. Maki and F. Ikeda, Bioorg. Med. Chem. Lett., 2008, 18, 1474–1477; (c) M. Tomishima, H. Ohki, A. Yamada, K. Maki and F. Ikeda, Bioorg. Med. Chem. Lett., 2008, 18, 2886–2890.
128. M. Tomishima, H. Ohki, A. Yamada, H. Takasugi, K. Maki, S. Tawara and H. Tanaka, J. Antibiot., 1999, 52, 674–676.
129. (a) W. Kim and J. M. Egan, Pharmacol. Rev., 2008, 60, 470–512; (b) J. J. Holst, Physiol. Rev., 2007, 87, 1409; (c) J. J. Holst, T. Vilsbøll and C. F. Deacon, Mol. Cell. Endocrinol., 2009, 297, 127–136; (d) D. J. Drucker and M. A. Nauck, Lancet, 2006, 368, 1696–1705.
130. (a) W. Creutzfeld, Diabetologia, 1979, 16, 75–85; (b) S. Mudaliar and R. R. Henry, Diabetologia, 2012, 55, 1865–1868.
131. J. J. Meier, W. E. Schmidt and H.-H. Klein, Internist, 2007, 48, 698–707.
132. B. L. Furman, Toxicon, 2012, 59, 464–471.
133. A. J. Garber, Expert Opin. Invest. Drugs, 2012, 21, 45–57.
134. T. Vilsboll, H. Agerso, T. Krarup and J. J. Holst, J. Clin. Endocrinol. Metab., 2003, 88, 220–224.
135. (a) R. E. van Genugten, D. H. van Raalte and M. Diamant, Diabetes Res. Clin. Pract., 2009, 86S, S26–S34; (b) J. J. Meier, Nat. Rev. Endocrinol., 2012, 8, 728–742.
136. D. J. Drucker, A. Dritselis and P. Kirkpatrick, Nat. Rev. Drug Discovery, 2010, 9, 267–268.
137. J. Eng, W. A. Kleinman, L. Singh, G. Singh and J. P. Raufman, J. Biol. Chem., 1992, 267, 7402–7405.
138. J. P. Raufman, R. T. Jensen, V. E. Sutcliff, J. J. Pisano and J. D. Gardner, Am. J. Physiol., Gastrointest. Liver Physiol., 1982, 242, G470–G474.
139. J. P. Raufman, L. Singh, G. Singh and J. Eng, J. Biol. Chem., 1992, 267, 21432–21437.
140. R. Göke, H. C. Fehmann, T. Linn, H. Schmidt, M. Krause, J. Eng and B. Göke, J. Biol. Chem., 1993, 268, 19650–19655.
141. European Medicines agency, Byetta: EPAR-Scientific Discussion, 2006, http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Scientific_Discussion/human/000698/WC500051842.pdf.
142. H. Reuter and E. Erdmann, Dtsch. Med. Wochenschr., 2007, 132, 571–574.
143. (a) C. Vézina, A. Kudelski and S. N. Sehgal, J. Antibiot., 1975, 28, 721–726; (b) S. N. Sehgal, H. Baker and C. Vézina, J. Antibiot., 1975, 28, 727–732.
144. (a) D. C. N. Swindells, P. S. White and J. A. Findlay, Can. J. Chem., 1978, 56, 2491–2492; (b) J. A. Findlay and L. Radics, Can. J. Chem., 1980, 58, 579–590; (c) J. B. McAlpine, S. J. Swanson, M. Jackson and D. N. Whittern, J. Antibiot., 1991, 44, 688–690.
145. Review: S. R. Park, Y. J. Yoo, Y.-H. Ban and Y. J. Yoon, J. Antibiot., 2010, 63, 434–441.
146. Review: E. I. Graziani, Nat. Prod. Rep., 2009, 26, 602–609.
147. H. Baker, A. Sidorowicz, S. N. Sehgal and C. Vézina, J. Antibiot., 1978, 31, 539–545.
148. R. R. Martel, J. Klicius and S. Galet, Can. J. Physiol. Pharmacol., 1977, 55, 48–51.
149. (a) R. Y. Calne, D. S. Collier, S. Lim, S. G. Pollard, A. Samaan, D. J. White and S. Tiru, Lancet, 1989, 334, 227; (b) B. D. Kahan, J. Y. Chang and S. N. Sehgal, Transplantation, 1991, 52, 185–191; (c) C. J. E. Watson, P. J. Friend, N. V. Jamieson, T. W. Frick, G. Alexander, A. E. Gimson and R. Calne, Transplantation, 1999, 67, 505–509; (d) R. Y. Calne, Transplant. Proc., 2003, 35, S15–S17.
