Cryphonectric acid and other minor metabolites from a hypovirulent strain of Cryphonectria parasitica

Article abstract of DOI:10.1021/np0103012

Investigations carried out on secondary metabolites produced in culture by a hypovirulent strain of Cryphonectria parasitica allowed the isolation of several compounds which were characterized by NMR analysis and derivatization reactions. The most abundant metabolite was a new compound, called cryphonectric acid (1). Other metabolites were diaporthin, the only known phytotoxic compound isolated from both virulent and hypovirulent strains of C. parasitica, (+)-orthosporin, and L-p-hydroxyphenyllactic acid (HOPLA). Root growth activity of the purified compounds was evaluated both on tomato seedlings and maize subapical segments.

Full text of DOI:10.1021/np0103012

48
J . Nat. Prod. 2002, 65, 48-50
Notes
Cr yp h on ectr ic Acid a n d Oth er Min or Meta bolites fr om a Hyp ovir u len t Str a in of
Cr yph on ectr ia pa r a sitica 1
Alberto Arnone,‡ Gemma Assante,*,† Gianluca Nasini,*,‡ Sara Strada,† and Annamaria Vercesi†
Istituto di Patologia Vegetale, Universita` degli Studi, 20123 Milano, Italy, and CNR, Centro di Studio per le Sostanze
Organiche Naturali, Dipartimento di Chimica, Politecnico di Milano, Via Mancinelli 7, I 20133 Milano, Italy
Received J une 14, 2001
Investigations carried out on secondary metabolites produced in culture by a hypovirulent strain of
Cryphonectria parasitica allowed the isolation of several compounds which were characterized by NMR
analysis and derivatization reactions. The most abundant metabolite was a new compound, called
cryphonectric acid (1). Other metabolites were diaporthin, the only known phytotoxic compound isolated
from both virulent and hypovirulent strains of C. parasitica, (+)-orthosporin, and L-p-hydroxyphenyllactic
acid (HOPLA). Root growth activity of the purified compounds was evaluated both on tomato seedlings
and maize subapical segments.
Cryphonectria (formerly Endothia) parasitica (Murr.)
Barr is the causal agent of chestnut blight, responsible for
the chestnut decline in many areas of Europe and North
America, where very susceptible western Castanea species
(C. sativa Mill. and C. dentata Borkh.) are present.2 The
healing and nonlethal cankers discovered on C. sativa in
Europe led to the isolation of C. parasitica strains char-
acterized by a reduced virulence when inoculated on
susceptible chestnuts.3,4 The aim of the present work is
either to isolate and characterize secondary metabolites
produced by a hypovirulent isolate showing a broad range
of vegetative compatibility or to assess their biological
activity on selected test plants.
The EtOAc crude extracts obtained from cultures of a
hypovirulent strain of C. parasitica designated E13, grown
on MPGA, were separated using a series of silica gel
chromatography columns and stepwise elution with organic
solvents. Four of the several compounds obtained were
completely characterized: metabolite 1, a new aromatic
acid, along with diaporthin, previously isolated from cul-
tures of virulent strains of C. parasitica,5 a trace of (+)-
orthosporin,6 and L-p-hydroxyphenyllactic acid (+)-HOPLA,
also produced by Candida spp. and various bacterial
species.7
Acetylation of 1 gave the tetraacetate derivative 2, which
showed a molecular peak at m/z 486 (EIMS). The H NMR
1
spectrum revealed a broad signal at δ 9.60 (1′-CO2H) (two
meta-coupled aromatic protons H-4 and -6, J ) 2.0 Hz), of
a broad singlet (H-7), which was correlated with an sp3
oxygen-bearing carbon; also present were two more signals
attributable to acetate groups (3′- and 5′-OAc) and to two
aromatic protons (H-2′ and -6′), which sharpened by
heating at ∼50 °C, indicating restricted rotation of the
phenyl group about the C(7)-C(4′) bond. The 13C NMR
spectrum gave four acetate signals and two carbonyl
signals (C-2 and -7′) and confirmed that one of the two
tetrasubstituted aromatic rings has a symmetry axis
passing through the C(1′), C(4′) carbons, since C-2′ and -6′
and C-3′ and -5′ presented double intensity.
