Inverse Electron Demand DielsâAlder Reactions of Heterocyclic

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Inverse electron demand Diels-Alder reactions of heterocyclic azadienes, 1-aza-1,3-butadienes, cyclopropenone ketals and related systems. A retrospective Jiajun Zhang, Vyom Shukla, and Dale L. Boger J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00834 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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The Journal of Organic Chemistry

Inverse Electron Demand Diels–Alder Reactions of Heterocyclic Azadienes, 1-Aza-1,3-Butadienes, Cyclopropenone Ketals and Related Systems. A Retrospective Jiajun Zhang,† Vyom Shukla,† and Dale L. Boger* Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States Abstract. A summary of the investigation and applications of the inverse electron demand Diels–Alder reaction is provided that have been conducted in our laboratory over a period that now span more than 35 years. The work, which continues to provide solutions to complex synthetic challenges, is presented in the context of more than 70 natural product total syntheses in which the reactions served as a key strategic step in the approach. The studies include the development and use of the cycloaddition reactions of heterocyclic azadienes (1,2,4,5-tetrazines; 1,2,4-, 1,3,5- and 1,2,3-triazines; 1,2-diazines; and 1,3,4-oxadiazoles), 1-aza-1,3-butadienes, α-pyrones, and cyclopropenone ketals. Their applications illustrate the power of the methodology, often provided concise and non-obvious total syntheses of the targeted natural products, typically were extended to the synthesis of analogues that contain deep-seated structural changes in more comprehensive studies to explore or optimize their biological properties, and highlight a wealth of opportunities not yet tapped.

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1. Introduction The opportunities to provide solutions to complex biological problems using complex natural products have grown as the methods to identify their biological targets have improved and as the techniques used to study, depict, and directly determine their target bound structures have advanced. In addition to the natural compounds themselves, the examination of key synthetic partial structures, compounds that contain deep-seated structural modifications, and even their unnatural enantiomers provide an added and powerful complement to such studies. Well-designed structural modifications in the natural products may be used to address the structural basis of their interaction with biological targets and to define fundamental relationships between structure, activity, functional reactivity, and biological properties. Natural products incorporate multiple properties and functions integrated into a highly functionalized, compact molecule. In many instances, each structural feature and substituent is thought to contribute to the biological activity. However, we recently highlighted examples where even single atom changes to a series of natural products were found to substantially improve on their biological properties. 1 In this work, a challenging problem with natural products is first to understand the subtle design elements that nature has provided and then to rationally extend them to provide more selective, more efficacious, or more potent compounds. Central to such studies and a major challenge for natural products is the development of synthetic strategies and new synthetic methodology tailored to their structures that permit their preparation and that of key analogues incorporating the deep-seated structural changes. In this perspective, we summarize our efforts on the development and applications of inverse electron demand Diels–Alder reactions that innocently began nearly 40 years ago and continue to provide solutions to challenging synthetic problems.2 The collective efforts have provided a series of unique, non-obvious total syntheses capable of not only affording the targeted natural products, but also key or a comprehensive set of structural analogues. Often, this was used to explore the origin of the biological properties of a natural product and to discover compounds with improved activity. Because an extraordinary amount of work has been conducted on both the mechanistic and synthetic aspects of the Diels–Alder reaction, a full overview is outside the scope of this perspective. However, a series of comprehensive treatments and reviews have chronicled aspects of its remarkable evolution and central role in synthesis.3 The Diels–Alder reaction4 may be classified into one of three types of π2s + π4s cycloadditions that reflect the interacting frontier molecular orbitals governing the rate, regioselectivity, and diastereoselectivity of the reactions: the normal HOMOdiene-controlled reaction, the neutral reaction, and the inverse electron demand or LUMOdienecontrolled reaction5 (Figure 1). Shortly after its discovery, the normal Diels–Alder reaction was quickly integrated into the field because of its ability to generate all-carbon six-membered ring systems with predictable regio- and stereochemical control. In contrast, the inverse electron demand Diels–Alder reaction was slower to emerge. The recognition that conjugated diene systems containing nitrogen are electron-deficient led to the investigation of the inverse electron demand Diels–Alder reaction and a demonstration of its utility in the preparation of six-membered heterocyclic ring systems. This was first disclosed in 19596 when Carboni and Lindsey published the

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synthesis of pyridazines and dihydropyridazines through the reaction of 1,2,4,5-tetrazines with dienophiles. In this work, the recognition of the intrinsic electron-deficient character of the heteroaromatic azadienes led to the demonstrated rate acceleration that accompanies the reversal of the traditional electronic properties of diene/dienophile reaction partners. This work also provided the basis for the examination of additional heterocyclic azadienes. Most notable of these are the early studies of Sauer and Neunhoeffer with six-membered heterocyclic azadienes,7 and the early examination of oxazoles8 and related five-membered heterocyclic azadienes.9 Herein, we summarize many of our contributions to the field, consisting of the investigations of heterocyclic and acyclic azadienes, cyclopropenone ketals, and related systems and their participation in inverse electron demand Diels–Alder reactions. Initiated at a time the inverse electron demand Diels Alder reaction was relatively obscure, these studies now span more than 35 years, and some represent the first examples of the application of the reaction to the total synthesis of complex natural products. In most instances, it was the natural product targets and the underlying biological properties that were first chosen for study and that in turn inspired the methodology discovery, development, or application. However, and for the purpose of this perspective, the work is presented in the context of a series of natural product total syntheses where the methodology served as a key strategic step in the approach and is organized under the type of electron-deficient diene used. In doing so we hope to illustrate the potential of the methodology for the preparation of complex molecules and to highlight the wealth of transformations yet to be discovered or exploited.

Figure 1. Classifications of Diels–Alder reactions. Our initial successes with the approach were sufficiently stunning (streptonigrin, 1983; lavendamycin, 1985; colchicine, 1986; prodigiosin, 1987; CC-1065, 1988)10 that they inspired our continued examination of new variations on the reactions throughout my career. It led to the introduction of a powerful strategy for divergent (total) synthesis (1984), consisting of late-stage aryl annulations from a common ketone intermediate, that we now routinely use. A unique transition state anomeric effect was delineated (1991) that is responsible for the remarkable cycloaddition diastereoselectivity of our 1-


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azadienes and we disclosed the first Diels–Alder reactions (1982) that entailed catalytic generation of enamine dienophiles and their in situ reaction with 1,2,4-triazines. The studies led to the discovery of the reversible thermal generation of πdelocalized vinylcarbenes from cyclopropenone ketals (1984) and the development of their ensuing cycloaddition reactions. A powerful tandem intramolecular cycloaddition cascade of 1,3,4-oxadiazoles was introduced (2002) that we presently use extensively. Even today, we continue to discover new, as yet, untapped potential for the methodology that highlights its remarkable scope. Our recent discovery of the first general method for catalysis of the inverse electron demand Diels– Alder reactions of heterocyclic azadienes (HFIP hydrogen bonding, 2016) likely expands their scope far beyond what we highlight herein. Similarly, our exploration of previously untapped (e.g., 1,2,3-triazines, 2011) or presently unknown (e.g., 1,2,3,5-tetrazines, unpublished) heterocyclic azadienes promise to provide new opportunities not yet envisioned by the field. We also like to think that our work, which highlighted the remarkable rates and efficiencies of many heterocyclic azadiene Diels–Alder reactions, helped inspire the tetrazine ligation11 and its related biorthogonal conjugation and labeling technology introduced by others and widely used today. 2. Heterocyclic Azadienes 2.1 1,2,4,5-Tetrazines Electron-deficient heterocyclic azadienes participate in welldefined inverse electron demand Diels–Alder reactions with electron-rich dienophiles, providing useful reagents for the preparation of highly substituted and functionalized heterocycles that are not as easily accessed by other means.12 Among them, 1,2,4,5-tetrazine is the most widely employed heterocyclic azadiene, acting as the 4π-electron component in [4+2] cycloadditions with a wide range of dienophiles, due to its remarkable reactivity, surprisingly good stability, synthetic accessibility, and broad synthetic applications (Figure 2). 13

diazine products by elimination of an amine, alcohol or thiol, which is commonly the rate-limiting step. A wide variety of dienophiles are capable of participation in Diels–Alder reactions with the electron-deficient 1,2,4,5-tetrazines and include the following five general categories:13 (1) electron-rich dienophiles, e.g. enamines, enol ethers and acetates, enols and enolates, ketene acetals, ynamines, selected heteroaromatics; (2) activated or strained dienophiles, e.g. norbornenes, benzynes, cyclopropenes, cyclobutenes, allenes, transcyclooctenes; (3) electron-neutral dienophiles, e.g. simple olefins, styrenes, alkynes; (4) electron-deficient dienophiles, e.g. acrylates, maleic anhydrides, propiolates; (5) heterodienophiles, e.g. imidates, amidines, thioimidates, hydrazones, imines, cyanamides (Figure 3). Based on the nature of inverse electron demand Diels–Alder reactions, electron-rich and strained dienophiles, both of which possess an inherent high lying HOMO, usually participate in 1,2,4,5-tetrazine [4+2] cycloaddition reactions at room temperature or even lower, whereas neutral olefinic or typical electron-deficient dienophiles require higher reaction temperatures (50–200 ºC) and more extended reaction times. Consistent with their impact on lowering the diene LUMO, the rate of 1,2,4,5-tetrazine participation in [4+2] cycloaddition reactions with electron-rich dienophiles increases as the electron-withdrawing capabilities of its substituents increase. For instance, Diels–Alder reactions of dimethyl 1,2,4,5tetrazine-3,6-dicarboxylate (1) with electron-rich or strained dienophiles are often conducted at room temperature or lower. 14 Moreover, it is sufficiently reactive to undergo reactions with unactivated and electron-deficient dienophiles (Figure 3).10, 13

Figure 2. Inverse electron demand Diels–Alder reactions of 1,2,4,5-tetrazines with dienophiles. Since the initial disclosure in 1959,6 extensive investigations have defined the scope of 1,2,4,5-tetrazine [4+2] cycloaddition reactions. Generally, their inverse electron demand Diels–Alder reactions with (hetero)dienophiles proceed through three steps: (1) a [4+2] cycloaddition reaction to form an initial bicyclic cycloadduct; (2) a retro [4+2] cycloaddition reaction with loss of nitrogen to afford a dihydro-1,2-diazine intermediate; and (3) final aromatization of the resulting intermediate to provide 1,2-

Figure 3. Representative dienophiles that participate in 1,2,4,5tetrazine [4+2] cycloaddition reactions and reactivity patterns. Predictable tuning of both the reactivity as well as the reaction regioselectivity may be made by adjusting substituents on either the 1,2,4,5-tetrazine or the dienophile. The only mode of cycloaddition observed with 1,2,4,5-tetrazines occurs across



C3/C6, and we are not aware of any example of a N1/N4 cycloaddition. Early studies of the 1,2,4,5-tetrazines have been comprehensively reviewed elsewhere,10b,13 including our own work, and focused on symmetrically substituted derivatives. More recent studies have provided approaches to the synthetically more challenging unsymmetrical 1,2,4,5tetrazines, expanding their synthetic utility.11c In our studies, the unsymmetrical 1,2,4,5-tetrazines (3, 5–12) were prepared and shown to participate in well-defined and predictably regioselective cycloaddition reactions with a wide range of electron-rich and unactivated dienophiles, providing the corresponding 1,2-diazines in excellent yields (Figure 4).14,15 However, there are examples of cycloaddition reactions that proceeded with unexpected regioselectivities. For instance, the [4+2] cycloaddition reaction of the unsymmetrical 1,2,4,5tetrazine 6 with ketene diethyl acetal 13 afforded the single regioisomeric product 14 as anticipated. However, the analogous sulfoxide-substituted 1,2,4,5-tetrazine 10 proceeded with a cycloaddition regioselectivity opposite predictions based on simple zwitterionic models or FMO analysis of the [4+2] cycloaddition reaction (Figure 4).16 Although the origin of the reversed regioselectivity remains undefined, it is possible that this may be attributed to a destabilizing steric or electronic interaction of the dienophile substituents with the larger and more electronegative methylsulfinyl substituent.

Figure 4. Regioselective Diels–Alder unsymmetrical 1,2,4,5-tetrazines.

reactions

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Scheme 1. Intramolecular Diels–Alder reactions of 1,2,4,5tetrazines utilizing tethered unactivated acetylenes.

Among the first problems addressed in my academic career, we recognized the powerful synthetic utility that the Diels– Alder reactions of 1,2,4,5-tetrazines possessed for the preparation of highly substituted, functionalized heterocyclic systems not easily accessed by traditional approaches. Initially conducted at a time the inverse electron demand Diels–Alder reaction was an obscure novelty relative to the more conventional normal Diels–Alder reaction, we began an extensive series of investigation that now span more than 35 years to explore and expand the heterocyclic azadiene reaction scope while implementing their use in the total synthesis of complex biologically active natural products. This began at a time when the total synthesis of a natural product required a team of students and still often concluded with the development of an approach to, but not the completion of, the projected total synthesis. As a result, I still find it stunning that our first applications of the methodology (steptonigrin in 1983, CC1065 in 1988, and prodigiosin in 1987) each entailed the effort of a single, albeit exceptionally talented, graduate student. The efforts also resulted in the delineation of three general strategies for implementing this powerful transformation for the preparation of highly functionalized pyridines, benzenes and pyrroles, respectively (Figure 5).

of

The intermolecular Diels–Alder reactions of 1,2,4,5tetrazines proceed slowly with unactivated dienophiles and typically require higher reaction temperatures and extended reaction times. For example, vigorous reaction conditions (140 ºC, 60 h) are required for [4+2] cycloaddition of 1,2,4,5tetrazine 3 with phenylacetylene (Scheme 1).14b By contrast, the intramolecular variants of such reactions proceed under much milder conditions (23–80 ºC, 6–16 h). 1,2,4,5-Tetrazine 17, possessing a terminal unactivated acetylene linked by a threecarbon tether, underwent near quantitative conversion to the bicyclic 1,2-diazine 18 with formation of a fused six-membered ring when warmed in dioxane at 80 ºC (6 h). 14b Likewise, the tethered alkyne 19 underwent a sequential N-acylation and [4+2] cycloaddition with formation of a fused five-membered ring at room temperature (16 h), affording 20 (96%).15 Figure 5. Three general synthetic strategies utilizing the inverse electron demand Diels–Alder reactions of 1,2,4,5-tetrazines.


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Since the products of inverse electron demand Diels–Alder reactions of 1,2,4,5-tetrazines are 1,2,4-triazines or 1,2-diazines that may also serve as 4π components for a second [4+2] cycloaddition reaction, it was envisioned that highly substituted or functionalized heterocyclic systems found in complex natural products could be assembled by sequential inverse electron demand Diels–Alder reactions. Central to the approach to the total synthesis of streptonigrin was the implementation of a 1,2,4,5-tetrazine→1,2,4-triazine→pyridine Diels–Alder strategy for convergent synthesis of the natural product through formation of the central and fully substituted pyridine ring. Similarly, we first developed and then applied a 1,2,4,5tetrazine→1,2-diazine→benzene Diels–Alder strategy for the total synthesis of natural products bearing highly functionalized indole or indoline moiety including PDE-I and II, CC-1065, cisand trans-trikentrin and the Amaryllidaceae alkaloids. Finally, and starting with its use in a total synthesis of prodigiosin, we developed a 1,2,4,5-tetrazine→1,2-diazine→pyrrole reaction sequence, including a unique penultimate reductive ring contraction reaction of an electron-deficient 1,2-diazine. The latter strategy proved ideally suited for preparation of the densely functionalized pyrrole cores found in a wide range of additional natural products, including isochrysohermidin, ningalins, roseophilin and lycogalic acid. Streptonigrin. Streptonigrin (30) is a historically important potent antitumor antibiotic that contains central to its structure a fully substituted and highly functionalized pyridine. 17 Our convergent total synthesis of streptonigrin was based on the 1,2,4,5-tetrazine→1,2,4-triazine→pyridine sequential Diels– Alder strategy for construction of this central pentasubstituted pyridine and assemblage of the full skeleton of the natural product. It was the targeted natural product and our interest in defining the source of its biological properties that inspired this methodology development, permitting the incorporation of structural modifications easily within each key region of the molecule.

Figure 6. Diels–Alder reactions of dimethyl 1,2,4,5-tetrazine3,6-dicarboxylate (1) with heterodienophiles. The first of two sequential Diels–Alder reactions was the [4+2] cycloaddition reaction of dimethyl 1,2,4,5-tetrazine-3,6dicarboxylate (1) with a quinolyl S-methylthioimidate to provide a substituted 1,2,4-triazine. In studies that led to the development of this reaction, no identifiable products could be isolated from the reaction of dimethyl 1,2,4,5-tetrazine-3,6dicarboxylate (1) with amidines 25 despite an initial exothermic

reaction that was accompanied by the evolution of nitrogen (Figure 6).18 This suggested that a subsequent undesired Diels– Alder reaction of the resulting dihydro-1,2,4-triazine products 21 with unreacted amidine was occurring faster than the aromatization of the penultimate intermediate 21. Moreover, the imidate 24 was found to provide the desired 1,2,4-triazine product 22, albeit in modest yield. In sharp contrast, the [4+2] cycloaddition reaction of S-methylthioimidate 23 with 1,2,4,5tetrazine 1 provided the 1,2,4-triazine cycloadduct 22 in superb yield (70%) under mild reaction conditions (80 ºC, dioxane). 18 Beautifully, the Diels–Alder reaction of quinolyl Smethylthioimidate 26 with 1,2,4,5-tetrazine 1 used in the total synthesis of streptonigrin smoothly afforded the 1,2,4-triazine 27 in an excellent yield (82%) (Scheme 2).19 In this case, the success of the Diels–Alder reaction proved to be dependent on the nature of heterodienophile and attributable to an optimal combination of nucleophilic character of the C=N dienophile (– NR2 > –OEt = –SMe) balanced against the leaving group ability of its X-substituent (–SMe > –OEt >> –NR2) such that aromatization of the penultimate intermediate effectively competes with further cycloaddition of the intermediate dihydro-1,2,4-triazine. Scheme 2. Key steps in the total synthesis of streptonigrin.

The second [4+2] cycloaddition reaction was that of the 1,2,4-triazine 27 with the pyrrolidine enamine 28 in which addition occurred selectively across C3/C6 of the 1,2,4-triazine with the nucleophilic carbon of the dienophile attached to C3, providing the fully functionalized pyridine core and the entire tetracyclic framework (29) of streptonigrin (Scheme 2).19 The work culminated in a convergent total synthesis of streptonigrin, requiring only 17 steps. This proved to be roughly half the number of steps used in contemporary efforts at the time (34 steps (Weinreb)20 and 27 steps (Kende)21) and remains one of the most concise of the four total syntheses of streptonigrin disclosed to date,17, 22 highlighting the power of the sequential cycloaddition strategy. In these studies, we prepared and examined a series of key partial structures and analogues of streptonigrin. 23 These and studies of others have provided insights into the structural features of streptonigrin required for expression of its biological activity. Most recently, and with the samples prepared nearly 30 years earlier, we were able to define its biological target, protein arginine deiminase 4 (PAD4), and elucidate the role of these



key structural features. We showed that the quinoline-5,8-dione portion of streptonigrin (A and B rings) is required for enzyme inactivation and forms covalent adducts with the enzyme, that the pyridyl C ring and its substituents can significantly impact potency, and that rings C and D convey isozyme selectivity (vs PAD1–3).24 PDE-I, PDE-II and (+)-CC-1065. (+)-CC-1065 (40), an antitumor-antibiotic isolated from cultures of Streptomyces zelensis, was shown to possess exceptionally potent cytotoxic activity, broad-spectrum antimicrobial activity, and promising in vivo antitumor activity. It is one of the most potent compounds yet identified, being approximately 1000 times more active than typical cytotoxic agents. Today, it is one of the most widely recognized and best characterized DNA-alkylating agents, although none of these features were defined at the time we initiated our studies. The noncovalent DNA binding selectivity and its DNA alkylation reaction maybe attributed to two complementary structural features: the identical central and right-hand subunits of CC-1065 are responsible for the high affinity, sequence-selective DNA minor groove binding, and the 4,4-spirocyclopropylcyclohexa-2,5-dienone unit present in the left-hand segment functions as a selective alkylating agent effectively delivered to double-stranded DNA. PDE I (38) and PDE II (39), two 3’,5’-cAMP phosphodiesterase inhibitors isolated from Streptomyces strain MD769-C6, possess the identical 1,2-dihydro-3H-pyrrolo[3,2e]indole structure found in CC-1065. The total synthesis of PDE I, PDE II, (+)-CC-1065 and its unnatural enantiomer, ent-(–)CC-1065, was accomplished utilizing a 1,2,4,5-tetrazine→1,2diazine→benzene Diels–Alder strategy. We envisioned that the central benzene ring of the core 1,2-dihydro-3H-pyrrolo[3,2e]indole structure could be constructed by an intramolecular Diels–Alder reaction of an alkyne-1,2-diazine which in turn would be derived from the product of an intermolecular inverse electron demand [4+2] cycloaddition reaction of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (1).

Figure 7. Intramolecular Diels–Alder reactions of alkyne-1,2diazines. The potential and scope of the intramolecular alkyne-1,2diazine Diels–Alder reaction was first examined. The results of the studies indicated that 1,2-diazine and alkyne substitution have only subtle effects on the rate of cycloaddition, whereas the length of the diene/dienophile linker chain (n = 1) and the nature of heteroatoms (X = NCO2Me) in the chain are the primary determinants of the success of the reaction (Figure 7). 25 Notably, such unactivated 1,2-diazines are typically reluctant diene participants in intermolecular Diels–Alder reactions and the entropic assistance provided by the tethered dienophile is sufficient to overcome the barrier to reaction.

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Scheme 3. Total synthesis of PDE-I, PDE-II and (+)-CC-1065.