150. (a) S. J. Collier, Curr. Opin. Immunol., 1990, 2, 854–858; (b) S. L. Schreiber, Science, 1991, 251, 283–287.
151. J. Heitman, N. R. Movva and M. N. Hall, Science, 1991, 253, 905–909.
152. (a) E. J. Brown, M. W. Albers, T. B. Shin, K. Ichikawa, C. T. Keith, W. S. Lane and S. L. Schreiber, Nature, 1994, 369, 756–758; (b) D. M. Sabatini, H. Erdjument-Bromage, M. Lui, P. Tempst and S. H. Snyder, Cell, 1994, 78, 35–43; (c) M. I. Chiu, H. Katz and V. Berlin, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 12574–12578; (d) C. J. Sabers, M. M. Martin, G. J. Brunn, J. M. Williams, F. J. Dumont, G. Wiederrecht and R. T. Abraham, J. Biol. Chem., 1995, 270, 815–822.
153. J. Choi, J. Chen, S. L. Schreiber and J. Clardy, Science, 1996, 273, 239–242.
154. Reviews: (a) S. N. Sehgal, Clin. Biochem., 1998, 31, 335–340; (b) X. M. Ma and J. Blenis, Nat. Rev. Mol. Cell Biol., 2009, 10, 307–318.
155. C. P. Eng, S. N. Sehgal and C. Vézina, J. Antibiot., 1984, 37, 1231–1237.
156. J. J. Gibbons, R. T. Abraham and K. Yu, Semin. Oncol., 2009, 36, S3–S17.
157. J. P. Boni, B. Hug, C. Leister and D. Sonnichsen, Semin. Oncol., 2009, 36, S18–S25.
158. Review: O. Grzybowska-Izydorczyk and P. Smolewski, Future Med. Chem., 2012, 4, 487–504.
159. Review: M. L. Maddess, M. N. Tackett and S. V. Ley, in Progress in Drug Research, ed. F. Petersen and R. Amstutz, Birkhäuser-Verlag, Basel, 2008, vol. 66, pp. 15–186.
160. (a) M. L. Maddess, M. N. Tackett, H. Watanabe, P. E. Brennan, C. D. Spilling, J. S. Scott, D. P. Osborn and S. V. Ley, Angew. Chem., Int. Ed., 2007, 46, 591–597; (b) S. V. Ley, M. N. Tackett, M. L. Maddess, J. C. Anderson, P. E. Brennan, M. W. Cappi, J. P. Heer, C. Helgen, M. Kori, C. Kouklovsky, S. P. Marsden, J. Norman, D. P. Osborn, M. A. Palomero, J. B. J. Parvey, C. Pinel, L. A. Robinson, J. Schnaubelt, J. S. Scott, C. D. Spilling, H. Watanabe and M. C. Willis, Chem.–Eur. J., 2009, 15, 2874–2914.
161. (a) W. W. Ma and A. Jimeno, Drugs Today, 2007, 43, 659–669; (b) B. Rini, S. Kar and P. Kirkpatrick, Nat. Rev. Drug Discovery, 2007, 6, 599–600.
162. M. B. Atkins, U. Yasothan and P. Kirkpatrick, Nat. Rev. Drug Discovery, 2009, 8, 535–536.
163. S. Brugaletta, F. Burzotta and M. Sabaté, Expert Opin. Pharmacother., 2009, 10, 1047–1058.
164. Reviews: (a) P. Wu and Y. Hu, Med. Chem. Commun., 2012, 3, 1337–1355; (b) D. A. Fruman and C. Rommel, Cancer Discovery, 2011, 1, 562–572; (c) Q. Zhou, V. W. Y. Lui and W. Yeo, Future Oncol., 2011, 7, 1149–1167; (d) S. J. Shuttleworth, F. A. Silva, A. R. L. Cecil, C. D. Tomassi, T. J. Hill, F. I. Raynaud, P. A. Clarke and P. Workman, Curr. Med. Chem., 2011, 18, 2686–2714; (e) E. Ciraolo, F. Morello and E. Hirsch, Curr. Med. Chem., 2011, 18, 2674–2685; (f) S. Schenone, C. Brullo, F. Musumeci, M. Radi and M. Botta, Curr. Med. Chem., 2011, 18, 2995–3014; (g) S. K. Pal, K. Reckamp, H. Yu and R. A. Figlin, Expert Opin. Invest. Drugs, 2010, 11, 1355–1366.
