Total synthesis of viridicatumtoxin B and analogues thereof: Strategy evolution, structural revision, and biological evaluation

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Total Synthesis of Viridicatumtoxin B and Analogues Thereof:

Strategy Evolution, Structural Revision, and Biological Evaluation

K. C. Nicolaou,*^,†,‡,#Christopher R. H. Hale,^{†,‡,⊥,#}Christian Nilewski,^{†,‡,⊥,#}Heraklidia A. Ioannidou,^†,‡,#

Abdelatif ElMarrouni,^†,‡,#Lizanne G. Nilewski,^†,‡,#Kathryn Beabout,^§Tim T. Wang,^§

and Yousif Shamoo^§,∥

§

∥

^†Department of Chemistry, Department of Biochemistry and Cell Biology, and Department of Ecology and Evolutionary Biology,

Rice University, 6100 Main Street, Houston, Texas 77005, United States

^‡Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States

S

* Supporting Information

ABSTRACT: The details of the total synthesis of viridicatumtoxin B

(1) are described. Initial synthetic strategies toward this intriguing

tetracycline antibiotic resulted in the development of key alkylation

and Lewis acid-mediated spirocyclization reactions to form the

hindered EF spirojunction, as well as Michael−Dieckmann reactions

to set the A and C rings. The use of an aromatic A-ring substrate,

however, was found to be unsuitable for the introduction of the

requisite hydroxyl groups at carbons 4a and 12a. Applying these

previous tactics, we developed stepwise approaches to oxidize

carbons 12a and 4a based on enol- and enolate-based oxidations,

respectively, the latter of which was accomplished after systematic

investigations that revealed critical reactivity patterns. The herein

described synthetic strategy resulted in the total synthesis of viridicatumtoxin B (1), which, in turn, formed the basis for the

revision of its originally assigned structure. The developed chemistry facilitated the synthesis of a series of viridicatumtoxin

analogues, which were evaluated against Gram-positive and Gram-negative bacterial strains, including drug-resistant pathogens,

revealing the ﬁrst structure−activity relationships within this structural type.

rather than bacterial, origins. The subject of this article is the

pursuit of viridicatumtoxin B (1) by total synthesis, its full

structural elucidation, and investigation of its antibacterial

properties as well as those of selected synthetic analogues. The

following brief historical overview places the present work and

its aims in perspective within the ﬁeld of tetracycline antibiotics.

The discovery of chlortetracycline (4, Chart 2a), the ﬁrst

tetracycline antibiotic, by B. M. Duggar of the American

Cyanamid Corporation in the late 1940s ushered in a new

subclass of antibacterial agents at the dawn of the golden era of

antibiotics.⁴The widespread success of tetracyclines in curing

previously high-mortality-rate diseases bestowed on them the

status of “wonder drug” shortly after their introduction into the

clinic.⁵Since the discovery of chlortetracycline and other ﬁrst-

generation tetracyclines [e.g., oxytetracycline (5) and tetracy-

cline (6), Chart 2a], second-generation tetracyclines, including

minocycline (7) and doxycycline (8) (Chart 2a), emerged with

improved properties. More recently, third-generation tetracy-

clines such as tigecycline (9)⁶and eravacycline (TP-434, 10)⁷

(Chart 2a) that overcome certain bacterial resistance

mechanisms have been introduced.⁸

INTRODUCTION

■

Within the class of tetracycline antibiotics, viridicatumtoxin B

(1),¹viridicatumtoxin A (2),²and spirohexaline (3)³(Chart 1)

are unique in that they include in their structures a geranyl-

derived subunit in the form of a spirobicyclic system (ring

system EF). In contrast to the majority of tetracyclines, these

members of the group are also distinguished by their fungal,

Chart 1. Molecular Structures of Viridicatumtoxins 1−3

Received: June 27, 2014

A

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Chart 2. Molecular Structures of (a) Bacterial Tetracyclines

and Designed Analogues and (b) Fungal Tetracyclines

Scheme 1. Proposed Biosynthesis of the Terpene-Derived

Spirocyclic Region of Viridicatumtoxin A (2)

In 2008, Kim et al.¹reported the isolation of viridicatumtoxin

B in small quantities along with viridicatumtoxin A (2) from

Penicillium sp. FR11 and, on the basis of NMR spectroscopic

analysis, assigned the hydroxy-epoxide structure 1′ (Chart 1) to

the former. These investigators observed potent activities for

viridicatumtoxins A and B against Gram-positive bacteria,

including methicillin-resistant Staphylococcus aureus (MRSA)

(MIC = 0.25 and 0.5 μg/mL, respectively). Interestingly, a

recent report suggested that the viridicatumtoxins exert their

antibacterial properties through inhibition of UPP synthase, an

important enzyme for bacterial peptidoglycan biosynthesis.^3,27

This stands in contrast to the mode of action of other

tetracyclines (e.g., 4−10, Chart 2a), which inhibit bacterial

protein synthesis by binding to the 30S subunit of the

ribosome.

Most naturally occurring tetracyclines are produced by

bacterial strains, although a few have been isolated from

fungi. Thus, in addition to those shown in Chart 1 (1−3),

hypomycetin (11),⁹anthrotainin (TAN-1652, 12),¹⁰TAN-

1612 (13),¹¹and BMS-192548 (14)¹²(Chart 2b) are fungal

metabolites.

Due to their complex structures and important biological

activities, tetracyclines have been the subject of numerous

synthetic campaigns since the 1950s. Noteworthy achievements

in tetracycline synthesis include those recorded by Woodward/

Pﬁzer,¹³Shemyakin,¹⁴Muxfeldt,¹⁵Barton,¹⁶Wasserman/

Scott,¹⁷Stork,¹⁸Tatsuta,¹⁹and, more recently, Myers²⁰and

Evans.²¹

First isolated in 1973 from a Penicillium strain in South

Africa, viridicatumtoxin A (2) yielded to X-ray crystallographic

analysis in 1976.^2,22The biosynthesis of this antibiotic was

studied by the groups of Vleggaar²³and, more recently, Tang

and co-workers,²⁴the latter of whom proposed a complete

biosynthetic pathway. Speciﬁcally, as shown in Scheme 1, it was

suggested that the EF-spirosystem is formed from polyketide

15 and a unit of geranyl pyrophosphate (16) as facilitated by

VrtC, a polyketide prenyltransferase.²⁵This reaction is followed

by oxidative cyclization catalyzed by another Vrt enzyme (VrtK,

a cytochrome P450-type enzyme)²⁶to aﬀord viridicatumtoxin

A (2) through transient intermediates 19−21 (on the basis of

computational studies), as shown in Scheme 1.

In view of the scarcity of viridicatumtoxin B, its interesting

but suspect structural assignment (i.e., 1′, Chart 1), and its

important biological activity, we initiated a program directed

toward its total synthesis. Herein, we describe details of our

investigations that led to important new knowledge and insights

with regard to this intriguing bioactive molecule.²⁸

RESULTS AND DISCUSSION

■

First-Generation Approach: Development of an

Anthrone Alkylation and Lewis Acid-Mediated Spirocyc-

lization. The intrigue surrounding the originally assigned

structure of viridicatumtoxin B (1′) is derived from its

uncommon epoxy-hemiacetal structural motif, its lipophilic

spirocyclic domain, and its high oxygenation, particularly on

ring B. Although the ﬁrst and rather curious structural feature

may have an explanation in this instance originating from the

surrounding functionalities of the molecule, from the synthetic

point of view, it was reasoned that it could be resolved by

B

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targeting either structure 1′ or 1 (Chart 1) and allowing their

potential thermodynamic equilibration to provide an answer.

The spirocycle provided a synthetic challenge that we decided

to face following the proposed biosynthesis, which inspired a

Friedel−Crafts-type approach (see Scheme 1). The remaining

major challenge of a synthesis of viridicatumtoxin B was initially

relegated to a rather speculative singlet oxygen [4 + 2]

cycloaddition to an appropriately substituted aromatic ring (B).

Finally, the construction of the enol−amide structural motif

characteristic of most tetracyclines was to be derived from an

isoxazole ring through hydrogenolysis, a well-tested and reliable

tactic introduced by Stork and Hagedorn.^18a

Scheme 3. Precedent for Arene−Endoperoxide

Rearrangements Based on (a) Thermal-, (b) Redox-, and (c)

Photo-Induced O−O Bond Cleavage

These considerations led to our ﬁrst retrosynthetic analysis of

viridicatumtoxin B as depicted in Scheme 2. Thus, it was

Scheme 2. First-Generation Retrosynthetic Analysis through

Endoperoxide 23

endoperoxide 34 by an iron(II) species, leading to bis-epoxide

38, as shown in Scheme 3b.^29kThis rearrangement is presumed

to proceed through the sequential one-electron transfers

involving transient species 35−37.^30,31Photochemically-

induced homolytic rupture of endoperoxides is also possible

as demonstrated in Scheme 3c.^29cSpeciﬁcally, endoperoxide 40,

generated from naphthalene derivative 39, formed bis-epoxide

42, presumably via diradical 41. Further elaboration of 42 led

ﬁrst to syn diol 43 and thence dihydroxy quinone 44,

demonstrating the accessibility and versatility of the endoper-

oxide moiety.³²

The synthesis of allylic chloride fragment 27 began with

geranic acid (45) and followed a modiﬁed sequence based on a

literature process,³³as shown in Scheme 4. Thus, acid-mediated

cationic cyclization (H₃PO₄) of 45 followed by methylation

(MeI, K₂CO₃) of the resulting acid produced methyl ester 46.

Epoxidation of the latter with m-CPBA furnished a mixture of

epoxide isomers which was treated with NaOMe to give the

desired allylic alcohol methyl ester 47 (70% for four steps).

Protection of the hydroxyl group of 47 (TBSCl, imid.) and

subsequent reduction (DIBAL-H) then led to allylic alcohol 48

reasoned that 1″ or 1 could be derived from bis-epoxide

structure 22 (R = selectively cleavable protecting group), which

could be traced to endoperoxide 23 through a radical-based

rearrangement and thence to aromatic system 24 via selective

oxygen addition to the electron-rich B-ring. This scenario was

inspired by a number of previous relevant studies, highlights of

which are shown in Scheme 3.²⁹Thus, endoperoxide 29

(generated from singlet oxygen and tetracene) was reported to

undergo a thermally-induced rearrangement to bis-epoxide 31,

presumably via diradical 30, and the latter was trapped with N-

methylmaleimide (32) to aﬀord adduct 33 (Scheme 3a).^29e

The regioselectivity of the latter reaction is of note in that it

reﬂects diﬀerent reactivities for the benzene and naphthalene

systems. A similar type of fragmentation was induced within

C

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Scheme 4. Synthesis of Allylic Chloride 27

Scheme 5. Synthesis of Spirocycle 25 and Conformational

Rationale for Its Stereoselective Formation

a

Reagents and conditions: (a) H₃PO₄(0.2 equiv), toluene, reﬂux, 90

min; (b) MeI (3.9 equiv), K₂CO₃(2.0 equiv), acetone, 25 °C, 15 h;

(c) m-CPBA (1.2 equiv), CH₂Cl₂, 0 → 25 °C, 3 h; (d) NaOMe (1.5

equiv), MeOH, reﬂux, 17 h, 70% for four steps; (e) TBSCl (1.6 equiv),

imidazole (2.0 equiv), CH₂Cl₂, 25 °C, 12 h; (f) DIBAL-H (2.7 equiv),

CH₂Cl₂, −78 → 0 °C, 70 min, 91% for two steps; (g) MsCl (1.2

equiv), Et₃N (1.5 equiv), DMAP (0.05 equiv), CH₂Cl₂, 25 °C, 14 h;

then LiCl (1.0 equiv), 42 h, 48%. m-CPBA = meta-chloroperox-

ybenzoic acid, TBS = tert-butyldimethylsilyl, DIBAL-H = diisobuty-

laluminum hydride, Ms = methanesulfonyl, DMAP = 4-dimethylami-

nopyridine.

