Total synthesis and biological evaluation of the tetramic acid based natural product harzianic acid and its stereoisomers

Article abstract of DOI:10.1021/ol503717r

The bioactive natural product harzianic acid was prepared for the first time in just six steps (longest linear sequence) with an overall yield of 22%. The identification of conditions to telescope amide bond formation and a Lacey-Dieckmann reaction into one pot proved important. The three stereoisomers of harzianic acid were also prepared, providing material for comparison of their biological activity. While all of the isomers promoted root growth, improved antifungal activity was unexpectedly associated with isomers in the enantiomeric series opposite that of harzianic acid.

Full text of DOI:10.1021/ol503717r

Letter
pubs.acs.org/OrgLett
Total Synthesis and Biological Evaluation of the Tetramic Acid Based
Natural Product Harzianic Acid and Its Stereoisomers
Alan R. Healy,† Francesco Vinale,‡ Matteo Lorito,‡,§ and Nicholas J. Westwood*,†
†School of Chemistry and Biomedical Sciences Research Complex, University of St. Andrews and EaStCHEM, North Haugh, St.
Andrews, Fife KY169ST, U.K.
‡CNR − Istituto per la Protezione Sostenibile delle Piante (IPSP-CNR), 80055 Portici, Italy
§Dipartimento di Agraria, Universita
̀
degli Studi di Napoli “Federico II”, 80138 Portici, Italy
S
* Supporting Information
ABSTRACT: The bioactive natural product harzianic acid was prepared for the first time in just six steps (longest linear
sequence) with an overall yield of 22%. The identification of conditions to telescope amide bond formation and a Lacey−
Dieckmann reaction into one pot proved important. The three stereoisomers of harzianic acid were also prepared, providing
material for comparison of their biological activity. While all of the isomers promoted root growth, improved antifungal activity
was unexpectedly associated with isomers in the enantiomeric series opposite that of harzianic acid.
Scheme 1. Retrosynthetic Analysis of Harzianic Acid (1a)
arzianic acid (1a) is a member of a subfamily of
tetramic acid containing natural products that is defined
H
by the presence of an unnatural 4,4-disubstituted glutamic
acid unit. Despite the unique and potent biological activities
displayed by members of this subfamily,1 there has been little
synthetic activity until our recently reported total synthesis of
JBIR-22.5
As a means of exploring further our synthetic approach to
members of this subfamily, we next looked to prepare
harzianic acid (1a), a secondary metabolite isolated from
Trichoderma harzianum.2 This filamentous fungus is used as a
biopesticide and biofertilizer due to its growth-promoting and
antifungal properties, and the production of 1a by the fungus
has been implicated in this biological activity.3,4 The relative
and absolute stereochemistry of 1a was assigned by Vinale et
al. using small-molecule X-ray crystallography (CCDC
745241) after an initial report by Sawa et al.2,6 More recently,
the C5′-isomer of 1a, referred to as isoharzianic acid (1b), has
also been isolated from the same fungus.7
Here, we report the first total synthesis of 1a and its three
stereoisomers, including isoharzianic acid (1b). To the best of
our knowledge, no synthetic routes to 1a and 1b have been
reported to date. All of the stereoisomers were assessed for
their antifungal and plant growth-promoting activities. Our
synthetic approach used the masked 4,4,-disubstitued glutamic
acid 3 as a core fragment that could be combined with the
appropriate polyene fragment 4 (Scheme 1). A late-stage
Lacey−Dieckmann condensation of 2 to form the tetramic
acid core in 1a would limit the requirement for protecting
groups and remove any issues associated with carrying the
tetramic acid core through the synthesis, as this unit is both
base labile and a powerful chelator of metals.8,9 The synthetic
route began with the synthesis of the masked 4,4-disubstituted
glutamic acid 3 using our recently reported methodology
Received: December 25, 2014
© XXXX American Chemical Society
A
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(summarized in Scheme 2).5 A highly diastereoselective aldol
condensation of the tert-butanesulfinamide imine derived from
Scheme 4. Total Synthesis of Harzianic Acid (1a) and 5′-
Isoharzianic Acid (1b)
Scheme 2. Synthesis of Masked 4,4-Disubstituted Glutamic
Acid Fragment 35
ethyl pyruvate 5 and ethyldimethyl pyruvate followed by a
subsequent N-methylation gave lactone 6. A one-pot
diastereoselective reduction and cleavage of the N-sulfinyl
group then provided 3, the stereochemistry of which was
assigned on the basis of X-ray crystallographic analysis of 610
and NOE studies on 3.5 This methodology has proved very
robust with access to 3 being achieved rapidly on multigram
scale.