The total synthesis of PDE-I, PDE-II26 and CC-106527 began with the inverse electron demand Diels–Alder reaction of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (1) with 4,4dimethoxy-3-buten-2-one (31) (Scheme 3). Even with the modest reactivity of dienophile 31, the reaction still only required mild reaction conditions (60 ºC, 22 h) and provided 1,2-diazine 32 in good yield (71%).26 Reduction of the methyl ketone 32 with sodium borohydride afforded the lactone 33 directly. Subsequent hydrolysis of the remaining C3 methyl ester followed by acidification led to an unexpectedly facile but welcomed room temperature decarboxylation. This two-step sequence provided 1,2-diazine 34 and completed differentiation of the C3/C6 methoxylcarbonyl groups present in 32. Following attachment of the alkyne side chain, the alkyne-1,2-diazine 35 proved reluctant to undergo the desired intramolecular Diels– Alder reaction necessary for indoline ring construction, perhaps because of the geometrical constraints of the alkyne side chain. Thus, hydrolytic ring opening of the N-alkyloxazinone system with potassium hydroxide followed by benzylic alcohol oxidation and N-acetylation afforded the alkyne-1,2-diazine 36 in which the alkyne side chain was no longer constrained. The intramolecular Diels–Alder reaction of 36 proceeded smoothly and provided the indoline 37 in 87% yield. Overall, the sequential Diels–Alder strategy constituted a novel approach by which to construct the key indoline moiety of the natural products. Additional key steps in the synthesis include the implementation of a Hemetsberger indole annulation to install


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the fused indole-2-carboxylate and a Lewis acid-catalyzed benzylic hydroperoxide rearrangement for installation of the C4 phenol.28 This approach to CC-1065 was also used for the preparation of an extensive series of analogues bearing deep-seated structural modifications in the natural product with the intention of defining the origin of its characteristic DNA alkylation selectivity, establishing the link between DNA alkylation and the ensuing biological properties, and determining the relationships between structure, chemical reactivity, and biological activity.29 These studies now include not only our work on CC-1065,26-27,30 but also duocarmycin A31 and SA,32 and yatakemycin33 where we not only conducted total syntheses of the natural products,34 defining their absolute stereochemistry and correcting a misassigned structure (yatakemycin),33 but we also characterized their DNA alkylation properties.35 In these studies, the DNA alkylation selectivity, rates, reversibility,36 and stereoelectronicallycontrolled reaction regioselectivity37 of both their natural and unnatural enantiomers were defined, and we characterized their isolated adenine N3 adducts (Figure 8).38 In fact, the effective and unprecedented behavior of the unnatural enantiomers was key to understanding the source of their DNA alkylation selectivity, which we showed arises from their noncovalent binding selectivity preferentially in the narrower, deeper ATrich minor groove (shape selective recognition). 39 We also defined the beautiful source of catalysis for the DNA alkylation reaction, which arises from a DNA binding induced conformational change that disrupts the vinylogous amide conjugation stabilizing the alkylation subunit (shape dependent catalysis).40 NMR-derived high-resolution structures of the natural products and their unnatural enantiomers bound to a common site in DNA were obtained in collaboration with Walter Chazin (Figure 8),41 and the studies helped established that the compounds undergo a “target-based activation” (induced conformational change) for DNA alkylation. 42 More than 135 publications and more than 2000 analogues of the natural products (e.g., CBI) have been disclosed in our efforts that were used to define fundamental relationships between structure, reactivity, and activity,43 and their impact on the DNA alkylation properties and biological activity of the natural products.44 Included in these, we recently demonstrated the key role the hydrophobic properties of the compounds play, driving the inherently reversible DNA alkylation reaction (hydrophobic binding-driven-bonding), and established the stunning magnitude of the effect.45 Data from more than 30 alkylation subunit analogues was used to established a predictive parabolic relationship between reactivity and cytotoxic potency that spans a 106 range of reactivity and activity.46 This defined the optimal balance between reactivity and stability and provided a fundamental design feature that was used to improve the potency of CC-1065 nearly 10-fold with a single atom exchange.47 In retrospect and especially given the time frame when such studies were uncommon, it is remarkable that such striking structural simplifications could be made to this class of natural products while still maintaining or even improving their extraordinary potency or selectivity.48 Today, the simplified structures and alkylation subunits that we introduced (e.g., CBI)44 are being used in the development of targeted therapies including antibody–drug conjugates,49 highly selective sequence selective DNA alkylating agents based on Dervan hairpin polyamides,50 and unique prodrug designs subject to tumor-selective free drug release.51 The powerful and now

widely used fluorescent intercalator displacement (FID) assay52 for establishing DNA binding selectivity or affinity was introduced in these studies and a convenient and more efficient M13-derived alternative to 32P-end-labeling of restriction fragments for DNA cleavage studies was developed for use in these studies.53

Figure 8. Top: Characteristics of the CC-1065 DNA alkylation reaction. Bottom: Structures of DNA bound (+)-duocarmycin SA and ent-(–)-duocarmycin SA established by NMR. Cis- and trans-trikentrin A. In initial studies of the intramolecular Diels–Alder reactions of alkyne-1,2-diazines, the [4+2] cycloaddition required reaction conditions (230 ºC) and extended reaction times (12–22 h) that we strove to improve. This led to the examination of intramolecular allene1,2-diazine Diels–Alder reactions and the development of much milder reaction conditions for complementary cycloadditions. 15 Cis- and trans-trikentrin A (47 and 48), isolated from the marine



sponge trikentrion flabelliforme, are highly substituted indoles representative of a class of alkaloids that possess antimicrobial activity.54 Their total synthesis was accomplished in only five steps based on the implementation of a sequential 1,2,4,5tetrazine→allene-1,2-diazine→indole Diels–Alder strategy.55 The 1,2-diazine cycloadducts derived from the [4+2] cycloaddition of 3,6-bis(thiomethyl)-1,2,4,5-tetrazine (2) were found to serve as direct precursors to substituted allene-1,2diazines that undergo a subsequent intramolecular Diels–Alder reaction (Scheme 4).15 Moreover, such allene-1,2-diazines participate in [4+2] cycloaddition reactions under substantially milder thermal conditions (120 ºC) than the corresponding alkyne-1,2-diazines (230 ºC). This may be attributed to both the increased reactivity of an allene dienophile and its entropically more favored adoption of a productive conformation for the intramolecular Diels–Alder reaction. The products of the allene-1,2-diazine intramolecular Diels–Alder reaction were designed to be indoles versus indolines by virtue of a cycloadduct tautomerization and a unique thermal elimination of methanesulfinic acid for aromatization. With direct access to the requisite allene-1,2-diazines from the products of the inverse electron demand Diels–Alder reactions of 3,6bis(thiomethyl)-1,2,4,5-tetrazine (2), this 1,2,4,5tetrazine→allene-1,2-diazine→indole iterative Diels–Alder strategy constitutes a general approach to synthesis of highly substituted indoles.

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conditions provided N-acetyl cis-trikentrin A (46). In addition, trans-trikentrin A was prepared by an identical sequence following deliberate epimerization of the methyl substituent in 44 under basic conditions. Scheme 5. Total synthesis of cis- and trans-trikentrin A.

Scheme 4. Sequential 1,2,4,5-Tetrazine→1,2-diazine→ benzene Diels–Alder strategy featuring an intramolecular Diels–Alder reaction of an allene-1,2-diazine.

These studies provided the basis of an exceptionally concise total synthesis of cis- and trans-trikentrin A. Treatment of 3,6bis(thiomethyl)-1,2,4,5-tetrazine (2) with the cis/trans mixture of pyrrolidine enamine 41 at room temperature cleanly provided dihydro-1,2-diazine 42 as a single diastereomer, possessing exclusively the cis-dimethyl stereochemistry regardless of the stereochemical integrity of the starting dienophile (Scheme 5).55 This selectivity may be attributed to the kinetically preferred Diels–Alder reaction of the cis-isomer through a less sterically encumbered [4+2] cycloaddition transition state. Subsequent acid-catalyzed elimination of pyrrolidine provided stereochemically pure 1,2-diazine 43 in superb yield (85% overall). Following oxidation to bis-sulfone 44 and introduction of a single allene side chain through an easily controlled S NAr displacement reaction, the intramolecular allene-1,2-diazine Diels–Alder reaction of 45 conducted under mild thermal

Anhydrolycorinone, Hippadine, Anhydrolycorinium Chloride. In efforts that extended the synthetic applications of the 1,2,4,5-tetrazine→1,2-diazine→benzene Diels–Alder strategy, an intramolecular inverse electron demand Diels– Alder reaction of an unsymmetrical 1,2,4,5-tetrazine was employed in the total synthesis of Amaryllidaceae alkaloids (52–54). Their characteristic pyrrolophenathridine skeleton presents an ideal target for the application of unsymmetrical 1,2,4,5-tetrazine→alkene-1,2-diazine→indoline sequential Diels–Alder strategy.56 The unsymmetrical 1,2,4,5-tetrazine 19 was prepared by clean, selective displacement of one methylthio group in 3,6bis(thiomethyl)-1,2,4,5-tetrazine (2) with 1-amino-3-butyne (Scheme 6). The first intramolecular [4+2] cycloaddition was accomplished smoothly at room temperature in superb yield (96%) under N-acylation conditions and the intermediate NBoc derivative prior to cyclization was not detected. The cycloadduct 20 was N-Boc deprotected and used directly in the amide coupling reaction with 49 to afford 1,2-diazine 50. The second intramolecular [4+2] cycloaddition reaction of 50 and subsequent aromatization via elimination of methanol was achieved under more vigorous conditions (265 ºC, 20 h) in quantitative yield to provide anhydrolycorinone precursor 51 bearing fully functionalized pentacyclic skeleton of Amaryllidaceae alkaloids.


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Scheme 6. Total synthesis of anhydrolycorinone, hippadine, anhydrolycorinium chloride.

Scheme 8. Synthesis of OMP.

Scheme 9. Total synthesis of prodigiosin.

OMP. In 1980, Kornfeld and co-workers described the first and a single example of the reductive ring contraction of a dimethyl 1,2-diazine-3,6-dicarboxylate, prepared by a tetrazine Diels–Alder reaction, effected by treatment with Zn/HOAc in the preparation of a dimethyl pyrrole-2,5-dicarboxylate.57 The reaction proceeds through initial reduction to a 1,4dihydrodiazine, subsequent reductive N–N bond cleavage, and final pyrrole cyclization (Scheme 7). Therefore, and in our work, the inverse electron demand Diels–Alder reactions of 1,2,4,5-tetrazines were projected to also provide access to pyrroles through this reductive ring contraction reaction. In our initial exploration of the scope of this reaction, it proved surprisingly tolerant of additional functionality (e.g. ethers, esters, carbamates and ketones), suggesting widespread utilization in organic synthesis.58 Scheme 7. Reductive ring contraction of electron-deficient 1,2diazines to pyrroles.

Our first demonstration of this 1,2,4,5-tetrazine→1,2diazine→pyrrole Diels–Alder strategy provided a simple preparation of octamethylporphyrin (OMP, 58).58 The synthesis began with a room temperature inverse electron demand Diels– Alder reactions of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (1) with 2-triethylsilyloxy-2-butene (55) to afford 1,2-diazine 56 in excellent yield (87%). Treatment of 56 with Zn/HOAc at room temperature smoothly provided the fully substituted pyrrole 57 that serves as the monomeric building block of OMP (Scheme 8). This study was a prelude to use of this strategy in the total synthesis of a family of polypyrroles possessing a common, characteristic pyrrolylpyrromethene skeleton.

Prodigiosin. Prodigiosin (66), a blood-red pigment, was the initial member of this class of natural products and displayed useful antimicrobial and potent cytotoxic activity and immunosuppressive properties that we wanted to explore. 59 It was envisioned that its central pyrrole B ring could be prepared by this 1,2,4,5-tetrazine→1,2-diazine→pyrrole Diels–Alder strategy.60 The room temperature [4+2] cycloaddition of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (1) with 1,1dimethoxyethylene (94%) followed by reductive ring contraction of the resulting 1,2-diazine 59 provided dimethyl 3methoxy-pyrrole-2,5-dicarboxylate (60) (Scheme 9). A threestep sequence achieved the effective differentiation of the C2/C5 carboxylates and selective removal of the electronically and sterically more accessible C5 methoxycarbonyl group, cleanly affording methyl 3-methoxypyrrole-2-carboxylate (61). A subsequent intramolecular palladium(II)-promoted 2,2’bipyrrole oxidative coupling reaction conducted with polymersupported palladium(II) acetate provided the unsymmetrical 2,2’-bipyrrole AB ring system of prodigiosin.60 Both the method developed for the synthesis of the starting unsymmetrical N,N’-dipyrrole carbonyl 63, employing pyrrole1-carboxylic acid and its in situ generated acid chloride 62,61 and its use in an intramolecular unsymmetrical 2,2’-bipyrrole



coupling through two sequential electrophilic palladations and subsequent reductive elimination were unique at the time of our work (1987) and are still innovative in todays’ era of undirected C–H activation reactions for unsymmetrical biaryl couplings. Finally, completion of the prodigiosin synthesis was accomplished through the condensation of aldehyde 65 derived from 64 with 2-methyl-3-pentylpyrrole (Scheme 9). In addition to prodigiosin, desmethoxyprodigiosin (67) and prodigiosene (68) were also prepared by this approach. Their comparisons demonstrated that the sequential removal of the peripheral substituents diminished the in vitro cytotoxic activity (Figure 9).60

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concise total synthesis of isochrysohermidin, but a non-obvious one as well where the core of a non-aromatic five-membered heterocycle was assembled with a heterocyclic azadiene Diels– Alder reaction. Scheme 10. Total synthesis of isochrysohermidin.

Figure 9. Comparison of the cytotoxic activity of prodigiosin, desmethoxyprodigiosin and prodigiosene. Isochrysohermidin. In a unique application of the 1,2,4,5tetrazine→1,2-diazine→pyrrole Diels–Alder strategy, we applied this approach to the total synthesis of d,l- and mesoisochrysohermidin (72), which we projected were ideal candidates for interstrand DNA cross-linking. Central to our approach was the implementation of two consecutive inverse electron demand Diels–Alder reactions of dimethyl 1,2,4,5tetrazine-3,6-dicarboxylate (1) with 1,1,4,4-tetramethoxy-1,3butadiene (69) followed by a double reductive ring contraction reaction for the construction of a fully functionalized 3,3’bipyrrole key intermediate.62 Treatment of 1 with 69 provided the double Diels–Alder cycloadduct 70 in good yield (65%). In a detailed study of this remarkable transformation, the first of the two [4+2] cycloaddition reactions was found to proceed rapidly at room temperature. The second was observed to be slower and required more vigorous, albeit still mild, thermal reaction conditions (60 ºC) due to the increased steric hindrance or decreased nucleophilic character of the intermediate dienophile. Although the aromatization steps requiring the loss of methanol proved sluggish, these were promoted by added 4Å molecular sieves. Double reductive ring contraction of symmetrical 4,4’-bis-1,2-diazine 70 upon treatment with activated zinc in glacial acetic acid smoothly afforded the symmetrical pyrrole dimer 71 in good yield (68%). Significantly, this strategy established the key dimeric skeleton of isochrysohermidin through a simple two-step sequence and constitutes a general approach to highly functionalized 3,3’bipyrroles (Scheme 10). With the dimeric pyrrole skeleton constructed, the completion of the synthesis of isochrysohermidin proved equally concise. Following N-methylation of 3,3’-bipyrrole 71, effective differentiation of the internal and external methyl esters was accomplished through exhaustive ester hydrolysis, cyclic seven-membered anhydride formation, diazomethane esterification of the remaining terminal carboxylic acids, and deliberate anhydride hydrolysis. A final low temperature 1O2 [4+2] cycloaddition across each of the pyrroles followed by in situ oxidative decarboxylation with endoperoxide fragmentation63 generated isochrysohermidin as a readily separable mixture of d,l- and meso-isochrysohermidin. It is likely that most readers, like us, will view this as not only a

With material prepared by this efficient 8-step synthesis, both d,l- and meso-isochrysohermidin were shown to exhibit the projected interstrand DNA cross-linking properties through reversible nucleophilic trap at each of the carbinolamides.62 Not only did this represent the discovery of only the fourth class of natural products to exhibit interstrand DNA cross-linking properties, but it also defined a new and potentially general approach to the development of synthetic DNA cross-linking agents. For instance, incorporation of a single carbinolamide subunit of isochrysohermidin into distamycin, an A–T rich duplex DNA minor groove binding agent, was explored (Figure 10).64

Figure 10. Isochrysohermidin-distamycin hybrids.


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Scheme 11. Total synthesis of ent-(–)-roseophilin.

Roseophilin. Roseophilin (80), a novel antitumor antibiotic, possesses a topologically unique pentacyclic skeleton that consists of a potentially strained macrocycle incorporated in an ansa-bridged azafulvene conjugated with a heterocyclic ring system composed of a substituted furan and pyrrole.59 Our approach to roseophilin featured a 1,2,4,5-tetrazine→1,2diazine→pyrrole Diels–Alder strategy for construction of an appropriately functionalized pyrrole ring, a ring-closing olefin metathesis reaction for introduction of the 13-membered macrocycle and a 5-exo-trig acyl radical-alkene cyclization for formation of the cyclopentanone found in the upper tricyclic core structure of roseophilin.65 The key inverse electron demand Diels–Alder reaction of dimethyl 1,2,4,5-tetrazine-3,6dicarboxylate (1) with the optically active enol ether 73 proceeded effectively at room temperature to provide 1,2diazine 74 in excellent yield (91%). Reductive ring contraction of 74 effected by treatment with Zn/TFA gave the highly functionalized pyrrole 75 for further incorporation into the precursor 76. Ring closing metathesis of 76 afforded 77, completing the introduction of the macrocycle. Following the transformation to phenyl selenoester 78, acyl radical–alkene 5exo-trig cyclization of 78 cleanly formed the strained cyclopentanone and generated 79 as a single disastereomer in 83% yield (Scheme 11). Aside from the cycloaddition studies, it was our introduction of phenyl selenoesters as effective precursors to acyl radicals, their slow competitive reduction relative to alkyl radicals, and our demonstration of their synthetic utility and scope of use in inter- and intramolecular alkene addition reactions that preceded its use in this key step in the total synthesis of roseophilin.66 Notably, the studies culminated in the total synthesis of ent(–)-roseophilin, which along with the concurrently disclosed synthesis of Tius and Harrington67 permitted the assignment of the absolute configuration of natural (+)-roseophilin. Remarkably, our unnatural enantiomer was found to be 2–10

fold more potent than the natural enantiomer in cancer cell growth inhibition assays, providing an unexpected conclusion to synthetic efforts that provided the unnatural enantiomer.65 It also provides an additional valuable tool for defining the mechanism of action and biological target(s) for not only roseophilin, but also the structurally related prodigiosins. Incapable of promoting the metal-dependent DNA damage and cleavage reactions observed with the prodigiosins and championed by some,68 the observations suggest the antimicrobial activity, cell growth inhibition activity, and the immunosuppressive properties of the prodigiosins and roseophilin are derived from alternative and perhaps independent targets. Thus, although the pyrrolylpyrromethene core of the prodigiosins can generate reactive oxygen species when bound by copper and can also function as a chloride ion symporter,69 the recent discovery by Harran that tailored fragments of roseophilin and unique roseophilin/prodigiosin hybrids can selectively antagonize Mcl-1 provides an attractive alternative biological target that entails inhibition of a protein– protein interaction.70 Ningalins. Beginning in 1997, members of a growing ningalin family of marine natural products have been isolated, which possess potent Pgp inhibitory activity, effective multidrug resistance (MDR) reversal properties and potent cytotoxic activity against sensitive and resistant tumor cell lines.71 Each of these natural products contains a central highly substituted pyrrole nucleus bearing diverse aryl substituents, which make them ideal targets for the 1,2,4,5-tetrazine→1,2diazine→pyrrole Diels–Alder strategy (Figure 11). Both the unique reactivity of dimethyl 1,2,4,5-tetrazine-3,6dicarboxylate (1) and the electron-rich character of the required diaryl alkynes combine to provide well matched cycloaddition partners for each of the ningalins and the related natural products.72–74

Figure 11. Unified strategy for total synthesis of ningalin natural products.



Ningalin A. The total synthesis of ningalin A (84), the simplest member of this family of marine natural products, began with the inverse electron demand Diels–Alder reaction of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (1) with the electron-rich diaryl acetylene 81 to afford the 1,2-diazine 82 in excellent yield (87%).72 The relative effectiveness of this transformation may be attributed to the electron-donating properties of the dienophile aryl alkoxy groups which increase the nucleophilic character of the acetylene and improve what is a typically poor reactivity of unactivated alkynes towards 1,2,4,5-tetrazines. Subsequent reductive ring contraction of the 1,2-diazine by treatment with Zn/HOAc smoothly afforded the pyrrole 83 bearing all requisite functionality and the key skeleton of ningalin A (Scheme 12).

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Scheme 13. Total synthesis of lamellarin O and lukianol A.

Scheme 12. Total synthesis of ningalin A.

Lamellarin O and Lukinol A. The total synthesis of lamellarin O (89) and lukianol A (90) employed a Diels–Alder reaction of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (1) with the alternative diaryl acetylene 85 to assemble the fully substituted 1,2-diazine 86 (85%), which was followed by a Znmediated reductive ring contraction reaction to provide the corresponding pyrrole core 87 in a highly efficient 2-step sequence.72 N-alkylation of 87 with 2-bromo-4’methoxyacetophenone gave the pentasubstituted pyrrole 88. Selective hydrolysis of the symmetrical diester 88 with 1.3 equiv LiOH followed by acid-promoted decarboxylation of the resulting monoacid afforded the appropriately substituted and functionalized pyrrole core found in both lamellarin O and lukianol A (Scheme 13).

Permethyl Storniamide A. Similarly, the first of the two key transformations in the total synthesis of permethyl storniamide A (95) relied on the Diels–Alder reaction of dimethyl 1,2,4,5tetrazine-3,6-dicarboxylate (1) with the acetylene 91 in toluene to give 1,2-diazine 92 in superb yield (90%).72 The unusual facility with which this [4+2] cycloaddition reaction occurs may be attributed to the six methoxy groups donating electron density into the alkyne dienophile. Subsequent zinc-promoted reductive ring contraction of 92 afforded the pyrrole 93, which was further converted to the core structure 94 found in the natural product via N-alkylation (Scheme 14). Scheme 14. Total synthesis of permethyl storniamide A.


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Ningalin B. Complementary to the studies described above, the approach to ningalin B (100) also employed the 1,2,4,5tetrazine→1,2-diazine→pyrrole Diels–Alder strategy. The inverse electron demand Diels–Alder reaction of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (1) with the unsymmetrical diaryl acetylene 96 proceeded smoothly and afforded 1,2diazine 97 in excellent yield (92%).73 Reductive ring contraction of 97 afforded the pyrrole core 98 and the subsequent N-alkylation of 98 followed by MOM deprotection with concomitant lactonization generated lactone 99, containing the full skeleton of ningalin B. The total synthesis of ningalin B was completed by the selective hydrolysis of methyl ester 99, decarboxylation and global deprotection (Scheme 15).

that entailed Curtius rearrangement of a dicarboxylic acid and subsequent in situ air oxidation to directly provide the biphenylene quinone methide found central to the structure of ningalin D.74 Scheme 16. Total synthesis of ningalin D.

Scheme 15. Total synthesis of ningalin B.