(91% for two steps). Attempted mesylation of 48 with MsCl/

Et₃N led to partial formation of allylic chloride 27, presumably

via the initially formed mesylate 49. It was then found that

treatment of 48 with MsCl/Et₃N followed by addition of LiCl

produced the allylic chloride 27 in satisfactory yield (48%).

With allylic chloride 27 readily available, we next turned our

attention to the construction of anthrone 28 (Scheme 5).

Bromo-benzyl juglone 51 was prepared in 66% overall yield

following a two-step literature procedure starting from juglone

(50).³⁴Known Brassard diene 53 was prepared in one step

starting from methyl 3-methoxybut-2-enoate (52) via formation

of the corresponding enolate and subsequent trapping with

TMSCl (99%).³⁵With these building blocks in hand, their

fusion through a Diels−Alder reaction was performed by

mixing the dienophile 51 with 3 equiv of diene 53 at −30 °C

and allowing the reaction mixture to reach ambient temper-

ature. The TMS ether of the initially formed Diels−Alder

adduct 54 was cleaved with silica gel, which caused collapse of

the ketal and spontaneous elimination of HBr (see 55, Scheme

5) followed by tautomerization, ultimately producing anthra-

quinone 56 in 90% overall yield. Similar types of cascade

Diels−Alder/elimination sequences have been performed

previously.³⁶Methylation of the free phenolic group of 56

(MeI, K₂CO₃) followed by regioselective deoxygenation

(SnCl₂, HCl/AcOH) and concomitant debenzylation then

furnished anthrone 28 (86% yield for two steps).³⁷

Previous reports have detailed the successful alkylation of the

methylene position of anthrones with simple alkyl halides.³⁸

These studies also provided precedent for alkylation at the

methylene position in preference to the free phenolic

position(s), the latter being deactivated by hydrogen bonding

with the adjacent carbonyl group. Nevertheless, the alkylation

of an anthrone with a more complex electrophile was

unprecedented and had not been utilized previously in the

context of tetracycline synthesis. It was, therefore, pleasing to

ﬁnd that union of anthrone 28 with chloride 27 was successful

when performed at 50 °C, employing Na₂CO₃as the base in

the presence of catalytic quantities of KI, providing the desired

a

Reagents and conditions: (a) Br₂(1.03 equiv), AcOH, 25 °C, 30 min;

then EtOH, reﬂux, 15 min; (b) BnBr (2.1 equiv), Ag₂O (2.1 equiv),

CH₂Cl₂, 25 °C, 18 h, 66% for two steps; (c) LDA (1.05 equiv), THF,

−78 °C, 1 h; then TMSCl (1.2 equiv), −78 → 25 °C, 100 min, 99%;

(d) 51 (1.0 equiv), 53 (3.0 equiv), CH₂Cl₂, −30 → 25 °C, 70 min;

then SiO₂(excess), 1 h, 90%; (e) MeI (5.0 equiv), K₂CO₃(5.0 equiv),

DMF, 65 °C, 14 h; (f) SnCl₂(7.0 equiv), AcOH:HCl 10:1, 50 °C,

86% for two steps; (g) 27 (1.0 equiv), KI (0.1 equiv), Na₂CO₃(2.5

equiv), acetone, 50 °C, 18 h, 51%, ca. 1:1 dr; (h) ZnI₂(0.78 equiv),

CH₂Cl₂, reﬂux, 25 h, 34%. Bn = benzyl, THF = tetrahydrofuran, LDA

= lithium diisopropylamide, TMS = trimethylsilyl.

product 58 in 51% yield (ca. 1:1 dr). It is worth noting that the

success of this reaction depended on degassing the solution and

conducting the reaction in the absence of light³⁹to prevent

oxidative radical dimerization of the anthrone substrate.⁴⁰

Initial attempts to form the EF spirosystem were based on

precedents that typically employed unprotected allylic alco-

hols⁴¹or allylic acetates⁴²as the cationic precursors. However,

most attempts to remove the TBS group of intermediate 58

D

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a

were complicated with side-products, including elimination or

allylic substitution products (e.g., allylic methyl ether with

HCl/MeOH). These observations led us to hypothesize that it

might be possible to directly ionize the TBS ether of 58,

thereby eliminating the need for the deprotection step. After a

brief screen of acidic conditions, it was found that ZnI₂indeed

promoted the desired transformation in 34% yield. As shown in

Scheme 5, portionwise treatment of allylic TBS ether 58 in

reﬂuxing CH₂Cl₂with ZnI₂produced spirocyclic compound 25

as a single detectable diastereomer. This reaction presumably

proceeds via ionization of the TBS ether to generate allylic

cation 59a/b. Then, intramolecular Friedel−Crafts-type re-

action with the more electron-rich arene (ring D) followed by

loss of a proton produces the observed product 25. We did not

isolate any product resulting from Friedel−Crafts reaction on

the B-ring, although some elimination products were observed.

While the yield of this transformation was, for the time, rather

modest, it was suﬃcient to allow us to probe the following

steps. The stereochemical conﬁguration of 25 was assigned by

comparison with subsequent intermediates whose structures

were unambiguously deduced from X-ray crystallographic

studies (see below). The observed diastereoselectivity of the

spirocyclization (58 → 25) can be rationalized by invoking the

transition state resulting from conformer 59a as having the

lowest energy barrier, as shown in Scheme 5. The transition

state resulting from alternative conformer 59b, required to

aﬀord the other (15-epi-25, undesired) diastereomer, suﬀers

from unfavorable interactions between one of the methyl

groups and the arene, as indicated in structure 59b. These

interactions are apparently not present in the transition state

leading to the observed product (59a → 25).

Scheme 6. Attempted Phenolic Oxidation of 25 and 63

a

Reagents and conditions: (a) PhI(OAc)₂(3.0 equiv), MeOH, 25 °C,

43 h, 37% for 62 + 26% recovered 25; (b) K₂CO₃(2.4 equiv), MeOH,

25 °C, 10 min, 54%; (c) PhI(OAc)₂(2.5 equiv), CH₂Cl₂:H₂O 7:1, 0

→ 25 °C, 55 min, 4% for 64, 13% for 62, 45% recovered 63. PIDA =

phenyliodonium diacetate.

As shown in Scheme 6, we next needed to perform a

phenolic oxidation to render the B-ring of the growing

molecule electrophilic, and thereby susceptible, to nucleophilic

attack from a negatively charged A-ring isoxazole (i.e., anion of

26, see Scheme 2). The prototypical sequence of events for

achieving this objective would involve PIDA [PhI(OAc)₂]- or

PIFA [PhI(TFA)₂]-mediated oxidation of the phenolic moiety

of 25 in MeOH to give intermediate 60, which may undergo a

second oxidation to yield the desired quinone monoketal 61.⁴³

In reality, however, 25 proved intransigent to these projected

transformations, leaving quinone monoketal 61 and its relatives,

60 and 64, elusive under a variety of conditions tested. These

included hypervalent iodine-, molecular oxygen-, and metal-

based oxidative conditions (see Supporting Information for

details). Instead, benzylic oxidation leading to quinomethide 62

was observed in several cases, as well as decomposition under

diﬀerent conditions. In a few cases, undesired products

involving oxidation of the F-ring were also observed.

Eventually, it was found that spirocycle 25 could be

tautomerized to its desmotropic form 63 under basic conditions

(i.e., K₂CO₃, Scheme 6).⁴⁴When the latter compound was

treated with PIDA in CH₂Cl₂/H₂O the desired quinone 64

could be isolated in only 4% yield, along with substantial

quantities of benzylic oxidation product 62 (13%) and

recovered starting material (45%). A number of diﬀerent

oxidants (i.e., Fremy’s salt, O₂/salcomine catalyst, PIFA) were

examined in eﬀorts to improve the eﬃciency of the trans-

formation of 63 to quinone 64, but unfortunately to no avail.

Unable to access quinone 64 in suﬃcient quantities, it was

decided to focus our attention on an approach that would

directly bring in the A-ring from an earlier stage, which would

circumvent the problematic B-ring phenolic oxidation barriers

we faced. Despite its failure to break through these barriers,

however, the initial approach provided us with useful

information and methods for the construction of the anthrone

and allylic halide substrates and established the required

spirocyclization reaction.

Second-Generation Approach: Attempts To Install

the C4a and C12a Hydroxyl Groups via Dearomatization

Pathways. The second-generation retrosynthesis of substrate

24, required for our endoperoxide rearrangement approach

(see Scheme 2), is shown in Scheme 7. Thus, to avoid the

oxidation problems discussed above, the subtarget molecule 24

was dissected through the C-ring, which retrosynthetically

deﬁned three fragments: allylic chloride 27, known homo-

phthalic anhydride 66,⁴⁵and AB-enone 65. The latter could be

further disassembled into known quinone monoketal 67⁴⁶and

Stork−Hagedorn isoxazole 26.^18a

The synthesis of requisite AB-enone 65 commenced from

the known Stork−Hagedorn isoxazole 26, which was prepared

using a modiﬁcation of the literature procedure (see Scheme 8).

Thus, the process for the synthesis of 26 involved acylation of

dimethyl malonate (68) (MgCl₂, Et₃N, AcCl, 96%),⁴⁷

methylation of the resulting enol (69) with Me₂SO₄(54%),

cyclization of 70 with H₂NOH to aﬀord compound 71 (48%),

and benzylation of the resulting hydroxy-isoxazole (BnBr,

Ag₂O, 67%). The best conditions we found for the benzylation

reaction were those employing Ag₂O which provided the O-Bn

in preference to the N-Bn product in ratios of up to 11:1.

Alternative conditions gave unacceptably low selectivity ratios

(Cs₂CO₃/BnBr: O:N ca. 1.25:1; TfOH/BnTCA: O:N ca. 1:7.5)

in agreement with Kornblum’s rule for ambident nucleo-

philes.⁴⁸A procedure using phenyldiazomethane as the

electrophile as used by Stork and co-workers^18awas considered

E

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Scheme 7. Second-Generation Retrosynthetic Analysis of

Viridicatumtoxin B (1 or its isomer 1″) through

Endoperoxide Precursor 24

Scheme 9. First Synthesis of AB-Quinone Monoketal 65

a

Reagents and conditions: (a) LiHMDS (2.2 equiv), THF −78 °C, 30

min; then MeOCOCl (1.0 equiv), −78 °C, 45 min, 96%; (b) 67 (1.0

equiv), NaOMe (1.0 equiv), MeOH, 25 °C, 18 h, 71%, ca. 3:1 dr; (c)

NaH (3.9 equiv), toluene, 0 → 90 °C, 2.5 h; (d) Me₃SnOH (9.0

equiv), ClCH₂CH₂Cl, 80 °C, 21 h, 39% for two steps; (e) PhSeCl (1.9

equiv), py (2.1 equiv), CH₂Cl₂, 0 °C, 1.5 h; (f) H₂O₂(excess),

CH₂Cl₂, 0 °C, 54% for two steps; (g) BnBr (3.0 equiv), K₂CO₃(4.0

equiv), acetone, 25 → 60 °C, 6.5 h, 94%. py = pyridine.