The relative disposition of substituents around the
aromatic rings was deduced along the following lines. The
Cryphonectric acid (1) is the metabolite most abundantly
produced by E13, representing more than 20% in weight
of the crude extract; 1 is a highly polar solid, slightly
soluble in organic solvents. Cryphonectric acid 1, isolated
as a pale yellow powder, mp >300 °C (dec), [R]D +9.2° (c
0.1, MeOH), analyzed for C15H10O8, and the formula was
supported by EIMS (M+, 318). Its IR spectrum (KBr)
showed bands at 3430 cm-1 (OH) and at 1750 cm-1
,
attributable to an ester-like function; the absorption at
1720 cm-1 indicated a further carbonyl function; the UV
spectrum (EtOH) showed absorptions at 206, 256, and 293
nm (ꢀ 26350, 9600, and 3500).
* To whom correspondence should be addressed. Tel: +39 2 23993046.
Fax: +39 2 23993080. E-mail: nasini@dept.chem.polimi.it; assante@unimi.it.
† Istituto di Patologia Vegetale.
‡ CNR, Centro di Studio per le Sostanze Organiche Naturali.
10.1021/np0103012 CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/08/2001

Notes
J ournal of Natural Products, 2002, Vol. 65, No. 1 49
and 62.9 MHz for 13C NMR; chemical shifts are in ppm (δ)
from TMS as internal standard. HPLC analyses were per-
formed on a LiChrograph L-6000A (Merck-Hitachi) equipped
with a L-4000 detector (λ ) 225 nm) and D-2500 integrator,
using a Merck RP-18, 0.45 × 25 cm column with CH3CN-
H2O (1-1.5, v/v) as eluent at a nominal flow rate of 0.5 cm3
carboxylic moiety was placed at C-1′, since irradiation of
H-2′ and -6′ caused the removal of three-bond couplings
from C-7′; C-7 was linked to both the aromatic rings since
H-7 presented long-range couplings with C-2a, -6, -6a, -3′,
-4′, and -5′, while one of the two meta protons was placed
at C-6 since it presented mutual NOEs with H-7 (3%). On
the basis of these results, the four acetoxy functions were
located at C-3, -5, -3′, and -5′ and the C-2 lactone carbon
was joined to C-2a to complete the molecular formula of 2.
Compound 2 was reacted subsequently with a solution of
diazomethane, giving the ester 3. The structure 1 proposed
for cryphonectric acid was confirmed by reaction of the fully
methylated compound 4 with LAH, to obtain the trihydroxy
derivative 6 via the opening of the lactone moiety and the
reduction of the two carbonyl ester functions. Long-range
coupling of 4.0 Hz between C-6 and H-8 and not between
C-6 and H2-1 in the corresponding triacetate derivative 7,
attributable to a three-bond interaction between the two
atoms, placed C-8 at C-7.8 Finally, the mutual NOEs
observed between H-8 and H2-1 (Experimental Section)
indicated that C-1 and C-8 are ortho-positioned.
As far as the stereochemistry of C-7 is concerned, the
CD values of 1 were very low (Experimental Section) and
the [R] value changed with time from +9.2° (0 time) to
+27.7° (1 day). Moreover in compound 6, CD and [R] values
were null; in our opinion the C-7 center must be racemic
and the optical activity must be attributed to atropoisom-
erism around the chirality axis C7-C4′ due to the steric
hindrance of the two substituents in the ortho position of
ring C. This hypothesis is in agreement with the broaden-
ing of the NMR signals of 2, which disappears by heating
at ∼50 °C. Cryphonectric acid (1) is unusual, but nor-
wedelic acid, a benzofuranic acid isomer of 1, occurs in
Wedelia calendulaceae.9
Metabolites 1, diaporthin, and (()-HOPLA, tested using
a tomato seedling bioassay up to 2 mM, did not induce any
necrotic symptoms on leaves. However, formation of tomato
seedlings was totally inhibited by 100 µM 1 and 200 µM
diaporthin, while a 75 and 50% inhibitory effect was
observed by using 100 and 10 µM diaporthin, respectively.