Ningalin D. The most complex of the ningalin marine natural products is ningalin D (105), incorporating a biphenylene quinone methide superimposed on a now oxidized pentasubstituted pyrrole core. The key element of our synthetic approach to ningalin D was a heterocyclic azadiene Diels–Alder reaction of 1 followed by reductive ring contraction of the resulting 1,2-diazine, affording the fully substituted pyrrole core central to the ningalin D structure. The key [4+2] cycloaddition of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (1) with the symmetrical alkyne 101 proceeded smoothly in refluxing toluene to provide the 1,2-diazine 102 in superb yield (87%) and was followed by treatment with Zn/TFA to provide the pyrrole core 103.74 Although such reductive ring contraction reactions are typically conducted with Zn/HOAc, we disclosed that the overall reaction is much faster (4–7 vs 24 h for 102, 25 ºC) with Zn/TFA and often much cleaner (Scheme 16). Additional highlights of the synthesis include a double Dieckmann cyclization of 104 to close the C and D aryl rings, and a subsequent highly effective and sterically demanding Suzuki coupling of the corresponding C and D phenol triflates for introduction of the F and G aryl rings. Perhaps the most innovative of the additional key steps was a penultimate and unusually effective formal oxidative decarboxylation reaction

In the course of the efforts on the ningalins, a large series of structural analogues were prepared by this approach in a concise and efficient manner (5–9 steps, 20–50% overall yield). These studies enabled us to conduct a systematic biological evaluation of the ningalin family and their synthetic analogues.72-75 Although most possess modest to low inherent cytotoxic activity, many were found capable of reversing the multidrugresistant (MDR) phenotype, resensitizing resistant tumor cell lines such as HCT116/VM46 to vinblastine and doxorubicin at a lower dose than the prototypical agent verapamil. Notably, each of active compounds was found to incorporate three hydrophobic domains that characterize the Pgp binding pharmacophore models, but unique in ningalin series is the lack of a basic amine central to the structure. As such, they not only



constitute efficacious compounds exhibiting unusually potent MDR reversal activity for a lead series, but they also depart from structural features considered central to common pharmacophore models of Pgp binding. Lycogarubrin C and Lycogalic acid. Lycogalic acid (aka chromopyrrolic acid, 113), first isolated from Lycogala epidendrum, has been identified as a common intermediate in the biosynthesis of the indolo[2,3-a]carbazole alkaloids, including rebeccamyin and staurosporine. Complementary to the synthetic studies on the ningalin family, we envisioned that lycogalic acid and its methyl ester, lycogarubin C (112), would be readily accessible through use of a 1,2,4,5-tetrazine→1,2diazine→pyrrole Diels–Alder strategy.76 An initial approach explored the inverse electron Diels–Alder reaction of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (1) with acetylene 106 and provided 1,2-diazine 107 in good yield (65%). However, the reaction was slow, requiring 15 d in refluxing toluene (110 ºC). Compared to the productive dialkoxyphenyl acetylenic dienophiles that were effectively used in the total synthesis of ningalins, the lower reactivity of 106 may be attributed to its less electron-rich character. Therefore, the more electron-rich acetylene, 1,2-bis(tributylstannyl)acetylene (108), was adopted in an alternative approach. The Diels–Alder reaction of 108 with dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (1) proceeded smoothly under remarkably mild conditions (45 ºC, dioxane, 24 h), providing 1,2-diazine 109 in exceptional yield (97%). Stille coupling of 1,2-diazine 109 with 110 proceeded effectively and twice, affording the same key 4,5-bis(indol-3yl)-1,2-diazine 107 in superb yield (90%). Treatment of 107 with Zn/HOAc cleanly promoted the reductive ring contraction reaction, providing pyrrole 111 containing the core structure of the natural products and completing the 1,2,4,5-tetrazine→1,2diazine→pyrrole conversions originally envisioned (Scheme 17).

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2.2. 1,2,4-Triazines Similar to 1,2,4,5-tetrazines, 1,2,4-triazines participate effectively as electron-deficient heterocyclic azadienes in inverse electron demand Diels–Alder reactions with electronrich dienophiles, such as enamines, ynamines, and strained olefins.77 The [4+2] cycloaddition of the parent 1,2,4-triazine occurs across C3/C6 with the nucleophilic carbon of the electron-rich olefins attached to C3. Loss of N 2 from the initial [4+2] cycloadduct followed by aromatization provides substituted pyridines in which the C3 and C4 substituents are derived from the dienophile. The only exceptions to this mode of cycloaddition are ynamines, which preferentially add across C5/N2 to provide pyrimidines.78 Typically, the Diels–Alder reactions proceed under mild conditions (25–80 ºC), and the rate-limiting step of the overall reaction is often not the [4+2] cycloaddition or subsequent loss of nitrogen, but rather the aromatization step.79 Early in our studies, we developed a “catalytic” inverse electron demand Diels–Alder reaction of in situ generated enamines with 1,2,4-triazines, which constitutes a novel variant of the conventional Diels–Alder reaction. Catalytic and in situ generation of a pyrrolidine enamine in the presence of a 1,2,4-triazine precedes the ensuing inverse electron demand Diels–Alder reaction which is followed by the immediate loss of nitrogen and finally aromatization with regeneration of the catalytic pyrrolidine, completing a mild, one-flask pyridine annulation (Figure 12).80 Additives (e.g., 4Å MS) used to promote in situ enamine formation often also improved the slow aromatization step such that the catalytic version of the reaction provided higher reaction yields than the stoichiometric reaction. Notably, it also represented one of the first examples (1982) of the enamine activation mode of organocatalysis and the first employed for a cycloaddition.

Scheme 17. Total synthesis of lycogarubrin C and lycogalic acid.

Figure 12. 1,2,4-Triazine–enamine [4+2] cycloaddition. Added electron-withdrawing substituents on the 1,2,4triazine nucleus affect the rate of cycloaddition, influence the mode of cycloaddition (C3/C6 vs C5/N2), and may control or alter the cycloaddition regioselectivity (Figure 13). 3,5,6Tris(ethoxycarbonyl)-1,2,4-triazine (114) behaves in a manner analogous to 1,2,4-triazine itself, but it is more reactive by virtue of its enhanced electron-deficient character.81 However, the increased reactivity and the noncomplementary substitution with C3 and C5 electron-withdrawing substituents does result in the occasional observation of lower reaction regioselectivity. The single noncomplementary C3 substitution with a strong electron-withdrawing substituent serves to reverse the [4+2] cycloaddition regioselectivity without altering the mode of cycloaddition (C3/C6 vs C5/N2), but it does diminish the rate of reaction. Similarly, the complementary substitution with a


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single C6 electron-withdrawing substituent increases the rate, regioselectivity, and mode (C3/C6) of cycloaddition.

50% yield from the reaction of 27 with 28 (6.2 kbar, 25 ºC) (Figure 14).19 Today, this would be a reaction especially wellsuited for H-bonding catalysis with HFIP or TFE. Based on the sequential implementation of two inverse electron demand Diels–Alder reactions, our convergent synthesis of streptonigrin (17 steps) remains one of the shortest total synthesis disclosed to date.17, 20-22

Figure 13. 1,2,4-Triazine substituent effects. Streptonigrin. As highlighted earlier, streptonigrin (30) is a potent antitumor antibiotic that contains a fully substituted and highly functionalized pyridine. Central to our synthetic approach to streptonigrin was a 1,2,4,5-tetrazine→1,2,4triazine→pyridine iterative Diels–Alder strategy for construction of this fully functionalized pentasubstituted pyridine core.19 The first of two Diels–Alder reactions, the [4+2] cycloaddition of thioimidate 27 with dimethyl 1,2,4,5tetrazine-3,6-dicarboxylate (1), was presented in the section on 1,2,4,5-tetrazines. The second [4+2] cycloaddition reaction was that of the product 1,2,4-triazine 27 with the enamine 28 for the pyridyl C ring construction. In our preliminary investigation of this transformation, 3,5,6-tris(ethoxycarbonyl)-1,2,4-triazine (114) underwent rapid reaction with the pyrrolidine enamine 28 under mild thermal conditions (CHCl3, 45 ºC, 3 h) with addition preferentially occurring across C3/C6 of the 1,2,4-triazine nucleus and the nucleophilic carbon of the dienophile attached to C3 (Figure 14).82 This reaction provided the fully substituted pyridine 115 in good yield and regioselectivity (59%, 9:1). However, the Diels–Alder reaction of the pyrrolidine enamine 28 with 1,2,4-triazine 27 under thermal conditions failed to deliver the desired product due in part to the thermal instability of the dienophile. Although the morpholino enamine 116 provided a sluggish addition with 1,2,4-triazine 27 and the desired product was obtained, the vigorous reaction conditions (CHCl3, 120 ºC, 42 h) required for complete reaction reduced the observed regioselectivity (68%, 1:1). All efforts to promote the [4+2] cycloaddition of the morpholino or pyrrolidine enamines 116 and 28 with 1,2,4-triazine 27 with the conventional Lewis acids were unsuccessful and only led to the consumption of the electron-rich olefin with no evidence of Diels–Alder catalysis. However, it was found that the use of pressure-promoted Diels–Alder reaction conditions were useful in increasing the rate of cycloaddition at mild reaction temperatures (25 ºC). As summarized in Figure 14, the pressure-promoted (6.2 kbar, 25 ºC) cycloaddition of either enamine 28 or 116 with 1,2,4-triazine 27 provided the desired cycloadducts 29/117 with a preference for the desired regioisomer 29 (1.4:1 and 2.8:1 29/117, respectively) in acceptable yields (58% and 65%). Under the best conditions examined, the desired product 29 bearing the full skeleton with the requisite functionality of streptonigrin was isolated in nearly

Figure 14. 1,2,4-Triazine–enamine cycloaddition in the total synthesis of streptonigrin. Lavendamycin. Lavendamycin (122) is a highly substituted and highly functionalized quinolone-5,8-quinone structurally and biosynthetically related to streptonigrin.83 It was envisioned that the central pyridine C ring could be prepared through an inverse electron demand Diels–Alder reaction of 1,2,4-triazine 114 with the pyrrolidine enamine 118.84–85 Cycloaddition of the enamine 118 occurred across C3/C6 of 3,5,6tris(ethoxycarbonyl)-1,2,4-triazine (114) with the nucleophilic dienophile carbon attached to C3, and provided the fully substituted 4-arylpyridine 119 central to the structure of lavendamycin in good yield (50%). It is notable that the 1,2,4triazine triester substitution permitted the reaction to be conducted at room temperature and proceeded with the good regioselectivity (6–8:1) (Scheme 18). The completion of the total synthesis of lavendamycin, complementary to four others that emerged near simultaneously,17 was based on a clever differentiation of the three ethyl esters, an innovative Pd(0)mediated amine aromatic substitution reaction for closure of the β-carboline (121)85 and a late-stage Friedlander condensation. Among these key transformations, the Pd(0)-mediated amination for construction of the β-carboline (121) is one we are especially proud of, having been conducted while I was still



a beginning faculty member in a small program. At a time the mechanism of even the Ullmann and Goldberg coupling reactions were unresolved, the reaction was designed to proceed by Pd(0) oxidative insertion into the aryl bromide (120), formation of a six-membered Pd(II) complex with coordination of the C3 pyridyl amine, and subsequent reductive elimination of Pd(0) with nitrogen–carbon bond formation.85 This constituted the first example of a palladium-catalyzed preparation of aryl amine with carbon–nitrogen bond formation, demonstrated that reductive elimination of nitrogen with nitrogen–carbon bond formation was possible, defined the viable Pd(0)/Pd(II) catalytic cycle, and ultimately provided the foundation for the development of the Buchwald–Hartwig reaction in studies initiated 10 years later.

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addition of the A-ring to the C-ring C6 carboxylate followed by Friedel–Crafts closure of the B-ring provided the fully functionalized phomazarin skeleton. Our synthetic work confirmed the latest structural assignment for phomazarin and provided a preliminary assessment of the natural product and the closely related synthetic analogues. The biological evaluation in a cancer cell growth inhibition assay revealed that phomazarin was inactive, but that two of its immediate precursors, ester 126 and 127, exhibited potent cytotoxic activity.

Scheme 18. Key steps in the total synthesis of lavendamycin.

Figure 15. Key step in the total synthesis of phomazarin and biological evaluation of phomazarin and its analogues. 2.3 1,3,5-Triazines

Phomazarin. Phomazarin (125) is the most widely known and extensively studied aza anthraquinone whose original structural assignment was revised twice. Our total synthesis, which served to unambiguously establish the structure of phomazarin, was based on the implementation of a heteroaromatic azadiene Diels–Alder reaction (1,2,4triazine→pyridine) for preparation of the fully substituted and appropriately functionalized pyridine C ring. 86 The [4+2] cycloaddition reaction of 3,5,6-tris(ethoxycarbonyl)-1,2,4triazine (114) with 1,1,2-trimethoxy-ethylene (123) proceeded effectively at room temperature and typically provided a mixture of the both unaromatized and aromatized cycloadduct, which was fully converted to the pyridine product 124 at higher reaction temperatures (100 ºC, dioxane, 30 min) and by the brief Et3N treatment (25 ºC, 1 h). The pyridine product 124 was obtained in excellent yield (85%) and was ideally functionalized for elaboration to the pyridine C-ring of phomazarin (Figure 15). The dienophile methoxy substituents not only served to enhance its nucleophilic character, but also served to introduce the phomazarin C3 and C4 hydroxy groups. In a complementary manner, the three electron-withdrawing ester substituents on the 1,2,4-triazine nucleus increased its [4+2] cycloaddition reactivity toward such electron-rich dienophiles and provided the necessary functionality for introduction of the natural product C2 carboxylic acid and C9/C10 quinone carbonyls. Regioselective nucleophilic

1,3,5-triazines are electron-deficient six-membered heteroaromatic rings that participate in well-defined [4+2] cycloaddition reactions with electron-rich dienophiles, such as ynamines, enamines, N,O-ketene acetals and amidines (Scheme 19).87 Typically, the initial Diels–Alder reaction is rapid and occurs at room temperature, and the slow step in the overall reaction cascade is the retro Diels–Alder loss of a nitrile followed by aromatization. The addition of electronwithdrawing substituents to the 1,3,5-triazine nucleus serves to accelerate both the rate of the initial Diels–Alder reaction as well as this subsequent retro Diels–Alder reaction of the cycloadduct. 2,4,6-Tris(ethoxycarbonyl)-1,3,5-triazine (128), like 1,3,5triazine itself, participates in [4+2] cycloaddition reactions at room temperature with the more reactive electron-rich dienophiles. The [4+2] cycloaddition of 128 with ynamines proceeds at room temperature and is followed by a retro Diels– Alder reaction (40–100 ºC) with loss of ethyl cyanoformate to provide the corresponding substituted pyrimidines.88 In contrast, the [4+2] cycloaddition reaction of 128 with enamines (25–100 ºC) provides cycloadducts that more slowly undergo a retro Diels–Alder reaction and subsequent aromatization. Although efforts to promote the retro Diels–Alder reaction/aromatization proved modestly successful, the reaction may be effectively carried out by either subsequent acid treatment (4 N HCl or HOAc) or even by simply conducting the reaction under acidic conditions (CH2Cl2–HOAc), that serve to facilitate the retro Diels–Alder reaction and subsequent aromatization (Scheme 19).88


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Scheme 19. 1,3,5-Triazine–ynamine and 1,3,5-triazine– enamine Diels–Alder reactions.

In addition, we found that amidines effectively participate in [4+2] cycloaddition reactions with 1,3,5-triazines.89 The unique cycloaddition reaction of amidines with 1,3,5-triazines proceeds through in situ amidine to 1,1-diaminoethene tautomerization and its [4+2] cycloaddition with the 1,3,5triazine, and is followed by imine generation through loss of ammonia from the Diels–Alder cycloadduct, imine to enamine tautomerization, and retro Diels–Alder loss of ethyl cyanoformate to provide the product pyrimidine. In our studies, this reaction proved general for a wide range of amidines and can be effectively conducted using either the 1,3,5-triazine or the amidine as the limiting reagent, indicating that the in situ trap of the 1,1-diamino-alkene derived from amidine tautomerization is unusually efficient. Moreover, in addition to the differences in the relative ease of preparation of amidines versus ynamines especially where R = H, amidines may be employed without deliberate introduction of an amine protecting group and permits the use of cyclic dienophiles not accessible with ynamines (Figure 16). Consistent with expectations, addition of electronwithdrawing substituents to the 1,3,5-triazine facilitates cycloaddition, and the more nucleophilic dienophiles derived from amidine tautomerization participate more readily than those derived from thioimidates or imidates.89 Moreover, it was shown that the presence of electron-withdrawing substituents serve to accelerate the retro Diels–Alder reaction of the [4+2] cycloadducts. The Diels–Alder cycloadducts of the parent 1,3,5-triazine or 2,4,6-tris(ethoxycarbonyl)-1,3,5-triazine (128) typically undergo the retro Diels–Alder reaction under mild thermal conditions (40–100 ºC). In contrast, the [4+2] cycloaddition reaction of ynamines with 2,4,6-tris(methylthio)1,3,5-triazine (129) provide stable [4+2] cycloadducts, which undergo the retro Diels–Alder reaction to provide the substituted pyrimidines under subsequent thermal (150–230 ºC) or acid-catalyzed (p-TsOH, 100 ºC) reaction conditions. In addition, the retro Diels–Alder reaction may be accelerated by treatment with m-CPBA to provide the methanesulfonyl derivatives that undergo in situ retro Diels–Alder reaction with loss of methanesulfonylcyanate at room temperature (Figure 16).88-89

Figure 16. 1,2,4-Triazine–amidine Diels–Alder reactions. 1,3,5-Triazine and dienophile substituent effects. Pyrimidoblamic Acid and Bleomycin A2. Pyrimidoblamic acid (135) is the heteroaromatic chromophore of bleomycin A2 (136), a potent and clinically effective antitumor agent that derives its properties through a metal-dependent sequenceselective cleavage of duplex DNA. Our initial approach to the pyrimidoblamic acid subunit was based on two complementary [4+2] cycloaddition reactions of 2,4,6-tris(ethoxycarbonyl)1,3,5-triazine (128) for the construction of a fully substituted pyrimidine nucleus.90 The first of these constitutes a Diels– Alder reaction of 1,3,5-triazine 128 with ynamine 130 in which the [4+2] cycloaddition occurs at room temperature, and it was the retro Diels–Alder collapse of the cycloadduct that required the more vigorous thermal conditions (95%, 100 ºC) (Scheme 20). Acid-catalyzed debenzylation of the resulting pyrimidine 131 (TfOH, CH2Cl2, 40 ºC) provided 4-aminopyridimine 132 in good yield (75%).90 Alternatively, 132 was derived directly and more conveniently in one step by treatment of 1,3,5-triazine 128 with propionamidine hydrochloride (133) (DMF, 100 ºC) in a reaction cascade that proceeded with thermal tautomerization of 133 to 1,1-diaminopropene and its [4+2] cycloaddition with 128 (Scheme 20).90 The sequential elimination of ammonia, imine to enamine tautomerization, and subsequent retro Diels–Alder loss of ethylcyanoformate under the reaction conditions



provided 132 directly in excellent yield (80%). The reaction was found to proceed best in the polar aprotic solvent DMF under modest thermal reaction conditions (100 ºC), and the amidine hydrochloride salts were shown to routinely provide higher yields of the product pyrimidines than the corresponding free base. It is likely that the tautomerization of 133, retro Diels–Alder reaction and the subsequent aromatization reaction were facilitated by both the presence of HCl derived from the amidine hydrochloride salts and the use of a polar, aprotic solvent under the thermal reaction conditions (>80 ºC). Scheme 20. Total synthesis of (–)-pyrimidoblamic acid and the key steps in total synthesis of bleomycin A2.

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The additional key strategic elements of our approach to bleomycin A2 include two highly diastereoseletive glycosylation reactions through the use of diphenyl glycosylphosphates as glycosyl donors in the synthesis of both the disaccharide and the glycosidated β-hydroxyl-L-histidine.92 Finally, sequential amide coupling of the glycosidated βhydroxyl-L-histidine subunit with tetrapeptide S and (–)pyrimidoblamic acid provided bleomycin A2 in a highly convergent manner. The intact sulfonium salt and only two protecting groups were incorporated into the final coupling substrates simplifying the final stages of the total synthesis. (+)-P-3A. Analogous to our approach to pyrimidoblamic acid, (+)-P-3A (139) and a series of structurally related agents were prepared using the [4+2] cycloaddition reaction of 2,4,6tris(ethoxycarbonyl)-1,3,5-triazine (128) with in situ generated 1,1-diaminoethylene derived from thermal tautomerization of acetamidine (85%).93 The corresponding ynamine [4+2] cycloaddition was not examined due to its synthetic inaccessibility. Analogous to the total synthesis of (–)pyrimidoblamic acid and following strategic differentiation of two ethyl esters and imine formation, the subsequent use of a diastereoselective N-acyloxazolidinone enolate-imine addition reaction provided the stereocontrolled introduction of the pyridimidine C2-acetamido side chain. Finally, without protection of the unreactive arylamine or hindered secondary amine of 137, direct amide coupling of 137 with the dipeptide 138 followed by acid-promoted global deprotection afforded (+)-P-3A (Scheme 21). Scheme 21. Total synthesis of (+)-P-3A.

A remarkably simple differentiation of the [4+2] cycloadduct ethyl esters was accomplished by treatment with NaBH4 (0 ºC) in which the sterically more accessible and electronically more reactive pyrimidine C2 ester was selectively reduced. Following conversion to the aldehyde and imine formation, diastereoselective addition of the stannous enolate of an Evans’ N-acyloxazolidinone provided predominately the anti imine addition product 134 bearing the absolute configuration of the natural product (anti:syn = 87:13).91 In this work, the absolute stereochemistry of the C2 benzylic center was unambiguously established and served to confirm the original but unconfirmed assignment of Umezawa.