Scheme 8. Synthesis and 1,2-Addition of Stork−Hagedorn

a

Isoxazole 26 to Quinone Monoketal 67

monoketal 67 was then achieved under basic conditions

(NaOMe) to give adduct 74 in 71% yield as an inconsequential

ca. 3:1 mixture of diastereomers. Dieckmann condensation

within 74 induced by NaH then led to tricycle 75 as a single

diastereomer (³J_4,4a= 11.2 Hz), presumably through epimeriza-

tion at C4 during the reaction. Demethylation and subsequent

decarboxylation of 75 was then carried out with Me₃SnOH⁴⁹at

elevated temperatures, providing 76 in 39% yield for the two

steps. Of note here is the observation that the use of LiOH

proved unsuccessful in promoting this transformation. Sub-

sequent oxidation of the latter substrate (i.e., 76) by way of

selenide formation/oxidation/syn-elimination furnished com-

pound 79 (54% for the two steps), through intermediate 77

and transient species 78. Finally, benzylation of the free

phenolic moiety within 79 (BnBr, K₂CO₃, 94%) aﬀorded the

desired AB-ring system fragment 65 (mp = 103 °C, Et₂O). The

structure of this key intermediate was unambiguously

conﬁrmed by X-ray crystallographic analysis (see ORTEP

representation, Figure 1).

a

Reagents and conditions: (a) MgCl₂(1.0 equiv), Et₃N (2.0 equiv),

AcCl (1.0 equiv), MeCN, 0 → 25 °C, 23 h, 96%; (b) Me₂SO₄(1.3

equiv), K₂CO₃(1.3 equiv), DMF, 0 → 25 °C, 17 h, 54%; (c) H₂NOH·

HCl (1.4 equiv), NaOMe (3.1 equiv), MeOH, 0 → 25 °C, 24 h, 48%;

(d) BnBr (1.2 equiv), Ag₂O (1.5 equiv), DMF, 25 °C, 18 h, 67%; (e)

LiHMDS (1.4 equiv), THF, −78 °C, 40 min; then 67 (1.0 equiv), −78

→ −30 °C, 45 min, 62%. Ac = acetyl, DMF = dimethylformamide,

HMDS = hexamethyldisilazide.

While the above route provided suﬃcient quantities of

fragment 65 for preliminary studies, scaling up the synthetic

sequence, especially the ester saponiﬁcation with Me₃SnOH,

proved inconvenient. To circumvent this problem, we elected

to change the C4-ester group to a more labile one. Alternative

moieties considered were the ethoxyethyl ester, originally

employed by Stork,^18aand the cyclic anhydride 80 (see Scheme

10), which was envisioned to undergo a one-pot Michael−

Dieckmann/decarboxylation cascade sequence. Eventually,

however, we selected a Teoc ester due to its anticipated

orthogonality to the rest of the functionalities within the

molecule. Thus, and as shown in Scheme 10, installation of the

Teoc ester at C4 of isoxazole 26 was accomplished using a

similar protocol as for the methyl ester (LiHMDS; then

TeocCl, 88%) to aﬀord derivative 81. Michael reaction of the

undesirable due to safety concerns. With isoxazole 26 readily

available, we next attempted its conjugate addition to quinone

monoketal 67, as shown in Scheme 8. Thus, deprotonation of

the methyl group of 26 with LiHMDS, followed by addition of

quinone monoketal 67 to the resulting anion (26′), however,

led to 1,2-addition product 72, rather than the desired 1,4-

product.

Faced with this predicament, we decided to install an

electron-withdrawing group at the isoxazole methyl group,

which we hoped would make the resulting nucleophile softer

due to its vinylogous diketone nature.^18aToward that end, and

as shown in Scheme 9, treatment of isoxazole 26 with LiHMDS

followed by quenching with methyl chloroformate provided 73

(96% yield). Michael addition of the latter to quinone

F

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Scheme 11. Preparation of Cyclic Anhydride Fragment 66

a

Reagents and conditions: (a) MeI (4.0 equiv), K₂CO₃(8.0 equiv),

acetone, reﬂux, 15 h, 91%; (b) NaH (6.0 equiv), diethyl malonate (4.0

equiv), THF, 0 °C, 2.5 h; then LDA (1.0 equiv), THF, 0 °C, 3.5 h,

65%; (c) MeOH:2 M NaOH 1:1, reﬂux, 12 h, 97%; (d) Ac₂O (4.2

equiv), toluene, reﬂux, 2 h, 99%. DEM = diethyl malonate.

Figure 1. ORTEP representation of AB-quinone monoketal 65.

Thermal ellipsoids at 30% probability. Gray = carbon, red = oxygen,

blue = nitrogen, green = hydrogen.

Scheme 12. Synthesis of Intermediate 88 through a

Michael−Dieckmann/Decarboxylation Cascade Sequence

a

and Spirocyclization of Hexacycle 90 to Heptacycle 24

Scheme 10. Improved Synthesis of AB-Quinone Monoketal

a

65 through the Use of a Teoc Ester

a

Reagents and conditions: (a) LiHMDS (2.2 equiv), THF, −78 °C, 30

min; then TeocCl (1.1 equiv), −78 °C, 1 h, 88%; (b) 67 (1.0 equiv), t-

BuOK (0.2 equiv), toluene, 25 °C, 4 h, 71%, ca. 2.4:1 dr; (c) TBAF

(1.2 equiv), THF, 25 °C, 45 min, 85%; (d) NaH (4.0 equiv), toluene,

0 → 110 °C, 4 h, 80%. TBAF = tetra-n-butylammonium ﬂuoride.

latter with 67 (t-BuOK) then gave adduct 82 (71% yield, ca.

2.4:1 dr, inconsequential). At this point, it was gratifying to

observe that the Teoc group could be removed in a

straightforward fashion using TBAF at 25 °C to furnish

decarboxylated product 83 (85% yield). Dieckmann cyclization

within 83, again induced by NaH, provided intermediate 76

(80% yield) which intersected the previously described route to

65 (see Scheme 9).

With AB-enone 65 in hand, our attention turned toward the

synthesis of the desired ABCD-ring fragment 88 (Scheme 12)

through union of the former with a suitable CD-ring precursor.

Due to the observed 1,2-addition of isoxazole anion 26′ to

quinone monoketal 67 (see Scheme 8), we anticipated the need

for a soft CD-ring nucleophile. Toward this end, we selected

the known homophthalic anhydride 66 [readily prepared in

four steps from 4-chlororesorcinol (84) through intermediate

85 as shown in Scheme 11].⁴⁵Anhydride 66 has traditionally

been used (after deprotonation with a strong base, typically

NaH or LDA) in Tamura-type Diels−Alder reactions with

quinones.⁵⁰However, we found that substrate 66 could be

united with AB-enone 65 using DBU as the base at 60 °C to

aﬀord pentacyclic system 88 (82% yield, see Scheme 12). A ¹H

NMR-tube experiment to monitor the reaction between 65 and

a

Reagents and conditions: (a) 65 (1.0 equiv), DBU (3.0 equiv); then

66 (1.3 equiv), MeCN, 25 → 60 °C, 18 h, 82%; (b) TFA (4.9 equiv),

CH₂Cl₂, 25 °C, 20 min, quant; (c) Et₃N (2.0 equiv), MsCl (1.7 equiv),

CH₂Cl₂, −50 °C, 1 h; then LiBr (3.5 equiv), THF, −50 → −20 °C, 1

h, quant; (d) 91 (1.5 equiv), Na₂CO₃(9.1 equiv), DMF, 25 °C, 45

min, 78%, ca. 6:1 dr; (e) BF₃·OEt₂(0.6 equiv), CH₂Cl₂, 0 °C, 1 h,

55%. DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, TFA = triﬂuoroacetic

acid.

66 suggested that it proceeds via a distinct stepwise mechanism

(intermediates 86 and 87 were tentatively observed; see

selected peaks, Figure 2), as opposed to a concerted [4 + 2]

cycloaddition followed by decarboxylation. Additionally, this

experiment indicated that the reaction required approximately

20 h for completion. Elimination of methanol from cyclization

product 88, induced by exposure to TFA in CH₂Cl₂, then led to

elaborated anthrone 89 in quantitative yield.

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BF₃·OEt₂, a catalyst that held promise to also induce the

pending spirocyclization at lower temperatures. Indeed,

exposure of 90 to substoichiometric amounts of this Lewis

acid at 0 °C led to the formation of the desired heptacycle 24 in

55% yield with minimum amounts of side-products. The

conﬁguration of 24 was conﬁrmed as shown in Scheme 12 by

NOESY experiments.

Scheme 13 depicts our attempts to realize the singlet oxygen

cascade sequence from 93 (derived from 24 via benzoate

Scheme 13. Preparation of Endoperoxide Precursor 93 and

a

Expected Pathway to Oxygenated Species 97

Figure 2. Monitoring the coupling of cyclic anhydride 66 and AB-

quinone monoketal 65 by ¹H NMR spectroscopy (MeCN-d₃, 500

MHz): (a) H NMR spectra of starting materials 65 and 66; (b) H

NMR spectra during reaction (0, 5.5, and 20 h); (c) ¹H NMR

spectrum of product 88 after puriﬁcation.

a

Reagents and conditions: (a) BzCl (15 equiv), Et₃N (25 equiv),

1

DMAP (0.06 equiv), CH₂Cl₂, 25 °C, 45 min, 79%; (b) Pd black (5.0

equiv), 1,4-dioxane:MeOH 1:1, H₂, 25 °C, 20 min, quant; (c) O₂, TPP

(cat.), CH₂Cl₂, sun lamp, −78 °C. Bz = benzoyl, TPP =

tetraphenylporphyrin.

Attempts to alkylate anthrone 89, however, with chloride 27

(Scheme 12) under the conditions employed for the alkylation

of the simpler anthrone 28 (Scheme 5) were thwarted by the

insolubility of 89 in acetone and the low reactivity of 27,

necessitating a search for a new protocol to achieve the desired

union of the two partners. Thus, with further experimentation,

it was found that upon using the corresponding allylic bromide

91 (prepared from allylic alcohol 48 by treatment with MsCl,

Et₃N, and LiBr in quantitative yield, Scheme 12) and changing

the solvent from acetone to DMF, the required alkylation

proceeded smoothly at ambient temperature and within 1 h to

provide the desired coupling product 90 in 78% yield.

With intermediate 90 available, and in an eﬀort to prepare

subtarget 24 (Scheme 12), we then proceeded to apply our

original spirocyclization conditions (i.e., ZnI₂, CH₂Cl₂, reﬂux,

see Scheme 5), but unfortunately encountered side-products,

presumed to arise from elimination,⁵¹demethylation,⁵²and/or

benzylic oxidation reactions. We reasoned that such side

reactions could be suppressed by using a nonmobile counterion

on the activating agent, a hypothesis that prompted us to test

derivative 92) to 97 through postulated intermediates 94−96.