As 3 was obtained in sufficiently high purity from 6, it was
decided to trap crude 3 directly with the required side chain 4
(Scheme 1) after a simple neutralization step. Assembly of the
polyene side chain 4 began with DDQ oxidation of
commercially available (E)-2-hexenol (7) to the correspond-
ing known aldehyde 8 (Scheme 3). In tandem, Meldrum’s
during the initial coupling of 3 and 4. Optimization of this
reaction by changing the solvent from THF to acetonitrile
resulted in the isolation of harzianic acid ethyl ester 12 in
good yield (75% from lactone 6) in just two steps (Scheme 4,
route B). This highly efficient procedure enables the synthesis
of harzianic acid ethyl ester 12 from lactone 6 in just 1 day.
Finally, hydrolysis of harzianic acid ethyl ester 12 with
aqueous NaOH (2N) using microwave irradiation provided a
readily separable 3:1 mixture of harzianic acid (1a) and 5′-
isoharzianic acid (1b), which resulted from partial epimeriza-
tion at the C5′ position, a common issue observed with
tetramic acids.9,15 Harzianic acid (1a) was synthesized in just
six steps (longest linear route from ethyl pyruvate) with an
overall yield of 22%. The NMR spectral data, HRMS, and the
specific rotation obtained for the synthetic harzianic acid (1a)
were in excellent agreement with the published data for the
natural sample (Figure 1A and Table S1, Supporting
Information).2 We next turned our attention to the synthesis
of the (R,R) enantiomer of harzianic acid 1c and its epimer
(S,R)-5′isoharzianic acid (1d). The described synthetic
sequence was repeated using (S)-tert-butanesulfinamide to
provide the (R,R) enantiomer of our key intermediate 3,
which was converted in an analogous manner to provide 1c
and 1d in high purity and quantities (Scheme S1 and Table
S2, Supporting Information).
Harzianic acid (1a) and isoharzianic acid (1b) have both
been reported to display antifungal activity and to increase
seed germination and shoot and root growth in canola and
tomato seedlings.6,7,16 The plant growth promoting activity of
harzianic acid (1a) is believed to be linked to its potent
siderophoric properties.16 Microbial siderophores are iron-
chelating agents involved in iron solubilization, which is a
crucial mechanism in plant nutrient regulation.17−19 Con-
sistent with this, harzianic acid (1a) has been shown to
increase seedling growth even under iron-deficient conditions
and also increased iron concentration in the plants.16 The
antifungal and plant growth promoting activities of harzianic
Scheme 3. Synthesis of β-Ketothioester Fragment 4
acid was condensed with diethylphosphonoacetic acid to give
9, which was refluxed with tert-butyl thiol in acetonitrile to
yield 10.11 Finally, a Horner−Wadsworth−Emmons reaction
of aldehyde 8 with the dianion generated from 10 provided
the required β-ketothioester 4 in good yield (Scheme 3).12
The synthesis of harzianic acid (1a) was completed via
modification of the silver trifluoroacetate mediated coupling of
4 and crude 3 following the protocol developed by the Ley
group.13,14 This approach initially gave the desired β-keto
amide 11 in good yield. Compound 11 was then cyclized
t
using BuOK to provide harzianic acid ethyl ester 12 as a
single diastereomer (Scheme 4, route A). However, it was also
observed that a small quantity (<10%) of 12 was formed
B
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Figure 1. (A) Comparison of selected 13C NMR signals of isolated
harzianic acid (1a)2 and synthetic 1a and 1b (see Scheme 1 for
1
numbering and Table S1 for a complete comparison of the H and
13C NMR spectral data). (a) 400 MHz, CD3OD; (b) 500 MHz,
1
CD3OD. (B) Section of the H NMR spectrum of synthetic 1a in
CDCl3 (see the Supporting Information for the full spectrum).