165. D. Benjamin, M. Colombi, C. Moroni and M. N. Hall, Nat. Rev. Drug Discovery, 2011, 10, 868–880.
166. Review: S. Gaali, R. Gopalakrishnan, Y. Wang, C. Kozani and F. Hausch, Curr. Med. Chem., 2011, 18, 5355–5379.
167. (a) J. P. Steiner, M. A. Connolly, H. L. Valentine, G. S. Hamilton, T. M. Dawson, L. Hester and S. H. Snyder, Nat. Med., 1997, 3, 421–428; (b) W. E. Lyons, E. B. George, T. M. Dawson, J. P. Steiner and S. H. Snyder, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 3191–3195; (c) R. E. Babine, J. E. Villafranca and B. G. Gold, Expert Opin. Ther. Pat., 2005, 15, 555–573.
168. B. Ruan, K. Pong, F. Jow, M. Bowlby, R. A. Crozier, D. Liu, S. Liang, Y. Chen, M. L. Mercado, X. Feng, F. Bennett, D. von Schack, L. McDonald, M. M. Zaleska, A. Wood, P. H. Reinhart, R. L. Magolda, J. Skotnicki, M. N. Pangalos, F. E. Koehn, G. T. Carter, M. Abou-Gharbia and E. I. Graziani, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 33–38.
169. (a) M. A. Gregory, H. Petkovic, R. E. Lill, S. J. Moss, B. Wilkinson, S. Gaisser, P. F. Leadlay and R. M. Sheridan, Angew. Chem., Int. Ed., 2005, 44, 4757–4760; (b) F. V. Ritacco, E. I. Graziani, M. Y. Summers, T. M. Zabriskie, K. Yu, V. S. Bernan, G. T. Carter and M. Greenstein, Appl. Environ. Microbiol., 2005, 71, 1971–1976; (c) R. J. M. Goss, S. Lanceron, A. D. Roy, S. Sprague, M. Nur-e-Alam, D. L. Hughes, B. Wilkinson and S. J. Moss, ChemBioChem, 2010, 11, 698–702.
170. Reviews: (a) K. J. Weissman, Trends Biotechnol., 2007, 25, 139–142; (b) S. R. Park, Y. J. Yoo, Y.-H. Ban and Y. J. Yoon, J. Antibiot., 2012, 63, 434–441.
171. M. V. Pasquetto, L. Vecchia, D. Covini, R. Diglio and C. Scotti, J. Immunother., 2011, 34, 611–628.
172. A. D. Ricart and A. W. Tolcher, Nat. Clin. Pract. Oncol., 2007, 4, 245–255.
173. P. F. Bross, J. Beitz, G. Chen, X. H. Chen, E. Duffy, L. Kieffer, S. Roy, R. Sridhara, A. Rahman, G. Williams and R. Pazdur, Clin. Cancer Res., 2001, 7, 1490–1496.
174. K. C. Nicolaou, J. S. Chen and S. M. Dalby, Bioorg. Med. Chem., 2009, 17, 2290–2303.
175. V. H. J. van der Velden, N. Boeckx, I. Jedema, J. G. Te Marvelde, P. G. Hoogeveen, M. Boogaerts and J. J. M. Van Dongen, Leukemia, 2004, 18, 983–988.
176. (a) R. A. De Claro, K. McGinn, V. Kwitkowski, J. Bullock, A. Khandelwal, B. Habtemariam, Y. Ouyang, H. Saber, K. Lee, K. Koti, M. Rothmann, M. Shapiro, F. Borrego, K. Clouse, X. H. Chen, J. Brown, L. Akinsanya, R. Kane, E. Kaminskas, A. Farrell and R. Pazdur, Clin. Cancer Res., 2012, 18, 5845–5849; (b) S. S. Minich, Ann. Pharmacother., 2012, 46, 377–383.