No root inhibition was detected in the presence of 10 µM 1
and 2 and 0.2 mM (()-HOPLA. In a second bioassay on
elongation of subapical segments of maize roots, 2 and 0.2
mM (()-HOPLA determined respectively a 16 and 35%
increase in the length of the treated samples, comparing
with the control. The results are in agreement with the
interference with cell differentiation shown by the culture
filtrates of E13 C. parasitica strain on chestnut calli of C.
sativa and C. mollissima.10 Further work is required in
order to characterize the physiological effects of the com-
pounds produced by E13 on the cultured cells of both
susceptible and resistant Castanea species.
min-1
.
Isola tion of 1, Dia p or th in , (+)-Or th osp or in , a n d (+)-
HOP LA. A hypovirulent strain of C. parasitica, E13, from the
Mycological Collection of the Plant Pathology Institute, Uni-
versity of Milan, was grown on MPGA (malt extract, peptone,
glucose, agar, 20-2-20-15 g L-1, pH 6.8) medium in 30 Roux
flasks at 24 °C in the dark for 14 days. The mycelia were
extracted twice with ethyl acetate containing 1% MeOH, and
the extracts (0.8 g) were chromatographed on a silica gel
column with stepwise elution using CH2Cl2-MeOH to obtain
diaporthin (30 mg), (+)-orthosporin (2 mg), (+)-HOPLA (15
mg), and cryphonectric acid (1) (220 mg). The known com-
pounds were further purified by PLC in hexanes-EtOAc (1:
1) or CH2Cl2-MeOH (15:1), while 1 was isolated using an RP-
18 silica gel HPLC column, using acetone-H2O (1:1) as the
eluent.
Dia p or th in a n d (+)-Or th osp or in . Both compounds were
identified by comparison of mp, [R]D, MS, and 1H NMR with
literature data.6
L-p -Hyd r oxyp h en ylla ctic a cid : mp 165-168 °C, [R]D +18°
(c 0.5, MeOH); 5 mg was treated with an ethereal solution of
CH2N2 to obtain the methylester, EIMS, m/z 196[M]+(10%),
178[M - 18]+(15), and 107(100); the compound was identified
by TLC, MS, and 1H NMR to a methyl ester of a commercial
sample of (()-HOPLA.
Cr yp h on ectr ic a cid (1): EIMS m/z 318[M]+(20%), 300[M
- 18]+(28), and 107(100); CD (c 0.1 mg/mL, MeOH) 208 and
223 nm (∆ꢀ -2.2 and -1.0); 1H NMR (acetone-d6) δ 9.10 (5H,
br signal, 4 × OH and 1′-CO2H), 7.14 (2H, s, H-2′ and -6′),
6.95 (1H, br s, H-7), 6.38 and 6.28 (2H, br d, J ) 2.0 Hz, H-4
and -6). anal. C 56.45%, H 3.10%, calcd for C15H10O8, C 56.61%,
H 3.17%.
Com p ou n d 2. Compound 1 (30 mg) was dissolved in dry
pyridine (0.5 mL) containing Ac2O (0.8 mL), and the solution
was kept at 0 °C for 12 h. The mixture was then poured into
ice-water and extracted with CH2Cl2. From PLC in hexanes-
EtOAc (1:1) of the residue, tetraacetate 2 (25 mg) was isolated
as a white solid: mp 160-165 °C, [R]D +48° (c 0.2, MeOH);
EIMS m/z 486 [M]+(4%), 444(50), 402(50), 384(40), 360(58),
1
318(65), and 300(40); H NMR (CDCl3) δ 9.60 (1H, br signal,
7′-OH), 7.73 (2H, br signal, H-2′ and -6′), 7.03 (1H, br d, J )
2.0 Hz, H-4), 6.99 (1H, dd, J ) 2.0 and 0.9 Hz, H-6), 6.52 (1H,
br s, H-7), 2.44 and 2.27 (6H, s, 3-and 5-OAc), 2.15 (6H, br
signal, 3′- and 5′-OAc); 13C NMR (CDCl3) δ 168.7 (s, 3′-and
5′-OCOMe), 168.6 (s, C-7′), 168.5 and 168.2 (s, 3-and 5-O-
COMe), 166.6 (s, C-2), 156.6 (s, C-5), 150.8 (s, C-6a), 150.1 (s,
C-3′ and -5′), 149.0 (s, C-3), 132.4 (s, C-1′), 124.8 (s, C-4′), 122.6
(d, C-2′ and C-6′), 117.4 (d, C-4), 115.6 (s, C-2a), 113.8 (d, C-6),
73.5 (d, C-7), 21.0 and 20.6 (q , 3- and 5-OCOMe), 20.4 (q, 3′-
and 5′-OCOMe). anal. C 56.53%, H 3.78%, calcd for C23H18O12
,
C 56.79%, H 3.73%.
