Total synthesis of thaxtomin A and its stereoisomers and findings of their biological activities

Article abstract of DOI:10.1021/ol4026556

The first and facile total synthesis of thaxtomin A and its three stereoisomers has been achieved. The synthetic approach involves intramolecular nucleophilic cyclization of an amide toward a ketoamide group to produce a C-hydroxydiketopiperazine scaffold

Full text of DOI:10.1021/ol4026556

ORGANIC
LETTERS
2013
Vol. 15, No. 22
5670–5673
Total Synthesis of Thaxtomin A and Its
Stereoisomers and Findings of Their
Biological Activities
Hongbo Zhang,†,‡ Xin Ning,‡ Hang Hang,‡ Xuyan Ru,‡ Haichen Li,§ Yonghong Li,†
Lizhong Wang,† Xiao Zhang,† Shujing Yu,† Yuanyuan Qiao,§ Xin Wang,*,†,‡ and
Peng George Wang*,†,‡
State Key Laboratory of Elemento-organic Chemistry, College of Pharmacy, and
College of Chemistry, Nankai University, Tianjin 300071, PR China
wangxinnk@nankai.edu.cn; pwang@nankai.edu.cn
Received September 13, 2013
ABSTRACT
The first and facile total synthesis of thaxtomin A and its three stereoisomers has been achieved. The synthetic approach involves intramolecular
nucleophilic cyclization of an amide toward a ketoamide group to produce a C-hydroxydiketopiperazine scaffold. The most amazing discovery was
that each of the four stereoisomers of TA exhibits different phytotoxic, fungicidal, and antiviral activities.
The growing need for new pesticides with new molecular
target sites and the desire for “greener” pest management
chemicals is increasingly the focus of interest in natural
products.1 Thaxtomins are a unique family of phytotoxins
generated as microbial metabolites possessing a unique
4-nitroindole moiety.2 Thaxtomin A (TA), the most pre-
dominant and active member of such a family, was initially
isolated in 1989.3 TA attracted extensive attention due to
its establishedroleasa virulencefactorinthe common scab
potato disease and its new herbicidal mode of action.4,5
Recent research indicated that TA is a green and safe
herbicide to rice6 and also may be used as a selective agent
to elevate the overall levels of common scab resistance in
the potato breeding populations.7 The most distinguish
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Pathol. 2002, 60, 1–8. (c) Tegg, R. S.; Melian, L.; Wilson, C. R.; Shabala,
S. Plant Cell Physiol. 2005, 46, 638–648. (d) Bischoff, V.; Cookson, S. J.;
Wu, S.; Scheible, W. R. J. Exp. Bot. 2009, 60, 955–965. (e) Brochu, V.;
Girard-Martel, M.; Duval, I.; Lerat, S.; Grondin, G.; Domingue, O.;
Beaulieu, C.; Beaudoin, N. BMC Plant Biol. 2010, 10, 272. (f) Tegg,
R. S.; Shabala, S. N.; Cuin, T. A.; Davies, N. W.; Wilson, C. R. BMC
Plant Biol. 2013, 13, 76.
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D. C.; Doricchi, L.; Perez Baz, M. J.; Millan, F. R.; Fernandez Chimeno, R. I.
WO 2010121079, 2010.
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Eyles, A.; Conner, A. Plant Pathol. 2009, 58, 137–144. (b) Hiltunen,
L. H.; Alanen, M.; Laakso, I.; Kangas, A.; Virtanen, E.; Valkonen, J.
Plant Pathol. 2011, 60, 426–435.
† State Key Laboratory of Elemento-organic Chemistry.
‡ College of Pharmacy.
§ College of Chemistry.
(1) (a) Cantrell, C. L.; Dayan, F. E.; Duke, S. O. J. Nat. Prod. 2012,
75, 1231–1242. (b) Dayan, F. E.; Owens, D. K.; Duke, S. O. Pest Manag
Sci. 2012, 68, 519–528.
(2) (a) Kers, J. A.; Wach, M. J.; Krasnoff, S. B.; Widom, J.; Cameron,
K. D.; Bukhalid, R. A.; Gibson, D. M.; Crane, B. R.; Loria, R. Nature
2004, 429, 79–82. (b) King, R. R.; Calhoun, L. A. Phytochemistry 2009,
70, 833–841. (c) Barry, S. M.; Kers, J. A.; Johnson, E. G.; Song, L.;
Aston, P. R.; Patel, B.; Krasnoff, S. B.; Crane, B. R.; Gibson, D. M.;
Loria, R.; Challis, G. L. Nat. Chem. Biol. 2012, 8, 814–816.
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J. Chem.Soc., Chem. Commun. 1989, 13, 849–850.
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Chem. 1992, 40, 834–837. (b) Park, D. H.; Yu, Y. M.; Kim, J. S.; Cho,
J. M.; Hur, J. H; Lim, C. K. Plant Dis. 2003, 87, 1290–1296. (c) Hill, J.;
Lazarovits, G. Plant Pathol. 2005, 27, 46–52. (d) Loria, R.; Kers, J.;
Joshi, M. Annu. Rev. Phytopathol. 2006, 44, 469–487.
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Zhang, L.; Pei, Y.; Jing, Y.; Hua, H. Chem. Biodivers. 2012, 9, 385–393.
r
10.1021/ol4026556
Published on Web 10/25/2013
2013 American Chemical Society