Thus, heptacyclic phenol 24 was converted to its benzoate 92

(BzCl, Et₃N, 79% yield) and thence to hexacyclic system 93

through hydrogenolysis (Pd black, H₂, quantitative yield) of the

N−O bond and the two benzyl ether moieties. Our reasoning

for the protection of the phenolic group of 24 was based on the

assumption that this change would promote the aromatization

of ring C within 92 by removing the possibility for a H-bond to

the C-ring carbonyl group. This assumption, however, proved

erroneous, as both 92 and 93 were found to exist exclusively in

their C-ring keto forms as opposed to their enol (phenolic)

forms. In retrospect, this outcome is not surprising in light of

previous reports, which indicated that conjugation of a

naphthalene ring (as in 92 and 93) with a ketone provides

thermodynamic stability greater than that of its phenolic

counterpart (as in the phenolic tautomers of 92 and 93).⁵³

Anticipating the singlet oxygen-initiated cascade sequence of

reactions shown in Scheme 13 (93 → 94 → 95 → 96 → 97)

based on precedented chemistry²⁹(see Scheme 3), we

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proceeded to react substrate 93 with singlet oxygen. This

transformation, however, could not be realized. Thus, bubbling

of O₂through a solution of substrate 93 in CH₂Cl₂in the

presence of catalytic quantities of TPP and with sun lamp

irradiation at −78 °C rapidly produced a product that proved,

upon warming, too labile for characterization. Prolonged

irradiation at −78 °C produced no detectable amounts of bis-

epoxide 97. We then attempted to tautomerize the C-ring of 93

to its corresponding phenolic form under basic conditions with

the hope that this maneuver (see above, Scheme 6) might lead

to a favorable result in the singlet oxygen reaction. However,

the previous conditions used for the corresponding keto−

phenol tautomerization (25 → 63, K₂CO₃, MeOH, Scheme 6)

failed to yield the desired product. Other eﬀorts to achieve the

desired outcome included attempts to form and rearrange the

endoperoxide using diﬀerent sensitizers on substrates with and

without the isoxazole ring, the latter scenario being inspired by

Myers’s hypothesis that the isoxazole moiety may act as a self-

sensitizer.^20aThese experiments failed to produce any of the

proposed intermediates in Scheme 13 (i.e., 94−97).

substrate decomposed, especially for the more electron-rich

substrate 98. Furthermore, phenol 101 was synthesized as a

possible substrate for C12a hydroxylation through a sequence

involving PIFA oxidation to the quinone, benzoyl ester

formation (BzCl, 65% for two steps), and regioselective

debenzylation (MgBr₂·OEt₂, 75%). Phenolic oxidation of this

substrate (i.e., 101), however, also failed to produce the desired

C12a oxidation product 102. The challenges faced here with

respect to installation of the C12a hydroxyl group are

reminiscent of previous synthetic eﬀorts toward tetracyclines,

including those of Woodward,¹³Muxfeldt,¹⁵Stork,¹⁸and, in

particular, Barton.⁵⁴

The failure to install the obligatory hydroxyl groups at C4a

and C12a through dearomatization pathways refocused our

attention on alternative tactics, the choice of which was based

on the intelligence gathered thus far. This information pointed

away from an aromatic A-ring as a precursor to the desired

oxygenation.

Third-Generation Approach: Stepwise Strategies To

Install the C4a and C12a Hydroxyl Groups. Our ﬁrst

attempts to install the coveted hydroxyl groups onto the

substrate via enol or enolate chemistry focused on the rather

simple AB-ring fragment 76 (Scheme 15). As shown in this

On the basis of these ﬁndings, we were forced to explore

alternative phenolic oxidation protocols in an eﬀort to install

oxidation at C12a (Scheme 14). Attempted phenolic oxidations

of substrate 24, or its corresponding hydrogenolyzed counter-

part 98, were unsuccessful. A number of other conditions,

including hypervalent iodine reagents, Fremy’s salt, Pb(OAc)₄,

and various metals with molecular oxygen, were also

unsuccessful. Typically either no reaction was observed or the

Scheme 15. Early Stage Installation of the C12a Hydroxyl

Group and Failed Attempts To Install Oxygenation at C4a

a

Scheme 14. Phenolic Oxidation Attempts To Hydroxylate at

a

C12a Heptacycle 24 and Derivatives 98 and 101

a

Reagents and conditions: (a) DMDO (1.7 equiv), acetone:H₂O 9:1,

25 °C, 1.5 h, 29% for 103, 50% for 12a-epi-103; (b) conditions

included several Lewis and Brønsted acids. DMDO = dimethyldiox-

irane.

scheme, treatment of 76 with DMDO gave a mixture of C12a-

hydroxylated compounds 103 and 12a-epi-103 (ca. 3:5 ratio,

79% yield combined, chromatographically separated, incon-

sequential) whose conﬁgurations were established through

analysis of their NOESY correlations and ^2,3J coupling

constants (see structure 12a-epi-103). We envisioned that the

extrusion of methanol from 103 might result in the formation

of methyl enol ether 105, whose selective epoxidation would

furnish 106. Such a strategy would be conceptually similar to

that of Myers, which features a fully functionalized AB-ring

enone system. However, the elimination of methanol (103 →

104 → 105, Scheme 15) turned out to be surprisingly

problematic. A variety of Lewis- or Brønsted-acidic conditions

failed to promote this transformation, leading instead to various

decomposition side-products, including lactone ring expansion

products involving the C12a hydroxyl group. We reasoned that

these failures may be due to mismatched electronics which did

not allow for the formation of the oxonium moiety adjacent to

a

Reagents and conditions: (a) Pd black (5.0 equiv), H₂, 1,4-

dioxane:MeOH, 1:1, 25 °C, 30 min, quant.; (b) PIFA (1.1 equiv),

DMF:H₂O 9:1, 40 °C, 2 h; (c) BzCl (4.4 equiv), Et₃N (10 equiv),

DMAP (0.06 equiv), CH₂Cl₂, 25 °C, 5 min, 65% for two steps; (d)

MgBr₂·OEt₂(1.0 equiv), Et₂O:benzene 1:7, 25 °C, 2 h, 75%. PIFA =

phenyliodonium bis(triﬂuoroacetate).

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a

the enone system. We then speculated that the presence of an

aromatic ring adjacent to the oxonium system (see structure

104, Scheme 15) might facilitate the formation of such a

species.

Scheme 17. Synthesis of BCDEF Pentacycle 60

With these postulates in mind, a new strategy was devised

based on the retrosynthetic analysis shown in Scheme 16,

Scheme 16. Third-Generation Retrosynthetic Analysis of

Viridicatumtoxin B Structure 1″

a

Reagents and conditions: (a) 66 (1.0 equiv), 67 (3.0 equiv), DBU

(3.0 equiv), MeCN, 60 °C, 15 h, 50%; (b) CSA (0.02 equiv), CH₂Cl₂,

25 °C, 30 min, quant.; (c) 91 (1.2 equiv), Na₂CO₃(10 equiv), DMF,

25 °C, 1 h, 75%, ca. 1:1 dr; (d) BF₃·OEt₂(0.2 equiv), CH₂Cl₂, 0 °C,

20 min, 82%. CSA = ( )-camphor-10-sulfonic acid.

of BF₃·OEt₂as before, producing the desired pentacyclic

compound 60 in 82% yield. An X-ray crystallographic analysis

of 60 [mp = 114−116 °C (EtOAc:CHCl₃1:1), see ORTEP,

Figure 3] unambiguously proved its spectroscopically derived

structural assignment.⁵⁵

which most notably circumvents an aromatic A-ring and

postpones the C4a/C12a oxygenations to the later stages of the

projected synthesis. Thus, viridicatumtoxin B structure 1″ was

disconnected through the A-ring with a Michael−Dieckmann

reaction similar to that which had successfully been deployed in

the second-generation strategy. This analysis unveiled isoxazole

fragment 81 and BCDEF pentacycle 61 as potential key

building blocks. The latter was envisioned to be derived from

fragments 91, 66, and 67 through a pathway similar to that of

the second-generation approach involving homophthalic

anhydride/quinone monoketal cyclization, anthrone alkylation,

and Lewis acid-mediated spirocyclization (see Scheme 12).

As shown in Scheme 17, a similar set of reaction conditions

as those used to prepare 24 (see Scheme 12) were employed to

synthesize spirocyclic compound 60. Thus, DBU-promoted

Michael−Dieckmann/decarboxylation cascade (107, 50%

yield) and CSA-catalyzed aromatization (quant.) gave anthrone

108. Anthrone alkylation with the allylic bromide 91 then gave

the alkylated compound 109 (75%, ca. 1:1 dr, inconsequential).

The steric congestion around the newly generated C−C bond

of the latter compound manifested itself in some unusual NMR

spectroscopic features. Thus, all three methyl groups of the F-

ring in 109 showed very broad signals, which suggested

hindered rotation around this region of the molecule at 298 K,

thereby forcing the methyl groups to experience a number of

diﬀerent magnetic environments. Supporting this notion was

the fact that when the ¹H NMR spectrum of 109 was acquired

at 338 K in DMSO-d₆, the methyl signals sharpened

considerably. Spirocyclization of 109 proceeded in the presence

Figure 3. ORTEP representation of spirocycle 60. Thermal ellipsoids

at 30% probability. Gray = carbon, red = oxygen, green = hydrogen.

With key intermediate 60 now accessible, we turned next to

its phenolic oxidation. Although initially concerned, due to the

previous recalcitrance of substrate 25 (see Scheme 6) to

undergo phenolic oxidation, these trepidations proved to be

unfounded.

Thus, in an initial experiment, treatment of spirocycle 60

with PIDA (3.0 equiv) led to the formation of desired

quinomethide−quinone monoketal 111 (54% yield) and side-

product 112 (22%, Scheme 18). The stereochemical conﬁg-

uration of the latter species was initially assigned on the basis of

a NOESY experiment and was later unambiguously conﬁrmed

through X-ray crystallographic analysis [mp = 201−203 °C

(EtOAc:CH₂Cl₂1:1), see ORTEP representation, Figure 4].

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product 112 to the desired compound 111 under acidic, basic,

or thermal conditions failed.

Scheme 18. Optimization of Phenolic Oxidation of Substrate

60 to p-Quinomethide 111

a

Eﬀorts to optimize the conversion of 60 to 111 led to the

preferred three-step sequence (via 61 and 110) shown in

Scheme 18. Thus, employment of limited quantities of PIDA

(1.2 equiv), MeOH:CH₂Cl₂(1:1) as solvent, and lower

temperature (0 → 25 °C), generated intermediate 61 in high

yield. The crude product in CH₂Cl₂was then treated with

catalytic quantities of CSA at 0 °C to provide p-quinomethide

110, which was subjected to further phenolic oxidation with

PIDA in MeOH to aﬀord 111 in 78% overall yield for the three

steps. It should be mentioned here that substrate 61 proved

intransigent to the desired pending reactions (i.e., Michael−

Dieckmann cascade), leading instead to MeOH-elimination

product 110. Access to substrate 111 opened the way forward,

despite the need for reduction of the p-quinomethide to the

desired phenolic structural motif at a later stage. The feasibility

of such a transformation was brieﬂy explored at this stage,

leading to the ﬁnding that it could be achieved with NaCNBH₃

(Scheme 18, 111 → 61, 29% yield, unoptimized).

The next task was the union of pentacyclic enone 111 with

isoxazole 81 through the Michael−Dieckmann sequence of

reactions as indicated in Scheme 19. On the basis of our success

with this sequence on the simpler systems discussed above (i.e.,

Schemes 9 and 10), we were rather optimistic for favorable

results in this instance as well. However, initial experimentation

with methyl ester 81 as the isoxazole equivalent proved

a

Reagents and conditions: (a) PhI(OAc)₂(3.0 equiv), MeOH, 25 °C,

2.8 h, 54% for 111 + 22% for 112; (b) PhI(OAc)₂(1.2 equiv),

MeOH:CH₂Cl₂1:1, 0 → 25 °C, 1 h; (c) CSA (0.07 equiv), CH₂Cl₂, 0

°C, 30 min; (d) PhI(OAc)₂(1.2 equiv), MeOH, 25 °C, 1.5 h, 78%

overall for b, c, and d; (e) NaCNBH₃(1.2 equiv), MeOH, 0 → 25 °C,

45 min, 29%, unoptimized.

Scheme 19. Failed Attempts To Close the A-Ring through

Dieckmann Condensations

a

Figure 4. ORTEP representation of side-product 112. Thermal

ellipsoids at 30% probability. Gray = carbon, red = oxygen, green =

hydrogen.

The formation of both 111 and 112 in the presence of excess

PIDA in MeOH at 25 °C is apparently a consequence of the

competing pathways shown in Scheme 18. Thus, initial

phenolic oxidation of 60 aﬀords 61, which suﬀers elimination

of MeOH induced by the in situ-generated AcOH leading to

phenol p-quinomethide 110 (isolated and characterized),

whose phenolic oxidation furnishes 111. Intermediate 61,

however, may also undergo phenolic oxidation, furnishing 112.