Investigations on the potential angiogenesis inhibition
by 1,11 carried out at two different concentrations, 50 and
12.5 µM, showed neither inhibition of the migration of
endothelial cells nor cytotoxicity or morphological dif-
ferentiation of bovine endothelial cells into capillary-like
structures, suggesting that 1 does not represent a potential
angiogenic inhibitor.
Com p ou n d 3. Compound 2 (10 mg) dissolved in dry CH2-
Cl2 (5 mL) was treated with an ethereal solution of CH2N2 for
10 min, and evaporation of the solution gave 3: EIMS, m/z
501[MH]+, MALDI, m/z 593.5[M + K]+, and 523.3[M + Na]+;
1H NMR (CDCl3) δ 7.71 (2H, s, H-2′ and -6′), 7.03 and 6.99
(2H, br d, J ) 2.0 Hz, H-4 and -6), 6.49 (1H, br s, H-7), 3.92
(3H, s, 7′-OMe), 2.43 and 2.26 (6H, s, 3-and 5-OAc), 2.17 (6H,
br signal, 3′- and 5′-OAc); HREIMS m/z 500.0939 (calcd for
C
24H20O12, 500.0948).
Com p ou n d 4. A solution of compound 1 (100 mg), in CH2-
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r e. Flash CC was per-
formed with Merck silica gel (0.04-0.06 mm) and TLC with
Merck HF254 and RP-18 F254. IR values were determined on
Perkin-Elmer 177 spectrophotometer, MS on a Finnigan-MAT-
TSQ70 spectrometer, and optical rotations on a J ASCO-500
DIP-181 polarimeter. NMR spectra were recorded on a Bruker
AC 250L spectrometer operating at 250.1 MHz for 1H NMR
Cl2-MeOH (9:1, v/v), was reacted repeatedly with a solution
of CH2N2 in dry CH2Cl2, until only one spot appeared on TLC
plates; PLC of the residue on CH2Cl2-MeOH (15:1) gave
compound 4 as white solid: mp 170-175 °C; [R]D -7.6° (c 0.2,
MeOH); IR (KBr) νmax 1753, 1726 and 1614 cm-1; UV(EtOH)
λmax 212, 255, 290, and 310sh nm (ꢀ 40850, 25500, 7900, and
4250); EIMS, m/z 388 [M]+(50%), 357(100), 343(35), 193(50),

50 J ournal of Natural Products, 2002, Vol. 65, No. 1
Notes
and 165(100); 1H NMR (CDCl3) δ 7.22 (2H, br signal, H-2′ and
-6′), 6.89 (1H, dd, J ) 1.0 and 0.5 Hz, H-7), 6.40 (1H, dd, J )
2.0 and 0.5 Hz, H-4), 6.19 (1H, dd, J ) 2.0 and 1.0 Hz, H-6),
3.98 (3H, s, 3-OMe), 3.93 (3H, s, 7′-OMe), 3.80 (3H, s, 5-OMe),
3.75 (6H, br signal, 3′- and 5′-OMe); NOE experiments (CDCl3),
{H-4} enhanced 3-OMe (3%) and 5-OMe (1%), {H-6} enhanced
H-7 (6%) and 5-OMe (2.5%), {H-7} enhanced H-6 (4%), {H-2′
and -6′} enhanced 3′- and 5′-OMe (4%), {3-OMe} enhanced H-4
(19%), {5-OMe} enhanced H-4 (5%) and H-6 (17%); 13C NMR
(CDCl3) δ 169.2 (s, C-2), 166.5 (s, C-5), 166.4 (s, C-7′), 159.1
(s, C-3), 158.9 (s, C-3′ and -5′), 155.2 (s, C-6a), 132.3 (s, C-1′),
116.8 (s, C-4′), 107.7 (s, C-2a), 105.6 (d, C-2′ and -6′), 98.5 and
97.1 (d, C-4 and -6), 73.2 (d, C-7), 56.3 (q, 3′- and 5′-OMe),
56.0 and 55.8 (q, 3- and 5-OMe), 52.4 (q, 7′-OMe). anal. C
62.10%, H 5.10%, calcd for C20H20O8 C 61.85%, H 5.19%.