Bleomycin A2 acts through the sequence selective cleavage of DNA by a reaction that is both metal-ion and O2 dependent.96 Approximately 70 analogues of the natural product 90, 92-94 that probed each subunit and nearly every substituent in the structure were prepared through use of this modular total synthesis of


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bleomycin A2.95 Our studies revealed that nearly every substituent and functional group in the natural product is integral to its activity.95 Key among the studies were those that helped establish that a G triplex-like H-bonding in the minor groove is responsible for the DNA cleavage selectivity, 97 and that fundamental conformational properties within the linker region of bleomycin contribute to the efficiency of DNA cleavage.98 A NMR-derived high resolution structure of DNA bound deglycobleomycin A2 was established in collaboration with JoAnne Stubbe (Figure 17).99

Figure 17. Top: Functional roles of bleomycin A2 subunits. Middle: DNA cleavage selectivity of bleomycin A2 derived from G-triplex-like H-bonding. Bottom: Structure of CoIII-OOH bleomycin A2 bound to DNA determined by NMR

into a compact biologically active compound.95 It is the pyrimidine C4 amino group and pyrimidine N2 that H-bonds with G at a cleavage site, providing the anchoring DNA interaction and recognition responsible for the DNA cleavage selectivity.97 The combined linker substituents restrict the compound to a now predictable dominant compact conformation, preorganizing the molecule into a single rigid conformation productive for DNA cleavage by the bound complex (Figure 17).98,100 2.4. 1,2,3-Triazines 1,2,3-Triazines, or v-triazines, were unknown until Smolinsky’s synthesis of 4,5,6-triphenyl-1,2,3-triazine through the thermolysis of an azidocyclopropene in the course of his studies on nitrenes in the 1960s.101 1,2,3-Triazines are electrondeficient, six-membered aromatic heterocycles containing three contiguous nitrogen atoms. It represents the least explored of the isomeric triazines. In part this may be attributed to the early challenges on their preparation combined with their poor cycloaddition scope in initial studies.102 Igeta provided a convenient synthesis,103 entailing the oxidative (NaIO4) ring expansion of N-aminopyrazoles, but the scope of their cycloaddition reactions remained limited. In 2011, there were only a handful of substituted monocyclic 1,2,3-triazines known at the time we initiated our efforts.101-104 We conducted a systematic study of the reactivity of 1,2,3-triazines in inverse electron demand Diels–Alder reactions, including an examination of the impact of a C5 substituent. 105 The C5 substituents were found to predictably exhibit a remarkable impact on the 1,2,3-triazine cycloaddition reactivity and further enhanced their intrinsic C4/N1 cycloaddition regioselectivity. Moreover, the impact of the addition of a complementary C5 electron-withdrawing substituent (R = NO 2 > CO2Me > Ph > H)105 was of a magnitude that it converted the modest cycloaddition scope of the parent 1,2,3-triazine into a heterocyclic azadiene system with a broad synthetic utility, substantially expanding the range of participating dienophiles and enhancing the rate of productive cycloaddition. Ynamine, enamine, ketene acetal, enol ether and enol acetate cycloadditions proved effective, the 5-carbomethoxy and especially the 5-nitro-1,2,3-triazine display an even larger scope of productive dienophiles, and a newly discovered amidine or imidate cycloaddition105-106 was found to proceed with extraordinary rates and efficiencies. These studies were extended to systematic examinations of C5 substituted electronrich and electron-poor 1,2,3-triazines bearing noncomplementary substitution patterns,106 providing a comprehensive understanding of a substituent’s impact on the scope, reactivity, and regioselectivity of 1,2,3-triazine cycloaddition reactions. Notably, even the electron-rich 1,2,3triazines participate in the newly discovered and effective cycloaddition reactions with amidines. Combined, the studies provided an additional new heterocyclic azadiene, complementary to the isomeric 1,2,4-triazines and 1,3,5triazines, that participates in powerful inverse electron demand Diels–Alder reactions and extends the heterocyclic ring systems accessible with the methodology. Additional observations led to the development of a method for the synthesis of βaminoenals through ring-opening of the parent 1,2,3-triazine with secondary amines (Figure 18).107

These studies defined a remarkable integration of the functional and conformational features of the natural product



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Figure 19. HFIP-promoted inverse electron demand Diels– Alder reactions of triazines. Yields in parentheses are those obtained with chloroform as solvent.

Figure 18. 1,2,3-Triazine Diels–Alder reactions. Finally, in the course of recent work that provided a total synthesis of methoxatin, we discovered the first general method for catalysis of the inverse electron demand Diels–Alder reactions of heterocyclic azadienes that entails hydrogen bonding activation by hexafluoroisopropanol (HFIP, Figure 19).108 This development likely expands the scope of all heterocyclic azadiene cycloadditions far beyond what we highlight herein, analogous to the impact Lewis acid catalysis had on the normal Diels–Alder reaction.

Pyrimidoblamic Acid and P-3A. Bleomycin A2 is the major component of the clinical anticancer drug Blenoxane. Although currently used in the clinic,95-96, 109 treatment with bleomycin is often limited by dose-dependent pneumonitis, lung fibrosis, and skin toxicities.110 The discovery of bleomycin-based analogues that lack off-target toxicity would improve their tolerability. Each structural unit of bleomycin plays an important role in the DNA binding and cleavage reaction that is responsible for its biological effects (see Figure 17). From a series of studies, (–)pyrimidoblamic acid and P-3A were identified as key subunits for the preparation of improved and/or simplified bleomycin analogues.111 Three prior total syntheses of (–)-pyrimidoblamic acid had been disclosed, including our own, and typically suffered from an inability to (fully) control the stereochemistry at the benzylic pyrimidine C2 tertiary center.90, 112 Our synthesis, like that of P3A, utilizing a 1,3,5-triazine cycloaddition (see Scheme 20) relied on a late-stage introduction of this center in a reaction that proceeded with modest diastereoselectivity (87:13). We envisioned second-generation total syntheses of (–)pyrimidoblamic acid and P-3A in which all necessary stereochemistry would be installed prior to a late-stage 1,2,3triazine [4+2] cycloaddition for introduction of the pyrimidine.113 This would address the limitation inherent in previous approaches, while also permitting late-stage divergent modification of both the natural product side chain or core heterocycle. Our second-generation total synthesis of (–)-pyrimidoblamic acid enlisted a [4+2] cycloaddition reaction between a trisubstituted 1,2,3-triazine and an appropriately functionalized amidine (Scheme 22). Consistent with our prior studies, the cycloaddition reaction was found to proceed at room temperature or lower and the amidine was found to add exclusively across C4/N1 (C6/N3) with no evidence of a redirected C5/N2 mode of cycloaddition. Cycloadduct 142 was produced as a single diastereomer with full control of the side chain stereochemistry in 54% yield (5 °C, 14 h then 25 °C, 6 h). The synthesis of (–)-pyrimidoblamic acid was completed in


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seven steps, involving protection of the secondary amine, hydrolysis and Curtius rearrangement of the ethyl ester, removal of the acetonide protecting group followed by Jones oxidation, amidation of the resulting carboxylic acid with ammonia and HOBt/EDCI, and a final global deprotection.

Scheme 23. Total synthesis of P-3A.

Scheme 22. Total synthesis of (–)-pyrimidoblamic acid.

Application of the key 1,2,3-triazine cycloaddition to the synthesis of P-3A might appear straightforward, but it was not clear what the impact of the 1,2,3-triazine C5 substituent might be. A study of the relative cycloaddition reactivity of the C5 substituted and unsubstituted triazines was conducted and the unsubstituted 1,2,3-triazine 144 was found to be even more reactive than its C5-methyl counterpart, providing the product pyrimidine at a faster rate and in a higher yield, with appreciable conversions observed in as little as 5 min. Importantly and despite the lack of a C5 substituent, 144 showed no evidence of competitive cycloaddition across C5/N2 (Scheme 23).113

The key cycloaddition reaction of 144 with amidine 140 proceeded smoothly, providing the desired pyrimidine in 76% yield as a single diastereomer (25 °C, CH 3CN, 12 h, Scheme 23).113 Pyrimidine 145 was elaborated to P-3A in a route modeled on that used for (–)-pyrimidoblamic acid. Amine Bocprotection, saponification of the ethyl ester, Curtius rearrangement and removal of the acetonide protecting group afforded amino alcohol 147, which was transformed to the primary carboxamide in two steps. Saponification of the t-butyl ester and coupling with NIm-Boc-L-His-L-Ala-Ot-Bu provided the fully assembled and protected P-3A, which was converted to the natural product in a global deprotection using TFA– CH2Cl2 (3:1). Dihydrolysergic Acid and Dihydrolysergol. Among the pharmacologically active ergot alkaloids,114 lysergic acid115 and its semisynthetic derivative lysergic acid diethylamide (LSD) are the most widely recognized members and several other derivatives are used clinically in the treatment of a range of neurological disorders.116 Due to the wide range of activities exhibited by this class of natural products and the interesting structural features inherent in the conformationally-restricted phenethylamine pharmacophore, the ergot alkaloids have been the subject of extensive study over the last half century.117 We reported a total synthesis of dihydrolysergic acid and dihydrolysergol,118 the latter of which had been prepared only by semisynthesis from lysergic acid prior to our work. The synthesis relied on two key steps. In addition to a powerful Diels–Alder reaction of 5-carbomethoxy-1,2,3-triazine with a conjugated enamine,105b we used an intramolecular Pd(0)catalyzed indole annulation,119 which we had developed earlier



in other studies, for the formation of a tricyclic ketone isomeric with those117,120 widely used in accessing the ergot alkaloids. By design, the advanced ketone-derived conjugated enamine and a series of late-stage heterocyclic azadiene inverse electron demand [4+2] cycloaddition reactions were used for divergent synthesis of ergot alkaloid analogues bearing deep-seated structural changes not readily accessible by conventional approaches.

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Scheme 25. Total synthesis of dihydrolysergic acid and dihydrolysergol.

Scheme 24. Synthesis of tricyclic ketone 150 enroute to dihydrolysergic acid and dihydrolysergol.

The synthesis of dihydrolysergic acid and dihydrolysergol began with the preparation of ketone 150 (Scheme 24). This sequence rests on the intramolecular Pd(0)-catalyzed Larock cyclization of 148, forming indole 149 in excellent yield (90%). Our prior studies on the intramolecular Larock annulation targeting the chloropeptins121 revealed that catalysts such as PdCl2(PPh3)2, Pd(PPh3)4 and Pd2(dba)3 were effective in the reaction while PdCl2, PdCl2(PhCN)2 or PdCl2(PPh3)2 were not. Pd2(dba)3 was first found to be especially successful in a challenging Suzuki coupling reaction in our vancomycin total synthesis, arising from an unusually rapid oxidative addition step with an analogous electron-rich aryl bromide.122 Optimization of our conditions produced the desired indole from a hindered o,o’-substituted aryl bromide substrate in 90% yield on gram scale (15% Pd2(dba)3, DtBPF, Et3N, DMF, 130 °C, 3 h). Further elaboration of 149 over 4 steps provided the tricyclic ketone 150.

The key inverse electron demand Diels–Alder reaction of 150 with 5-carboxmethoxy-1,2,3-triazine (152) (Scheme 25) was found to proceed in excellent yield upon conversion of ketone 150 to enamine 151 (pyrrolidine, 4Å MS, CHCl3, 25 °C, 8 h) and subsequent treatment with 152 (0.1 M CHCl3, 25 °C, 3 h, 75% over 2 steps).118 This cycloaddition proceeded rapidly to provide pyridine 153 as a single regioisomer at room temperature without detection of intermediates. The exclusive N1/C4 regioselectivity and azadiene reactivity were in accordance with expectations and benefitted from the complementary azadiene substitution (C5-CO2Me). Tetracycle 153 was elaborated to the natural products in a sequence involving N-methylation of the pyridine, exhaustive reduction with NaCNBH3 to provide almost entirely a single diastereomer, indoline deprotection and oxidation to the indole, and final ester hydrolysis (dihydrolysergic acid) or reduction (dihydrolysergol). In addition, the enamine derived from ketone 150 was employed in additional heterocyclic azadiene cycloaddition reactions for the late-stage, divergent preparation of a series of heterocyclic derivatives without optimization. This included the reaction of a series of substituted 1,2,3triazines, each of which displayed the expected C4/N1 regioselectivity and provided differentially substituted pyridines, 1,3,5-triazine, which provided the pyrimidine, and 3,6-dicarbomethoxy-1,2,4,5-tetrazine, which provided a substituted pyridazine and access to the pyrrole through a zincmediated reductive ring contraction (Scheme 26). 118


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Scheme 26. Synthesis of heterocyclic ergot alkaloid analogues.

Methoxatin. In studies that led to the development of the first general method for the catalysis of the inverse electron demand Diels–Alder reactions of heterocyclic azadienes, we targeted the total synthesis of methoxatin, a redox active quinone that serves as a cofactor for methanol dehydrogenase and is found in a variety of methylotrophic bacteria.123 Although the discovery of methoxatin, also known as pyrroloquinoline quinone (PQQ), led to a series of studies devoted to the discovery of a methoxatin-dependent mammalian enzyme, no such protein has yet been identified.124 Nevertheless, PQQ has been shown to play a role in a variety of mammalian processes,125 is offered as a dietary supplement, and has been shown to reduce the risk of injury in heart attacks and strokes.126 Due to its potentially important role in human health, its putative role as a vitamin, and its unusual structure, PQQ has been the target of a number of increasingly concise total syntheses.127 We recently disclosed the shortest total synthesis of methoxatin reported to date that relied on the discovery and development of a powerful hydrogen bonding activation of heterocyclic azadiene cycloaddition reactions.108 The approach was designed to permit the late-stage divergent synthesis of PQQ analogues through use of a series of complementary heterocyclic azadiene cycloaddition reactions. The synthesis of methoxatin began with the known ketone 156 (Scheme 27). Treatment with pyrrolidine provided enamine 157, which was used without further purification in the key cycloaddition. Initial optimization studies revealed that the addition of trifluoroacetic acid (1.5 equiv) improved reaction yields, suggesting that acid-promoted aromatization or hydrogen bonding may be contributing to the improvement. Unaromatized cycloadduct was not detected as a stalled intermediate, indicating that the additive was not serving as a catalyst for the final aromatization step, but was accelerating the cycloaddition itself. Systematic studies led to the discovery that hexafluoroisopropanol (HFIP) was a remarkably effective solvent or additive for the cycloaddition (Figure 20), promoting the reaction through H-bonding to the heterocyclic azadiene, and providing the desired cycloadduct in a stunning 95% yield over 2 steps (0.1 M HFIP, 60 °C, 24 h). 108

Scheme 27. Total synthesis of methoxatin.

Figure 20. H-bonding effect of solvent on cycloaddition. Methoxatin was accessed in only 3 steps from cycloadduct 159. DDQ oxidation (4.0 equiv, 0.004 M C6H6, 90 °C, 48 h) followed by treatment with RuO4 using modified conditions that generated RuO4 in situ (0.4 equiv RuO2, 5.0 equiv NaIO4, 0.004 M H2O–CH2Cl2–CH3CN 1:1:1, 23 °C, 30 min) cleanly provided the desired o-quinone in 63% yield over two steps. Lastly, a global saponification (0.01 M THF–0.5 M aq. LiOH, 23 °C, 6 h) provided methoxatin in 94% yield. Finally, and as designed, a series of representative heterocyclic azadienes were found to undergo cycloaddition with enamine 157 in HFIP without optimization to provide the corresponding methoxatin analogues (Scheme 28).108



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Scheme 28. Divergent synthesis of methoxatin analogues.

2.5. 1,3,4-Oxadiazoles The tandem Diels–Alder/1,3-dipolar cycloaddition reactions of 1,3,4-oxadiazoles have been studied by a limited number of groups, generally employing symmetrical oxadiazoles bearing strong electron-withdrawing substituents in intermolecular reactions.128,129 These five-membered aromatic heterocycles were first shown by Vasil’ev9b to undergo inverse electron demand Diels–Alder reactions that trigger a cycloaddition cascade proceeding through a [4+2] cycloaddition, loss of nitrogen (N2) to generate a carbonyl ylide, and a final 1,3dipolar cycloaddition. While this cycloaddition cascade is potentially powerful and was exemplified several times, including with the preparation of complex norbornadiene structures,130 limitations of the reaction precluded its widespread adoption. These limitations include the lack of differentiation between the dienophile and dipolarophile since the ensuing 1,3-dipolar cycloaddition is faster than the initiating Diels–Alder reaction and the necessity for use of symmetrical oxadiazoles and olefins/alkynes (Figure 21). Informed by our study of the reactivity of electron-deficient heterocyclic azadienes and because of our interest in the Vinca alkaloids, we designed and developed an intramolecular [4+2]/[3+2] cycloaddition cascade inspired by the structure of vindoline, the lower half of vinblastine.131 Representing the first example of a tandem intramolecular Diels–Alder/1,3-dipolar cycloaddition cascade of 1,3,4-oxadiazoles, this methodology differentiates the dienophile and dipolarophile, extends the scope of oxadiazoles used in such reactions, and provides regio- and stereocontrol over the resultant cycloadducts (Figure 21). We recently reviewed the emerging scope of this intramolecular cycloaddition cascade.132

Figure 21. 1,3,4-Oxadiazole cycloaddition reactions and tandem intramolecular cycloaddition cascade. Anhydrolycorinone. Although our work with this heterocycle primarily focused on the tandem cycloaddition cascade detailed above, the first use of a 1,3,4-oxadiazole in a natural product total synthesis was exemplified with anhydrolycorinone.133 The basis for the development of this reaction cascade came from the observation that tethered alkyne dienophiles and olefins bearing a leaving group generated high yields of furan products after an initial Diels–Alder cycloaddition (Figure 21). This reactivity was recognized as being complementary to the cycloadditions of oxazoles but entails a more facile loss of N2 (vs nitrile) in the retro Diels– Alder aromatization event, and was exploited in a concise synthesis of a highly substituted benzene system. The use of a methyl enol ether as the initiating dienophile provided a furan through loss of methanol that was appropriately set up to react further in a second Diels–Alder reaction with a tethered olefin, generating the pentacyclic core of anhydrolycorinone in a single step (Scheme 29). Sequential ester saponification and decarboxylation provided the natural product.


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Scheme 29. Total synthesis of anhydrolycorinone.

Vindoline, Vinblastine, and Vincristine. The inspiration for our development of the intramolecular [4+2]/[3+2] cascade cycloaddition reaction of 1,3,4-oxadiazoles was the dimeric Vinca alkaloids vinblastine and vincristine. Thus, among the first total synthesis to be published utilizing this methodology was that of vindoline, in 2005.134 Since then, we have disclosed two additional total syntheses of vindoline, a series of syntheses of related Aspidosperma alkaloids, the divergent total syntheses of four different natural product alkaloid classes from a common cascade cycloadduct, as well as the total syntheses135 of vinblastine, vincristine, and an extensive series of analogues including ultra-potent vinblastines. Vinblastine and vincristine were isolated in 1958 and 1961, respectively, in trace quantities from Catharanthus roseus (L.) G. Don136 and are two of the most important and widely recognized antitumor drugs currently in use in the clinic. 137 Their clinical efficacy has resulted in their use in combination therapies for the treatment of Hodgkin’s disease, testicular cancer, breast cancer, ovarian cancer, head and neck cancer, and non-Hodgkin’s lymphoma (vinblastine) and in curative treatments of childhood lymphocytic leukemia and Hodgkin’s disease (vincristine), and has earned them positions in the World Health Organization’s List of Essential Medicines.

Figure 22. Synthesis of vinblastine. Highlighted regions are those targeted in our medicinal chemistry efforts. Because of their clinical importance, low natural abundance, and complex dimeric structure, vinblastine and vincristine have remained the subject of extensive investigations, including our own interest in the molecules over the last two decades.138,139

Scheme 30. 11-Step total synthesis of vindoline.

In the course of these efforts, we comprehensively defined the importance and role of nearly every atom and substituent in vinblastine and vincristine, including the vindoline C4 acetate,140 C5 ethyl substituent,141 C6–C7 double bond, and the vindoline core structure itself142 and have systematically explored the upper catharanthine-derived C20′ ethyl substituent,143 C16′ methyl ester,144 added C10′ or C12′ indole substitutions,145 and, most recently, the C20’ alcohol.146 We have shown that the latter is remarkably tolerant to replacement, resulting in the discovery of both ultra-potent analogues of the natural products146b as well as those that match the potency of the natural products but do not suffer from overexpressed Pgpderived resistance146a (Figure 22). Because each of three classes of improved analogues that we discovered 145-146 not only incorporate added structural features not found in the natural products, but also substantially surpass their properties, we like to think of them as prototypical examples of “supernatural” products.147 These discoveries were enabled by our development of the powerful intramolecular [4+2]/[3+2] cascade cycloaddition reaction of 1,3,4-oxadiazoles and its use in concise syntheses of vindoline, the lower subunit of vinblastine. Our first-generation synthesis of (–)-vindoline (11 steps) rested on the cycloaddition cascade of either 161 or 162.134,148 This reaction established the full pentacyclic core of the natural product, generated six contiguous stereocenters about the newly formed central sixmembered ring, and introduced nearly all the functionality found in vindoline in a single step. Although the key features of this reaction have been detailed in our work, it is important to note that the difference in the facility of the cycloaddition of 161 and 162 arises from the electronic character of the benzyloxy substituent of 161, which decelerates the typically fast 1,3-dipolar cycloaddition. This rate deceleration arises from a destabilizing electrostatic interaction in the transition state between the central oxygen of the 1,3-dipole and the (Z)OBn substituent. Both cycloadducts were elaborated to vindoline in 7 or 10 steps, providing efficient syntheses of the natural product (Scheme 30). This observation of the importance of the initiating dienophile in the cascade cycloaddition reaction led to the examination of additional functionalized dienophiles and the discovery that an allene could be used to initiate the reaction to provide the desired cascade cycloadduct in exceptional yields (Scheme 31).149 The initial Diels–Alder reaction affords a



cycloadduct that in turn undergoes loss of N 2 to provide an unusual cross-conjugated 1,3-dipole that proved to be extraordinarily effective in the ensuing 1,3-dipolar cycloaddition, providing the cycloadduct as a single diastereomer in 92% yield. Corey dihydroxylation and subsequent Pb(OAc)4-mediated diol cleavage followed by lactam α-hydroxylation and in situ silyl ether formation provided an intermediate in our first-generation synthesis of (– )-vindoline, completing a formal total synthesis of the natural product.

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total syntheses of (–)-vindoline that complement our efforts utilizing direct chromatographic chiral resolution.

Scheme 31. Total synthesis of vindoline through an alleneinitiated oxadiazole cycloaddition cascade.

Figure 23. Asymmetric total syntheses of vindoline.

Finally, while the enantiomers of late-stage intermediates in the syntheses detailed above were directly and chromatographically resolved to provide access to both (–)vindoline and ent-(+)-vindoline, we also developed a complementary asymmetric total synthesis.150 This was based on a cycloaddition cascade in which the dienophile linking tether bears a chiral substituent that sets the absolute stereochemistry of all six stereocenters about the central sixmembered ring in the resulting cycloadduct (Figure 23). The length of the linking tether was shortened to permit effective control of the cycloaddition facial selectivity, enlist milder cycloaddition reaction conditions (140 oC, 16 h), and required a ring expansion later in the synthesis. This ring expansion was accomplished by two approaches. The first relied on the hydrolytic ring expansion of a N,O-ketal generated through a unique oxidative deformylation reaction while the second employed generation of an intermediate aziridinium ion in a thermodynamically-controlled ring expansion. Together these two approaches provided two distinct and concise asymmetric

Our work culminated in a 12-step total synthesis of vinblastine that featured a biomimetic iron-mediated coupling of vindoline and catharanthine 151 and a further iron-mediated hydrogen atom transfer (HAT) olefin functionalization for introduction of the C20’ alcohol.135,152-154 These two key reactions have now been leveraged for the synthesis of hundreds of informative analogues of vinblastine. Related Alkaloids. Complementary to our work on vindoline, we disclosed the total syntheses of several related Aspidosperma alkaloids.148 These efforts were conducted in parallel with our work on the methodology itself, allowed for the synthesis of a series of natural products of increasing complexity, and defined the scope and key features of the cycloaddition cascade. These investigations not only culminated in our synthesis of vindoline, but many also served to address longstanding questions on the absolute configuration and characterization of the targeted natural products. The first set of natural products were accessible from a common cycloadduct intermediate, highlighting the use of the methodology in divergent synthesis (Figure 24).