Figure 2. (A) Antibiotic activity of 1a−d against S. rolfsii and (B) P.
ultimum. Concentrations ranged from 1 to 1000 μg plug−1.
acid (1a) have highlighted it as a promising bioactive
compound which could be used as an alternative to living
antagonists. With samples of all four stereoisomers 1a−d in
hand, we decided to assess their relative biological activity in a
series of assays.
ASSOCIATED CONTENT
■
S
* Supporting Information
When tested in an assay to assess the effect of the
compounds on the root length of tomato seedlings, all of the
stereoisomers significantly promoted root length at concen-
trations above 0.001 mM (Figure S2, Supporting Informa-
tion). As it seems likely that 1a and 1b can interconvert under
typical assay conditions (hence complicating interpreta-
tion9,15), results which demonstrate significant differences in
behavior between the two enantiomeric series were also
sought. In this context, assessment of the activity of
stereoisomers 1a−d against the pathogens Sclerotinium rolfsii
and Pythium ultimum showed that while all the stereoisomers
were able to inhibit the pathogens, the two isomers in the
enantiomeric series R,R-1c and S,R-1d were significantly more
active than S,S-1a and R,S-1b (Figure 2).
In summary, we have completed the first total synthesis of
harzianic acid 1a and its stereoisomers 1b−d via a short,
stereoselective route involving the key masked 4,4-disubsti-
tuted glutamic acid 3. Variation of the aldehyde 8 and the
pyruvate starting material would facilitate the synthesis of a
wide range of analogues through this convergent route. The
antifungal activity of the two isomers in the enantiomeric
series (R,R-1c and S,R-1d) were significantly more active than
the natural harzianic acid (1a) and isoharzianic acid (1b).
Detailed chemical and biological experimental procedures,
1
spectroscopic data, and copies of H and 13C NMR spectra
for all new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: njw3@st-andrews.ac.uk.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support for this project was provided by Cancer
Research UK (CRUK Grant No. C21383/A6950), Italian
Ministry of Education, University and Research (MIUR),
PON R&C 2007-2013 Programma Operativo Nazionale
̀
Ricerca & Competitivita 2007-2013 (GenoPOM-pro
PON02_00395_3082360, Linfa PON03PE_00026_1 and
SIcurezza e innovazione teCnologica Utile alla salvaguardia e
valorizzazione dei prodotti tipici di oRigine Animale - Sicura),
Campania Region, Piano di Sviluppo Rurale Misura 124
(Progetto Integrato Limone − PIL, Ministry of Economic
C
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1981−2001. (d) Jeong, Y.-C.; Moloney, M. G. Synlett 2009, 2009,
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(16) Vinale, F.; Nigro, M.; Sivasithamparam, K.; Flematti, G.;
Ghisalberti, E. L.; Ruocco, M.; Varlese, R.; Marra, R.; Lanzuise, S.;
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(17) Hofte, M. Classes of Microbial Siderophore. In Iron Chelation
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Development, Italy (Grape and Health Wine − GHW)). We
also wish to thank Carolyn Horsburgh and Tomas Lebl
(University of St. Andrews), the EPSRC National Mass
Spectrometry Service Centre, Swansea, and Roberta Marra,
Roberta Quarto, and Roberta Panza (IPSP-CNR and
Universita
assistance.
̀
degli Studi di Napoli “Federico II”) for technical
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