The facility by which substrate 60 undergoes phenolic

oxidation as compared to its simpler counterpart 25 mentioned

above (see Scheme 6) is attributed to the extra methoxy group

residing on ring B of the former. Attempts to convert side-

a

Reagents and conditions: (a) 81 (1.1 equiv), t-BuOK (0.2 equiv),

toluene, 25 °C, 45 min, 74%, mixture of four diastereomers; (b) TBAF

(1.2 equiv), THF, 25 °C, 10 min, 60%, ca. 2:1 dr; (c) NaCNBH₃(5.0

equiv), THF, 0 °C, 2 min, 82%, ca. 2:1 dr; (d) BnBr (10 equiv), NaH

(10 equiv), TBAI (0.15 equiv), toluene, 25 → 100 °C, 3.75 h, 85%, ca.

2:1 dr. TBAI = tetra-n-butylammonium iodide.

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otherwise. Thus, although the ﬁrst step (Michael reaction,

Scheme 19) of the anticipated fusion of 111 with 81 proceeded

well, leading to Michael adduct 113 (t-BuOK cat., 74% yield,

mixture of four diastereoisomers), conditions for the

subsequent step (Dieckmann reaction) proved elusive despite

considerable experimentation (a variety of basic conditions

typically led to recovery of unreacted 113 or decomposition).

Removal of the Teoc group from 113 led to 115 and its C15-

epimer (15-epi-115, ca. 2:1 dr)⁵⁶(TBAF, 60% yield), which,

however, also failed to undergo the coveted Dieckmann

cyclization (to 116, Scheme 19). Reduction of the quinome-

thide moiety of 115/15-epi-115 furnished 117/15-epi-117

(NaCNBH₃, 82% yield, ca. 2:1 dr). The dibenzyl ether (119/

15-epi-119) of the latter was also prepared (NaH, BnBr, TBAI

cat., 85% yield, ca. 2:1 dr). Neither 117/15-epi-117 nor 119/

15-epi-119 entered the desired Dieckmann reaction to aﬀord

the expected heptacyclic systems 118/15-epi-118 or 120/15-

epi-120, respectively. Similar problems were reported by Stork

and Kahne.⁵⁷

Dieckmann cascade to aﬀord the heptacyclic product (111 →

114 + 15-epi-114) is a testament to the activating power of the

phenyl ester moiety in such circumstances.

We initially explored the C12a hydroxylation of substrate

114, with the Teoc group still intact, as shown in Scheme 21.

a

Scheme 21. C12a Hydroxylation of Substrate 114

Faced with these diﬃculties and inspired by the work of

White⁵⁸and Myers,^20dwe opted to employ the phenyl ester

counterpart of isoxazole 81 (see 123, Scheme 20a). Isoxazole

Scheme 20. Synthesis of Phenyl Ester 123 (a) and Its

Successful Michael−Dieckmann Cyclization with Enone 111

a

(b)

a

Reagents and conditions: (a) DMDO (2.8 equiv), acetone, −78 °C,

3.5 h, 21% for 125 (ca. 2:1 dr), 9% for 124 (ca. 2:1 dr); (b) m-CPBA

(2.7 equiv), CH₂Cl₂, −78 °C, 1.5 h, 55% for 124 (71% brsm, ca. 2:1

dr) + 24% recovered 114; (c) HF·Et₃N (100 equiv), DMSO, 60 °C, 2

h, 43%, ca. 2:1 dr. DMSO = dimethyl sulfoxide.

Thus, hydroxylation of substrate 114 at C12a with DMDO

(conditions a) at −78 °C produced the desired compound 124

(together with its C15 epimer 15-epi-124, ca. 2:1 dr) but only

as a minor product (9% combined yield). The major product in

this reaction was seven-membered lactone 125 formed together

with its C15-epimer (15-epi-125, 21% combined yield, ca. 2:1

dr) through rearrangement/ring expansion of presumed

transient intermediate 126 + 15-epi-126 (see Scheme 21).

C12a hydroxylation of substrate 114 + 15-epi-114 was also

achieved with m-CPBA, this time leading exclusively to the

desired hydroxy compound 124 + 15-epi-124 (ca. 2:1 dr) in

71% combined yield based on 24% recovered starting material.

However, attempts to remove the Teoc group from the latter

mixture with HF·Et₃N led to product 127 + 15-epi-127 from

which the Teoc had been removed and the B-ring had been

expanded to a seven-membered lactone moiety, presumably

through rearrangement of ﬂeeting intermediate 128 + 15-epi-

128 (or its Teoc counterpart which suﬀers Teoc removal

concomitantly or subsequently), as depicted in Scheme 21.

Exposure of 124 (+ 15-epi-124) to TBAF led to decomposition

and partial recovery of starting material.

a

Reagents and conditions: (a) NaOH (1.9 equiv), H₂O:EtOH 3:10,

25 °C, 3 h, 99%; (b) PPh₃(1.05 equiv), PhOH (1.05 equiv), DIAD

(1.05 equiv), THF, reﬂux, 3 h, 78%; (c) LiHMDS (2.2 equiv), THF,

−78 °C, 30 min; then TeocCl (2.2 equiv), −78 °C, 2 h, 86%; (d) 123

(1.1 equiv), t-BuOK (1.2 equiv), toluene, 25 °C, 15 min, 94%, ca. 1:1

dr. DIAD = diisopropyl azodicarboxylate.

phenyl ester 123 was prepared from methyl ester 26 via 121

and 122 through saponiﬁcation (NaOH, 99% yield),

esteriﬁcation with PhOH under Mitsunobu conditions (PPh₃,

DIAD, 78% yield),⁵⁹and Teoc attachment (LiHMDS, TeocCl,

86% yield) as shown in Scheme 20a. It is of note that several

standard ester-forming reactions (e.g., mixed anhydride, acid

chloride, DCC) failed to bring about the desired esteriﬁcation

between 121 and PhOH. Gratifyingly, quinone monoketal 111

reacted smoothly with phenyl ester isoxazole 123 in the

presence of a slight excess of t-BuOK to give directly the

desired heptacyclic compound 114 as a ca. 1:1 mixture with its

C-15 diastereoisomer (15-epi-114) in 94% yield (Scheme

The diﬃculties of removing the Teoc group in the presence

of the C12a hydroxyl group led us to explore the alternative

sequence in which the Teoc moiety was removed prior to the

hydroxylation step. To this end, and as shown in Scheme 22,

114 (+ 15-epi−114) was treated with excess TBAF in THF,

furnishing small amounts of the desired product 116 (+ 15-epi-

20b).⁶⁰The stereochemical relationship of H4 and H4a within

114 was evident from the observed coupling constant J_4,4a

9.9 Hz, indicative of the diaxial disposition of these protons.

The impressive rapidity (<15 min at 25 °C) of the Michael−

3

=

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132/15-epi-132 (ca. 2:1 dr) as shown in Scheme 22. Both

diastereomers were of the syn stereochemical conﬁguration

with regard to the C12a hydroxyl and C4a hydrogen residues,

presumably as a consequence of the concavity of the AB ring

system. It should be noted at this point that at temperatures

above −40 °C, epoxidation of the F-ring oleﬁnic site began to

occur. Other oxidizing agents such as m-CPBA, Davis

oxaziridine,⁶⁶and magnesium monoperoxyphthalate (MMPP)

proved unsuccessful in providing the desired C12a-hydroxy-

lated compound (i.e., 132 +15-epi-132).

Scheme 22. Teoc Removal from 114 and C12a

Hydroxylation of 116

a

With the C12a hydroxylation problem solved, the challenge

of installing the C4a hydroxyl group became the next task, a

mission that was to prove even more intransigent than the one

before it. Our initial attempts to achieve this goal are shown in

Scheme 23. Thus, reduction of the quinomethide moiety of

a

Scheme 23. Initial Strategy for C4a Hydroxylation

a

Reagents and conditions: (a) TBAF (5.3 equiv), THF, 25 °C, 1 h,

16% for 116 (ca. 2:1 dr) + 33% recovered 114; (b) HF·Et₃N (excess),

DMSO, 25 → 60 °C, 3 h, 28% for 116 (ca. 2:1 dr) + 30% for 131; (c)

TBAF (10 equiv), NH₄F (20 equiv), THF, 25 °C, 5 min, 98%, ca. 2:1

dr; (d) DMDO (7.6 equiv), [Ni(acac)₂] (0.2 equiv), acetone, −78 →

−65 °C, 6.5 h, 61%, ca. 2:1 dr. acac = acetylacetonate.

116, 16% combined yield) and unreacted starting material

(33%). Presumably, this failure was caused by deprotonation of

the diketone moiety adjacent to the isoxazole (see 129) that

rendered the desired loss of TMSF, ethylene, and carbon

dioxide less facile (see 129 → 130). Faced with this

predicament, we undertook a thorough investigation of

conditions to achieve the desired Teoc group removal from

114 (+ 15-epi-114), including ZnCl₂, ZnF₂, MgBr₂, NH₄F, and

SiF₄,^61,62but unfortunately to no avail. Disappointingly, the use

of HF·Et₃N led to the desired product 116 (+ 15-epi-116) in

28% combined yield, albeit with substantial amounts of side-

product 131 (30% yield), presumably formed through an air-

oxidation process.⁶³Indeed, it was not until we combined

a

Reagents and conditions: (a) NaCNBH₃(10 equiv), THF, −78 °C,

Furstner’s TBAF−NH F conditions⁶⁴with solvent degassing

1.5 h, 51% for 133 + 19% for 15-epi-133, chromatographically

separated; (b) various Lewis and Brønsted acids: see the Supporting

Information; (c) THF:2 M aq. HCl 10:1, 25 °C, 3.5 h, 98%; (d)

various conditions: see the Supporting Information.

̈

4

(to prevent air oxidation of the A-ring) that we reached a

solution to this problem, obtaining a pleasing 98% yield of the

coveted deprotected product 116 (+15-epi-116) (Scheme 22).

Having solved the Teoc removal problem, we turned our

attention to the C12a hydroxylation task using 116/15-epi-116

as a substrate. Our initial use of DMDO in acetone at low

temperature (i.e., −78 °C) proved irreproducible, gave low

conversion to product due to substrate insolubility, and suﬀered

from TLC monitoring problems. To improve the reaction, we

resorted to the use of Ni(acac)₂, an additive known to facilitate

DMDO oxidations through coordination.⁶⁵Indeed, portion-

wise addition of DMDO to a stirred mixture of 116/15-epi-116

and catalytic amounts of Ni(acac)₂in acetone at −78 to −65

°C led to 61% combined yield of C12a hydroxylated product

132/15-epi-132 with NaCNBH₃led to the now chromato-

graphically separable derivatives 133 and 15-epi-133. Gratify-

ingly, one of the two isomers (more polar) crystallized readily

from EtOAc [mp = 213−215 °C (decomp)] and yielded to X-

ray crystallographic analysis, establishing its stereochemical

conﬁguration as 15-epi-133 (see ORTEP representation, Figure

5), and as a consequence, that of its less polar epimer as the

desired diastereomer 133.⁵⁵Several attempts to prepare

methylenol ether 134 as a potential precursor of the desired

4a-hydroxylated compound 136 through selective epoxidation

M

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Scheme 24. Attempts To Generate C4a,5-Oleﬁn 141 as a

a

Substrate for Dihydroxylation

Figure 5. X-ray-derived ORTEP representation of compound 15-epi-

133. Thermal ellipsoids at 30% probability. Gray = carbon, blue =

nitrogen, red = oxygen, green = hydrogen.

