Total synthesis of brevianamide A

Article abstract of DOI:10.1038/s41557-020-0442-3

The fungal-derived bicyclo[2.2.2]diazaoctane alkaloids are of interest to the scientific community for their potent and varied biological activities. Within this large and diverse family of natural products, the insecticidal metabolite (+)-brevianamide A is particularly noteworthy for its synthetic intractability and inexplicable biogenesis. Despite five decades of research, this alkaloid has remained an elusive target for chemical synthesis due to insurmountable issues of reactivity and selectivity associated with all previously explored strategies. We herein report the chemical synthesis of (+)-brevianamide A (seven steps, 7.2% overall yield, 750 mg scale), which involves a bioinspired cascade transformation of the linearly fused (?)-dehydrobrevianamide E into the topologically complex bridged-spiro-fused structure of (+)-brevianamide A. [Figure not available: see fulltext.]

Full text of DOI:10.1038/s41557-020-0442-3

Articles
https://doi.org/10.1038/s41557-020-0442-3
Total synthesis of brevianamide A
Robert C. Godfrey, Nicholas J. Greenꢀ ꢀ, Gary S. Nichol and Andrew L. Lawrenceꢀ ꢀ
ꢀ✉
The fungal-derived bicyclo[2.2.2]diazaoctane alkaloids are of interest to the scientific community for their potent and varied
biological activities. Within this large and diverse family of natural products, the insecticidal metabolite (+)-brevianamide A is
particularly noteworthy for its synthetic intractability and inexplicable biogenesis. Despite five decades of research, this alka-
loid has remained an elusive target for chemical synthesis due to insurmountable issues of reactivity and selectivity associated
with all previously explored strategies. We herein report the chemical synthesis of (+)-brevianamide A (seven steps, 7.2% over-
all yield, 750ꢀmg scale), which involves a bioinspired cascade transformation of the linearly fused (−)-dehydrobrevianamide E
into the topologically complex bridged-spiro-fused structure of (+)-brevianamide A.
he Diels–Alder cycloaddition is one of the most important
Despite decades of detailed chemical and biochemical stud-
reactions in synthetic organic chemistry, allowing the ste- ies, the biosynthetic origins of (+)-brevianamide A (1) and B (2)
T
reoselective construction of six-membered rings through remain unknown. We herein propose a modified biosynthetic
the concerted formation of two new bonds (Fig. 1a)1. The limited hypothesis that builds on the pioneering work of several research
appearance of Diels–Alder reactions in biosynthetic pathways has groups7,8. To appreciate the origins of this modified biosynthetic
always fascinated chemists2–6. The bicyclo[2.2.2]diazaoctane alka- proposal it is important first to outline key details of the elegant
loids, which are a vast group of natural products isolated from work of Williams and co-workers26–29. Their biomimetic synthetic
various marine and terrestrial fungi, have played a key role in the studies have mainly focused on an early proposed pathway (Fig. 2,
development of our understanding of biosynthetic Diels–Alder pathway 1)33, which was informed by the seminal work of Birch and
reactions7,8. They are also of broader interest to both chemists and Sammes20,34–36. The pathway begins with a stereoablative oxidation
biologists alike due to their important biological activities, diverse of (+)-deoxybrevianamide E (3) to give achiral azadiene 4, which
biosynthetic origins and synthetically daunting structures. There then undergoes enantioselective Diels–Alder cycloaddition to give
are two distinct families of bicyclo[2.2.2]diazaoctane alkaloids, a scalemic mixture of bicyclo[2.2.2]diazaoctane enantiomers, 5 and
the monooxopiperazine-type structures (Fig. 1b), which include ent-5. Both enantiomers then undergo (R)-selective indole oxida-
the anthelmintic paraherquamides9–11
,
calmodulin-inhibiting tion and a [1,2]-alkyl shift, so that the major enantiomer (5) gives
malbrancheamides12 and neuroprotective chrysogenamides13; and brevianamide A (1) and the minor enantiomer (ent-5) gives brevi-
the dioxopiperazine-type structures (Fig. 1c), which include the anamide B (2). Williams’s synthetic studies, however, revealed that
cytotoxic stephacidins and notoamides14,15, and the insecticidal this proposed Diels–Alder reaction (4 to 5/ent-5) actually produces
brevianamides16,17
.
an unwanted diastereomer as the major product29. Furthermore,
Brevianamides A (1) and B (2) were originally isolated by Birch biosynthetic feeding experiments with isotopically labelled 5 failed
and Wright in 1969 from the fungus Penicillium brevicompac- to show notable incorporation into (+)-brevianamide A (1)37.