Com p ou n d 5. A solution of 1 (15 mg), in CH2Cl2-MeOH,
was treated with CH2N2 for 5 min. From PLC of the residue 5
was isolated as an oil: 1H NMR (acetone-d6) δ 9.28 and 8.41
(2H, br s, 3- and 5-OH), 9.05 (2H, br s, 3′- and 5′-OH), 7.08
(2H, s, H-2′ and -6′), 6.92 (1H, dd, J ) 1.0 and 0.5 Hz, H-7),
6.36 (1H, dd, J ) 1.9 and 0.5 Hz, H-4), 6.25 (1 H, dd, J ) 1.9
and 1.0 Hz, H-6), 3.83 (3H, s, 7′-OMe); HREIMS m/z 332.0531
(calcd for C16H12O8 , 332.0528).
(12.5%), {3′-and 5′-OMe} enhanced H-2′ and -6′ (13%); 13C
NMR (acetone-d6) δ 171.8, 171.5, and 170.6 (s, 1-, 8-, and 7′-
OCOMe), 162.0 and 161.0 (s, C-3 and -5), 160.3 (s, C-3′ and
-5′), 144.9 and 140.6 (s, C-6a and -1′), 116.7 and 114.4 (s, C-2
and -4′), 106.3 (d, C-6), 105.7 (d, C-2′ and -6′), 98.0 (d, C-4),
67.6 (d, C-8), 67.0 (t, C-7′), 58.7 (t, C-1), 56.9 (q, 3′- and 5′-
OMe), 56.8 and 56.2 (q, 3- and 5-OMe), 21.6, 21.4, and 21.3
(q, 1-, 8-, and 7′-OCOMe). anal. C 60.95%, H 6.20%, calcd for
C
25H30O10, C 61.21%, H 6.16%.
Tom a to Seed lin g Bioa ssa y. Seedlings of tomato cv. Super
Marmande, grown until the fourth fully expanded leaf, were
cut and transferred to 50 mL of distilled H2O in glass vials
placed in a growth chamber at 24 °C, 70% relative humidity,
and 14/10 photoperiod (light/dark). After 24 h conditioning,
H2O was replaced with test solutions. Compound 1, diaporthin,
and (()-HOPLA (Sigma) were tested for phytotoxic effects and
root growth inhibition at 24, 48, and 72 h, at 2, 0.2, 0.1, and
0.01 mM. All the metabolite solutions were filter sterilized
through 0.2 µm filters. Each assay was performed twice on 10
replicates of tomato seedlings.
Su ba p ica l Ma ize Root Segm en t Bioa ssa y. Maize seed-
lings (Dekalb cv. DK 300), washed in tap H2O for 2 h, were
incubated in 2 L beakers containing 0.5 M CaSO4 for 24 h.
Root apical tips were then eliminated, and the subapical root
segments (0.6 cm) were conditioned in 0.5 M CaSO4 for 2 h.
Afterward, the segments were measured on millimeter paper
and transferred to buffer solution (0.5 mM CaSO4, 0.5 mM KCl,
0.1 mM Mes-Na, pH 6), and buffer solution was added with
0.2 and 2 mM (()-HOPLA. At the end of incubation period
carried out for 4 h on an orbital shaker (80 rpm) at 26 °C, the
segments were remeasured in order to assess their elongation.
The mean values were evaluated on three replicate trials
carried out on 40 segments.