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Figure 24. Divergent total synthesis of Aspidosperma natural products from a common cycloadduct. Minovine. Minovine, isolated from Vinca minor L., is implicated in the biosynthetic pathway of alkaloids bearing the vincadifformine core structure.155 The naturally occurring material was reported to possess an [α] D = 0 in alcoholic solvents, an unusual feature that had not been addressed in previous racemic syntheses of the natural product. Our synthesis of optically active minovine, proceeding in 6 steps from cycloadduct 171 derived from the tandem cycloaddition cascade of 170 (74%), addressed this issue (Scheme 32).156

naturally occurring minovine is not racemic, nor does it racemize, but it does exhibit an unusual solvent dependent optical rotation. For our own purposes, the synthesis of minovine allowed us to probe the reactivity of the lactam and oxido bridge present in cycloadduct 171 and was instrumental in our development of effective ring opening and lactam reduction reactions. The discovery that these steps could be conducted in either order expanded the range of targets that could be pursued using the methodology (Scheme 32). N-Methylaspidospermidine. The enantiomer of the cycloadduct used in the total synthesis of optically active minovine was used for the first total synthesis of (+)-Nmethylaspidospermidine and served to confirm the absolute configuration of the natural product. Isolated from Evatamia peduncularis, Vallesia dichotoma, Aspidosperma quebracho blanco, and Vinca minor L.,157 (+)-N-methylaspidospermidine and the closely related aspidospermidine possess an absolute configuration opposite that found in vindoline and the Vinca alkaloids. Cycloadduct 171 was elaborated to the target compound in just 5 steps, provided further insight into the optional order of lactam reduction/oxido bridge opening, and established an approach for the removal of the methyl ester, expanding the scope of future targets (Scheme 33).148 Scheme 33. Total synthesis of (+)-N-methylaspidospermidine.

Scheme 32. Total synthesis of minovine.

Optically active minovine was found to exhibit a solvent, but not concentration, dependent optical rotation: natural 172 [α]23D –17 (c 0.35, CHCl3), +16 (c 0.40, MeOH), and 0 ± 3 (c 0.28, EtOH). In addition, studies were carried out to determine whether a reversible Diels–Alder reaction, previously suggested to be responsible for racemization and the observed optical rotation of 0, was possible with the natural product. Attempts at such a reaction showed that it is not observed. Thus,

4-Desacetoxyvindorosine. This same cycloadduct was used in the concise total synthesis of 4-desacetoxyvindorosine. Although it is a key late-stage biosynthetic intermediate enroute to vindorosine, it had never been fully characterized and no total synthesis of this alkaloid had been reported. Differing from our ultimate target vindoline only by lack of the C16 methoxy and C4 acetoxy substituents, 4-desacetoxyvindorosine allowed investigation of the introduction of the C6–C7 double bond found in both natural products. The strategy chosen for the introduction of this functionality involved α-hydroxylation of the lactam present in 171. Following carbonyl excision, secondary alcohol elimination was expected to provide the Δ6,7olefin, following precedent of Kuehne on similar intermediates. Initial efforts to use this second transformation on a C7 alcohol focused on the conditions first developed by Kuehne 158 (Ph3P–CCl4, Et3N) and later by Fukuyama159 (Ph3P–CCl4). Although a thorough study was conducted, the reaction proved to be more difficult to implement than anticipated. A sensitivity



to moisture was observed, leading to recovery of starting material with the potential intermediacy of an aziridinium ion, and use of excess reagent resulted in competitive in situ chloride displacement or addition (Figure 25). Variations on the reaction conditions provided small improvements but were ultimately ineffective with the more highly functionalized substrates required for vindoline or vindorosine. However, we found that the Mitsunobu reagent160 Ph3P–DEAD was uniquely effective, providing high conversions under mild conditions, due to its ability to activate the secondary alcohol for aziridinium ion formation or elimination without release of a nucleophile or the necessity of added base. Compound 171 was elaborated to (+)and ent-(–)-4-desacetoxyvindorosine in 7 steps (Scheme 34).148

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and it had only been characterized by MS and 1H NMR.162 In addition and since desacetoxyvinblastine, lacking only the vindoline C4 acetoxy group, is also a natural product and had been shown to be equally or more efficacious in vivo than vinblastine itself,163 we undertook the synthesis of 177 and 4desacetoxyvinblastine by employing the tandem cycloaddition cascade of 175 (Scheme 35). This approach proved to be straightforward and used the more reactive disubstituted (vs trisubstituted for vindoline) tethered dienophile in the key cycloaddition cascade that proceeded in excellent yield and with perfect diastereoselectivity (TIPB, 230 °C, 18 h, 83%). A further 7 steps provided 4-desacetoxyvindoline (177), which was fully characterized for the first time.148 It was also further incorporated in 4-desacetoxyvinblastine in a single additional step, permitting its contemporary assessment alongside additional analogues of vinblastine.135 Scheme 35. Total synthesis of (+)-4-desacetoxyvindoline.

Figure 25. Introduction of the Δ6,7-olefin. Scheme 34. Total synthesis of (+)-4-desacetoxyvindorosine.

4-Desacetoxyvindoline and 4-Desacetoxyvinblastine. Concurrent with our efforts towards vindoline, a total synthesis of its 4-desacetoxy variant was conducted and illustrated the ease of preparation of simpler derivatives of the natural product. 4-desacetoxyvindoline, a natural product in its own right, is the penultimate biosynthetic precursor to vindoline. 161 Prior to our work, no total synthesis of the compound had been reported,

Vindorosine. A concise total synthesis of (–)- and ent-(+)vindorosine was developed that allowed us to probe the key cycloaddition reaction of a substrate bearing the trisubstituted dienophile required for the introduction of the C4 acetoxy group.164 Vindorosine is identical in structure to vindoline, lacking only the C16 methoxy substituent. Its densely functionalized structure and similarity to vindoline have resulted in it being the focus of several beautiful and historically important total syntheses.165 Our efforts focused on the use of (Z)- and (E)-178, bearing isomeric trisubstituted electron-rich enol ethers as the tethered initiating dienophiles. Trisubstitution of the dienophile generally slows initiation of the intramolecular [4+2]/[3+2] cycloaddition cascade, although recent studies have defined reaction conditions that permit the use of even more difficult trisubstituted electron-deficient and unactivated alkenes.140 The exceptions to this generalization are trisubstituted olefins bearing an electron-donating substituent that serves to activate the dienophile for participation in an 1,3,4-oxadiazole inverse electron demand Diels–Alder reaction. The electron rich olefins in (Z)- and (E)-178 were anticipated to be ideally suited for initiation of the cycloaddition cascade and introduction of the C4 alkoxy substituent (Figure 26). Moreover, (Z)-178 not only directly provides the natural β-


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stereochemistry but was also expected to be more effective in the key cycloaddition cascade due to decreased steric interactions in the ensuing 1,3-dipolar cycloaddition transition state (Figure 26). While (E)-178 was shown to undergo the cycloaddition cascade in excellent yield (95%, TIPB, 230 °C, 1 mM, 18 h), (Z)-178 proved more challenging, requiring considerable optimization to provide the desired cycloadduct in good yield (78%, TIPB, 230 °C, 0.1 mM, 60 h). 148

Both were shown to be equally effective in the cycloaddition cascade, indicating that electronic and not steric features were contributing to the difference in the behavior of (Z)- vs (E)-178. A second but less likely explanation involves the preferred stereochemistry of putative cyclobutene epoxides that may act as reversibly generated intermediates for the highly reactive carbonyl ylides. It is plausible that they may possess epoxide stereochemistries that influence the rate of the ensuing 1,3dipolar cycloaddition. The enantiomers of cycloadduct 179 were elaborated to (–)- and ent-(+)-vindorosine in 7 steps (Scheme 36).148 Scheme 36. Total synthesis of (–)-vindorosine.

Figure 26. Cycloaddition cascades enroute to vindorosine. Considerable work was conducted to define the subtleties of the effects responsible for the observations. To summarize these studies, it was observed that the 1,3-dipolar cycloaddition, generally the fast step in the tandem cycloaddition cascade, was now the rate-limiting step in the case of (Z)-178. Two explanations may account for this observation. The first is that the transition state of the ensuing 1,3-dipolar cycloaddition of (Z)-178 suffers from a destabilizing, and thus decelerating, electrostatic interaction of its central oxygen with the OBn substituent of the reacting dienophile (Figure 26). This was confirmed by comparing the reactions of (Z)- and (E)-181, each bearing a methyl group in place of the OBn substituent (Figure 27).

Spegazzinine and Aspidospermine. Finally we undertook the synthesis of (–)-aspidospermine and (+)-spegazzinine, in which we examined the effect of aryl substitution on the key [4+2]/[3+2] cascade.166 Spegazzinine, isolated in 1956 from Aspidosperma chakensis Spegazzini,167 has been shown to be a potent inhibitor of mitochondrial photophosphorylation oxidative phosphorylation.168 Aspidospermine, isolated in the late 1800s from the Aspidosperma quebrancho,169 exhibits a wide range of biological activities.170 At the time of our work, spegazzinine had not been targeted by total synthesis whereas aspidospermine has been one of the most attractive synthetic targets among the Aspidosperma alkaloids.171 Our approach enlisted the cycloaddition cascade of 183, which proceeded in excellent yield to provide 184 as a single diastereomer (71%, o-Cl2C6H4, 180 °C, Scheme 37).166 (–)Aspidospermine and (+)-spegazzinine could be accessed from 184 by a sequence of oxido bridge ring opening, lactam carbonyl excision, removal of the C3 methyl ester, benzyl deprotection, acetylation and, in the case of the former, phenol methylation and C3 alcohol removal. This work served to establish the absolute configuration of natural (+)-spegazzinine and unambiguously determine its relative C3 alcohol stereochemistry. As remarkable as it may seem, this synthesis of aspidospermine also was the first not to rely on a late-stage Fisher indole synthesis first introduced by Stork. 171a

Figure 27. Model systems for the key cycloaddition cascade.



Scheme 37. Total synthesis of (+)-spegazzinine and (–)aspidospermine.

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and the assignment of the absolute configuration remained to be established. The synthesis of these compounds was accomplished in 9 and 10 steps, respectively, from cycloadduct 188 and established the absolute configuration of the natural products. 175 In addition to the cycloaddition cascade, which proceeded in excellent yield (71%, o-Cl2C6H4, 180 °C), the synthesis featured a key tetrahydrofuran ring introduction with direct formation of the C19 N,O-ketal (Scheme 38). This transformation, which takes advantage of the inherent C19 oxidation state, was affected in a single step through treatment of 193 with HF/pyridine to generate a stable cyanohydrin. Direct trap of the reversibly generated iminium ion, generated through oxido bridge ring opening, is facilitated by the two quaternary centers flanking C19. Scheme 38. Tetrahydrofuran ring formation enroute to a total synthesis of (+)-fendleridine.

Divergent Total Synthesis: Diverse Natural Product Families from a Common Intermediate. The cycloadduct derived from the cycloaddition cascade of 187 was used as a common intermediate in the divergent total synthesis of natural products from four different alkaloid classes. These syntheses enlisted four different late-stage strategic bond formations embedded in each natural product core structure (Figure 28).

Kopsinine. Kopsinine was first isolated from Kopsia Longiflora Merr.176 and differs from the closely related Aspidosperma alkaloids by the addition of a bond joining C21 to C2 to provide a bicyclo[2.2.2]octane system embedded in its hexacyclic core. Our interest in the molecule stemmed from the potential of its access through a late-stage C2–C21 strategic bond formation that would be distinct from previous syntheses of Kopsia alkaloids, all of which employ Diels–Alder reactions of unnatural Aspidosperma-like pentacyclic C2–C5 dienes.177 Scheme 39. Strategic C2–C21 bond formation in the total synthesis of (–)-kopsinine.

Figure 28. Divergent total synthesis of four different alkaloid natural product classes from a common cycloadduct. Fendleridine. Fendleridine was isolated in 1964 from Aspidosperma fendleri WOODSON and is the parent compound of the aspidoalbine family of alkaloids.172 Unique to this class of natural products is the oxidized C19 N,O-ketal embedded in the Aspidosperma alkaloid pentacyclic ring system. Although a handful of total and formal syntheses of fendleridine (189)173 and its congener 1-acetylaspidoalbidine174 were disclosed prior to our work, all provided racemic material

Kopsinine was accessed in 8 steps from cycloadduct 188.178 Key to the synthesis was a late-stage C2–C21 bond formation mediated by SmI2 in 10:1 THF–HMPA, proceeding through a radical-mediated conjugate addition cyclization and subsequent protonation of the further reduced ester enolate from the less hindered convex face to provide the product in excellent yield (75%) and as a single diastereomer (Scheme 39). Kopsifoline D. Kopsifolines A–F, first isolated from K. fruticosa (Ker) A. DC. in 2003,179 constitute the first members of a new a family of alkaloids that possesses a unique core hexacyclic ring system and a previously unprecedented C3– C21 bond. The central six-membered ring contains five or six stereogenic centers, of which three or four are quaternary. Our synthesis of kopsifoline D relied on a biomimetic late-stage C21–C3 strategic bond formation via a transannular enamide alkylation of a C21 iodide within the Aspidosperma skeleton, directly introducing the correct C2 oxidation state.180 The key C21–C3 bond formation was accomplished by treatment of 198 with BF3•OEt2 and Me2S to affect Cbz deprotection, followed by subjection to Et3N in EtOAc to affect the intramolecular ring


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closure and provide the natural product (Scheme 40). This not only provided the first total synthesis of a naturally occurring kopsifoline and established the absolute configuration for the alkaloid class, but it remains the only reported total synthesis still today. Scheme 40. C3–C21 bond formation in the total synthesis of (– )-kopsifoline D.

Deoxoapodine. As part of these studies, we were also able to access (–)-deoxoapodine, isolated from Tabernae armeniaca,181 through a late-stage C21–O–C6 bond formation, providing the first synthesis of the natural product in optically active form. C21–O–C6 bond formation to provide access to deoxoapodine was accomplished by treatment of 200 with Bu4NF, which resulted in clean silyl ether deprotection with concomitant conjugate addition (single diastereomer) without detection or isolation of the intermediate alcohol (Scheme 41). Carbamate deprotection provided deoxoapodine, identical in all respects to authentic material.180

effect on the rate of the reaction, and the reaction displays a characteristic pressure-induced endo diastereoselectivity.182 Significantly, the reaction is not only regiospecific but also endo specific, typically exhibiting unusually high levels of diastereoselectivities (≥20:1) often to the extent that a single cycloadduct is observed. This remarkable diastereoselectivity may be attributed to two complementary features of the reaction: (1) a stabilizing secondary orbital interaction between diene C2 and the dienophile OR substituent that preferentially stabilizes the endo transition state, and (2) a transition state anomeric effect in which the nitrogen lone pair and the electronrich σ bond of the enol ether are trans periplanar in the endoboat transition state.182a Both effects enhance the characteristic endo diastereoselectivity and both are absent in the alternative exo transition state. The latter effect is also unique to 1-aza-1,3butadienes and not found in the all-carbon Diels–Alder reaction, accounting for the superior diastereoselectivity.

Scheme 41. Tetrahydrofuran ring formation enroute to a total synthesis of (–)-deoxoapodine.

3. 1-Aza-1,3-Butadienes At the time we initiated our studies, the Diels–Alder reactions of the acyclic azadienes, such as α,β-unsaturated imines, were rare since their intrinsic instability, competitive 1,2-imine addition or imine tautomerization precludes successful [4+2] cycloaddition.3d Moreover, the cyclic enamine products often proved unstable under the reaction conditions. In our studies, we addressed these limitations by incorporation of suitable substituents on the acyclic 1-aza-1,3-butadienes. The complementary N1 and C3 substitution of α,β-unsaturated imines with electron-withdrawing substituents enhance the intrinsic electron-deficient nature of the azadiene and thus accelerate its rate of reaction with electron-rich dienophiles in inverse electron demand Diels–Alder reactions. In addition, a bulky electron-withdrawing N-1 substituent preferentially decelerates 1,2-imine addition through effective steric shielding and conveys product stability to [4+2] cycloadduct by affording a deactivated enamine. Through such design principles, we established approaches to control and accelerate the 4π participation of acyclic 1-aza-1,3-butadienes in [4+2] cycloaddition reactions.182 In our studies, N-sulfonyl and N-phosphinyl α,β-unsaturated imines were shown to constitute stable, easily accessible, substituted 1-aza-1,3-butadienes suitable for use in wellbehaved Diels–Alder reactions (Figure 29).182 The reaction exhibits characteristics of a concerted [4+2] cycloaddition. The dienophile and diene geometry are conserved in the reaction products, trans-1,2-disubstituted dienophiles react faster than cis-1,2-disubstituted dienophiles, there is little or no solvent

Figure 29. reaction.

N-Sulfonyl-1-aza-1,3-butadiene

Diels–Alder

By virtue of lowering the diene LUMO, both the complementary C3 addition and the noncomplementary C2 or C4 addition of an electron-withdrawing substituent to the Nsulfonyl 1-azadiene substantially accelerate their reaction rate in inverse electron demand Diels–Alder reactions, while maintaining or enhancing the superb regioselectivity and endo diastereoselectivity.182 Typically, a single [4+2] cycloadduct is generated at satisfactory rates at 25 °C or lower and 1-aza-1,3butadienes bearing complementary C3 electron-withdrawing groups react faster than 1-azadienes bearing noncomplementary C2 and C4 electron-withdrawing groups (Figure 30).



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The acyclic 1-aza-1,3-butadienes have also been examined in intramolecular Diels–Alder reactions.182e, 184 Similar to observations made in the intermolecular cycloadditions, the reaction displays a strong endo diastereoselectivity (>20:1). Representative of this, the product 203 was derived from cycloaddition of 202 through an anti-endo transition state as depicted in Figure 32. In addition, the entropic assistance provided by the intramolecular nature of such reactions has also allowed the use of less reactive azadienes, including O-alkyl oximes and less reactive electron neutral or electron-deficient dienophiles, including tethered terminal or 1,2-disubstituted alkenes and alkynes.

Figure 30. Complementary and noncomplementary substituent effects. We have also shown that the thermal reactions of N-sulfonyl1-aza-1,3-butadienes with enol ether dienophiles bearing chiral auxiliaries provide highly diastereoselective (endo and facial diastereoselection) inverse electron demand Diels–Alder cycloadditions.183 This is largely the result of the exquisitely organized [4+2] cycloaddition transition state. Vinyl ethers have been shown to preferentially react with dienes through an s-trans conformation. As the transition state model in Figure 31 illustrates, when the dienophile approaches the diene in this strans conformation in the highly organized endo boat transition state, the dienophile preferentially adopts a conformation in which the H is oriented toward and its large group (RL) is oriented away from the diene. The chiral auxiliary comes into proximity to the sterically demanding and electronegative sulfonyl groups, requiring the sulfonyl phenyl group to orient itself on the diene face opposite the approaching dienophile.

Figure 32. Representative intramolecular 1-aza-1,3-butadiene Diels–Alder reactions. The inverse electron demand Diels–Alder reactions of acyclic N-sulfonyl-1-aza-1,3-butadienes provide a stabilized highly substituted cyclic enamine, which can be further converted to a fully substituted pyridine (Figure 33). Based on the studies that defined the scope of this powerful transformation, we have applied this strategy in the total synthesis of a series of complex natural products containing a fully substituted pyridine or pyridone ring as a key structural element, including streptonigrone, fredericamycin A, nothapodytine, mappicine, camptothecin, rubrolone aglycon and piericidin A1and B1.

Figure 33. Pyridine synthesis based on the inverse electron demand Diels–Alder reactions of acyclic 1-azadienes. Figure 31. Transition-state model of an asymmetric Diels– Alder cycloaddition of N-sulfonyl-1-aza-1,3-butadienes with enol ethers bearing chiral auxiliaries. In our studies, 18 optically active enol ethers were examined.183 Among them, three new, readily accessible, and previously unexplored γ-lactone and γ-lactam chiral auxiliaries were found to provide remarkable selectivities (up to 49:1 endo:exo and 48:1 facial selectivity). The transition state model for these reactions places the electronegative sulfonyl oxygen on the same face as the approaching dienophile. This directs the electronegative ester, lactone, or lactam carbonyls of the effective dienophiles, although sterically undemanding, away from the sulfonyl group, behaving as effective RL groups. X-ray crystal structures of the cycloadducts, albeit being ground-state structures of the reaction products, embody each of these features.

Streptonigrone. Streptonigrone (211) is a highly substituted and densely functionalized quinolone-5,8-quinone related to the potent antitumor antibiotic streptonigrin that contains a fully substituted pyridone C ring central to its structure. 17 A welldesigned inverse electron demand Diels–Alder cycloaddition of a fully functionalized N-sulfonyl-1-aza-1,3-butadiene bearing a complementary C3 electron-withdrawing substituent was anticipated to provide a cycloadduct that could be easily elaborated to the pyridone.185 The C3 carboxylate incorporated into the azadiene as a lactone serves three strategic functions. First, it would further accelerate the rate of diene participation in the LUMOdiene-controlled Diels–Alder reaction and reinforced the inherent cycloaddition regioselectivity. Second, it offered a convenient manner to differentiate and protect the D ring phenol. Finally, it was designed to serve as suitable functionality for the introduction of the pyridone C5 amine. As expected, the inverse electron demand Diels–Alder reaction of


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1-azadiene 206 with ketene acetal 207 occurred at room temperature to afford cycloadduct 208 in only 30 min. Treatment of 208 with tBuOK followed by DDQ provided the fully substituted pyridine 210.185 This unusual aromatization sequence proceeded with intermediate generation of imidate 209 derived from methanesulfonamide deprotonation, loss of sulfene facilitated by vinylogous amide activation of the departing amine, and finally loss of methoxide. Subsequent oxidative aromatization was accomplished upon DDQ treatment of the intermediate imidate, providing the full skeleton 210 of streptonigrone (Scheme 42). As far as we are aware, our total synthesis of streptonigrone represents the only total synthesis disclosed to date.17

Scheme 43. Key step in the total synthesis of fredericamycin A.

Scheme 42. Key step in the total synthesis of streptonigrone.

Fredericamycin A. Fredericamycin A (217), a structurally unique and potent antitumor antibiotic, features an unusual spiro[4.4]nonene (CD ring system) and four aromatic rings including a pyridone (F ring). A key step in our total synthesis of fredericamycin A was the room temperature inverse demand Diels–Alder reaction of N-sulfonyl-1-aza-1,3-butadiene 212, bearing a noncomplementary C2 electron-withdrawing group, with 213 for the construction of the appropriately substituted pyridone F ring.186 The noncomplementary addition of the strong electron-withdrawing C2 ethoxycarbonyl group lowers the inherent low lying azadiene LUMO to the extent that even the modestly reactive dienophile 213 was found to react effectively at 25 °C (95%, Scheme 43). The cycloadduct 214 was converted directly to pyridine 215 by simple treatment with DBU. This was followed by a single-step Michael addition/intramolecular acylation upon LDA deprotonation and reaction of the anion with cyclopentenone and subsequent DDQ aromatization for annulation of the DE ring system.186 Notably, the synthesis of the carbon skeleton of the DEF ring system (216) was accomplished in only four steps.