177. G. R. Pettit, Y. Kamano, C. L. Herald, A. A. Tuinman, F. E. Boettner, H. Kizu, J. M. Schmidt, L. Baczynskyj, K. B. Tomer and R. J. Bontems, J. Am. Chem. Soc., 1987, 109, 6883–6885.
178. G. R. Pettit, S. B. Singh, F. Hogan, P. Lloyd-Williams, D. L. Herald, D. B. Burkett and P. J. Clewlow, J. Am. Chem. Soc., 1989, 111, 5463–5465.
179. (a) S. O. Doronina, B. E. Toki, M. Y. Torgov, B. A. Mendelsohn, C. G. Cerveny, D. F. Chace, R. L. DeBlanc, R. P. Gearing, T. D. Bovee, C. B. Siegall, J. A. Francisco, A. F. Wahl, D. L. Meyer and P. D. Senter, Nat. Biotechnol., 2003, 21, 778–784; (b) P. D. Senter and E. L. Sievers, Nat. Biotechnol., 2012, 30, 631–637.
180. C. Deng, B. Pan and O. A. O'Connor, Clin. Cancer Res., 2013, 19, 22–27.
181. G. D. Lewis Phillips, G. Li, D. L. Dugger, L. M. Crocker, K. L. Parsons, E. Mai, W. A. Blättler, J. M. Lambert, R. V. J. Chari, R. J. Lutz, W. L. T. Wong, F. S. Jacobson, H. Koeppen, R. H. Schwall, S. R. Kenkare-Mitra, S. D. Spencer and M. X. Sliwkowski, Cancer Res., 2008, 68, 9280–9290.
182. (a) P. M. LoRousso, D. Weiss, E. Guardino, S. Girish and M. X. Sliwkowski, Clin. Cancer Res., 2011, 17, 6437–6447; (b) H. A. Burris III, J. Tibbitts, S. N. Holden, M. X. Sliwkowski and G. D. Lewis Phillips, Clin. Breast Cancer, 2011, 11, 275–282.
183. (a) C. A. Hudis, N. Engl. J. Med., 2007, 357, 39–51; (b) C. L. Arteaga, M. X. Sliwkowski, C. K. Osborne, E. A. Perez, F. Puglisi and L. Gianni, Nat. Rev. Clin. Oncol., 2012, 9, 16–32.
184. Reviews: (a) J. M. Cassady, K. K. Chan, H. G. Floss and E. Leistner, Chem. Pharm. Bull., 2004, 52, 1–26; (b) A. Kirschning, K. Harmrolfs and T. Knobloch, C. R. Chim., 2008, 1523–1543.
185. S. Remillard, L. I. Rebhun, G. A. Howie and S. M. Kupchan, Science, 1975, 189, 1002–1005.
186. M. Lopus, E. Oroudjev, L. Wilson, S. Wilhelm, W. Widdison, R. Chari and M. A. Jordan, Mol. Cancer Ther., 2010, 9, 2689–2699.
187. A. Kawai, H. Akimoto, Y. Kozai, K. Ootsu, S. Tanida, N. Hashimoto and H. Nomura, Chem. Pharm. Bull., 1984, 32, 3441–3451.
188. (a) R. V. J. Chari, B. A. Martell, J. L. Gross, S. B. Cook, S. A. Shah, W. A. Blättler, S. J. McKenzie and V. S. Goldmacher, Cancer Res., 1992, 52, 127–131; (b) W. S. Widdison, S. D. Wilhelm, E. E. Cavanagh, K. R. Whiteman, B. A. Leece, Y. Kovtun, V. S. Goldmacher, H. Xie, R. M. Steeves, R. J. Lutz, R. Zhao, L. Wang, W. A. Blättler and R. V. J. Chari, J. Med. Chem., 2006, 49, 4392–4408.
189. S. J. Isakoff and J. Baselga, J. Clin. Oncol., 2011, 29, 351–354.
190. A. Mullard, Nat. Rev. Drug Discovery, 2013, 12, 329–332.
191. T. F. Molinski, D. S. Dalisay, S. L. Lievens and J. P. Saludes, Nat. Rev. Drug Discovery, 2009, 8, 69–85.
192. B. M. Olivera, W. R. Gray, R. Zeikus, J. M. McIntosh, J. Varga, J. Rivier, V. de Santos and L. J. Cruz, Science, 1985, 230, 1338–1343.
