Synthesis and evaluation of structural requirements for antifungal activity of cyrmenin B1 analogues

Article abstract of DOI:10.1016/j.tetlet.2011.11.023

In a study aiming to define the structural elements essential to cyrmenin B₁ antifungal activity, the synthesis of a series of analogues was carried out. The structural modification introduced stepwise in distinct areas of the parent molecule a

Full text of DOI:10.1016/j.tetlet.2011.11.023

Tetrahedron Letters 53 (2012) 228–231
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and evaluation of structural requirements for antifungal activity
of cyrmenin B1 analogues
Narayan S. Chakor a, Sabrina Dallavalle a, , Loana Musso a, Paola Sardi b
⇑
a Dipartimento di Scienze Molecolari Agroalimentari, Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy
b Dipartimento di Protezione dei Sistemi Agroalimentare e Urbano e Valorizzazione delle Biodiversità, Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In a study aiming to define the structural elements essential to cyrmenin B1 antifungal activity, the syn-
thesis of a series of analogues was carried out. The structural modification introduced stepwise in distinct
areas of the parent molecule allowed a deeper insight to be gained into the most relevant structural fea-
tures affecting the activity of the natural compound. The bioassay results clearly indicate the relevance of
each portion of the parent molecule, only the modification of the conjugated double bond geometry being
tolerated.
Received 12 September 2011
Revised 27 October 2011
Accepted 4 November 2011
Available online 10 November 2011
Keywords:
Cyrmenin
Ó 2011 Elsevier Ltd. All rights reserved.
Natural products
Dehydropeptides
Antifungal activity
Synthesis
Chemical crop protection has become an essential tool to guar-
antee product yields and quality and to meet the ever-growing de-
mands of humans.1
of natural products. They exhibit high antifungal activity, showing
at the same time an exceptionally low toxicity for animal cell
cultures.4a
Nature has been a very fruitful source of new compounds and
natural substances still exert a strong influence on modern crop
protection development, by serving as lead structures for the dis-
covery of active molecules, often featuring novel and unique
modes of action.2
In a previous paper we reported the first total synthesis of cyr-
menin B1 (9a) and of its (8E,10E)-geometrical isomer (9b).5
Due to its modular nature, the developed synthetic route al-
lowed the preparation of some representative derivatives and ana-
logues for structure–activity relationship studies. This Letter
discusses the approaches to the optimization of the lead structure,
to gain a deeper insight into the most relevant structural features
affecting the antifungal activity of cyrmenin B1.
Promising biological activity and broad spectrum against differ-
ent plant pathogens promoted the choice of fungicides originating
from microbial sources. However, microbial fungicides have well
known limitations in their development and practical use. Disad-
vantages are for example the low productivity of fungicidal metab-
olites by microorganisms when compared to the yields of synthetic
chemicals, and often the low stability in the conditions of use in
agriculture.3
Our strategy to modulate the activity focused on modifications
in three distinct areas of the molecule: the lipophilic unsaturated
Recently, Sasse et al.4 have isolated novel antifungal metabo-
lites, named cyrmenin A, B1 and B2 (Fig. 1), from the culture broth
of strains of the myxobacteria Cystobacter armeniaca and Archan-
gium gephyra.
R
O
H
9
8
COOMe
N
11
N
10
H
O
MeO
The cyrmenins are modified N-acyldipeptide esters containing a
dehydroalanine,
a 2-amino-3-methoxyacrylate moiety, and a
R =
Cyrmenin A
Cyrmenin B1
Cyrmenin B2
(2E,4Z)-undecadienoic or dodecadienoic acid residue. With their
two adjacent dehydroaminoacids and an N-substituted 2-amino-
3-methoxyacrylate unit, the cyrmenins represent a unique series
R =
R =
⇑
Corresponding author. Tel.: +39 2 50316818; fax: +39 2 50316801.
E-mail address: sabrina.dallavalle@unimi.it (S. Dallavalle).
Figure 1. Structures of the cyrmenins.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.11.023