structural feature of TA is the C-hydroxydiketopiperazine
scaffold. In fact, such a scaffold exists in several classes of
natural products (Figure 1).8 Thus, a facile synthetic approach
to produce the C-hydroxydiketopiperazine scaffold is very
desirable for total synthesis and further structural modifica-
tion of these natural products. Furthermore, the chemical
synthesis may shed light on how the stereochemical properties
of chiral pesticide TA have played a role in its biological
activities, which has never been answered previously.
itself were accomplished to date.9a,b Protocol B provided
hemiaminal product via intramolecular nucleophilic cycli-
zation of an amide toward a ketoamide group. Despite of
the scattering appearance of protocol B in literature,10 it
has never been well studied, especially on factors affecting
the cyclization and stereochemistry.
Scheme 1. Protocols for Constructing C-Hydroxydiketopiper-
azine9,10
In this work, we report the first total synthesis of TA and
its three stereoisomers through protocol B (Scheme 1) by
investigating the cyclization reaction and its stereoselectiv-
ity in great detail. Moreover, the most amazing discovery was
that each stereoisomer of TA exhibits different bioactivities,
indicating the versatile role of C-hydroxydiketopiperazine
scaffold for developing new biologically active chemicals.
Figure 1. Typical natural products containing C-hydroxydike-
topiperazine.8
The total synthesis of TA has not been achieved. This
might be attributed to the challenge of stereocontrolled
formation of the C-hydroxydiketopiperazine.9 When con-
structing a 2,5-diketopiperazine scaffold bearing an N-acyl
hemiaminal chiral center which was observed in many
nature products,8 there were two established protocols as
shown in Scheme 1. Protocol A or A0 features the dehy-
drated intermediate a2. However, when applying this
protocol for the synthesis of TA, considering the incom-
patibility of the nitro group toward most of the reduction
manipulations, and the high epimerization risk of the
hemiaminal chiral center in basic or acidic media, it is
difficult to find proper protecting groups, which are not
only suitable for the preparation of a2 from a1 or a10 but
also orthogonal in the final global deprotection. Such
restrictions might well explain why only the synthesis of
some structurally simplified TA derivatives rather than TA
Scheme 2. Synthesis of Compound 8
(9) (a) Gelin, J.; Mortier, J.; Moyroud, J.; Chene, A. J. Org. Chem.
1993, 58, 3473–3475. (b) Moyroud, J.; Gelin, J.; Chene, A.; Mortier, J.
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6585–6596.
Our synthesis started with the known dimethyl N-Boc-
glutamate 111 which was readily prepared from L-glutamic
acid in two steps. Then, the N-methylation of 1 with MeI
and Ag2O in DMF produced 2 in a yield of 90%. The
diester 2 was reduced by DIBAL-H, followed by the
ꢀ
(11) Kokotos, G.; Padron, J. M.; Martı
´
n, T.; Gibbons, W. A.;
Martın, V. S. J. Org. Chem. 1998, 63, 3741–3744.
´
Org. Lett., Vol. 15, No. 22, 2013
5671