Our convergent total synthesis of fredericamycin A featured two additional key transformations, a regiospecific intermolecular chromium carbene benzannulation reaction for AB ring construction and a simple aldol closure for the introduction of the spiro[4.4]nonene CD ring system.186-187 With this approach, ent-fredericamycin A, the key partial structure 218, constituting the fully functionalized ABCDE ring system,187 and 219, constituting the fully functionalized DEF ring system,186 were prepared to address the origin of its biological properties. The important observations were made that both enantiomers of fredericamycin A exhibit potent and indistinguishable cytotoxic activity and that each fragment (ABCDE and DEF) contributes significantly to the biological properties of fredericamycin A. Piericidin A1 and B1. An analogous 1-azadiene bearing a noncomplementary C2 electron-withdrawing group was utilized in a total synthesis of piericidin A1 (227) and B1 (228). The piericidins are an important class of biologically active natural products. They are among the most potent inhibitors of the mitochondrial electron transport chain protein NADHubiquinone reductase (complex I, Ki = 0.6–1.0 nM) and have contributed extensively to the elucidation of the enzyme properties. It has been suggested that piericidins mimic the structure of enzyme cofactor ubiquinone (coenzyme Q) and competitively bind complex I.188 In addition, the piericidins were identified as a class of highly selective antitumor agents, displaying a greater selectivity and potency than the comparison standards.189 Central to our approach to the piericidins was an inverse electron demand Diels–Alder reaction of the N-sulfonyl-1-aza1,3-butadiene 220, bearing a noncomplementary C2 carboxylate, with tetramethoxyethene (221) followed by Lewis acid-promoted aromatization for synthesis of the fully functionalized pyridine core.190 Treatment of 220 with tetramethoxyethene (221) smoothly afforded the [4+2] cycloadduct 222 (64%) at 50 °C, even though 221 is tetrasubstituted and sterically demanding (Scheme 44). The enhanced reactivity of 220 in the Diels–Alder reaction may be attributed to the lower inherent azadiene LUMO derived from the incorporation of a C2 electron-withdrawing group into the azadiene. Efforts to induce aromatization under a variety of



basic conditions were unsuccessful, whereas the use of BF3•OEt2 cleanly affected this transformation and provided the fully substituted pyridine 223. Reduction of the ethyl ester followed by protection of the resulting primary alcohol provided 224. It was envisioned that directed lithiation of C4’ position on the pyridine ring followed by a borylation/oxidation sequence would permit incorporation of the remaining C4’ hydroxyl group. Interestingly, treatment of 224 with excess of butyllithium followed by trimethylborate and peracetic acid unexpectedly afforded the C-silylated product 225, resulting from both the desired C4’ hydroxylation and a competitive reverse brook rearrangement of the benzylic TIPS ether under the strongly basic conditions. The disilylation of 225 followed by the Appel reaction of the resulting primary alcohol cleanly provided a surprisingly stable heterobenzylic bromide 226 and the left-hand fragment of piericidins.190 Scheme 44. Key step in the total synthesis of piericidins.

Additional key steps in this synthesis include the use of an asymmetric anti-aldol reaction for installation of the C9 and C10 relative and absolute stereochemistry, convergent assemblage of the side chain through formation of the C5–C6 trans double bond with a modified Julia olefination, and a surprisingly effective penultimate benzylic Stille crosscoupling reaction of the pyridyl core with the fully functionalized side chain. Notably, this strategic late-stage, convergent coupling approach was anticipated to permit routine access to the analogues in which each half of the molecule could be systematically modified.190 Consequently and in addition to the natural products, a series of key analogues were prepared.190b Their evaluation permitted a scan of the key structural features of the piericidins, providing new insights into the importance and the role of each substituent found in both the pyridyl core as well as the side chain. Nothapodytine B, (–)-Mappicine and (+)-Camptothecin. Camptothecin (238) is the parent member of a clinically important class of DNA topoisomerase I inhibitors that exhibit efficacious antitumor activity.191 Nothapodytine B (236), an oxidized derivative of mappicine (237), has been identified as an antiviral lead with selective activities against HSV-1, HSV2, and human cytomegalovirus (HCMV). Since all three

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alkaloids possess the same ABCD ring system, we designed a single unified strategy for their total syntheses.192 Central to our approach was the implementation of a room temperature, inverse electron demand Diels–Alder reaction of the Nsulfonyl-1-aza-1,3-butadiene 229, bearing a noncomplementary C4 carboxylate, with two different dienophiles for the introduction of the pyridone D ring with assemblage of the full carbon skeletons of either nothapodytine B and (–)mappicine or (+)-camptothecin (Scheme 45). Scheme 45. Key step in the total synthesis of nothapodytine B, (–)-mappicine and (+)-camptothecin.

Treatment of 229 with ketene acetal 207 at room temperature led to the formation of the sensitive [4+2] cycloadduct 230. Notably, the deliberate incorporation of the noncomplementary C4 electron-withdrawing substituent resulted in a Diels–Alder cycloaddition that proceeded at 25 °C without altering the inherent [4+2] cycloaddition regioselectivity. Subsequent aromatization of the cycloadduct under basic conditions provided the highly substituted pyridine 231 in 64% yield without intermediate isolations.192b Addition of EtMgBr to 231 in the presence of a tertiary amine proceeded cleanly to give the corresponding ethyl ketone 232 without tertiary alcohol formation by virtue of tertiary amine-promoted ketone enolization. Finally, deprotection of both the benzylic and pyridone methyl ethers with in situ benzylic bromination and subsequent cyclization afforded nothapodytine B. Reduction of nothapodytine B with (S)-BINAL-H provided (–)-mappicine (73%, 99.9% ee).192b


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This strategy was also applied to a total synthesis of camptothecin.192a The inverse electron demand Diels–Alder cycloaddition of the same 1-azadiene 229 with ketene acetal 235 proceeded at room temperature within 4 h to give the desired [4+2] cycloadduct 236. Addition of sodium ethoxide to the sensitive Diels–Alder cycloadduct 236 resulted in the sequential elimination and aromatization to form the highly substituted pyridine 237 (70%) under mild conditions without intermediate isolations. Significantly, based on this unified strategy, concise total syntheses of nothapodytine B, (–)mappicine and (+)-camptothecin were all accomplished in only 5–9 steps from a readily available α,β-unsaturated ketone as the precursor to the N-sulfonyl-1-aza-1,3-butadiene 229. Rubrolone. Rubrolone (239), a red tropoloalkaloid, possesses the unique azuleno[2.3-c]pyridine-2,5,13-trione aglycon 240 characteristic of a class of structurally related agents. Key to our total synthesis of rubrolone was the implementation of a rare 4π participation of an O-alkyl α,βunsaturated oxime in an intramolecular Diels–Alder reaction for construction of a tetrasubstituted pyridine and assemblage of the AB ring system.184,193 In our studies, the intramolecular Diels–Alder reaction of 204 was observed at 170 °C and the [4+2] cycloadduct gradually converted to the desired pyridine 205 through in situ elimination of methanol (Scheme 46). No reaction was found occur at temperatures lower than 140 °C, and a productive rate of reaction was observed at 170–200 °C to provide 205 (70%).184

selective Diels–Alder reaction of the cyclopropenone ketal followed by in situ generation of a norcaradiene and roomtemperature electrocyclic rearrangement.193 The details of these transformations are elaborated in the ensuing paragraphs. 4. 3-Carbomethoxy-2-Pyrones Much of the work we have described has involved the formation of an electron-rich olefin from an aliphatic carbonyl group followed by inverse electron demand [4+2] cycloaddition with a series of electron-deficient azadienes for the late-stage divergent synthesis of a wide range of heteroaromatics. Complementary to this, we developed an approach to a full range of aromatics bearing selectively protected and positionally varied oxygen substituents.196 This process involves conversion of the carbonyl to an electron-deficient diene, a 3-carbomethoxy-2-pyrone, and its reaction in a series of inverse electron demand Diels–Alder reactions with electron-rich dienophiles bearing oxygen substituents. In this manner, a single aliphatic substrate bearing a carbonyl group provides a point of divergence for a full range of aryl annulations (Figure 34).

Scheme 46. Key step in the total synthesis of rubrolone aglycon.

Precedent for this Diels–Alder reaction may be found in the use of α,β-unsaturated N,N-dimethylhydrazones in intermolecular194 and intramolecular195 [4+2] cycloaddition reactions. However, the previously reported failure of α,βunsaturated oxime cycloadditions suggested that their use may not prove viable. Therefore, the disclosure that the O-alkyl α,βunsaturated oxime 204 participates as an effective 4π component of an intramolecular Diels–Alder reaction with an electron-deficient dienophile indicates that the introduction of an alkoxy electron-donating substituent (OR) on the nitrogen atom of the inherently electron-deficient 1-aza-1,3-butadiene system, like the dimethylamino group of the unsaturated dimethylhydrazones (NMe2), may be sufficient to promote its participation in a normal, HOMOdiene-controlled Diels–Alder reaction.184 Additional highlights in our total synthesis of rubrolone aglycon include the introduction of the C-ring oxygenated troplone through a room-temperature intermolecular, exo

Figure 34. Divergent late-stage aryl annulation. Although the synthetic utility of α-pyrones in normal Diels– Alder reactions was well established prior to our work, their use in inverse demand reactions was less developed. C3 substitution of the pyrone with an electron-withdrawing group accelerates its participation in inverse electron demand [4+2] cycloadditions and enhances the observed regioselectivity. This observation was exploited for the divergent preparation of a full range of oxygen substituted aromatics and was extended to the total synthesis of a number of natural products, including sendaverine,196a juncusol,197 6,7-benzomorphans,196a and the azafluoranthene alkaloids, including rufescine, norrufescine, and imeluteine (Figure 35).198 Unlike conventional approaches in which the synthesis typically begins with a precursor containing the aromatic ring, the late-stage divergent aryl



annulation from a common precursor ketone permits the rapid and comprehensive examination of a key series of aryl and heteroaromatic natural products and analogues.198

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of electron-rich and electron-deficient dienes (Figure 36).199 In these investigations, we also discovered a remarkable thermal [3+4] cycloaddition reaction of cyclopropenone ketals with αpyrones that provided a complementary approach to tropone annulation.199 This additional reactivity was found to be derived from a reversible, thermal generation of a π-delocalized singlet vinylcarbene and its 2π participation in a π2s + π4s cycloaddition. The full scope of the cycloaddition reactivity of such thermally generated π-delocalized singlet vinylcarbenes was defined and was found to extend to a range of additional [3+2] and [1+2] cycloaddition reactions with electron-deficient olefins and carbonyls (Figure 36).199-200

Figure 36. Cyclopropenone ketal cycloaddition reactions.

Figure 35. 3-Carbomethoxy-2-pyrone synthetic applications. 5. Cyclopropenone Ketals Structural or electronic features that accelerate a normal Diels–Alder reaction would be expected to decelerate the corresponding inverse electron demand [4+2] cycloaddition. The exception to this generalization is the behavior of strained olefins, which react effectively with both electron-rich and electron-deficient dienes. This unique reactivity can be attributed to both their raised HOMO and lowered LUMO energy levels as well as the release of ring strain that accompanies their cycloaddition. In our studies on the total synthesis of tropoloalkaloids, we found that α-pyrones react with cyclopropenone ketals in Diels–Alder reactions, and were able to extend this observation to their reaction with a variety

Recently, we disclosed the intramolecular cycloaddition reactions of cyclopropenone ketals.201 Such ketals tethered to olefins bearing a single electron-withdrawing substituent were found to undergo [3+2] cycloadditions in excellent yields under mild conditions (80 °C) while others displayed a bifurcated reactivity, providing intermediate cyclopropanes derived from an initial endo [1+2] cycloaddition of the thermally generated π-delocalized singlet vinylcarbene under mild thermal conditions (80–100 °C) and the [3+2] cycloadducts at higher reaction temperatures (170–180 °C, Figure 37). The ease of accessing the [3+2] cycloadducts directly under mild conditions was found to be dependent on the nature of the linking chain as well as the nature of the electron-withdrawing substituent. The corresponding intramolecular reactions with electron-rich, electron-deficient, and neutral 1-substituted dienes were also investigated and found to proceed under mild conditions and provide exclusive exo [4+2] cycloaddition products in normal, inverse electron demand, and neutral Diels–Alder reactions. These reactions do not depend on reaction conditions, diene substituents, or nature of the linking tethers and proceed without the intervention or trap of the vinylcarbenes.


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The Journal of Organic Chemistry Our approach to colchicine was complementary to a series of pioneering total syntheses203 and was based on a remarkable thermal [3+4] cycloaddition reaction of a cyclopropenone ketal with Eschenmoser’s α-pyrone, a reaction that proceeds by reversible thermal generation of a π-delocalized singlet vinylcarbene and its 2π participation in a π2s + π4s cycloaddition (Figure 38). More than 35 years after our disclosure of this synthesis, it remains one of the most concise and efficient total synthesis disclosed to date 203 and likely could be further improved with implementation of modern advances in the field. Azafluoranthene and Tropoloisoquinoline Alkaloids. In efforts on the total synthesis of the azafluoranthene alkaloids, 198 we envisioned the development of a divergent synthesis of the tropoloisoquinoline alkaloids, including imerubrine, isoimerubrine, grandirubrine, and granditropone. 204 These efforts, in which the powerful concept of “divergent total synthesis” was introduced as a synthetic strategy, also inspired our development of methodology for late-stage aromatic annulation (Figure 39).198,205 Key to the approach to the tropoloisoquinoline alkaloids was the use of a room temperature pressure-promoted [4+2] cycloaddition of a cyclopropenone ketal with a common α-pyrone also utilized for the total syntheses of the azafluoranthene alkaloids. Additional highlights include the development of a modified isoquinoline synthesis,206a the use of a direct synthesis of 2-canyoquionlines via N-tosyl Reissert intermediates,206b the implementation of an effective α-pyrone synthesis utilizing Meldrum’s acid, and a regioselective hydroxylation of granditropone.

Figure 37. Intramolecular cycloaddition reactions. Colchicine. The cycloaddition reactions of cyclopropenone ketals first found application in the total synthesis of colchicine.202 The first of the tropolone alkaloids to be identified, colchicine is a historically important antimitotic agent that inhibits cell division through tubulin binding and exhibits antitumor activity. Even today, new therapeutic applications of colchicine are emerging that would be exciting to probe, including its use in the treatment of cerebrovascular ischemia, auto-inflammatory diseases, atherosclerosisassociated inflammation, dermatology, and atrial fibrillation.

Figure 39. Divergent synthesis of azafluoranthene and tropoloisoquinoline alkaloids.

Figure 38. Key step in the total synthesis of colchicine.

Rubrolone. The [4+2] cycloaddition reaction of cyclopropenone ketals was also utilized in a total synthesis of the rubrolone aglycon.193,207 Rubrolone, isolated from



Streptomyces enchinoruber,208 was identified in a single-crystal X-ray structure determination and shown to possess a unique aglycon characteristic of a class of structurally related agents. 208 Our approach, which is only one of two total syntheses disclosed to date,209 centered on two key steps. The AB ring system of the natural product was constructed by an intramolecular [4+2] reaction of an O-alkyl α,β-unsaturated oxime and a tethered alkyne, and the C ring was introduced with the use of a Diels–Alder reaction between a cyclopropenone ketal and a highly oxygenated AB ring system as a prelude to C-ring tropolone introduction (Scheme 47). Scheme 47. Tropolone introduction and completion of the total synthesis of the rubrolone aglycon.

The key cyclopropenone ketal Diels–Alder reaction proceeded at room temperature to provide a single diastereomer in superb yield (97%), derived from exclusive cycloaddition through the less sterically encumbered exo transition state (Figure 40). Bromination, selective deprotection of the dimethyl ketal, base-promoted elimination of HBr followed by acid-catalyzed hydrolysis of the mixed ketal provided tropolone 256. This reaction sequence proceeds through mixed ketal hydrolysis, enolization with norcaradiene generation, electrocyclic rearrangement to the cycloheptatrienone ketal, and tautomerization to 256.

A remarkable series of more than 70 total syntheses of complex natural products are summarized, each of which enlisted a key inverse electron demand Diels–Alder reaction central to the synthetic approach and emerged from our studies spanning nearly 40 years. Underlying these efforts are fundamental studies on the discovery, development, or scope of the cycloaddition reaction of heterocyclic azadienes, 1-aza-1,3butadienes, cyclopropenone ketals, α-pyrones, and related systems. Perhaps the single most remarkable feature of the studies is the number of key cycloaddition reactions that were conducted at room temperature. In practice, we have found that it is easier to match the complementary diene/dienophile reactivity in the LUMOdiene-controlled cycloadditions than is typically accessible to the normal Diels–Alder reaction. The rate, predictable intrinsic reaction regioselectivity, mode of cycloaddition, and diastereoselectivity of the inverse electron demand Diels–Alder reactions rival or surpass those of normal Diels–Alder reactions. Our recent discovery of the solvent hydrogen bonding catalysis (HFIP or TFE) of heterocyclic azadiene cycloadditions now not only allows the reactions to be conducted under milder conditions with a broader scope than previously realized, but also offers a general fundamental feature for future use analogous to the impact of Lewis acid catalysis on the normal Diels–Alder reaction. The applications of these methods in the total syntheses of the series of natural products highlight the power of the methodology, especially for the synthesis of complex heterocyclic ring systems. In the context of our work, the advent of the synthetic methodology not only permitted the preparation of a series of targeted biologically important natural products, but enabled the divergent syntheses of key partial structures, and analogues incorporating deep-seated structural modifications. This provided the opportunity to gain valuable insights into the structure–function relationships of the natural products, to explore the origin of their biological properties, and to design compounds with improved potency and selectivity. In most instances, it was the targeted natural products that inspired the methodology development or discovery. The development of previously unknown heterocyclic azadienes including the 1,2,3,5-tetrazines, extensions of the recent studies on solvent hydrogen bonding catalysis of the Diels–Alder reaction, alternative tethering strategies for the intramolecular cascade cycloaddition reactions of 1,3,4-oxadiazoles for accessing additional alkaloid scaffolds, key applications of the late-stage divergent aryl annulation strategy, and the total synthesis of additional natural product classes utilizing these approaches are ongoing and will be detailed in due course. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID Dale L. Boger: 0000-0002-3966-3317 Author Contributions

Figure 40. Exo [4+2] cycloaddition of a cyclopropenone ketal enroute to the rubrolone aglycon. 6. Conclusion

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†

JZ and VS contributed equally to this work.

Biographies


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Dale Boger received his BSc in Chemistry from the University of Kansas, his PhD in Chemistry from Harvard University with E. J. Corey, and served on the faculty at the University of Kansas (Medicinal Chemistry) and Purdue University (Chemistry) before joining The Scripps Research Institute in 1990 where he is the Richard and Alice Cramer Professor of Chemistry.

Vyom Shukla received his BA in Chemistry from Reed College (2014) where he worked with Professor Alan Shusterman and Professor Summer Gibbs of Oregon Health & Science University on the synthesis and application of photoswitchable fluorophores for super-resolution microscopy. He is currently a Ph.D. candidate at The Scripps Research Institute (2014present) with Professor Boger, working on the total synthesis and bioorganic investigation of alkaloid natural products. ACKNOWLEGEMENTS The work summarized herein has been the collective efforts throughout my career by a remarkably gifted, spirited, and talented group of graduate students and postdoctoral colleagues with whom I have had the opportunity to work with. It is their personal and professional progression as scientists that provided the true pleasure from the conduct of the work. It remains a privilege to work at such a great institution for nearly 30 years now (TSRI), and earlier at the University of Kansas where the work first started, with such wonderful student and faculty colleagues. We are especially grateful to NIH for the financial support of our studies (CA042056, CA041101). REFERENCES

Jiajun Zhang received his BSc in Chemistry from Peking University, where he conducted research in total synthesis of Schisandraceae triterpenoids and Vibsane diterpenoids under the supervision of Professor Zhen Yang and Professor Jiahua Chen. In 2013, he moved to The Scripps Research Institute for graduate studies with Professor Boger, focusing on the new generation total synthesis of vinblastine and previously in accessible analogues.

(1) Boger, D. L. The difference a single atom can make: Synthesis and design at the chemistry–biology interface. J. Org. Chem. 2017, 82, 11961-11980. (2) Boger, D. L. Cycloaddition reactions of azadienes, cyclopropenone ketals, and related systems: Scope and applications. Chemtracts: Org. Chem. 1996, 9, 149-189. (3) (a) Alder, K. The diene synthesis. In Newer Methods of Preparative Organic Chemistry, Wiley: New York, 1948; Vol. 1, pp 381-511. (b) Huisgen, R.; Grashey, R.; Sauer, J. Cycloaddition reactions of alkenes. In Chemistry of Alkenes, Patai, S., Ed. Wiley: New York, 1964; pp 878-953. (c) Oppolzer, W. Intramolecular [4+2] and [3+2] cycloadditions in organic synthesis. Angew. Chem. Int. Ed. Engl. 1977, 16, 10-



23. (d) Boger, D. L. Diels–Alder reactions of azadienes. Tetrahedron 1983, 39, 2869-2939. (e) Corey, E. J. Catalytic enantioselective Diels–Alder reactions: Methods, mechanistic fundamentals, pathways, and applications. Angew. Chem. Int. Ed. 2002, 41, 1650-1667. (4) Diels, O.; Alder, K. Synthesen in der hydroaromatischen reihe. Justus Liebigs Ann. Chem. 1928, 460, 98-122. (5) (a) Huisgen, R. Cycloadditions – definition, classification, and characterization. Angew. Chem. Int. Ed. Engl. 1968, 7, 321328. (b) Sauer, J. Diels–Alder-reactions part I: New preparative aspects. Angew. Chem. Int. Ed. Engl. 1966, 5, 211-230. (c) Sauer, J. Diels–Alder reactions II: The reaction mechanism. Angew. Chem. Int. Ed. Engl. 1967, 6, 16-33. (d) Sauer, J.; Sustmann, R. Mechanistic aspects of Diels–Alder reactions: A critical survey. Angew. Chem. Int. Ed. Engl. 1980, 19, 779-807. (6) Carboni, R. A.; Lindsey, R. V. Reactions of tetrazines with unsaturated compounds. A new synthesis of pyridazines. J. Am. Chem. Soc. 1959, 81, 4342-4346. (7) (a) Sauer, J.; Wiest, H. Diels–Alder additions with “inverse” electron demand. Angew. Chem. Int. Ed. Engl. 1962, 1, 269-269. (b) Steigel, A.; Sauer, J. (4+2)-Cycloadditionen 6gliedriger heterocyclen mit inaminen. Tetrahedron Lett. 1970, 11, 3357-3360. (c) Neunhoeffer, H.; Werner, G. Reaktion von pyrazinen mit 1-diäthylamino-propin. Justus Liebigs Ann. Chem. 1972, 761, 39-49. (d) Neunhoeffer, H.; Werner, G. Cycloadditionen mit azabenzolen, VII. Reaktion von pyrimidinen mit N,N-diäthyl-1-propinylamin. Justus Liebigs Ann. Chem. 1974, 1190-1194. (e) Neunhoeffer, H.; Bachmann, M. Cycloadditionen mit azabenzolen, X. Cycloadditionen mit 1,3,5-triazinen. Chem. Ber. 1975, 108, 3877-3882. (8) (a) Kondrat’eva, G. Y. Condensation of homologs of oxazole with maleic anhydride. Khim. Nauka Prom. 1957, 2, 666-667. (b) Karpeiskii, M. Y.; Florent’ev, V. L. Condensation of oxazoles with dienophiles – a new method for the synthesis of pyridine bases. Russ. Chem. Rev. (Engl. Ed.) 1969, 38, 540546. (c) Turchi, I. J.; Dewar, M. J. S. Chemistry of oxazoles. Chem. Rev. 1975, 75, 389-437. (9) (a) Jacobi, P. A.; Weiss, K. T.; Egbertson, M. Bis heteroannulation. 6. The first example of a Diels–Alder reaction involving a thiazole ring with an acetylenic dienophile. Geometrical control of reaction pathway. Heterocycles 1984, 22, 281-286. (b) Vasil’ev, N. V.; Lyashenko, Y. E.; Kolomiets, A. F.; Sokol’skii, G. A. Cycloaddition of 2,5bis(trifluoromethyl)-1,3,4-oxadiazole to olefins. Chem. Heterocycl. Compd. 1987, 23, 470-470. (c) Eddaïf, A.; Laurent, A.; Mison, P.; Pellissier, N. Reaction de Diels–Alder intramoleculaire de 3H-pyrroles. Tetrahedron Lett. 1984, 25, 2779-2782. (10) (a) Boger, D. L. Diels–Alder reactions of heterocyclic azadienes. Scope and applications. Chem. Rev. 1986, 86, 781793. (b) Boger, D. L.; Weinreb, S. M. Hetero Diels–Alder Methodology in Organic Synthesis. Academic: San Diego, 1987. (11) (a) Blackman, M. L.; Royzen, M.; Fox, J. M. Tetrazine ligation: Fast bioconjugation based on inverse-electron-demand Diels–Alder reactivity. J. Am. Chem. Soc. 2008, 130, 1351813519. (b) Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A. Tetrazine-based cycloadditions: Application to pretargeted live cell imaging. Bioconjugate Chem. 2008, 19, 2297-2299. (c) Wu, H.; Devaraj, N. K. Advances in tetrazine bioorthogonal chemistry driven by the synthesis of novel tetrazines and dienophiles. Acc. Chem. Res. 2018, 51, 1249-1259.