tum16, and were the first known bicyclo[2.2.2]diazaoctane alkaloids Williams, therefore, proposed an alternative biosynthetic pathway
(Fig. 1c)7,8. Brevianamide A (1) exhibits potent antifeedant activ- (Fig. 2, pathway 2), which begins with a diastereoselective indole
ity against the larvae of the insect pests Spodoptera frugiperda (fall oxidation of (+)-deoxybrevianamide E (3) to give hydroxyindo-
armyworm) and Heliothis virescens (tobacco budworm)17, and is the lenine 637,38. A stereospecific [1,2]-alkyl shift then gives indoxyl 7,
major isolated diastereomer (d.r. ≳90:10)18,19. In 1970, Porter and which undergoes a diketopiperazine oxidation and Diels–Alder
Sammes proposed that the bicyclo[2.2.2]diazaoctane cores of brevi- cycloaddition to give brevianamides A (1) and B (2)37,38. Williams
anamides A (1) and B (2) could be biosynthesized through an intra- and co-workers reasoned that “[t]he preponderance of 1 over
molecular hetero-Diels–Alder cycloaddition (Fig. 1d)20, a proposal 2 would be due either to the relative activities of two different
that has been extended to encompass all bicyclo[2.2.2]diazaoctane [Diels–Alderase] enzymes or the affinity of a single enzyme active
alkaloids7,8. Recently, Diels–Alderase enzymes have been identi- site for the individual conformers”37. All efforts to substantiate this
fied in the biosynthetic gene clusters responsible for the malbran- second-generation pathway in vitro, however, have failed, largely
cheamide and paraherquamide monooxopiperazine-type alkaloids21. due to the instability of indoxyl 77. Furthermore, hydroxyindolenine
However, no Diels–Alderase enzyme has yet been identified for the 6 is likely to undergo rapid 5-exo-trig cyclization to give the known
brevianamides, or for any other dioxopiperazine-type alkaloid22,23
.
shunt-metabolite brevianamide E (8)16,34, which is proposed to be a
Furthermore, despite five decades of research, the chemical synthesis biosynthetic dead-end37.
of brevianamide A (1) has remained an elusive target, although sev-
Our modified biosynthetic hypothesis for (+)-brevianamide
eral syntheses of the minor diastereomer, brevianamide B (2), have A (1) involves an alternative biosynthetic precursor, (+)-dehydro
been reported (for a full summary, see Supplementary Section 1)24–32
.
deoxybrevianamide E (9), which is a known natural product isolated
Despite the similar structures of the two natural products, none fromvariousPenicilliumandAspergillusspecies(Fig.3)39–41.Crucially,
of these strategies have been successfully applied to the synthesis the diketopiperazine ring in (+)-dehydrodeoxybrevianamide E (9)
of brevianamide A (1) due to insurmountable issues of reactivity is already at the oxidation level required for a subsequent Diels–
and selectivity.
Alder reaction. Our pathway involves a point-to-point chirality
✉
EaStCHEM School of Chemistry, University of Edinburgh, Edinburgh, UK. e-mail: a.lawrence@ed.ac.uk
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a
d
Diels–Alder
[π4s π2s]
O
+
N
N
OH
Diene
O
Dienophile
b
c
Diastereomers
O
O
O
Cl
HO
NH
O
NH
HN
HN
N
NH
HN
O
O
H
H
O
H
H
Cl
O
O
H
H
HN
O
H
HN
O
H
HN
O
O
NH
HN
O
O
HN
MeN
O
N
N
N
O O
N
N
N
O
N
HO
1
2
(+)-Brevianamide A (+)-Brevianamide B
(–)-Paraherquamide
Anthelmintic
(+)-Malbrancheamide
Calmodulin inhibitor
(+)-Chrysogenamide A
Neuroprotective
(+)-Stephacidin A
Cytotoxic
(–)-Notoamide A
Cytotoxic
Major
Minor
Insecticidal
Fig. 1 | Diels–Alder cycloaddition and representative examples of bicyclo[2.2.2]diazaoctane alkaloids, which are proposed to be biosynthesized
via intramolecular hetero-Diels–Alder reactions. a, Diels–Alder cycloaddition. b, Representative monooxopiperazine-type bicyclo[2.2.2]diazaoctane
alkaloids (bicyclo[2.2.2]diazaoctane cores highlighted in blue). c, Representative dioxopiperazine-type bicyclo[2.2.2]diazaoctane alkaloids (bicyclo[2.2.2]
diazaoctane cores highlighted in green). d, Sammes’ proposed biosynthetic hetero-Diels–Alder reaction. Coloured circles indicate general substituents for
the Diels–Alder reaction components.