Table 1. Explorations on the Intramolecular Nucleophilic Cyclization
conditions
results
conditions
LiOH, MeOH
DABCO, MeOH
DIEA, MeOH
conversion (%)
C-10 dea (%)
CF3COOH, THF or MeOH
CH3COOH, THF or MeOH
TsOH, THF or MeOH
LiCl, THF or MeOH
ZnCl2, THF or MeOH
FeCl3, THF or MeOH
CuCl2, THF or MeOH
AlCl3, THF or MeOH
no reaction (ꢀ)
100 (yield: 90%)
<5
<5
<5
<5
<5
<5
<5
67
90
93
99
99
99
99
99
99
99
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Et3N, MeOH
Et3N, LiCl, MeOH
Et3N, ZnCl2, MeOH
Et3N, FeCl3, MeOH
Et3N, CuCl2, MeOH
Et3N, AlCl3, MeOH
MgBr2 Et2O, THF or MeOH
NaH, THF
<5
3
Et3N, MgBr2 Et2O, MeOH
100 (yield: 90%)
3
a C-10 de = (TA ꢀ m-TA)/(TA þ mTA).
treatment with PCC togivealdehyde 3 in 85% yield. Direct
couplingofaldehyde3with3-nitro-2-iodoaniline412 under
standard Pd-catalyzed indole synthesis condition provided
the 4-nitrotryptophan derivative 5 in 80% yield. By using
methylamine, the ester moiety was converted into the
secondary amide, which was then deprotected by trifluor-
oacetic acid, the 4-nitrotryptophan derivatives 6 could be
obtained in a 90% yield over these two steps. After
extensive exploration, the fragment arylpyruvic acid 713
was introduced to the specific secondary amide with the
coupling agent T3P in 80% yield (Scheme 2).
Scheme 3. Preparation of TA and Iso-TA
With the product 8 in hand, the next task was to achieve
the intramolecular cyclization, a well-recognized challenge
to achieve. First, to avoid the epimerization of C-10, we
have screened a variety of acids to catalyze such transfor-
mation, but such acid conditions did not catalyze the
reaction. Second, although the C-hydroxydiketopipera-
zine was constructed under inorganic base condition, the
epimerization of C-10 occurred, and the C-10 de values
were all below 70%. Third, some organic bases were used
to catalyze this reaction. Luckily, these did result in a
significance increase in C-10 de values (>90%). Especially
when treatment of compound 8 with Et3N in methanol, the
C-10 de value was up to 99% with a ratio of TA/iso-TA
(i-TA) of 2.6:1. However, the reaction conversion was only
5% under this condition. To increase the reaction conversion
rate while maintaining high stereoselectivity, we screened a
variety of Lewis acid into the Et3N/MeOH reaction system.
Ultimately, it was found that the treatment of compound 8
satisfactory. It was later found that the mixture of diaste-
reoisomers from treatment withE3N inMeOHcould reach
an equilibrium state quantitatively and without C-10 de
lost. The ratio of TA/i-TA was 2.6:1. After recrystalliza-
tion from methanol, pure TA was obtained. Treatment of
mother liquid with E3N/MeOH converse the ratio of TA/i-
TA to 2.6:1 again. Thus, this method may allow facile
large-scale access to TA (Scheme 3).
The structure of TA and i-TA were confirmed by single-
1
crystal X-ray analysis (Figure 2). The H NMR and 13C
NMR spectra of synthetic TA were in good agreement
with those of authentic samples.3 Through the same syn-
thetic route, mirror-TA (m-TA) and mirror-iso-TA (mi-TA)
were synthesized using D-glutamic acid to replace
L-glutamic acid as the starting material showed in Scheme 2.
Phytotoxic activity, fungicidal activity, and antiviral
activity of these four TA stereoisomers were evaluated
using reported procedures.14 Generally, stereoisomers of
chiral pesticides may differ with respect to biological
with Et3N/MgBr2 Et2O in Methanol under room tempera-
3
ture for 7 days could produce TA and its C-12 epimer i-TA in
1
90% yield with a ratio of TA/i-TA of 1:2.5 by H NMR
analysis. The C-10 de value of TA is 99% (Table 1).
Although this represented a truly significant increase in
synthetic efficiency for TA, the diastereoselectivity is not
(13) Wong, H. N.; Xu, Z. L.; Chang, H. M.; Lee, C. M. Synthesis
1992, 793–797.
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P.; Wang, Z.; Song, H.; Zou, X.; Yang, H. J. Agric. Food Chem. 2008, 56,
9535–9542. (b) Wang, Z.; Xu, H.; Yu, S.; Feng, Q.; Wang, S.; Li, Z.
J. Agric. Food Chem. 2010, 58, 3651–3660. (c) Wu, M.; Han, G.; Wang,
Z.; Liu, Y.; Wang, Q. J. Agric. Food Chem. 2013, 61, 1030–1035.
(12) Sienkowska, M.; Benin, V.; Kaszynski, P. Tetrahedron 2000, 56,
165–173.
5672
Org. Lett., Vol. 15, No. 22, 2013