193. Review: R. Halai and D. J. Craik, Nat. Prod. Rep., 2009, 26, 526–536.
194. (a) B. M. Olivera, L. J. Cruz, V. De Santos, G. W. LeCheminant, D. Griffin, R. Zeikus, J. M. McIntosh, R. Galyean, J. Varga, W. S. Gray and J. Rivier, Biochemistry, 1987, 26, 2086–2090; (b) K. Valentino, R. Newcomb, T. Gadbois, T. Singh, S. Bowersox, S. Bitner, A. Justice, D. Yamashiro, B. B. Hoffman, R. Ciaranello, G. Miljanich and J. Ramachandran, Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 7894–7897; (c) L. Nadasdi, D. Yamashiro, D. Chung, K. Tarczy-Hornoch, P. Adriaenssens and J. Ramachandran, Biochemistry, 1995, 34, 8076–8081.
195. W.-H. Xiao and G. J. Bennett, J. Pharmacol. Exp. Ther., 1995, 274, 666–672.
196. A. Schmidtko, J. Lötsch, R. Freynhagen and G. Geisslinger, Lancet, 2010, 375, 1569–1577.
197. C. Cuevas and A. Francesch, Nat. Prod. Rep., 2009, 26, 322–337.
198. M. M. Sigel, L. L. Wellham, W. Lichter, L. E. Dudeck, J. L. Gargus and A. H. Lucas, in Food-Drugs from the Sea: Proceedings 1969, ed. H. W. Youngken, Jr., Marine Technol. Soc., Washington, DC, 1970, pp. 281–294.
199. K. L. Rinehart, T. G. Holt, N. L. Fregeau, J. G. Stroh, P. A. Keifer, F. Sun, L. H. Li and D. G. Martin, J. Org. Chem., 1990, 55, 4512–4515.
200. R. Sakai, K. L. Rinehart, Y. Guan and A. H.-J. Wang, Proc. Natl. Acad. Sci. U. S. A., 1992, 89, 11456–11460.
201. E. J. Corey, D. Y. Gin and R. S. Kania, J. Am. Chem. Soc., 1996, 118, 9202–9203.
202. E. J. Martinez and E. J. Corey, Org. Lett., 2000, 2, 993–996.
203. D. Mendola, Biomol. Eng., 2003, 20, 441–458.
204. D. Mendola, S. A. Naranjo Lozano, A. R. Duckworth and R. Osinga, in Frontiers in Marine Biotechnology, ed. P. Proksch and W. E. G. Müller, Horizon Bioscience, Wymondham, UK, 2006, pp. 21–72.
205. C. Cuevas, M. Pérez, M. J. Martín, J. L. Chicharro, C. Fernández-Rivas, M. Flores, A. Francesch, P. Gallego, M. Zarzuelo, F. De la Calle, J. García, C. Polanco, I. Rodríguez and I. Manzanares, Org. Lett., 2000, 2, 2545–2548.
206. WHO World Malaria Report 2012, http://www.who.int/malaria/publications/world_malaria_report_2012/en/.
207. M. Warsame, P. Olumese and K. Mendis, Drug Dev. Res., 2010, 71, 4–11.
208. L. H. Miller and X. Su, Cell, 2011, 146, 855–858; R. J. Maude, C. J. Woodrow and L. J. White, Drug Dev. Res., 2010, 71, 12–19.
209. F. Liao, Molecules, 2009, 14, 5362–5366.
210. Coordinating Group for Research on the Structure of Qinghaosu, Kexue Tongbao, 1977, 3, 142.
211. P. M. O'Neill and G. H. Posner, J. Med. Chem., 2004, 47, 2945–2964.
212. J. N. Burrows, K. Chibale and T. N. C. Wells, Curr. Top. Med. Chem., 2011, 11, 1226–1254.
213. N. J. White, Science, 2008, 320, 330–334.
214. T. Gordi, Expert Rev. Clin. Pharmacol., 2012, 5, 157–162.
215. Guidelines for the treatment of malaria, World Health Organization, Geneva, 2nd edn, 2010.
216. R. J. Maude, C. J. Woodrow and L. J. White, Drug Dev. Res., 2010, 71, 12–19.
217. Management of severe malaria – a practical handbook, World Health Organization, Geneva, 3rd edn, 2013.
218. G. Schmid and W. Hofheinz, J. Am. Chem. Soc., 1983, 105, 624–625.
219. (a) W.-S. Zhou and X.-X. Xu, Acc. Chem. Res., 1994, 27, 211–216; (b) J. S. Yadav, B. Thirupathaiah and P. Srihari, Tetrahedron, 2010, 66, 2005–2009; (c) H.-D. Hao, Y. Li, W.-B. Han and Y. Wu, Org. Lett., 2011, 13, 4212–4215 ; and references cited therein.