Enantiomers
Diastereomers
H
N
NH
HN
N
N
(R)
Diastereoselective
and enantioselective
[4 + 2]
Enantiodivergent
[O]
(R)
H
H
OH
OH
NH
N
N
OH
O
O
O
O
(e.r. ≥90:10)
Diels–Alderase
required?
(R)-selective
HN
NH
HN
N
N
N
N
O
O
O
O
O
4
5
ent-5
Minor
[1,2]-shift
Major
H
N
Pathway 1
[O]
NH
O
HO
HN
H
N
HN
O
O
H
Shunt
metabolite37
O
H
H
N
O
H
O
H
O
H
O
N
NH
HN
N
Pathway 2
N
N
O
O
3
8
(+)-Deoxybrevianamide E
(–)-Brevianamide E
1
2
(+)-Brevianamide A
Major
(+)-Brevianamide B
Minor
N
O
O
Diastereoselective
[1,2]-shift
[O]
[4 + 2]
H
H
HO
N
O
NH
N
N
OH
NH
H
N
O
H
(d.r. 90:10)
Diels–Alderase
required?
H
O
H
N
O
O
N
6
7
10
Fig. 2 | Previous biosynthetic proposals for brevianamides A and B. In 1989, building on the earlier work of Birch and Sammes, Williams and co-workers
proposed pathway 1 as a plausible biosynthetic pathway towards brevianamides A and B33. In 1993, following their synthetic and biosynthetic studies,
Williams and co-workers proposed an alternative order of biosynthetic transformations, as shown in pathway 237. Biosynthetic feeding experiments with
isotopically labelled 5 and (−)-brevianamide E (8), however, show no notable incorporation into 1 or 2.
transfer via a sequential diastereoselective indole oxidation, a ste- intermediate 11 is likely to undergo reversible 5-exo-trig cycli-
reospecific [1,2]-alkyl shift and tautomerization, to give enantio- zation to give pentacycle 12, this is not a known natural product
pure azadiene 10. Diels–Alder cycloaddition of azadiene 10 would and is therefore unlikely to represent a dead-end (cf. brevianamide
then give brevianamides A (1) and B (2)37,38. Although our proposed E (8) in pathway 2, Fig. 2). We herein report the total synthesis of
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N
ethanolamine and phenylhydrazine, resulted in unwanted cleavage
of the amide bond. Our search for less nucleophilic reagents eventu-
ally led us to ammonia in methanol47, which not only deprotected
the primary amine but also resulted in spontaneous cyclization to
give (+)-dehydrodeoxybrevianamide E (9) in 49% yield over the
three steps from methyl ester 15. Thus, by developing a new imine
acylation reaction and avoiding unnecessary protecting group
manipulations,thetotalsynthesisof(+)-dehydrodeoxybrevianamide
E (9) has been achieved, proceeding in a longest linear sequence
of five steps, in 34% overall yield, and requiring just two chro-
matographic purifications (cf. previous synthesis: 12 steps, 8%
overall yield)29.
H
N
Diastereoselective
[O]
H
N
HO
O
H
N
O
H
O
H
O
N
N
11
9
(+)-Dehydro-
[1,2]-shift
deoxybrevianamide E
O
H
N
NH
O
NH
Oxidation of (+)-dehydrodeoxybrevianamide E (9) using a
variety of oxidants (for example, dioxiranes43, oxaziridines23, sin-
glet oxygen48 and peroxy acids) gave dehydrobrevianamide E (12),
alongside diastereomer 20 (Fig. 4). Use of m-CPBA gave the high-
est diastereoselectivity (d.r. 64:36, 57% yield), with no appreciable
improvement observed at lower temperatures. The stereoselectivity
of related biosynthetic indole oxidations is known to be controlled
by flavin-dependent monooxygenase enzymes8. Synthetic access
to both diastereomers 12 and 20, however, enabled us to prepare
both the natural-(+) and unnatural-(−) enantiomers of brevian-
amides A (1) and B (2) (see below). Exposure of dehydrobrevian-
amide E (12) to LiOH in water at ambient temperature for 30 min
successfully gave (+)-brevianamide A (1) and (+)-brevianamide
B (2) in a combined 63% yield, presumably via the domino retro-
5-exo-trig/[1,2]-alkyl shift/Diels–Alder reaction sequence outlined
in Fig. 3. Keeping the reaction time to a minimum and avoiding
elevated temperatures was essential to prevent alkaline hydrolysis
of the products, as previously observed by Birch and co-workers34.