Table 2. Herbicidal Activity of Compounds (Percent Inhibition)
(Rate = 1500g/ha)a
BC
AR
EC
post pre post pre post
30.0 70.0 40.0 30.0 45.0 10.0
DS
compd
TA
pre
post
pre
25.0 80.0
20.0 10.0
10.0 10.0
i-TA
10.0 15.0 10.0 15.0
0
0
15.0
10.0
10.0
0
m-TA
mi-TA
10.0
5.0 10.0
0
0
0
0
5.0
20.0 100
10.0 15.0
0
0
dichlobenil 100
5.0 95.4
64.9
Figure 2. X-ray of TA and iso-TA.
a BC = B. campestris, AR = A. retroflexus, EC = E. crusgalli,
DS = D. sanguinalis; post = postemergence; pre = pre-emergence. All
treatments were performed in triplicate.
activity, plant metabolism, environmental fate, and eco-
toxicity.15 It has been previously shown that the 10S,12S
epimer of TA has reduced phytotoxic activity.16 Our bioassay
result showed that among four stereoisomers, only natural
product TA with the 10S,12R configuration showed obvious
growth inhibitory activity on dicotyledon weeds B. campestris
and A. retroflexus (dichlobenil, a commercial herbicide, was
used as a control, Table 2). This absolute stereoselectivity was
also important in the antifungi activity. All four stereoisomers
were tested in vitro against Cercospora arachidicola and
Physalospora piricola. Only TA’s enantiomer m-TA showed
excellent activity, while the other three stereoisomers have
weak or no inhibitory activity (Table 3). Furthermore, the
fungicidal activities for m-TA and commercial fungicide
chlorothalonil were compared. The EC50 values of m-TA
against Cercospora arachidicola and Physalospora piricola
were 8.52 and 1.57 μg mLꢀ1, respectively, while the corre-
sponding values for chlorothalonil were 8.64 and 7.33 μgmLꢀ1,
respectively, indicating that m-TA has similar anti-Cercos-
pora arachidicola activity as chlorothalonil and obviously
better anti-Physalospora piricola activity than chlorothalonil.
For our antiviral activity investigation, anti-tobacco mosaic
virus (TMV) activities of these four TA stereoisomers were
compared with a benchmark antiviral drug ribavirin. The
assay results in Table 4 showed that i-TA displayed higher
activity than ribavirin and mi-TA exhibited the same level
of activity as ribavirin, while TA and m-TA did not
exhibit inhibitory activity at all. These results showed
that stereochemistry plays a determining role on their
antiviral activities.
Table 3. Fungicidal Activity of Compounds at a Dosage of
50 μg/mL (Percent Inhibition)a
fungi
TA
i-TA
m-TA
mi-TA
CH
CA
PP
0
7.1 ( 0.6 78.6 ( 1.6
0
78.0
43.9 ( 1.7 24.4 ( 0.6 96.0 ( 2.0 24.4 ( 1.3 89.0
a CA = Cercospora arachidicola, PP = Physalospora piricola,
CH = chlorothalonil. Values are mean ( SD (n = 3).
Table 4. Antiviral Activity of Compounds against TMV at
500 μg/mL (Percent Inhibition)
compd
inactivation efect
curative efect
protection efect
TA
10.4
42.3
6.4
0
8.6
44.2
0
i-TA
38.9
0
m-TA
mi-TA
ribavirin
34.8
37.6
36.1
38.4
40.6
38.2
that natural product TA has obvious growth inhibitory
activity on dicotyledon weeds B. campestris and A. retro-
flexus, m-TA has simmilar antifungi activity as fungicide
chlorothalonil, i-TA has higher anti-TMV activity than
antiviral drug ribavirin, and mi-TA exhibited the same
level of activity as ribavirin. The strict stereoselective
properties of the four stereoisomers indicated that the
C-hydroxydiketopiperazine scaffold represents a rich
resource for developing new biologically active chemicals.
In conclusion, natural product TA and its three stereo-
isomers have been synthesized for the first time. A con-
certed catalytic condition using Et3N/MgBr2 Et2O has
3
been developed to implement the construction of a
C-hydroxydiketopiperazine scaffold by an intramolecular
nucleophilicadditionstrategy. The convenientpurification
procedure of TA from diastereoisomeric mixtures and the
conversion of i-TA into TA in situ make large scale
production possible. The biological test results indicated
Acknowledgment. We thank the National Natural
Science Foundation of China (Grant Nos. 21102076 and
91013013) and the Natural Science Foundation of Tianjin
(Grant Nos. 10JCYBJC04100) for financial support.
Supporting Information Available. Experimental de-
tails, characterization data, 1H and 13C NMR spectra of
new compounds, and X-ray data for TA and iso-TA
(CIF). This material is available free of charge via the
Internet at http://pubs.acs.org.
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The authors declare no competing financial interest.
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