220. J.-M. Kindermans, J. Pilloy, P. Olliaro and M. Gomes, Malar. J., 2007, 6, 125–130.
221. R. Van Noorden, Nature, 2010, 466, 672.
222. P. S. Covello, Phytochemistry, 2008, 69, 2881–2885.
223. (a) P. S. Covello, K. H. Teoh, D. R. Polichuk, D. W. Reed and G. Nowak, Phytochemistry, 2007, 68, 1864–1871; (b) Review: G. D. Brown, Molecules, 2010, 15, 7603–7698.
224. B. Liu, H. Wang, Z. Du, G. Li and H. Ye, Plant Cell Rep., 2011, 30, 689–694.
225. Review: S. S. Chandran, J. T. Kealey and C. D. Reeves, Process Biochem., 2011, 46, 1703–1710.
226. V. Hale, J. D. Keasling, N. Renninger and T. T. Diagana, Am. J. Trop. Med. Hyg., 2007, 77(Suppl 6), 198–202.
227. H. Tsuruta, C. J. Paddon, D. Eng, J. R. Lenihan, T. Horning, L. C. Anthony, R. Regentin, J. D. Keasling, N. S. Renninger and J. D. Newman, PLoS One, 2009, 4, e4489.
228. D.-K. Ro, E. M. Paradise, M. Ouellet, K. J. Fisher, K. L. Newman, J. M. Ndungu, K. A. Ho, R. A. Eachus, T. S. Ham, J. Kirby, M. C. Y. Chang, S. T. Withers, Y. Shiba, R. Sarpong and J. D. Keasling, Nature, 2006, 440, 940–943.
229. C. J. Paddon, P. J. Westfall, D. J. Pitera, K. Benjamin, K. Fisher, D. McPhee, M. D. Leavell, A. Tai, A. Main, D. Eng, D. R. Polichuk, K. H. Teoh, D. W. Reed, T. Treynor, J. Lenihan, M. Fleck, S. Bajad, G. Dang, D. Diola, G. Dorin, K. W. Ellens, S. Fickes, J. Galazzo, S. P. Gaucher, T. Geistlinger, R. Henry, M. Hepp, T. Horning, T. Iqbal, H. Jiang, L. Kizer, B. Lieu, D. Melis, N. Moss, R. Regentin, S. Secrest, H. Tsuruta, R. Vazquez, L. F. Westblade, L. Xu, M. Yu, Y. Zhang, L. Zhao, J. Lievense, P. S. Covello, J. D. Keasling, K. K. Reiling, N. S. Renninger and J. D. Newman, Nature, 2013, 496, 528–32.
230. R. J. Roth and N. Acton, J. Nat. Prod., 1989, 52, 1183–1185.
231. J. Dhainaut, A. Dlubala, R. Guevel, A. Medard, G. Oddon, N. Raymond and J. Turconi, PCT Int. Appl., 2009 , WO2011026865 (Sanofi-Aventis SA).
232. R. Feling, A. Burgard, C. Lattemann and R. Göller, 25. Irseer Naturstofftage, 2013.
233. Institute for OneWorld Health, San Francisco: OneWorld Health Newsletter, 2011, Issue 5, http://www.a2s2.org/upload/5.ArtemisininConferences/1.2013Kenya/Presentations/Day1/3.SanofiSSpresentation.pdf.
234. (a) F. Lévesque and P. Seeberger, Angew. Chem., Int. Ed., 2012, 51, 1–5; (b) D. Kopetzki, F. Lévesque and P. Seeberger, Chem. Eur. J., 2013, 19, 5450–5456.
235. K. Kupferschmidt, Science, 2012, 336, 798.

---

Footnotes

† New address: Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig.

‡ Absorption, distribution, metabolism, excretion, toxicology

---