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(12) Boger, D. L. Diels–Alder reactions of azadienes: Scope and application. Bull. Soc. Chim., Belg. 1990, 99, 599-615. (13) (a) Neunhoeffer, H. Tetrazines and pentazines. In Comprehensive Heterocyclic Chemistry, Neunhoeffer, H., Ed. Pergamon: London, 1984; Vol. 3, pp 550-555. (b) Neunhoeffer, H.; Wiley, P. F. Chemistry of 1,2,3-Triazines and 1,2,4Triazines, Tetrazines, and Pentazin. Wiley: New York, 1978; Vol. 33, pp 1073-1283. (c) Sauer, J. 1,2,4,5-tetrazines. In Comprehensive Heterocyclic Chemistry II, Katritzky, A. R.; Rees, C. W.; Scriven, E. F. V., Eds. Pergamon Press: 1996; Vol. 6, pp 901-955. (d) Mayer, S.; Lang, K. Tetrazines in inverseelectron-demand Diels–Alder cycloadditions and their use in biology. Synthesis 2017, 49, 830-848. (14) (a) Boger, D. L.; Coleman, R. S.; Panek, J. S.; Huber, F. X.; Sauer, J. A detailed, convenient preparation of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate. J. Org. Chem. 1985, 50, 5377-5379. (b) Boger, D. L.; Schaum, R. P.; Garbaccio, R. M. Regioselective inverse electron demand Diels–Alder reactions of N-acyl 6-amino-3-(methylthio)-1,2,4,5-tetrazines. J. Org. Chem. 1998, 63, 6329-6337. (c) Sakya, S. M.; Groskopf, K. K.; Boger, D. L. Preparation and inverse electron demand Diels– Alder reactions of 3-methoxy-6-methylthio-1,2,4,5-tetrazine. Tetrahedron Lett. 1997, 38, 3805-3808. (15) Boger, D. L.; Sakya, S. M. Inverse electron demand Diels–Alder reactions of 3,6-bis(methylthio)-1,2,4,5-tetrazine. 1,2-Diazine introduction and direct implementation of a divergent 1,2,4,5-tetrazine→1,2-diazine→benzene (indoline/indole) Diels–Alder strategy. J. Org. Chem. 1988, 53, 1415-1423. (16) Hamasaki, A.; Ducray, R.; Boger, D. L. Two novel 1,2,4,5-tetrazines that participate in inverse electron demand Diels–Alder reactions with an unexpected regioselectivity. J. Org. Chem. 2006, 71, 185-193. (17) Bringmann, G.; Reichert, Y.; Kane, V. V. The total synthesis of streptonigrin and related antitumor antibiotic natural products. Tetrahedron 2004, 60, 3539-3574. (18) Boger, D. L.; Panek, J. S. 1,2,4-Triazine preparation via thermal cycloaddition of dimethyl 1,2,4,5-tetrazine-3,6dicarboxylate with aryl thioimidates. Tetrahedron Lett. 1983, 24, 4511-4514. (19) (a) Boger, D. L.; Panek, J. S. Formal total synthesis of streptonigrin. J. Org. Chem. 1983, 48, 621-623. (b) Boger, D. L.; Panek, J. S. Inverse electron demand Diels–Alder reactions of heterocyclic azadienes: Formal total synthesis of streptonigrin. J. Am. Chem. Soc. 1985, 107, 5745-5754. (20) (a) Weinreb, S. M.; Basha, F. Z.; Hibino, S.; Khatri, N. A.; Kim, D.; Pye, W. E.; Wu, T. T. Total synthesis of the antitumor antibiotic streptonigrin. J. Am. Chem. Soc. 1982, 104, 536-544. (b) Basha, F. Z.; Hibino, S.; Kim, D.; Pye, W. E.; Wu, T.-T.; Weinreb, S. M. Total synthesis of streptonigrin. J. Am. Chem. Soc. 1980, 102, 3962-3964. (21) Kende, A. S.; Lorah, D. P.; Boatman, R. J. A new and efficient total synthesis of streptonigrin. J. Am. Chem. Soc. 1981, 103, 1271-1273. (22) (a) Donohoe, T. J.; Jones, C. R.; Barbosa, L. C. A. Total synthesis of (±)-streptonigrin: De novo construction of a pentasubstituted pyridine using ring-closing metathesis. J. Am. Chem. Soc. 2011, 133, 16418-16421. (b) Donohoe, T. J.; Jones, C. R.; Kornahrens, A. F.; Barbosa, L. C. A.; Walport, L. J.; Tatton, M. R.; O’Hagan, M.; Rathi, A. H.; Baker, D. B. Total synthesis of the antitumor antibiotic (±)-streptonigrin: Firstand second-generation routes for de novo pyridine formation


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using ring-closing metathesis. J. Org. Chem. 2013, 78, 1233812350. (23) (a) Boger, D. L.; Yasuda, M.; Mitscher, L. A.; Drake, S. D.; Kitos, P. A.; Thompson, S. C. Streptonigrin and lavendamycin partial structures. Probes for the minimum, potent pharmacophore of streptonigrin, lavendamycin, and synthetic quinoline-5,8-diones. J. Med. Chem. 1987, 30, 19181928. (b) Yasuda, M.; Boger, D. L. Streptonigrin and lavendamycin partial structures. Preparation of 7-amino-2-(2’pyridyl)quinoline-5,8-quinone-6’-carboxylic acid: A probe for the minimum, potent pharmacophore of the naturally occurring antitumor-antibiotics. J. Heterocycl. Chem. 1987, 24, 12531260. (24) (a) Knuckley, B.; Jones, J. E.; Bachovchin, D. A.; Slack, J.; Causey, C. P.; Brown, S. J.; Rosen, H.; Cravatt, B. F.; Thompson, P. R. A fluopol-ABPP HTS assay to identify PAD inhibitors. Chem. Commun. 2010, 46, 7175-7177. (b) Dreyton, C. J.; Anderson, E. D.; Subramanian, V.; Boger, D. L.; Thompson, P. R. Insights into the mechanism of streptonigrininduced protein arginine deiminase inactivation. Bioorg. Med. Chem. 2014, 22, 1362-1369. (25) Boger, D. L.; Coleman, R. S. Intramolecular Diels–Alder reactions of 1,2-diazines: General indoline synthesis. Studies on the preparation of the central and right-hand segments of CC1065. J. Org. Chem. 1984, 49, 2240-2245. (26) (a) Boger, D. L.; Coleman, R. S. Diels–Alder reactions of heterocyclic azadienes: Total synthesis of PDE-II methyl ester. J. Org. Chem. 1986, 51, 3250-3252. (b) Boger, D. L.; Coleman, R. S. Diels–Alder reactions of heterocyclic azadienes: Total synthesis of PDE I, PDE II, and PDE I dimer methyl ester. J. Am. Chem. Soc. 1987, 109, 2717-2727. (c) Boger, D. L.; Coleman, R. S.; Invergo, B. J. Studies on the total synthesis of CC-1065: Preparation of a synthetic simplified 3-carbamoyl1,2-dihydro-3H-pyrrolo[3,2-e]indole dimer/trimer/tetramer (CDPI dimer/trimer/tetramer) and development of methodology for PDE-I dimer methyl ester formation. J. Org. Chem. 1987, 52, 1521-1530. (27) Boger, D. L.; Coleman, R. S. Total synthesis of (+)-CC1065 and ent-(–)-CC-1065. J. Am. Chem. Soc. 1988, 110, 13211323. (28) (a) Boger, D. L.; Coleman, R. S. Further observations on the Lewis acid-catalyzed benzylic hydroperoxide rearrangement: Use of a boron-trifluoride/hydrogen peroxide preformed, aged reagent. Tetrahedron Lett. 1987, 28, 10271030. (b) Boger, D. L.; Coleman, R. S. Benzylic hydroperoxide rearrangement: Observations on a viable and convenient alternative to the Baeyer-Villiger rearrangement. J. Org. Chem. 1986, 51, 5436-5439. (c) Boger, D. L.; Yohannes, D. Selectively protected L-dopa derivatives: Application of the benzylic hydroperoxide rearrangement. J. Org. Chem. 1987, 52, 5283-5286. (29) (a) Boger, D. L.; Johnson, D. S. CC-1065 and the duocarmycins: Understanding their biological function through mechanistic studies. Angew. Chem. Int. Ed. Engl. 1996, 35, 1438-1474. (b) Boger, D. L.; Johnson, D. S. CC-1065 and the duocarmycins: Unraveling the keys to a new class of naturally derived DNA alkylating agents. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3642-3649. (30) (a) Boger, D. L.; Coleman, R. S. Total synthesis of (±)N2-(phenylsulfonyl)-CPI, (±)-CC-1065, (+)-CC-1065, ent-(–)CC-1065, and the precise, functional agents (±)-CPI-CDPI2, (+)-CPI-CDPI2, and (–)-CPI-CDPI2 [(±)-(3bR*,4aS*)-, (+)(3bR,4aS)-, and (–)-(3bS,4aR)-deoxy-CC-1065]. J. Am. Chem.

Soc. 1988, 110, 4796-4807. (b) Boger, D. L.; Coleman, R. S. Total synthesis of (+)- and (–)-CPI-CDPI2: (+)-(3bR,4aS)- and (–)-(3bS,4aR)-deoxy-CC-1065. J. Org. Chem. 1988, 53, 695698. (31) (a) Boger, D. L.; McKie, J. A.; Nishi, T.; Ogiku, T. Enantioselective total synthesis of (+)-duocarmycin A, epi-(+)duocarmycin A, and their unnatural enantiomers. J. Am. Chem. Soc. 1996, 118, 2301-2302. (b) Boger, D. L.; McKie, J. A.; Nishi, T.; Ogiku, T. Total synthesis of (+)-duocarmycin A, epi(+)-duocarmycin A and their unnatural enantiomers: Assessment of chemical and biological properties. J. Am. Chem. Soc. 1997, 119, 311-325. (32) (a) Boger, D. L.; Machiya, K.; Hertzog, D. L.; Kitos, P. A.; Holmes, D. Total synthesis and preliminary evaluation of (+)- and ent-(–)-duocarmycin SA. J. Am. Chem. Soc. 1993, 115, 9025-9036. (b) Boger, D. L.; Machiya, K. Total synthesis of (+)-duocarmycin SA. J. Am. Chem. Soc. 1992, 114, 1005610058. (c) MacMillan, K. S.; Nguyen, T.; Hwang, I.; Boger, D. L. Total synthesis and evaluation of iso-duocarmycin SA and iso-yatakemycin. J. Am. Chem. Soc. 2009, 131, 1187-1194. (33) (a) Tichenor, M. S.; Kastrinsky, D. B.; Boger, D. L. Total synthesis, structure revision, and absolute configuration of (+)yatakemycin. J. Am. Chem. Soc. 2004, 126, 8396-8398. (b) Tichenor, M. S.; Trzupek, J. D.; Kastrinsky, D. B.; Shiga, F.; Hwang, I.; Boger, D. L. Asymmetric total synthesis of (+)- and ent-(–)-yatakemycin and duocarmycin SA: Evaluation of yatakemycin key partial structures and its unnatural enantiomer. J. Am. Chem. Soc. 2006, 128, 15683-15696. (c) Tichenor, M. S.; MacMillan, K. S.; Trzupek, J. D.; Rayl, T. J.; Hwang, I.; Boger, D. L. Systematic exploration of the structural features of yatakemycin impacting DNA alkylation and biological activity. J. Am. Chem. Soc. 2007, 129, 10858-10869. (d) Tichenor, M. S.; Boger, D. L. Yatakemycin: Total synthesis, DNA alkylation, and biological properties. Nat. Prod. Rep. 2008, 25, 220-226. (34) Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Goldberg, J. A. CC-1065 and the duocarmycins: Synthetic studies. Chem. Rev. 1997, 97, 787-828. (35) (a) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H.; Munk, S. A.; Kitos, P. A.; Suntornwat, O. Duocarmycin-pyrindamycin DNA alkylation properties and identification, synthesis, and evaluation of agents incorporating the pharmacophore of the duocarmycin-pyrindamycin alkylation subunit. Identification of the CC-1065 duocarmycin common pharmacophore. J. Am. Chem. Soc. 1990, 112, 8961-8971. (b) Boger, D. L.; Johnson, D. S.; Yun, W. (+)- and ent-(–)-duocarmycin SA and (+)- and ent-(–)-N-BOC-DSA DNA alkylation properties. Alkylation site models that accommodate the offset AT-rich adenine N3 alkylation selectivity of the enantiomeric agents. J. Am. Chem. Soc. 1994, 116, 1635-1656. (c) Boger, D. L.; Johnson, D. S.; Yun, W.; Tarby, C. M. Molecular basis for sequence selective DNA alkylation by (+)- and ent-(–)-CC-1065 and related agents: Alkylation site models that accommodate the offset ATrich adenine N3 alkylation selectivity. Bioorg. Med. Chem. 1994, 2, 115-135. (d) Boger, D. L.; Munk, S. A. DNA alkylation properties of enhanced functional analogs of CC-1065 incorporating the 1,2,9,9a-tetrahydrocyclopropa[1,2c]benz[1,2-e]indol-4-one (CBI) alkylation subunit. J. Am. Chem. Soc. 1992, 114, 5487-5496. (e) Parrish, J. P.; Kastrinsky, D. B.; Wolkenberg, S. E.; Igarashi, Y.; Boger, D. L. DNA alkylation properties of yatakemycin. J. Am. Chem. Soc. 2003, 125, 10971-10976. (f) Trzupek, J. D.; Gottesfeld, J. M.; Boger, D. L. Alkylation of duplex DNA in nucleosome core particles by duocarmycin SA and yatakemycin. Nat. Chem. Biol. 2006,



2, 79. (g) Boger, D. L.; Yun, W.; Terashima, S.; Fukuda, Y.; Nakatani, K.; Kitos, P. A.; Jin, Q. DNA alkylation properties of the duocarmycins: (+)-Duocarmycin A, epi-(+)-duocarmycin A, ent-(–)-duocarmycin A and epi,ent-(–)-duocarmycin A. Bioorg. Med. Chem. Lett. 1992, 2, 759-765. (36) Boger, D. L.; Yun, W. Reversibility of the duocarmycin A and SA DNA alkylation reaction. J. Am. Chem. Soc. 1993, 115, 9872-9873. (37) (a) Boger, D. L.; Mesini, P.; Tarby, C. M. Chemical and structural comparison of N-BOC-CBQ and N-BOC-CBI: Identification and structural origin of an unappreciated but productive stability of the CC-1065 and duocarmycin SA alkylation subunits. J. Am. Chem. Soc. 1994, 116, 6461-6462. (b) Boger, D. L.; Mesini, P. Design, synthesis, and evaluation of CC-1065 and duocarmycin analogs incorporating the 2,3,10,10a-tetrahydro-1H-cyclopropa[d]benzo[f]quinol-5-one (CBQ) alkylation subunit: Identification and structural origin of subtle stereoelectronic features that govern reactivity and regioselectivity. J. Am. Chem. Soc. 1994, 116, 11335-11348. (c) Boger, D. L.; Mesini, P. DNA alkylation properties of CC-1065 and duocarmycin analogs incorporating the 2,3,10,10atetrahydrocyclopropa[d]benzo[f]quinol-5-one alkylation subunit: Identification of subtle structural features that contribute to the regioselectivity of the adenine N3 alkylation reaction. J. Am. Chem. Soc. 1995, 117, 11647-11655. (d) Boger, D. L.; Turnbull, P. Synthesis and evaluation of CC-1065 and duocarmycin analogs incorporating the 1,2,3,4,11,11ahexahydrocyclopropa[c]naphtho[2,1-b]azepin-6-one (CNA) alkylation subunit: Structural features that govern reactivity and reaction regioselectivity. J. Org. Chem. 1997, 62, 58495863. (38) Boger, D. L.; Ishizaki, T.; Zarrinmayeh, H. Isolation and characterization of the duocarmycin-adenine DNA adduct. J. Am. Chem. Soc. 1991, 113, 6645-6649. See also: ref. 35(c) and 35(e). (39) (a) Boger, D. L.; Invergo, B. J.; Coleman, R. S.; Zarrinmayeh, H.; Kitos, P. A.; Thompson, S. C.; Leong, T.; McLaughlin, L. W. A demonstration of the intrinsic importance of stabilizing hydrophobic binding and non-convalent van der waals contacts dominant in the non-covalent CC-1065/B-DNA binding. Chem.-Biol. Interact. 1990, 73, 29-52. (b) Boger, D. L.; Coleman, R. S.; Invergo, B. J.; Sakya, S. M.; Ishizaki, T.; Munk, S. A.; Zarrinmayeh, H.; Kitos, P. A.; Thompson, S. C. Synthesis and evaluation of aborted and extended CC-1065 functional analogs: (+)- and (–)-CPI-PDE-I1, (+)- and (–)-CPICDPI1, and (±)-, (+)-, and (–)-CPI-CDPI3. Preparation of key partial structures and definition of an additional functional role of the CC-1065 central and right-hand subunits. J. Am. Chem. Soc. 1990, 112, 4623-4632. (c) Boger, D. L.; Zarrinmayeh, H.; Munk, S. A.; Kitos, P. A.; Suntornwat, O. Demonstration of a pronounced effect of noncovalent binding selectivity on the (+)CC-1065 DNA alkylation and identification of the pharmacophore of the alkylation subunit. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1431-1435. (d) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H. (+)-CC-1065 DNA alkylation: Key studies demonstrating a noncovalent binding selectivity contribution to the alkylation selectivity. J. Am. Chem. Soc. 1991, 113, 39803983. (e) Boger, D. L.; Johnson, D. S. Second definitive test of proposed models for the origin of the CC-1065 and duocarmycin DNA alkylation selectivity. J. Am. Chem. Soc. 1995, 117, 1443-1444. (40) (a) Boger, D. L.; Garbaccio, R. M. Shape-dependent catalysis: Insights into the source of catalysis for the CC-1065

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and duocarmycin DNA alkylation reaction. Acc. Chem. Res. 1999, 32, 1043-1052. (b) Boger, D. L.; Garbaccio, R. M. Catalysis of the CC-1065 and duocarmycin DNA alkylation reaction: DNA binding induced conformational change in the agent results in activation. Bioorg. Med. Chem. 1997, 5, 263276. (c) Boger, D. L.; Hertzog, D. L.; Bollinger, B.; Johnson, D. S.; Cai, H.; Goldberg, J.; Turnbull, P. Duocarmycin SA shortened, simplified, and extended agents: A systematic examination of the role of the DNA binding subunit. J. Am. Chem. Soc. 1997, 119, 4977-4986. (d) Boger, D. L.; Bollinger, B.; Hertzog, D. L.; Johnson, D. S.; Cai, H.; Mésini, P.; Garbaccio, R. M.; Jin, Q.; Kitos, P. A. Reversed and sandwiched analogs of duocarmycin SA: Establishment of the origin of the sequence-selective alkylation of DNA and new insights into the source of catalysis. J. Am. Chem. Soc. 1997, 119, 4987-4998. (e) Boger, D. L.; Garbaccio, R. M. Are the duocarmycin and CC-1065 DNA alkylation reactions acidcatalyzed? Solvolysis pH–rate profiles suggest they are not. J. Org. Chem. 1999, 64, 5666-5669. (f) Ambroise, Y.; Boger, D. L. The DNA phosphate backbone is not involved in catalysis of the duocarmycin and CC-1065 DNA alkylation reaction. Bioorg. Med. Chem. Lett. 2002, 12, 303-306. (g) Ellis, D. A.; Wolkenberg, S. E.; Boger, D. L. Metal cation complexation and activation of reversed CPyI analogues of CC-1065 and duocarmycin SA: Partitioning the effects of binding and catalysis. J. Am. Chem. Soc. 2001, 123, 9299-9306. (41) (a) Eis, P. S.; Smith, J. A.; Rydzewski, J. M.; Case, D. A.; Boger, D. L.; Chazin, W. J. High resolution solution structure of a DNA duplex alkylated by the antitumor agent duocarmycin SA. J. Mol. Biol. 1997, 272, 237-252. (b) Schnell, J. R.; Ketchem, R. R.; Boger, D. L.; Chazin, W. J. Binding-induced activation of DNA alkylation by duocarmycin SA: Insights from the structure of an indole derivative–DNA adduct. J. Am. Chem. Soc. 1999, 121, 5645-5652. (c) Smith, J. A.; Bifulco, G.; Case, D. A.; Boger, D. L.; Gomez-Paloma, L.; Chazin, W. J. The structural basis for in situ activation of DNA alkylation by duocarmycin SA. J. Mol. Biol. 2000, 300, 1195-1204. (42) (a) Wolkenberg, S. E.; Boger, D. L. Mechanisms of in situ activation for DNA-targeting antitumor agents. Chem. Rev. 2002, 102, 2477-2496. (b) Boger, D. L. The duocarmycins: Synthetic and mechanistic studies. Acc. Chem. Res. 1995, 28, 20-29. (43) MacMillan, K. S.; Boger, D. L. Fundamental relationships between structure, reactivity, and biological activity for the duocarmycins and CC-1065. J. Med. Chem. 2009, 52, 5771-5780. (44) (a) Boger, D. L.; Ishizaki, T.; Wysocki, R. J.; Munk, S. A.; Kitos, P. A.; Suntornwat, O. Total synthesis and evaluation of (±)-N-(tert-butoxycarbonyl)-CBI, (±)-CBI-CDPI1, and (±)CBI-CDPI2: CC-1065 functional agents incorporating the equivalent 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2e]indol-4-one (CBI) left-hand subunit. J. Am. Chem. Soc. 1989, 111, 6461-6463. (b) Boger, D. L.; Yun, W.; Teegarden, B. R. An improved synthesis of 1,2,9,9atetrahydrocyclopropa[c]benz[e]indol-4-one (CBI): A simplified analog of the CC-1065 alkylation subunit. J. Org. Chem. 1992, 57, 2873-2876. (c) Boger, D. L.; McKie, J. A. An efficient synthesis of 1,2,9,9atetrahydrocyclopropa[c]benz[e]indol-4-one CBI: An enhanced and simplified analog of the CC-1065 and duocarmycin alkylation subunits. J. Org. Chem. 1995, 60, 1271-1275. (d) Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Searcey, M. Synthesis of CC-1065 and duocarmycin analogs via