(to aﬀord 135) and thence hydrolysis, or through 137 via

hydrolysis, enolization, and oxidation, failed. Eﬀorts to generate

134 from 133 employing a variety of conditions, including

Lewis acids or protic acids, or under anhydrous conditions (see

Supporting Information), however, did not give the desired

product but led instead to ketone 137. Similarly, various

conditions to carry out the desired enol ether formation at the

quinomethide stage (compound 132/15-epi-132) also failed. In

addition, a number of transketalization reactions with dimethyl

ketal 133 directed at forming an oxa-seleno mixed ketal⁶⁷

(intended to be used as a precursor for a selenoxide/syn

elimination sequence to produce the desired methylenol ether

134) ended without success. Attempts to achieve C4a

hydroxylation of ketone 137 through enolization followed by

oxidation also failed, typically resulting in aromatization to

aﬀord 138 (Scheme 23) through β-elimination/tautomerization

(see Supporting Information for more details).

We then explored the possibility of achieving C4a

hydroxylation using dimethyl ketal 133 (single diastereomer)

to generate oleﬁnic compound 141, whose directed dihydrox-

ylation⁶⁸was expected to yield 142 (see Scheme 24). To this

end, exposure of 133 to NaCNBH₃in AcOH produced

chromatographically separable methyl ethers 140 (40%) and 5-

epi-140 (32%), presumably through reactive oxonium species

139.⁶⁹The conﬁguration of 140 (and by deduction, of 5-epi-

140) was assigned on the basis of 2D NMR (NOESY)

spectroscopic analysis and the observed ³J_4a,5= 8.7 Hz coupling

constant (see structure 140, bottom of Scheme 24). Numerous

attempts (e.g., Lewis and protic acids) to generate oleﬁn 141

from 140 or 5-epi-140 through elimination of MeOH, however,

failed. In several cases, the bis-elimination/tautomerization

product 143 was isolated instead. Attempted solvolysis of

isomer 5-epi-140 by heating in triﬂuoroethanol at 70 °C as a

means to reach oleﬁn 141 led, surprisingly, to triﬂuoroethyl

ester 144 (70% yield, Scheme 24), presumably through a retro-

Dieckmann ring opening reaction.⁷⁰

a

Reagents and conditions: (a) NaCNBH₃(4.3 equiv), AcOH, 25 °C,

40 min, 40% for 140, 32% for 5-epi-140, chromatographically

separated; (b) various Lewis and Brønsted acids; (c) CF₃CH₂OH,

70 °C, 2 h, 70%.

selective formation and regioselective reductive opening of

epoxide 146, as outlined in Scheme 25. As shown in Scheme

26, execution of this plan began with formation of presumed

trianion 148 (KHMDS, −78 °C) followed by quenching with

PhSeCl (−78 → −40 °C) to aﬀord phenyl selenide 149 as a

single isomer (46% yield + 26% recovered starting material).

The conﬁguration of the phenylseleno group within 149 was

tentatively assigned on steric grounds (i.e., concavity of the cis-

fused AB-ring system). Treatment of selenide 149 with DMDO

at −78 °C produced a complex mixture from which seven-

membered lactone 152 could be isolated (46% yield),

presumably formed through the indicated pathways involving

selenoxide 150, oleﬁn 145, and either acylketene 151 (pathway

A)^71,72or hydroxy-epoxide 153 (pathway B) (see Scheme 26).

Other oxidizing agents (e.g., H₂O₂, m-CPBA) led to similar

results. In an attempt to shut down the hypothesized hydroxy-

epoxide pathway, we prepared TMS ether 154 from 149

Unable to reach the Δ^4a,5-compound 141 (Scheme 24), we

turned to the Δ^4,4a-compound 145 as an alternative substrate

for the desired C4a hydroxylation. It was envisioned that 145

could be reached from 133 through an oxidative process and

then converted to the desired hydroxylated product 147 via

N

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tentatively support pathway A, rather than B, for the formation

of 152 from 154 (and 149), although due to the lability of the

TMS group the possibility of both pathways operating

concurrently cannot be excluded.

Scheme 25. Proposed Elaboration of 133 to C4a-

Hydroxylated Compound 147 through C4,4a-Oleﬁn 145 and

Epoxide 146

A number of other attempts to prepare Δ^4,4a-substrates such

as 157 (Scheme 27) and 145 (Scheme 28) were made. Thus,

a

Scheme 27. Attempted Preparation of Enone 157

a

Scheme 26. Attempted Preparation of Oleﬁn 145

a

Reagents and conditions: (a) TFA:CH₂Cl₂(1:2), 0 °C, 40 min, 52%;

(b) H₂O₂(4.5 equiv), CH₂Cl₂, 0 °C, 10 min, 35%.

TFA-induced hydrolysis of ketal 149 gave triketone 155 (52%

yield) which was expected to facilitate the formation of 157

through selenoxide formation (156) and syn-elimination (see

Scheme 27). Unfortunately, however, oxidation of selenide 155

(H₂O₂) did not lead to the expected oleﬁn 157, but rather to

the expanded keto-lactone 158 (35% yield), presumably

through selenoxide 156 (see Scheme 27). Similarly, hydrox-

ylation of 133 with Davis oxaziridine through its trianion

(KHMDS, −78 °C, 52% yield) followed by ketal hydrolysis

(TFA, 63% yield) furnished secondary alcohol 160, whose

attempted dehydration (e.g., through the corresponding

mesylate) also failed (see Scheme 28). Iodide 159 (Scheme

28) was also prepared from 133 (KHMDS; NIS, 37%) as a

potential precursor to oleﬁnic compound 145, through

oxidation and syn-elimination of the corresponding iodoso

compound.⁷³Attempts to eﬀect the latter transformation,

however, by m-CPBA oxidation failed. Moreover, attempted

Mukaiyama dehydration⁷⁴from the trianion derived from 133

also did not give the desired oleﬁn 145 (Scheme 28).

a

Reagents and conditions: (a) KHMDS (3.3 equiv), THF, −78 °C, 1

h; then PhSeCl (3.5 equiv), −78 → −40 °C, 1 h, 46% (62% brsm) +

26% recovered 133; (b) from 149: DMDO (1.0 equiv), THF, −78 °C,

20 min, 46%; from 154: DMDO (1.5 equiv), THF, −78 °C; then 25

°C, 14 h, 7%; (c) TMSOTf (30 equiv), Et₃N (40 equiv), CH₂Cl₂, 0

°C, 5 min, 62%. OTf = triﬂuoromethanesulfonate.

Faced with these obstacles toward Δ^4,4a-substrates 145 and

157, we decided to return to Δ^4a,5-substrate 141 which we had

previously considered but later abandoned (see Scheme 24).

Inspired by a report from the Tatsuta group,⁷⁵we reasoned that

a 1,3-directed reduction of the C5-carbonyl moiety at the 137

stage could give us access to the C5-hydroxy compound 162,

whose dehydration would lead to the targeted substrate 141

that could serve as a precursor to the desired trihydroxylated

intermediate 142 (Scheme 29). However, when 137 was

treated with NaBH(OAc)₃, we instead isolated the 1,2-directed

(TMSOTf, Et₃N, 62% yield, Scheme 26) and employed it as a

substrate in the DMDO oxidation, only to observe the same

ring expansion product 152, albeit in only 7% yield (with

concomitant loss of the TMS residue). This outcome seems to

O

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Scheme 28. Other Attempts To Synthesize Oleﬁnic

Compounds 141 and 153

Scheme 29. Attempted Preparation of C5-Hydroxy

Compound 162 through 1,3-Directed Reduction and

Observation of 1,2-Directed Reduction Product 164

a

Reagents and conditions: (a) KHMDS (3.8 equiv), THF, −78 °C, 1

h; then Davis oxaziridine (3.5 equiv), −78 → −40 °C, 1 h, 52%; (b)

TFA:CH₂Cl₂(1:3), 0 → 25 °C, 45 min, 63%; (c) KHMDS (3.6

equiv), THF, −78 °C, 80 min; then NIS, −78 → −35 °C, 1 h, 37%.

NIS = N-iodo-succinimide.

reduction product 164 (35% yield, Scheme 29), along with

other unidentiﬁable products. After further unsuccessful

attempts to secure the targeted C5-hydroxy derivative of our

heptacyclic compound, we opted to pursue the newly emerged

1,2-directed reduction product 164 as a possible substrate for

advancement to viridicatumtoxin B. The rationale behind this

choice included the expectation that this intermediate and its

derivatives could be resistant to elimination and aromatization

side reactions involving rings A and B as previously observed

(see Schemes 23 and 24). Brief experimentation led to an

improved protocol for the selective reduction of triketone 137

[NaBH(OAc)₃, EtOAc:acetone] that delivered alcohol 164 in

53% yield (Scheme 30). The newly generated hydroxy group

within the latter compound was protected as a TBS ether

through the use of excess TBSOTf (most likely needed due to

steric diﬃculties caused by the concavity of the AB-ring system)

to aﬀord compound 165 (64% yield). Conversion of the latter

compound to its trianion (166) with KHMDS followed by

quenching with Davis oxaziridine led to the coveted C4

hydroxylated product 167 in 29% yield (47% based on 39%

recovered starting material). Alternative bases (LiHMDS,

NaHMDS, or LDA) or equivalents of base did not improve

the outcome of this reaction. The conﬁguration of the newly

generated C4a hydroxyl moiety within 167 was deduced from

NOESY correlations as shown in Scheme 30 (bottom).

a

Reagents and conditions: (a) NaBH(OAc)₃(1.3 equiv), EtOH:THF

1:1, 0 → 25 °C, 3.25 h, 35%.

in addition to elimination of the OTBS group from its A-ring,

had also suﬀered removal of the TBS group from the phenolic

oxygen. These observations suggest that the presumed initially

formed dianion 169 is too reactive entering the proposed

cascade sequence shown in Scheme 31. Thus, attack of the

tertiary alkoxide within 169 on the adjacent carbonyl moiety

generates a new alkoxide anion that abstracts the TBS group

from the adjacent phenolic position to aﬀord epoxide species

170. Prompted by the adjacent enolate anion, the epoxide

within the latter species undergoes facile β-elimination/opening

with concomitant loss of the B-ring OTBS group to aﬀord

diketone dianion 171. The generation of the newly established

anion at C4 within 171 is facilitated by the isoxazole ring on

one side and the quinone moiety on the other. Species 171

then suﬀers OTBS elimination/ring A aromatization, leading to

observed product 172 upon protonation.

Taken together, these extensive investigations on the C4a

hydroxylation deﬁned the required structural features for the

substrate as (a) protected hydroxyl group at C1 (ring A), (b)

free hydroxyl group at C12a (rings A/B), and (c) free phenolic

moiety at C11 (ring C). Straying away from any one of these

key structural motifs led to either no conversion or rearranged

products of undesired structures.

In an eﬀort to improve the C4a hydroxylation reaction, we

targeted bis-TBS ether 168 (Scheme 31) as a substrate,

counting on its dianion to undergo the desired transformation

more eﬃciently. Bis-silylation of 164 with excess TBSOTf

required the use of Hunig’s base and led to 168 in 57% yield.