Following purification by column chromatography, which is only
the fourth purification in the entire seven-step synthesis, 750 mg of
(+)-brevianamide A (1) and 60 mg of (+)-brevianamide B (2) were
isolated. Chiral-HPLC analysis revealed both products (1 and 2)
were isolated in a 93:7 enantiomeric ratio, which is not surprising
given many of our synthetic intermediates may have undergone
partial racemization; recrystallization gave (+)-brevianamide A (1)
in an enantiomeric ratio of 99:1. The unnatural (−)-enantiomers of
brevianamides A (1) and B (2) were similarly accessed by subjecting
the minor diastereomer, compound 20, to the same reaction condi-
tions (Fig. 4).
HO
H
O
N
N
O
H
Tautomerization
O
N
O
12
NH
N
N
OH
O
O
HN
HN
O
O
10
H
H
Diastereoselective
[4 + 2]
O
NH
HN
N
N
O
O
(d.r. 90:10)
Diels–Alderase
required?
1
2
(+)-Brevianamide A
Major
(+)-Brevianamide B
Minor
Fig. 3 | A modified biosynthetic proposal for brevianamides A and B.
The known natural product (+)-dehydrodeoxybrevianamide E (9),
which has the diketopiperazine ring at the oxidation level required for
a subsequent Diels–Alder reaction, is invoked as an alternative biosynthetic
precursor towards brevianamides A (1) and B (2). This avoids the
intermediacy of indoxyl 7 (Fig. 2, pathway 2), the instability of which
has thwarted previous biomimetic approaches. Although oxidation
of (+)-dehydrodeoxybrevianamide E (9) is expected to give compound 12,
akin to the oxidative ring closure of 3 to give 8 (Fig. 2, pathway 2),
(−)-dehydrobrevianamide E (12) is not a known natural product and is
therefore less likely to represent a biosynthetic dead-end.
The Diels–Alder reaction produces (+)-brevianamide A (1) and
(+)-brevianamide B (2) in a 93:7 diastereomeric ratio (Fig. 4), which
brevianamide A (1), following a strategy inspired by our modified closely matches the ratio observed when they are isolated from
biosynthetic proposal.
Penicillium brevicompactum (for further details, see Supplementary
Section 2)18. Thus, we suggest that this Diels–Alder reaction might
be a spontaneous process that occurs naturally without direct
Results and discussion
The synthesis of (+)-dehydrodeoxybrevianamide E (9) commenced enzyme participation37. This is in contrast to the requirement of
with phthaloyl protection of commercially available l-tryptophan Diels–Alderase enzymes in the stereoselective biogenesis of the
methyl ester 13 (Fig. 4)42. The crude product from this reaction, closely related malbrancheamide and paraherquamide alkaloids21.
ester 14, was subjected directly to Danishefsky’s reverse prenylation During the review process for this manuscript, Williams, Sherman,
conditions using B-prenyl-9-borabicyclo[3.3.1]nonane to give inter- Li and co-workers reported their identification of the brevianamide
mediate 15 in 69% yield over the two steps43. Hydrolysis of methyl A biosynthetic gene cluster49. Their detailed biosynthetic studies,
ester 15 was accompanied by ring opening of the phthaloyl group to which included targeted gene disruption, heterologous expression,
give diacid 16, which presumably explains why a protecting group precursor incorporation studies and in vitro biochemical analysis
switch from phthaloyl to tert-butyloxycarbonyl or trityl has been also support a spontaneous, non-enzyme-mediated, biosynthetic
undertaken in related synthetic endeavours43,44. To avoid these extra- Diels–Alder reaction49.
neous two steps (deprotection/reprotection), we explored the pos-
In summary, 50 years after its discovery by Birch16, the chemical
sibility of an SN2-type demethylation of methyl ester 15. Although synthesis of brevianamide A (1) has been achieved (seven steps, 7.2%
LiI in EtOAc, as reported by Fisher and Trinkle45, gave the desired overall yield). Key to the success of this synthesis is a bioinspired
product 17, we found LiCl in DMF gave a cleaner reaction. Lithium one-step cascade transformation of the linearly fused pentacyclic
carboxylate 17 was then subjected to a one-pot acyl chloride forma- dehydrobrevianamide E (12) into the far more complex hexacy-
tion and imine acylation reaction with dehydroproline 18 to give clic structure of brevianamide A (1). This complexity-generating
N-acyl enamine 1946. Attempts to deprotect the primary amine sequence would probably not have been constructed from a purely
in 19 using conventional phthaloyl deprotection reagents, such retrosynthetic analysis of the target, demonstrating the unique
as hydrazine, ethylene diamine, methylamine, hydroxylamine, utility of the biomimetic approach in natural product synthesis.