Com p ou n d 6. LAH (40 mg) was added to a solution of 4
(50 mg) in dry THF and the mixture stirred for 1 h at 40 °C;
the reaction was quenched with EtOAc, then acidified with
diluted HCl and extracted with EtOAc; PLC of the residue gave
35 mg of compound 6: white crystals, mp 130-135 °C; UV
(EtOH)λmax 209 and 283 nm (ꢀ 48 700 and 4500); CIMS
(isobutane) m/z, 347[MH - 18]+(70%), 346(50), 329(100), and
179(60); 1H NMR (acetone-d6) δ 6.79 (2H, s, H-2′ and -6′), 6.50
(1H, br d, J ) 10.9 Hz, H-8), 6.49 and 6.25 (2H, br d, J ) 2.4
Hz, H-4 and -6), 4.92 (1H, d, J ) 10.9 Hz, 8-OH), 4.82 (1H,
dd, J ) 11.7 and 8.7 Hz, H-1a), 4.66 (2H, d, J ) 5.7 Hz, H2-7′),
4.65 (1H, dd, J ) 11.7 and 3.8 Hz, H-1b), 4.19 (1H, t, J ) 5.7
Hz, 7′-OH), 3.84 and 3.66 (6H, s, 3- and 5-OMe), 3.82 (6H, s,
3′- and 5′-OMe), 3.46 (1H, dd, J ) 8.7 and 3.8 Hz, 1-OH);
HREIMS m/z 364.1523 (calcd for C19H24O7, 364.1515).
Com p ou n d 7. Compound 6 was acetylated to obtain 7 as
Refer en ces a n d Notes
(1) Secondary Mould Metabolites Part 60. For part 59, see: Arnone, A.;
Merlini, L.; Nasini, G.; Vajna de Pava, O.; Zunino, F. J . Chem. Soc.,
Perkin Trans. 1 2001, 610-616.
(2) Anagnostakis, S. L. Adv. Pl. Pathol. 1988, 6, 123-137.
(3) Heiniger, U.; Rigling, D. Annu. Rev. Phytopathol. 1994, 32, 581-599.
(4) Griffin, G. J . J . Forestry 2000, 98, 22-27.
(5) Ga¨umann, E.; Naef-Roth, S. Pflanzensch. Ber. 1957, 19, 9-16.
(6) Ichihara, A.; Hashimoto, M.; Hirai, T.; Takeda, I.; Sasamura, Y.;
Sakamura, S.; Sato, R.; Tajimi A. Chem. Lett. 1989, 1495-1498.
(7) Narayanan, T. K.; Rao, G. R. Can. J . Microbiol. 1976, 22, 384-389.
(8) Hansen, M.; J acobson, J . J . Magn. Reson. 1975, 20, 520-529.
(9) Govindachari, T. R.; Premila, M. S. Phytochemistry 1985, 24, 3068-
3069.
1
an oil: CIMS (isobutane) m/z 491[MH]+, 431[MH - 60]+; H
NMR (acetone-d6) δ 7.58 (1H, dd, J ) 0.8 and 0.5 Hz, H-8),
6.86 (1H, dd, J ) 2.5 and 0.8 Hz, H-6), 6.67 (2H, br s, H-2′
and -6′), 6.49 (1H, dd, J ) 2.5 and 0.5 Hz, H-4), 5.06 (2H, br
s, H2-7′), 4.99 and 4.93 (2H, d, J ) 11.6 Hz, H2-1), 3.84 (3H, s,
5-OMe), 3.79 (3H, s, 3-OMe), 3.73 (6H, s, 3′- and 5′-OMe), 2.07,
2.05, and 1.71 (9H, s, 1-, 8-, and 7′-OAc); selected NOE
experiments (acetone-d6), {H2-1} enhanced H-8 (20%), {H-4}
enhanced 3-OMe (3%) and 5-OMe (1.5%), {H-6} enhanced H-8
(2%) and 5-OMe (2%), {H-8} enhanced H2-1 (4%) and H-6 (2%),
{H2-7′} enhanced H-2′ and -6′ (6.5%), {3-OMe} enhanced H2-1
(0.5%) and H-4 (19%), {5-OMe} enhanced H-4 (8.5%) and H-6
(10) Piagnani, C.; Faoro, F.; Sant, S.; Vercesi, A. Eur. J . For. Pathol. 1997,
27, 23-32.
(11) Falck, W.; Goodwin, R. H.; Leonard, E. J . Immunol. Methods 1980,
33, 239-247.
NP0103012