Non-phenolic dicinnamamides from Pholiota spumosa: Isolation, synthesis and antitumour activity

Article abstract of DOI:10.1002/ejoc.200700558

Two new amides derived from cinnamic acid, namely, (R)-2-hydroxyputrescine dicinnamamide (4) and pholiotic acid {(2R)-2-[(S)-3-hydroxy-3-methylglutaryloxy] putrescine dicinnamamide} (5), in addition to the known compound maytenine (N¹,N⁸

Full text of DOI:10.1002/ejoc.200700558

FULL PAPER
DOI: 10.1002/ejoc.200700558
Non-Phenolic Dicinnamamides from Pholiota Spumosa: Isolation, Synthesis
and Antitumour Activity[‡]
Marco Clericuzio,*[a] Silvia Tabasso,[b] Juan A. Garbarino,[c] Marisa Piovano,[c]
Venera Cardile,[d] Alessandra Russo,[e] and Giovanni Vidari[f]
Keywords: Natural products / Cinnamic acid / Amides / Structure elucidation / Total synthesis / Prostate cancer
Two new amides derived from cinnamic acid, namely,
(R)-2-hydroxyputrescine dicinnamamide (4) and pholiotic
acid {(2R)-2-[(S)-3-hydroxy-3-methylglutaryloxy]putrescine
dicinnamamide} (5), in addition to the known compound
maytenine (N1,N8-dicinnamoyl spermidine) (3) were isolated
from the fruiting bodies of the Basidiomycete Pholiota spu-
mosa. The absolute configuration of 4 was established as (R)
by its total synthesis starting from (S)-dimethylmalate,
whereas that of 5 was determined by conversion into the
known compound methyl (S)-4-[(S)-1-(1-naphthalen-1-yl)-
ethylcarbamoyl]-3-hydroxy-3-methylbutanoate (14). Com-
pounds 3 and 5 exhibited an inhibitory effect on cell growth
of the androgen-insensitive DU-145 prostate cancer cells,
which suggests that these fungal polyamine conjugates, like
other polyamine analogues, might have chemotherapeutic
potential against androgen-independent prostatic cancer.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
results in derivatives of the coumaric, caffeic and ferulic ac-
ids (hydroxycinnamoyl acid amides, HCA). Both mono-
and diamides of these phenylpropanoic acids have been re-
ported, and the amino moieties are derived from poly-
amines, such as putrescine[2] and spermidine,[6] or from de-
carboxylated amino acids such as tryptamine.[3,7] The phe-
nolic groups of plant amides are of primary importance for
their noteworthy biological roles, which include, among
others, the regulation of flowering mechanisms.[4,8] More-
over, some of these compounds are good inhibitors of α-
glucosidase[9] and some have been studied as potential hy-
pocholesterolemic agents.[10]
Introduction
Cinnamic acid amides 1 and 2 (often referred to with the
trivial name “dicinnamides”), recently isolated by us from
the fruiting bodies of the Basidiomycete Pholiota spumosa
(Fr.) Sing. (Strophariaceae),[1] belong to a new class of fun-
gal metabolites. In fact 1 and 2, as well as related com-
pounds 3–5 reported in this paper, are the first examples
of cinnamic acid amides occurring in the fungal kingdom.
Several strictly related metabolites are common constituents
of green plants (Angiospermae);[2–5] however, most of them
bear oxygenated substituents on the aromatic rings, which
Amides of aromatic acids different from cinnamic and
hydroxycinnamic acids are equally rarely represented
among fungal metabolites. Interestingly, Steglich and co-
workers[11] isolated a benzamide, named pistillarin [N1,N8-
bis(3,4-dihydroxy)benzoylspermidine], from the fruiting
bodies of Clavariadelphus and Ramaria (mushrooms be-
longing to the order Aphyllophorales s.l., Basidiomycetes),
which contains phenolic substituents like plant amides.
The biological role of the non-phenolic fungal diamides
is still largely unknown; dicinnamide 1 seems to be devoid
of most bioactivities showed by plants HCA,[9] although we
found that 1 inhibits the vitality of human prostate cancer
cells.[12] Therefore, in this work we describe the antigrowth
activity of compounds 3 and 5 against androgen-dependent
LNCaP prostate cancer cells and androgen-insensitive DU-
145 prostate cancer cells. Several biochemical parameters,
such as cell vitality (MTT assay), cell membrane integrity
(lactate dehydrogenase release) and genomic DNA frag-
mentation (COMET assay) were tested.
[‡] Fungal Metabolites, 53 (part 52: G. Gilardoni, M. Clericuzio,
S. Tosi, G. Zanoni, G. Vidari, J. Nat. Prod. 2007, 70, 137–139);
Studies on Chilean Fungi, 5 (part 4: ref.[12]). This work is part
of the doctoral thesis of S. T.
[a] Dipartimento di Scienze Ambientali e della Vita, Università del
Piemonte Orientale “A. Avogadro”,
via Bellini 25/G, 15100 Alessandria, Italy
Fax: +39-0131360390
E-mail: marco.clericuzio@mfn.unipmn.it
[b] Dipartimento di Chimica Generale ed Organica Applicata,
Università di Torino,
via P. Giuria 7, 10125 Torino, Italy
[c] Departamento de Quimica, Universidad Técnica F. S. Maria,