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O
H
N
H
N
H
N
H
N
O
LiOH, H2O/THF
O
(2) t-BuOCl, NEt3, THF
r.t., 3 h
(1)
O
H
N
NH2
N
NPhth
OMe
NEt3, toluene
111 °C, 18 h
94%
H
O
H
O
H
O
H
O
B(9-BBN)
O
O
CO2H
OMe
OMe
OH
Steps (1) and (2), 69%
First chromatographic
purification
14
13
15
[21 g]
16
Commercially
available
(3) LiCl, DMF
160 °C, 21 h
H
N
H
N
H
N
CO2Me
H
N
H
N
(5) NH3/MeOH
r.t., 18 h
(4) (COCl)2, DMF
NEt3, CH2Cl2
Commercially
available
O
NPhth
CO2Me
H
O
NPhth
OLi
N
H
O
Steps (3)–(5), 49%
N
CO2Me
NCS, NEt3
79%
H
O
Second chromatographic
purification
CH2Cl2, 0 °C, 3 h distillation
N
9
18
0 °C to r.t., 21 h
(+)-Dehydrodeoxy-
brevianamide E
[8.5 g]
N
19
17
CO2Me
18
57% (d.r. 36:64)
[15 g]
Third
(6) m-CPBA (1 equiv.)
chromatographic
purification
CHCl3, r.t., 6 h
Domino reaction:
retro-5-exo-trig,
[1,2]-shift,
O
HN
HN
O
H
H
Diels–Alder
NH
NH
+
(7) LiOH, H2O, r.t., 0.5 h
HO
HO
O
O
NH
HN
+
N
O
N
O
N
N
63% (d.r. 93:7)
Fourth chromatographic
purification
O
O
H
O
H
O
N
N
1
2
(+)-Brevianamide A
(+)-Brevianamide B
20
[850 mg]
12
[1.5 g]
[750 mg]
e.r. 93:7
99:1
[60 mg]
e.r. 93:7
Recrystallization
O
NH
NH
O
H
H
(7) LiOH, H2O, r.t., 0.5 h
+
O
X-ray (+)-1
O
HN
60% (d.r. 92:8)
Fourth chromatographic
purification
NH
N
N
O
O
ent-1
ent-2
(–)-Brevianamide A
(–)-Brevianamide B
[128 mg]
e.r. 5:95
[12 mg]
e.r. 8:92
Fig. 4 | Total synthesis of brevianamides A and B. The synthesis begins with the shortest reported total synthesis of (+)-dehydrodeoxybrevianamide
E (9) (five steps, 34% overall yield, 8.5 g scale). Oxidation of (+)-dehydrodeoxybrevianamide E (9) gives a diastereomeric mixture of dehydrobrevianamide
E (12) and 20. Exposure of 12 and 20 to LiOH in water gives the natural and unnatural enantiomers of brevianamides A (1) and B (2), respectively. 9-BBN,
9-borabicyclo[3.3.1]nonane; m-CPBA, meta-chloroperoxybenzoic acid; DMF, dimethylformamide; NCS, N-chlorosuccinimide; Phth, phthaloyl. The black,
blue and red spheres represent carbon, nitrogen and oxygen, respectively.
2. Stocking, E. M. & Williams, R. M. Chemistry and biology of biosynthetic
Online content
Diels–Alder reactions. Angew. Chem. Int. Ed. 42, 3078–3115 (2003).
Any Nature Research reporting summaries, source data, extended
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data, supplementary information, acknowledgements, peer review
information; details of author contributions and competing inter-
ests; and statements of data and code availability are available at
https://doi.org/10.1038/s41557-020-0442-3.
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Data availability
Author contributions
All the characterization data and experimental protocols are provided in this article
and its Supplementary Information. Crystallographic data have been deposited at the
Cambridge Crystallographic Data Centre, under deposition number CCDC 1918446
(compound 1). Copies of the data can be obtained free of charge via https://www.ccdc.
cam.ac.uk/structures/.
R.C.G., N.J.G. and A.L.L. conceived, designed and carried out the synthetic
experiments. G.S.N. performed the crystallographic studies. All authors discussed
and co-wrote the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Acknowledgements
Supplementary information is available for this paper at https://doi.org/10.1038/
s41557-020-0442-3.
This work was supported by an EPSRC First Grant (EP/N029542/1) and a Marie Curie
Career Integration Grant (631132, POSIN). We thank T. Herlt for assistance and advice
regarding chromatography, and acknowledge SIRCAMS at the University of Edinburgh
for mass spectrometry.
Correspondence and requests for materials should be addressed to A.L.L.
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