Interestingly, however, treatment of 168 with KHMDS at −78

°C led rapidly to aromatized product 172 (43% yield), which,

At this point, we decided to move forward and test the

remaining steps of the devised synthetic strategy toward

viridicatumtoxin B that required reinstallment of the carbonyl

̈

P

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group at C1, selective removal of the methyl group from the

C10 methyl ether, and rupture of the isoxazole moiety to

provide the required enol amide functionality at the terminus of

the molecule. To this end and as shown in Scheme 32, silyl

Scheme 30. Successful C4a Hydroxylation of 164 through

Oxidation of Its Trianion

a

Scheme 32. Synthesis of Triketone 136

a

Reagents and conditions: (a) NaBH(OAc)₃(1.2 equiv), EtOAc:ace-

tone 1:1, 40 °C, 3.5 h, 53%; (b) TBSOTf (35 equiv), 2,6-lut. (53

equiv), ClCH₂CH₂Cl, 5 → 25 °C, 1.8 h, 64%; (c) KHMDS (3.5

equiv), THF, −78 °C, 65 min; then Davis oxaziridine (4.0 equiv), −78

°C, 40 min, 29% for 167 (47% brsm) + 39% recovered 165. lut. =

lutidine.

a

Reagents and conditions: (a) HF·py (excess), MeCN, 0 → 55 °C, 25

h, 66%; (b) DMP (5.2 equiv), ClCH₂CH₂Cl, 0 → 40 °C, 4 h, 32%; (c)

BCl₃, BBr₃, AlCl₃/EtSNa, or 9-I-BBN. DMP = Dess−Martin

periodinane, BBN = borabicyclo[3.3.1]nonane.

Scheme 31. Synthesis and Unsuccessful Attempt To C4a

Hydroxylate Bis-TBS Ether 168

a

ether 167 was desilylated (HF·py, 66% yield) to aﬀord C1

hydroxy compound 173 which was assumed to exist in

equilibrium with its hemiacetal form 173′ based on its broad

¹H and ¹³C NMR spectroscopic features. Oxidation of this

mixture with DMP resulted in the formation of ketone 136

(32% yield, unoptimized) through reaction of 173, which

apparently drives the equilibrium in the right direction for

funneling both isomers (173 and 173′) toward the desired

product (136). The obligatory removal of the methyl group

from the C10 phenolic group, however, proved diﬃcult despite

the inspiring precedent for such deprotections in the

tetracycline series.^19,20bThus, attempts to achieve this goal

with BCl₃, BBr₃, AlCl₃/EtSNa, and 9-I-BBN⁷⁶failed, leading to

either decomposition and/or cleavage of the benzyl ether as

indicated by LCMS detection of hydroxy-isoxazole 174 (but

not 175).

In contrast to this disappointing result, the rupture of the

isoxazole ring planned as the ultimate step of the synthesis

proved feasible with substrate 136 under hydrogenation

conditions in the presence of Pd black, furnishing 10-O-

methylviridicatumtoxin B (176, 57% yield) as shown in Scheme

33. Interestingly, the spectroscopic data of this compound

conﬁrmed its hydroxy-keto form 176 rather than the originally

assigned¹(to viridicatumtoxin B) hydroxy-epoxide structure

(i.e., 1′, Chart 1).

a

Reagents and conditions: (a) TBSOTf (20 equiv), i-Pr₂EtN (30

equiv), CH₂Cl₂, 0 → 25 °C, 33 min, 57%; (b) KHMDS (2.5 equiv),

THF, −78 °C, 45 min, 43%.

Q

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are summarized in Scheme 35. Thus, regioselective demethy-

lation of the known diethyl ester 85 with BBr₃followed by

Scheme 33. Total Synthesis of 10-O-Methylviridicatumtoxin

B

a

Scheme 35. Synthesis of the Carbon Framework 187 of

a

Viridicatumtoxin B with a C10-OBn Protecting Group

a

Reagents and conditions: (a) H₂, Pd black (5.3 equiv), MeOH:1,4-

dioxane 1:1, 25 °C, 8 min, 57%.

Scheme 34. Final Retrosynthetic Analysis of

Viridicatumtoxin B (1)

The path-pointing results and intelligence discussed above

served as the basis for the ﬁnal strategy and drive toward

viridicatumtoxin B.

Total Synthesis and Structural Revision of Viridica-

tumtoxin B: Final Drive. Scheme 34 presents the ﬁnal

retrosynthetic analysis and successful strategy that led to the

total synthesis of viridicatumtoxin B. Thus, cyclic anhydride

177, quinone monoketal 67, allylic bromide 91, and isoxazole

123 were conﬁdently deﬁned as the required building blocks

for the intended route (based on the studies described above).

The high modularity of the strategy (i.e., four building blocks of

approximately equal sizes) and the accessibility of the starting

materials appeared attractive for further development and

designed-analogue construction. The adoption of the benzyl

ether protecting groups at both the anhydride (177) and

isoxazole (123) fragments oﬀered the advantage of their

concurrent removal as the last step in the synthesis. The overall

plan called for the union of the anhydride (i.e., 177) and

quinone monoketal (i.e., 67) fragments through (1) a

Michael−Dieckmann/decarboxylation cascade to cast the C

ring, (2) an S_N2 alkylation to attach the F ring, (3) a Lewis

acid-catalyzed spirocyclization to cast the E ring, and (4) a

Michael−Dieckmann sequence to fuse the A-ring as outlined in

Scheme 34.

a

Reagents and conditions: (a) BBr₃(1.35 equiv), CH₂Cl₂, −78 → 25

°C, 30 min; (b) BnBr (1.1 equiv), Ag₂O (1.9 equiv), DMF, 25 °C, 15

h, 66% for two steps; (c) NaOH (27 equiv), H₂O:EtOH 5:7, reﬂux, 15

h; (d) Ac₂O (1.1 equiv), toluene, reﬂux, 1 h, 90% for two steps; (e) 67

(3.0 equiv), NaH (3.0 equiv), THF, 0 °C, 45 min; then 25 °C, 1 h; (f)

DBU (5.0 equiv), toluene, 65 °C, 4.5 h, 54% for two steps; (g) CSA

(0.02 equiv), CH₂Cl₂, 25 °C, 30 min, 99%; (h) 91 (1.1 equiv),

Na₂CO₃(10 equiv), DMF, 25 °C, 1 h, 77%, ca. 1:1 dr; (i) BF₃·OEt₂

(0.10 equiv), CH₂Cl₂, 0 °C, 20 min, 73%; (j) PIDA (1.2 equiv),

MeOH:CH₂Cl₂1:1, 0 → 25 °C, 1 h; (k) CSA (0.07 equiv), CH₂Cl₂, 0

°C, 5 min, 85% for two steps; (l) PIDA (1.2 equiv), MeOH:CH₂Cl₂

10:1, 25 °C, 1.5 h, 90%; (m) 123 (1.1 equiv), t-BuOK (1.2 equiv),

toluene, 25 °C, 15 min, 91%, ca. 2:1 dr; (n) TBAF (10 equiv), NH₄F

(20 equiv), degassed THF, 25 °C, 5 min, 86%, ca. 2:1 dr.

benzylation (BnBr, Ag₂O) led to benzyl ether 178 in 66%

overall yield. Saponiﬁcation of the ester moieties of the latter

(aq NaOH) and heating of the resulting dicarboxylic acid (179)

with Ac₂O as previously reported⁴⁵furnished desired anhydride

177 in 90% overall yield.

Anhydride 177 was reacted with quinone monoketal 67

through the action of NaH (Michael−Dieckmann sequence)

followed by treatment of the coupling product with DBU

(decarboxylation) to aﬀord tricycle 180 in 54% overall yield.

Access to building blocks 67, 91, and 123 has already been

discussed above, leaving only the preparation of the benzyl-

protected anhydride fragment 177 to be described here. Its

synthesis and union with the other fragments (67, 91, and 123)

R

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Elimination of MeOH from the latter induced by CSA gave the

corresponding anthrone (99% yield), which was alkylated with

allylic bromide fragment 91 in the presence of Na₂CO₃to

furnish intermediate 181 (77% yield). Exposure of 181 to

catalytic amounts of BF₃·OEt₂aﬀorded spirocycle 182 in 73%

yield. The latter was then treated with PIDA in MeOH:CH₂Cl₂

to give the initially formed quinone monoketal 183 and thence

with CSA to aﬀord the phenol p-quinomethide 184 (85% yield

overall). Phenolic oxidation of the latter with PIDA in

MeOH:CH₂Cl₂then delivered 185 in 90% yield, as observed

previously with the corresponding C10-methyl series of

substrates (see Scheme 18). Coupling of 185 with isoxazole

phenyl ester fragment 123 was eﬀected with t-BuOK (Michael

reaction/Dieckmann condensation) furnishing heptacyclic

compound 186 together with its unseparable C15 epimer

(15-epi-186, ca. 2:1 dr, 91% combined yield). Removal of the

now-superﬂuous Teoc group was accomplished through

application of the developed desilylation/decarboxylation

procedure (TBAF/NH₄F) to furnish advanced intermediate

187 together with its unseparable isomer 15-epi-187 (ca. 2:1

dr) in 86% combined yield.

recovered starting material, yielding C12a-hydroxylated com-

pound 188 together with its 15-epimer (15-epi-188, 35% yield,

50% yield after one recycle, ca. 2:1 dr, Scheme 36). Employing

a solution of DMDO in CH₂Cl₂⁷⁷did not result in a noticeable

improvement of conversion or yield, which might be attributed

to the decreased stability of DMDO in that solvent. Exposure of

the quinomethide mixture (188 + 15-epi-188) to NaCNBH₃

gave chromatographically separable reduced products 189

(39% yield) and 15-epi-189 (19% yield) whose conﬁgurations

were assigned by comparisons of their NMR spectroscopic data

to those of their corresponding C10-OMe counterparts (as

discussed above, the structures of the latter were unambigu-

ously assigned through X-ray crystallographic analysis, see

Scheme 23 and Figure 5). Isomer 189 was then advanced to

TBS ether 192 as shown in Scheme 36. Thus, treatment of 189

with HCl in aqueous THF led quantitatively to triketone 190,

whose reduction with NaBH(OAc)₃furnished C1-hydroxy

compound 191 in 47% yield. The latter was then silylated with

TBSOTf in the presence of 2,6-lutidine to aﬀord 192 (61%

yield).

The ﬁnal stretch of the synthesis of viridicatumtoxin B is

depicted in Scheme 37. The challenging C4a hydroxylation of

The next step in the synthesis entailed hydroxylation of the

molecule at C12a (see Scheme 36), a process that was plagued

with increased insolubility issues of the benzyl-protected

substrate 187/15-epi-187 as compared to the methyl-protected

version employed in the earlier generation route. This

necessitated the use of DMDO in the presence of Ni(acac)₂

in CH₂Cl₂(instead of acetone); the eﬃciency of the reaction

remained similar as the previous result after recycling of

Scheme 37. Completion of the Total Synthesis of

a

Viridicatumtoxin B (1)

a

Scheme 36. Synthesis of Enolate-Oxidation Precursor 192

a

Reagents and conditions: (a) KHMDS (3.4 equiv), THF, −78 °C, 1

h; then Davis ox. (3.9 equiv), −78 °C, 1.7 h, 20% of 193 + 45%

recovered 193; (b) HF·py (excess), MeCN, 0 → 50 °C, 25 h, 61%; (c)

DMP (3.0 equiv), ClCH₂CH₂Cl, 0 → 50 °C, 7.5 h, 66%; (d) H₂, Pd

black (4.9 equiv), 1,4-dioxane:MeOH 1:1, 25 °C, 8 min, 98%.

a

Reagents and conditions: (a) [Ni(acac)₂] (0.2 equiv), DMDO (5.1

precursor 192 proceeded in the presence of KHMDS (added

ﬁrst at −78 °C to form the presumed trianion of 192) and

Davis oxaziridine (added thence at −78 °C) to aﬀord

hydroxylated product 193 (20% yield + 45% recovered starting

material). Desilylation of 193 with HF·py furnished poly-

hydroxy compound 194, whose equilibrium with its lactol form

equiv), CH₂Cl₂, −78 → −60 °C, 6.5 h, 36% (ca. 2:1 dr, 60% brsm),

50% after one recycle; (b) NaCNBH₃(10 equiv), THF, −78 → −60

°C, 90 min, 39% for 189, 19% for 15-epi-189, chromatographically

separated; (c) 2 N aq. HCl:THF 1:10, 25 °C, 5 h, quant.; (d)

NaBH(OAc)₃(1.2 equiv), EtOAc:acetone 1:1, 40 °C, 105 min, 47%;

(e) TBSOTf (40 equiv), 2,6-lut. (60 equiv), CH₂Cl₂, 0 → 25 °C, 1 h,

61%.