N. S. Chakor et al. / Tetrahedron Letters 53 (2012) 228–231
229
moiety was then obtained by the treatment of acetyl derivatives
8a–d with the weak Lewis acid LiClO4 and DBU at À15 °C.8 Com-
pounds 9c and 9d were obtained in moderate yields (50–55%).
Concerning Part B modifications, we prepared compound 14
with an alanine moiety in place of the dehydroserine found in
the natural cyrmenin, assuming that its removal could possibly
give advantages such as increased metabolic stability (Scheme 2).
Part B
C7H15
O
H
N
COOMe
N
H
O
MeO
L-Alanine methyl ester hydrochloride was coupled with (2E,4Z)-
2-methyldodeca-2,4-dienoic acid 10 in the presence of BOP and DI-
PEA to afford the amide ester 11 that was quantitatively converted
into acid 12 by alkaline hydrolysis. Subsequent coupling with D,L-
Part A
Cyrmenin B1
Part C
serine methyl ester hydrochloride furnished the dipeptide alcohol
13. IBX oxidation in ethyl acetate gave an unstable aldehyde, which
was immediately converted into b-methoxyacrylate 14 by treat-
ment with TMSCHN2.
Compounds 7a and 7b (Scheme 1), intermediates in the synthe-
sis of cyrmenin B1 and its geometrical isomer 9b, respectively, can
be considered as cyrmenin analogues modified at the central Part
B, as they bear a hydroxymethyl group in place of the exo double
bond.
Finally, we tried to verify if any antifungal activity could be re-
tained by cyrmenin derivatives lacking the methoxyacrylate moi-
ety (Part C modifications). The synthesis was achieved following
the pathway outlined in Scheme 3. Condensation of compound
1a with two units of D,L-serine methylester hydrochloride afforded
compound 16 that, after double acetylation and elimination, gave
compound 18 in a 52% yield.
The activity of cyrmenin B1 (9a), and of 7 analogues (9b–d, 14,
7a–b, 18) was tested against Bacillus subtilis (Ehrenberg) Cohn,
strain IPV 2430, Saccharomyces cerevisiae Meyen ex Ec. Hansen,
strain IPV 637, Aspergillus niger Tegh., strain IPV F303, Botrytis cine-
rea Pers., strain IPV F5.2, Cochliobolus miyabeanus, (S.Ito and Kurib)
Drechsler ex Dastur, and Pyricularia oryzae Cavara, strain IPV A1,
according to the method reported by Sasse et al.4a
Figure 2. Regions of cyrmenin B1 modified in the synthetic analogues.
chain (Part A, Fig. 2), the dehydroalanine moiety (Part B) and the b-
methoxyacrylate system (Part C).
To elucidate the role of the conjugated (8E,10Z) double bonds
(Part A), we prepared and tested, besides the natural compound
9a, the unnatural cyrmenin (9b) with (8E,10E) double bonds. The
synthetic pathway, summarised in Scheme 1, started from N-acyl-
serine derivatives 1a and 1b, prepared from (2E,4Z)-2-methyldo-
deca-2,4-dienoic acid and (2E,4E)-2-methyldodeca-2,4-dienoic
acid.5 Exploiting the same strategy, we prepared compounds 9c
and 9d bearing a phenyl ring and a saturated chain in the left hand
side, respectively. The synthesis of 9c started from benzoylserine
methyl ester 1c.6 The synthesis of 9d entailed the hydrogenation
of the N-acyl dipeptide alcohol 4b followed by oxidation of the
product by treatment with IBX in ethyl acetate at reflux, to give
the desired aldehyde 5d. The required b-methoxyacrylate group
was inserted while reacting 5a–d, as a tautomeric mixture of
keto/enol forms, with TMSCHN2 in methanol/toluene.7 Deprotec-
tion of the silyl ether by treatment with TBAF and acetylation gave
the dipeptide alcohol 7a–d in high yield. The dehydroaminoacid
OH
OTBDPS
COOR'
OTBDPS
O
O
O
H
d
a
c
COOCH3
N
R
R
N
H
COOCH3
R
N
H
R
N
H
O
OH
2a-c: R'= OCH3
3a-c: R'= OH
4a-c
g
1a-c
b
OR"
OTBDPS
H
OTBDPS
H
O
O
O
H
f
COOMe
N
N
COOMe
N
COOCH3
R
N
R
N
H
N
H
H
O
H3CO
O
O
O
H
H3CO
7a-d: R"= H
8a-d: R"= COCH3
6a-d
5a-d
e,d
h
i
4b
C7H15
O
H
N
COOMe
R
N
H
c
d
R =
R =
a
b
R =
R =
O
H3CO
C10H21
C7H15
9a (Cyrmenin B1),
9b-d
Scheme 1. Synthesis of compounds 9a–d. Reagents and conditions: (a) TBDPSCl, imidazole, CH2Cl2, 0 °C, 95–97%; (b) LiOH, THF/H2O; (c)
D,
L-serine methyl ester
hydrochloride, BOP, iPr2EtN, THF, 0 °C to rt, 53–76%; (d) IBX, EtOAc, reflux; (e) Pd/C (10%), EtOAc, 24 h, rt; (f)TMSCHN2, toluene/methanol, rt, 24–65%; (g) TBAF, THF, 0 °C to rt,
54–75%; (h) AcCl, Py, CH2Cl2, 0 °C to rt, 88–97%; (i) LiClO4, DBU, THF, À15 °C to rt, 50–58%.