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intramolecular aryl radical cyclization of a tethered vinyl chloride. Tetrahedron Lett. 1998, 39, 2227-2230. (e) Lajiness, J. P.; Boger, D. L. Asymmetric synthesis of 1,2,9,9atetrahydrocyclopropa[c]benzo[e]indol-4-one (CBI). J. Org. Chem. 2011, 76, 583-587. (f) Boger, D. L.; Wysocki, R. J.; Ishizaki, T. Synthesis of N-(phenylsulfonyl)-CI, N-((tertbutyloxy)carbonyl)-CI, CI-CDPI1, and CI-CDPI2: CC-1065 functional analog incorporating the parent 1,2,7,7atetrahydrocycloprop[1,2-c]indol-4-one (CI) left-hand subunit. J. Am. Chem. Soc. 1990, 112, 5230-5240. (g) Boger, D. L.; Santillán, A.; Searcey, M.; Brunette, S. R.; Wolkenberg, S. E.; Hedrick, M. P.; Jin, Q. Synthesis and evaluation of 1,2,8,8atetrahydrocyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one, the parent alkylation subunit of CC-1065 and the duocarmycins: Impact of the alkylation subunit substituents and its implications for DNA alkylation catalysis. J. Org. Chem. 2000, 65, 4101-4111 and references cited therein. (45) Wolfe, A. L.; Duncan, K. K.; Lajiness, J. P.; Zhu, K.; Duerfeldt, A. S.; Boger, D. L. A fundamental relationship between hydrophobic properties and biological activity for the duocarmycin class of DNA-alkylating antitumor drugs: Hydrophobic-binding-driven bonding. J. Med. Chem. 2013, 56, 6845-6857. (46) (a) Parrish, J. P.; Hughes, T. V.; Hwang, I.; Boger, D. L. Establishing the parabolic relationship between reactivity and activity for derivatives and analogues of the duocarmycin and CC-1065 alkylation subunits. J. Am. Chem. Soc. 2004, 126, 8081. (b) Boger, D. L.; Yun, W. Role of the CC-1065 and duocarmycin N2 substituent: Validation of a direct relationship between solvolysis chemical stability and in vitro biological potency. J. Am. Chem. Soc. 1994, 116, 5523-5524. (c) Boger, D. L.; Munk, S. A.; Ishizaki, T. (+)-CC-1065 DNA alkylation: Observation of an unexpected relationship between cyclopropane electrophile reactivity and the intensity of DNA alkylation. J. Am. Chem. Soc. 1991, 113, 2779-2780. (d) Boger, D. L.; Yun, W. CBI-TMI: Synthesis and evaluation of a key analog of the duocarmycins. Validation of a direct relationship between chemical solvolytic stability and cytotoxic potency and confirmation of the structural features responsible for the distinguishing behavior of enantiomeric pairs of agents. J. Am. Chem. Soc. 1994, 116, 7996-8006. (e) Boger, D. L.; Ishizaki, T. Resolution of a CBI precursor and incorporation into the synthesis of (+)-CBI, (+)-CBI-CDPI1, (+)-CBI-CDPI2: Enhanced functional analogs of (+)-CC-1065. A critical appraisal of a proposed relationship between electrophile reactivity, DNA-binding properties, and cytotoxic potency. Tetrahedron Lett. 1990, 31, 793-796. (47) Tichenor, M. S.; MacMillan, K. S.; Stover, J. S.; Wolkenberg, S. E.; Pavani, M. G.; Zanella, L.; Zaid, A. N.; Spalluto, G.; Rayl, T. J.; Hwang, I.; Baraldi, P. G.; Boger, D. L. Rational design, synthesis, and evaluation of key analogues of CC-1065 and the duocarmycins. J. Am. Chem. Soc. 2007, 129, 14092-14099. (48) Szpilman, A. M.; Carreira, E. M. Probing the biology of natural products: Molecular editing by diverted total synthesis. Angew. Chem. Int. Ed. 2010, 49, 9592-9628. (49) (a) Chari, R. V. J.; Miller, M. L.; Widdison, W. C. Antibody–drug conjugates: An emerging concept in cancer therapy. Angew. Chem. Int. Ed. 2014, 53, 3796-3827. (b) Chari, R. V. J.; Jackel, K. A.; Bourret, L. A.; Derr, S. M.; Tadayoni, B. M.; Mattocks, K. M.; Shah, S. A.; Liu, C.; Blättler, W. A.; Goldmacher, V. S. Enhancement of the selectivity and antitumor efficacy of a CC-1065 analogue through

immunoconjugate formation. Cancer Res. 1995, 55, 40794084. (c) Suzawa, T.; Nagamura, S.; Saito, H.; Ohta, S.; Hanai, N.; Yamasaki, M. Synthesis of a novel duocarmycin derivative DU-257 and its application to immunoconjugate using poly(ethylene glycol)-dipeptidyl linker capable of tumor specific activation. Bioorg. Med. Chem. 2000, 8, 2175-2184. (d) Jeffrey, S. C.; Torgov, M. Y.; Andreyka, J. B.; Boddington, L.; Cerveny, C. G.; Denny, W. A.; Gordon, K. A.; Gustin, D.; Haugen, J.; Kline, T.; Nguyen, M. T.; Senter, P. D. Design, synthesis, and in vitro evaluation of dipeptide-based antibody minor groove binder conjugates. J. Med. Chem. 2005, 48, 13441358. (e) Zhao, R. Y.; Erickson, H. K.; Leece, B. A.; Reid, E. E.; Goldmacher, V. S.; Lambert, J. M.; Chari, R. V. J. Synthesis and biological evaluation of antibody conjugates of phosphate prodrugs of cytotoxic DNA alkylators for the targeted treatment of cancer. J. Med. Chem. 2012, 55, 766-782. (f) Elgersma, R. C.; Coumans, R. G. E.; Huijbregts, T.; Menge, W. M. P. B.; Joosten, J. A. F.; Spijker, H. J.; de Groot, F. M. H.; van der Lee, M. M. C.; Ubink, R.; van den Dobbelsteen, D. J.; Egging, D. F.; Dokter, W. H. A.; Verheijden, G. F. M.; Lemmens, J. M.; Timmers, C. M.; Beusker, P. H. Design, synthesis, and evaluation of linker-duocarmycin payloads: Toward selection of HER2-targeting antibody–drug conjugate SYD985. Mol. Pharm. 2015, 12, 1813-1835. (g) Yu, L.; Lu, Y.; Yao, Y.; Liu, Y.; Wang, Y.; Lai, Q.; Zhang, R.; Li, W.; Wang, R.; Fu, Y.; Tao, Y.; Yi, S.; Gou, L.; Chen, L.;Yang, J. Promiximabduocarmycin, a new CD56 antibody-drug conjugate, is highly efficacious in small cell lung cancer xenograft models. Oncotarget. 2017, 9, 5197-5207. (50) (a) Chang, A. Y.; Dervan, P. B. Strand selective cleavage of DNA by diastereomers of hairpin polyamide-seco-CBI conjugates. J. Am. Chem. Soc. 2000, 122, 4856-4864. (b) Minoshima, M.; Bando, T.; Sasaki, S.; Shinohara, K.-i.; Shimizu, T.; Fujimoto, J.; Sugiyama, H. DNA alkylation by pyrrole–imidazole seco-CBI conjugates with an indole linker: Sequence-specific DNA alkylation with 10-base-pair recognition through heterodimer formation. J. Am. Chem. Soc. 2007, 129, 5384-5390 and references cited therein. (51) (a) Wolfe, A. L.; Duncan, K. K.; Parelkar, N. K.; Brown, D.; Vielhauer, G. A.; Boger, D. L. Efficacious cyclic N-acyl Oamino phenol duocarmycin prodrugs. J. Med. Chem. 2013, 56, 4104-4115. (b) Lajiness, J. P.; Robertson, W. M.; Dunwiddie, I.; Broward, M. A.; Vielhauer, G. A.; Weir, S. J.; Boger, D. L. Design, synthesis, and evaluation of duocarmycin O-amino phenol prodrugs subject to tunable reductive activation. J. Med. Chem. 2010, 53, 7731-7738. (c) Jin, W.; Trzupek, J. D.; Rayl, T. J.; Broward, M. A.; Vielhauer, G. A.; Weir, S. J.; Hwang, I.; Boger, D. L. A unique class of duocarmycin and CC-1065 analogues subject to reductive activation. J. Am. Chem. Soc. 2007, 129, 15391-15397. (d) Uematsu, M.; Boger, D. L. Asymmetric synthesis of a CBI-based cyclic N-acyl O-amino phenol duocarmycin prodrug. J. Org. Chem. 2014, 79, 96999703. (e) Wolfe, A. L.; Duncan, K. K.; Parelkar, N. K.; Weir, S. J.; Vielhauer, G. A.; Boger, D. L. A novel, unusually efficacious duocarmycin carbamate prodrug that releases no residual byproduct. J. Med. Chem. 2012, 55, 5878-5886. (52) (a) Tse, W. C.; Boger, D. L. A fluorescent intercalator displacement assay for establishing DNA binding selectivity and affinity. Acc. Chem. Res. 2004, 37, 61-69. (b) Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C.; Hedrick, M. P. A simple, high-resolution method for establishing DNA binding affinity and sequence selectivity. J. Am. Chem. Soc. 2001, 123, 5878-5891. (c) Boger, D. L.; Fink, B. E.; Hedrick, M. P. Total



synthesis of distamycin A and 2640 analogues: A solutionphase combinatorial approach to the discovery of new, bioactive DNA binding agents and development of a rapid, high-throughput screen for determining relative DNA binding affinity or DNA binding sequence selectivity. J. Am. Chem. Soc. 2000, 122, 6382-6394. (d) Tse, W. C.; Ishii, T.; Boger, D. L. Comprehensive high-resolution analysis of hairpin polyamides utilizing a fluorescent intercalator displacement (FID) assay. Bioorg. Med. Chem. 2003, 11, 4479-4486. (e) Ham, Y.-W.; Tse, W. C.; Boger, D. L. High-resolution assessment of protein DNA binding affinity and selectivity utilizing a fluorescent intercalator displacement (FID) assay. Bioorg. Med. Chem. Lett. 2003, 13, 3805-3807. (f) Boger, D. L.; Tse, W. C. Thiazole orange as the fluorescent intercalator in a high resolution FID assay for determining DNA binding affinity and sequence selectivity of small molecules. Bioorg. Med. Chem. 2001, 9, 2511-2518. (g) Yeung, B. K. S.; Tse, W. C.; Boger, D. L. Determination of binding affinities of triplex forming oligonucleotides using a fluorescent intercalator displacement (FID) assay. Bioorg. Med. Chem. Lett. 2003, 13, 3801-3804. (53) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.; Haught, J.; Bina, M. An alternative and convenient strategy for generation of substantial quantities of singly 5’-32p-end-labeled double-stranded DNA for binding studies: Development of a protocol for examination of functional features of (+)-CC-1065 and the duocarmycins that contribute to their sequenceselective DNA alkylation properties. Tetrahedron 1991, 47, 2661-2682. (54) Capon, R. J.; Macleod, J. K.; Scammells, P. J. The trikentrins: Novel indoles from the sponge Trikentrion flabelliforme. Tetrahedron 1986, 42, 6545-6550. (55) Boger, D. L.; Zhang, M. Total synthesis of (±)-cis and (±)-trans-trikentrin A: Diels–Alder reactions of heteroaromatic azadienes. J. Am. Chem. Soc. 1991, 113, 4230-4234. (56) Boger, D. L.; Wolkenberg, S. E. Total synthesis of Amaryllidaceae alkaloids utilizing sequential intramolecular heterocyclic azadiene Diels–Alder reactions of an unsymmetrical 1,2,4,5-tetrazine. J. Org. Chem. 2000, 65, 91209124. (57) Bach, N. J.; Kornfeld, E. C.; Jones, N. D.; Chaney, M. O.; Dorman, D. E.; Paschal, J. W.; Clemens, J. A.; Smalstig, E. B. Bicyclic and tricyclic ergoline partial structures. Rigid 3-(2aminoethyl)pyrroles and 3- and 4-(2-aminoethyl)pyrazoles as dopamine agonists. J. Med. Chem. 1980, 23, 481-491. (58) Boger, D. L.; Coleman, R. S.; Panek, J. S.; Yohannes, D. Thermal cycloaddition of dimethyl 1,2,4,5-tetrazine-3,6dicarboxylate with electron-rich olefins: 1,2-Diazine and pyrrole introduction. Preparation of octamethylporphin (OMP). J. Org. Chem. 1984, 49, 4405-4409. (59) Hu, D. X.; Withall, D. M.; Challis, G. L.; Thomson, R. J. Structure, chemical synthesis, and biosynthesis of prodiginine natural products. Chem. Rev. 2016, 116, 7818-7853. (60) (a) Boger, D. L.; Patel, M. Total synthesis of prodigiosin. Tetrahedron Lett. 1987, 28, 2499-2502. (b) Boger, D. L.; Patel, M. Total synthesis of prodigiosin, prodigiosene, and desmethoxyprodigiosin: Diels–Alder reactions of heterocyclic azadienes and development of an effective palladium(II)promoted 2,2’-bipyrrole coupling procedure. J. Org. Chem. 1988, 53, 1405-1415. (61) (a) Boger, D. L.; Patel, M. Indole N-carbonyl compounds: Preparation and coupling of indole-1-carboxylic acid anhydride. J. Org. Chem. 1987, 52, 3934-3936. (b) Boger, D.

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L.; Patel, M. Activation and coupling of pyrrole-1-carboxylic acid in the formation of pyrrole N-carbonyl compounds: Pyrrole-1-carboxylic acid anhydride. J. Org. Chem. 1987, 52, 2319-2323. (62) Boger, D. L.; Baldino, C. M. d,l- and mesoIsochrysohermidin: Total synthesis and interstrand DNA crosslinking. J. Am. Chem. Soc. 1993, 115, 11418-11425. (63) (a) Boger, D. L.; Baldino, C. M. Singlet oxygen mediated oxidative decarboxylation of pyrrole-2-carboxylic acids. J. Org. Chem. 1991, 56, 6942-6944. (b) Wasserman, H. H.; DeSimone, R. W.; Boger, D. L.; Baldino, C. M. Singlet oxygen oxidation of bipyrroles: Total synthesis of d,l- and mesoisochrysohermidin. J. Am. Chem. Soc. 1993, 115, 8457-8458. (64) Yeung, B. K. S.; Boger, D. L. Synthesis of isochrysohermidin–distamycin hybrids. J. Org. Chem. 2003, 68, 5249-5253. (65) Boger, D. L.; Hong, J. Asymmetric total synthesis of ent(–)-roseophilin: Assignment of absolute configuration. J. Am. Chem. Soc. 2001, 123, 8515-8519. (66) (a) Boger, D. L.; Mathvink, R. J. Acyl radicals: Functionalized free radicals for intramolecular cyclization reactions. J. Org. Chem. 1988, 53, 3377-3379. (b) Boger, D. L.; Mathvink, R. J. Phenyl selenoesters as effective precursors of acyl radicals for use in intermolecular alkene addition reactions. J. Org. Chem. 1989, 54, 1777-1779. (c) Boger, D. L.; Mathvink, R. J. Intramolecular acyl radical–alkene addition reactions: Macrocyclization reactions. J. Am. Chem. Soc. 1990, 112, 4008-4011. (d) Boger, D. L.; Mathvink, R. J. Tandem freeradical alkene addition reactions of acyl radicals. J. Am. Chem. Soc. 1990, 112, 4003-4008. (e) Boger, D. L.; Mathvink, R. J. Acyl radicals: Intermolecular and intramolecular alkene addition reactions. J. Org. Chem. 1992, 57, 1429-1443. (67) Harrington, P. E.; Tius, M. A. Synthesis and absolute stereochemistry of roseophilin. J. Am. Chem. Soc. 2001, 123, 8509-8514. (68) Fürstner, A. Chemistry and biology of roseophilin and the prodigiosin alkaloids: A survey of the last 2500 years. Angew. Chem. Int. Ed. 2003, 42, 3582-3603. (69) (a) Díaz de Greñu, B.; Hernández, P. I.; Espona, M.; Quiñonero, D.; Light, M. E.; Torroba, T.; Pérez-Tomás, R.; Quesada, R. Synthetic prodiginine obatoclax (GX15-070) and related analogues: Anion binding, transmembrane transport, and cytotoxicity properties. Chem. Eur. J. 2011, 17, 1407414083. (b) Seganish, J. L.; Davis, J. T. Prodigiosin is a chloride carrier that can function as an anion exchanger. Chem. Commun. 2005, 5781-5783. (70) Bracken, J. D.; Carlson, A. D.; Frederich, J. H.; Nguyen, M.; Shore, G. C.; Harran, P. G. Tailored fragments of roseophilin selectively antagonize Mcl-1 in vitro. Tetrahedron Lett. 2015, 56, 3612-3616. (71) (a) Fan, H.; Peng, J.; Hamann, M. T.; Hu, J.-F. Lamellarins and related pyrrole-derived alkaloids from marine organisms. Chem. Rev. 2008, 108, 264-287. (b) Young, I. S.; Thornton, P. D.; Thompson, A. Synthesis of natural products containing the pyrrolic ring. Nat. Prod. Rep. 2010, 27, 18011839. (72) Boger, D. L.; Boyce, C. W.; Labroli, M. A.; Sehon, C. A.; Jin, Q. Total syntheses of ningalin A, lamellarin O, lukianol A, and permethyl storniamide A utilizing heterocyclic azadiene Diels–Alder reactions. J. Am. Chem. Soc. 1999, 121, 54-62. (73) Boger, D. L.; Soenen, D. R.; Boyce, C. W.; Hedrick, M. P.; Jin, Q. Total synthesis of ningalin B utilizing a heterocyclic azadiene Diels–Alder reaction and discovery of a new class of


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heteroatom double bonds: Butenolide, furan, and γ-keto ester preparation. Tetrahedron Lett. 1984, 25, 5615-5618. (c) Boger, D. L.; Brotherton, C. E. Effective, thermal one-carbon + twocarbon cycloaddition of cyclopropenone ketals with electrondeficient olefins: Cyclopropane formation. Tetrahedron Lett. 1984, 25, 5611-5614. (d) Boger, D. L.; Wysocki, R. J. Thermal cycloaddition reactions of π-delocalized singlet vinylcarbenes: Three-carbon 1,1-/1,3-dipoles. The thermal three-carbon + twocarbon cycloaddition. J. Org. Chem. 1988, 53, 3408-3421. (201) (a) Patel, P. R.; Boger, D. L. Intramolecular [1 + 2] and [3 + 2] cycloaddition reactions of cyclopropenone ketals. J. Am. Chem. Soc. 2010, 132, 8527-8529. (b) Patel, P. R.; Boger, D. L. Intramolecular Diels–Alder reactions of cyclopropenone ketals. Org. Lett. 2010, 12, 3540-3543. (202) (a) Boger, D. L.; Brotherton, C. E. Thermal, four-carbon + three-carbon cycloaddition reaction of cyclopropenone ketals. Total synthesis of deacetamidocolchiceine: Formal total synthesis of colchicine. J. Org. Chem. 1985, 50, 3425-3427. (b) Boger, D. L.; Brotherton, C. E. Thermal reactions of cyclopropenone ketals. Application of the cycloaddition reactions of delocalized singlet vinylcarbenes: Three-carbon 1,1-/1,3-dipoles. An alternative synthesis of deacetamidocolchiceine: Formal total synthesis of colchicine. J. Am. Chem. Soc. 1986, 108, 6713-6719. (203) Graening, T.; Schmalz, H.-G. Total synthesis of colchicine in comparison: A journey through 50 years of synthetic organic chemistry. Angew. Chem. Int. Ed. 2004, 43, 3230-3256. (204) Boger, D. L.; Takahashi, K. Total synthesis of granditropone, grandirubrine, imerubrine, and isoimerubrine. J. Am. Chem. Soc. 1995, 117, 12452-12459. (205) Li, L.; Chen, Z.; Zhang, X.; Jia, Y. Divergent strategy in natural product total synthesis. Chem. Rev. 2018, 118, 37523832. (206) (a) Boger, D. L.; Brotherton, C. E.; Kelley, M. D. A simplified isoquinoline synthesis. Tetrahedron 1981, 37, 39773980. (b) Boger, D. L.; Brotherton, C. E.; Panek, J. S.; Yohannes, D. Direct introduction of nitriles via use of unstable Reissert intermediates: Convenient procedures for the preparation of 2-cyanoquinolines and 1-cyanoisoquinolines. J. Org. Chem. 1984, 49, 4056-4058. (207) Boger, D. L.; Zhu, Y. Diels–Alder reactions of cyclopropenone ketals: A concise tropolone annulation applicable to rubrolone C ring introduction. J. Org. Chem. 1994, 59, 3453-3458. (208) (a) Schuep, W.; Blount, J. F.; Williams, T. H.; Stempel, A. Production of a novel red pigment, rubrolone, by Streptomyces echinoruber sp. nov. II. Chemistry and structure elucidation. J. Antibiot. 1978, 31, 1226-1232. (b) Palleroni, N. J.; Reichelt, K. E.; Mueller, D.; Epps, R.; Tabenkin, B.; Bull, D. N.; Schuep, W.; Berger, J. Production of a novel red pigment, rubrolone, by Streptomyces echinoruber sp. nov. I. Taxonomy, fermentation and partial purification. J. Antibiot. 1978, 31, 1218-1225. (209) (a) Kelly, T. R.; Echavarren, A.; Whiting, A.; Weibel, F. R.; Miki, Y. Synthesis of the chromophore of rubrolone. Tetrahedron Lett. 1986, 27, 6049-6050. (b) Kelly, T. R.; Liu, H. A new pyridine synthesis. J. Am. Chem. Soc. 1985, 107, 4998-4999.


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