1

(194′) was evident from the H NMR spectra (CDCl₃, 500

S

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MHz) at ambient temperature (broad signals) and −40 °C

(two sets of sharp signals, ca. 2:1 ratio). (This was the same

phenomenon we encountered with their 10-methoxy counter-

parts 173/173′, Scheme 32, as discussed above). Oxidation of

the so-obtained mixture (194 + 194′) with DMP then gave

triketone 195 in 66% yield, with the lactol form 194′ being

funneled into the oxidation pathway by the equilibrium.

Pleasantly, hydrogenation of the latter compound (Pd black,

H₂) produced synthetic viridicatumtoxin B (1) in 98% yield

through cleavage of the two benzyl ethers, rupture of the

isoxazole N−O bond, and tautomerization of the resulting

hydroxy-imine to the desired primary amide.

not only ( )-viridicatumtoxin B [( )-1] but also a number of

analogues that are simpler and easier to synthesize for biological

evaluation (see Chart 3 for structures). Speciﬁcally, analogues

Chart 3. Molecular Structures of Natural Viridicatumtoxin A

(2), Synthetic Viridicatumtoxin B (1), Synthesized

Viridicatumtoxin Analogues (V2−V6), and Tetracycline

Drugs Minocycline (7) and Tigecycline (9)

The physical properties of synthetic viridicatumtoxin B were

consistent with those of the authentic natural product and our

proposed structure (1). The ¹H NMR data of synthetic

viridicatumtoxin B (1) were in good agreement with those

reported in the literature.¹However, we observed a ¹³C NMR

signal at δ = 194.1 ppm for C5, whereas the reported chemical

shift for the proposed C5 epoxy-hemiacetal in the originally

assigned structure was δ = 116.4 ppm. Therefore, a detailed re-

examination of the authentic carbon NMR spectrum (obtained

from Professor W. G. Kim) of natural viridicatumtoxin B was

conducted. Indeed, the signal near δ = 194 ppm was observable

in the authentic spectrum of the natural product. We assume

that, due in part to the minute amounts of this complex natural

product isolated, an erroneous interpretation of the HMBC

spectrum seemingly supported the original structural assign-

ment (see the Supporting Information for more details and

copies of the authentic spectra). We therefore revised the

originally proposed structure of viridicatumtoxin B (1′)¹to that

shown in Scheme 37 (i.e., 1). Furthermore, our synthetic

material crystallized from CH₂Cl₂/EtOH in suitable form for X-

ray crystallographic analysis [mp = 245−247 °C (decomp)]

and proved unambiguously its structure as shown in 1 (see

ORTEP representation, Figure 6).⁵⁵

Biological Evaluation of Synthetic ( )-Viridicatumtox-

in B and Analogues. Employing the developed synthetic

technologies in this research program, we were able to access

( )-V2, ( )-V3, ( )-V4, ( )-V5, and ( )-V6, all lacking the

C4a hydroxyl group so cumbersome to install, were synthesized

(see Supporting Information for their synthesis) and, together

with ( )-1, were tested against a number of bacterial strains

and compared to natural viridicatumtoxin B [(+)-1, reported

values¹], natural viridicatumtoxin A [(+)-2, obtained from

Professor Yi Tang], minocycline (Minocin, 7), and tigecycline

(Tygacil, 9) (see Chart 3 for structures).

As shown in Table 1, all of the viridicatumtoxins and

analogues tested exhibited antibacterial eﬃcacy against Gram-

positive bacteria [(E. faecalis S613, E. faecium 501, and

methicillin-resistant Staphylococcus aureus 371 (MRSA 371)]

but were largely inactive against Gram-negative bacteria (i.e., A.

baumannii AB210). Thus, synthetic viridicatumtoxin B [( )-1]

exhibited comparable antibacterial properties against these

strains (E. faecalis S613, E. faecium 501, and MRSA 371: MIC =

1, 0.5, and 4 μg/mL, respectively) to those reported for natural

viridicatumtoxin B [(+)-1] against similar strains (E. faecalis

KCTC5191, E. faecium KCTC3122, MRSA CCARM3167:

MIC = 2, 0.5, and 0.5 μg/mL, respectively), despite the racemic

nature of the former. The potencies of synthetic ( )-1 were

also comparable to those reported¹for natural viridicatumtoxin

A [(+)-2] against similar strains (see Table 1).

Viridicatumtoxin analogue ( )-V2, lacking the C4a hydroxyl

group, displayed high potency against the same strains (E.

faecalis S613: MIC = 0.5 μg/mL; E. faecium 501: MIC = 0.5 μg/

Figure 6. X-ray-derived ORTEP representation of synthetic

viridicatumtoxin B (1). Thermal ellipsoids at 30% probability. Gray

= carbon, blue = nitrogen, red = oxygen, green = hydrogen.

T

dx.doi.org/10.1021/ja506472u | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

Article

Table 1. Minimum Inhibitory Concentration (MIC) Data of Compounds against Gram-Positive and Gram-Negative Bacteria

and Comparison with Selected Literature Data

Gram-(+)

Gram-(−)

a

this study

ref 1

this study

ref 1

E. faecalis E. faecium MRSA

E. faecalis

KCTC5191

E. faecium

KCTC3122

MRSA

CCARM3167

A. baumannii

AB210

A. calcoaceticus

b

E. coli

CCARM1356

b

entry

S613

501

371

KCTC2357

(−)-7

(−)-9

( )-1

(+)-2⁷⁸

( )-V2

( )-V3

( )-V4

( )-V5

( )-V6

4

2

1

4

2

8

4

8

2

−

2

−

0.5

−

0.5

4

0.5

64

−

1

−

>64

0.5

1

0.5

1

c

1

4

1

−

0.25

2

>64

0.5

4

0.5

2

−

4

1

0.5

−

b

−

64

c

−

a

MIC assays were run in triplicate; data are given in units of μg/mL. Taken from ref 1 for comparison. Enantiopure material [(+)-1] isolated from

Penicillium sp. FR11 was used in ref 1.

mL; MRSA 371: MIC = 2 μg/mL) leading to the conclusion

that this functionality is not necessary for antibacterial activity

in this subclass of tetracyclines. Of note is the loss of

considerable potency in going from the natural to the opposite

C15 conﬁguration [analogues ( )-V3 and ( )-V4] as shown in

Table 1. Interestingly, methyl ethers ( )-V5 and ( )-V6, also

lacking the C4a hydroxyl moiety, demonstrated potent

antibacterial properties against E. faecalis S613 [( )-V5: MIC

= 1 μg/mL; ( )-V6: MIC = 0.5 μg/mL], E. faecium 501

[( )-V5: MIC = 1 μg/mL; ( )-V6: MIC = 0.5 μg/mL], and

MRSA 371 [( )-V5: MIC = 8 μg/mL; ( )-V6: MIC = 2 μg/

mL]. These results further support the conclusion that the C4a

hydroxyl group of the viridicatumtoxin analogues is not

necessary for biological activity (see Table 1). Finally, despite

the previously reported activity of viridicatumtoxins against

several Gram-negative bacterial strains,¹our tested compounds

were inactive against A. baumannii AB210, consistent with

previous reports suggesting that the C4-dimethylamino residue

is important for imparting the broad-spectrum activity observed

for both minocycline (7) and tigecycline (9).⁷⁹Incorporation

of such a moiety into the viridicatumtoxin scaﬀold could

expand their antibacterial proﬁle as well as improve their

pharmacological properties.⁷⁹

In preliminary experiments to probe the mode-of-action of

viridicatumtoxin analogues, time-kill assays were performed to

measure the killing of E. faecalis S613 by viridicatumtoxin A (2)

and ( )-V6 alongside tigecycline (9). The motivation for this

study was previous reports that tetracycline analogues with an

aromatic C-ring [e.g., viridicatumtoxin A (2) and ( )-V6] act

via a bactericidal mechanism as opposed to a bacteriostatic one

(i.e., inhibition of the bacterial ribosome).⁸⁰Our time-kill assays

clearly indicated that both viridicatumtoxin A (2) and ( )-V6

act bacteriostatically and not bactericidally (see Supporting

Information for additional data).⁸¹Although ( )-V6 did not

meet the criterion for bactericidal activity, the ability of ( )-V6

to kill E. faecalis S613 was similar to the clinically used and

bacteriostatic antibiotic tigecycline (9). If viridicatumtoxins

[i.e., viridicatumtoxin A (2) and ( )-V6] are indeed inhibitors

of the bacterial ribosome, as opposed to inhibitors of UPP

synthase as suggested by Tomoda and co-workers,³then based

on the structure of tigecycline (9) bound to the Thermus

thermophilus ribosome, the C4−C7 positions of viridicatumtox-

ins are likely ideal sites for further modiﬁcations, as those

positions do not directly interact with the ribosome.⁸²

CONCLUSIONS

■

In summary, we described herein the evolution of synthetic

strategies toward viridicatumtoxin B [( )-1] that ﬁnally led to

the development of a viable route for the total synthesis of this

intriguing natural product. During the course of our studies, we

discovered various chemical transformations and acquired

synthetic knowledge expected to prove valuable for the

synthesis of other fungal tetracyclines and related compounds.

In particular, we delineated tactics and strategic requirements

for the installation of the hydroxyl groups at C4a and C12a.

Other important ﬁndings include the anthrone alkylation/

spirocyclization reactions to create the congested EF-

spirosystem as well as the Michael−Dieckmann cascade

reactions that led to the construction of rings A and C. Most

notably, our synthetic eﬀorts led to structural revision and

conﬁgurational assignment of viridicatumtoxin B. Furthermore,

application of the developed synthetic technologies allowed the

preparation of a number of simpler analogues of the natural

product endowed with antibacterial properties comparable with

those of viridicatumtoxins A and B, despite their racemic

nature. The described chemistry paves the way for further

studies, including an asymmetric synthesis of viridicatumtoxins

and the design, synthesis, and biological evaluation of other

analogues for potential applications as antibacterial agents.⁸³

ASSOCIATED CONTENT

* Supporting Information

Experimental procedures and characterization data for key

compounds (pdf and cif ﬁles). This material is available free of

charge via the Internet at http://pubs.acs.org.

■

S

AUTHOR INFORMATION

Corresponding Author

■

kcn@rice.edu

Present Address

^#Department of Chemistry, Rice University, 6100 Main St.,

Houston, Texas 77005.

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Author Contributions

^⊥These authors contributed equally.

U

dx.doi.org/10.1021/ja506472u | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

Article

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Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

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We thank Drs. D. H. Huang and L. Pasternack for NMR

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from the National Institutes of Health (USA) (grant

AI055475), the Skaggs Institute of Research, the Cancer

Prevention & Research Institute of Texas (CPRIT), and The

Welch Foundation. Fellowships to C.R.H.H. (NSF Graduate

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dx.doi.org/10.1021/ja506472u | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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(83) For a recent review on the role of chemical synthesis in

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G. Angew. Chem., Int. Ed. 2014, DOI: 10.1002/anie.201310843.

X

dx.doi.org/10.1021/ja506472u | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Total synthesis of viridicatumtoxin B and analogues thereof: Strategy evolution, structural revision, and biological evaluation

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