230
N. S. Chakor et al. / Tetrahedron Letters 53 (2012) 228–231
C7H15
O
C7H15
C7H15
O
H
N
COOCH3
OH
COOH
N
H
N
H
COOR
a
c
O
O
10
11 R= OCH3
12 R= OH
13
d,e
b
C7H15
O
H
N
COOCH3
OCH3
N
H
14
Scheme 2. Synthesis of alanine derivative 14. Reagents and conditions: (a)
L
-Alanine methyl ester hydrochloride, BOP,iPr2NEt,THF, 0 °C to rt, 3h, 95%; (b) LiOHÁH2O, THF/H2O,
1 h; (c) -serine methyl ester hydrochloride, BOP, iPr2NEt,THF, 0 °C to rt, 3 h, 75%; (d) IBX, EtOAc, reflux, 7 h; (e) TMSCHN2, CH3OH/H2O, rt, 2 h, 66%.
D,L
OH
OH
COOH
C7H15
O
C7H15
O
H
N
COOCH3
OH
b
a
1a
N
H
N
H
O
16
15
OAc
C7H15
O
C7H17
O
H
H
N
c
COOCH3
OAc
N
COOCH3
d
N
N
H
H
O
O
17
18
Scheme 3. Synthesis of derivative 18. Reagents and conditions: (a) LiOH, THF/H2O; (b) D,L-serine methyl ester hydrochloride, BOP, HOBT, THF, 53%; (c) acetyl chloride,
pyridine, CH2Cl2, 85%; (d) LiClO4, DBU, THF, 52%.
Table 1
The data confirmed the activity of cyrmenin B1 against some fil-
amentous fungi such as Pyricularia and Aspergillus sp. However, in
our experiments we did not observe any activity of cyrmenin B1 to-
wards S. cerevisiae and B. cinerea. The biological data regarding the
analogues featured by modifications at Part A highlight that Z
geometry of the double bond at position 10 is more favourable
for the activity than the E geometry. Moreover, the replacement
of the conjugated double bonds with the corresponding saturated
chain (9a, 9b vs 9d) caused a complete loss of antifungal activity.
Compound 9c, bearing a phenyl group in place of the (2E,4Z)-dode-
cadienoic residue, was inactive as well. These results underline
that the conjugated diene, preferably with (8E,10Z) geometry, is
an essential moiety.
Percent inhibition of mycelial growth induced by compounds 9a and 9b
Concn
l
gm LÀ1
Aspergillus nigera
Pyricularia oryzaeb
9a
9b
9a
9b
25
50
100
200
5
5
0
5
32
40
52
65
28
31
40
44
11
27
9
11
a
Standard compound tetraconazole, 100% inhibition at 20 mg LÀ1
.
.
b
Standard compound azoxystrobin 100% inhibition at 100 mg LÀ1
The modifications introduced at the exo double bond of the par-
ent molecule (Part B) demonstrate that also this element is crucial
for the antifungal activity. In fact, both compounds 14, bearing an
alanine residue, and 7a and 7b, bearing a serine, were inactive
against all the tested strains.
The replacement of the methoxy group in the b-methoxyacry-
late group with a hydrogen atom (compound 18) again resulted
in a complete loss of antifungal activity.
Collectively, the cyrmenin B1 analogues, except for the geomet-
rical isomer 9b, showed a complete lack of antifungal activity.
Thus, only 9a and 9b were further investigated.
The growth inhibition of A. niger and P. oryzae induced by these
compounds was assayed by the commonly used ‘poisoned food’
technique. The activity at four different concentrations is reported
in Table 1 as percent inhibition of mycelial growth in comparison
to the untreated control (For experimental details see Supplemen-
tary data).
The data showed that cyrmenin B1 (9a) is effective for the
reduction of P. oryzae mycelial growth, with the reduction of spor-
ulation, while its effect against A. niger at the same concentration
was moderate. Compound 9b showed activity against both fungi
even if lower than the activity showed by the parent molecule.
In conclusion, a series of cyrmenin B1 analogues were synthe-
sized and assayed for antifungal activity and structure–activity
relationship studies. Only the modification of the conjugated dou-
ble bond geometry (9a vs 9b) was tolerated. The complete lack of
activity of compounds 9c and 9d underlines that simply maintain-
ing the lipophilicity of portion A in the molecule is not enough to
have active compounds. Replacement of the exo methylene group
with a simple methyl group was also detrimental. The modification

N. S. Chakor et al. / Tetrahedron Letters 53 (2012) 228–231
231
of the expectedly crucial (compare strobilurins9 and analogues) b-
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Acknowledgments
The authors gratefully acknowledge Mr. Elio Burrone for the
antifungal tests and the University of Milan (FIRST funds) for finan-
cial support.
Supplementary data
Supplementary data (experimental procedure and spectral data
for all compounds) associated with this article can be found, in the
online version, at doi:10.1016/j.tetlet.2011.11.023.