Casilla 110-V, Valparaiso, Chile
[d] Dipartimento di Scienze Fisiologiche, Università di Catania,
Viale A. Doria 6, 95125 Catania, Italy
[e] Dipartimento di Chimica Biologica, Chimica Medica e Biologia
Molecolare, Università di Catania,
Viale A. Doria 6, 95125 Catania, Italy
[f] Dipartimento di Chimica Organica, Università di Pavia,
via Taramelli 10, 27100 Pavia, Italy
Supporting information for this article is available on the
WWW under http://www.eurjoc.org/ or from the author.
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M. Clericuzio et al.
FULL PAPER
constant of ca. 16 Hz for the trans cinnamic systems (to-
gether with two aromatic broad multiplets at about δ = 7.4
and 7.5 ppm) were good markers of their presence. Note-
worthy, dicinnamamides 3–5 were partly or completely lost
on silica gel columns.
Compound 3 was the first dicinnamamide to elute. Its
MS (ESI+) spectrum showed an [M+1]+ pseudomolecular
ion at m/z = 406, which is indicative of an odd molecular
weight of 405 and suggests the presence of a third nitrogen
atom in comparison to the structures of 1 and 2. By means
of NMR spectroscopic analysis, 3 was unequivocally as-
signed the structure N1,N8-dicinnamoylspermidine, which is
a compound known by the name maytenine and previously
isolated from the roots and bark of the Amazonian tree
Maytenus chuchuasha.[13] Maytenine shows important bio-
activities, such as antipyretic and vasodilatation proper-
ties,[14] which have stimulated much synthetic work.[15] To
the best of our knowledge, compound 3 is the only example
of a non-phenolic cinnamamide found in plants. In the Ex-
perimental Section we report the complete NMR spectra of
maytenine (3) in two different solvents (CD3OD and [D8]-
THF/D2O), whereas in the original paper[13] the NMR
spectroscopic data were reported in CDCl3, in which 3 is
scarcely soluble.
The second dicinnamamide to elute was 4, and it was
more difficult to obtain in pure form. In fact, it constantly
coeluted with dicinnamamide 5 on RP-18 columns; how-
ever, it could be separated by employing Sephadex LH-20
columns or, alternatively, by silica gel column chromatog-
raphy after methylation of 5 to 5a (see Experimental Sec-
tion for details). Compound 4 was obtained in small
amounts (about 2 mg per gram of crude extract) as a fine
Results and Discussion
The fruiting bodies of Pholiota spumosa were soaked in powder that stuck to glassware, and it is partly soluble only
EtOH, which afforded a slightly better yield of extraction in 95% aqueous MeOH. The molecular formula
than acetone, previously employed.[1] Separation of the C22H24N2O3 was derived from the HRMS (ESI+) spec-
crude extract with an RP-18 column afforded three dicinnam- trum, which exhibited a pseudomolecular ion peak at
amides (3–5), which eluted before dicinnamide 1. The 387.16807 corresponding to C22H24N2O3Na. In compari-
spots of these compounds were strongly UV absorptive on son to those of 1, the NMR spectra of 4 showed the pres-
TLC plates but remained colourless upon exposure to sulfo- ence of two nonequivalent cinnamic units along with an
aldehydo reagents. The occurrence of dicinnamamides in additional secondary alcoholic resonance at δ = 69.2 ppm
1
the various chromatographic fractions could be easily con- in the 13C NMR spectrum and at δ = 3.79 ppm in the H
1
firmed by analysis of the H NMR spectra, and their pres- NMR spectrum (see Tables 1 and 2). Compound 4 thus cor-
ence was obvious even in mixtures; in fact, the two doublets responded to N1,N4-dicinnamamoyl 2-hydroxyputrescine,
at about δ = 6.6 ppm, which showed the typical coupling and it was found to be the dextrorotatory enantiomer. Be-
1
Table 1. H NMR (400 MHz, CD3OD) chemical shifts (in ppm) of compounds 4 and 5.
Proton
4
5
1
2
3
4
3.48 (m), 3.43 (m)
3.79 (m)
1.78 (m), 1.65 (m)
3.40 (m), 3.30 (m)
3.64 (dd, J = 13.6, 4.0 Hz), 3.45 (m)
5.11 (m)
1.90 (m)
3.40 (m)
2Ј, 2ЈЈ
3Ј, 3ЈЈ
6.65 (d, J = 16.0 Hz), 6.59 (d, J = 16.0 Hz) 6.63 (d, J = 15.8 Hz), 6.60 (d, J = 15.8 Hz)
7.50 (d, J = 16.0 Hz), 7.48 (d, J = 16.0 Hz) 7.57 (d, J = 15.8 Hz), 7.49 (d, J = 15.8 Hz)
5Ј, 9Ј, 5ЈЈ, 9ЈЈ
6Ј, 7Ј, 8Ј, 6ЈЈ, 7ЈЈ, 8ЈЈ
7.41–7.35 (m)
7.57–7.49 (m)
7.41–7.34 (m)
7.56–7.50 (m)
2ЈЈЈ
4ЈЈЈ
6ЈЈЈ
–
–
–
2.70 (d, J = 13.8 Hz)
2.56 (d, J = 14.5 Hz)
1.40 (s)
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Non-Phenolic Dicinnamamides From Pholiota Spumosa
cause crystals suitable for X-ray analysis could not be ob- precursor of dicinnamamide 12, was unfeasible by us. In
tained, the absolute configuration of (+)-(4) was established fact, the diborane reduction gave mainly the N1-mono-
on the basis of a homochiral sample prepared by total syn- amine instead of the desired diamine, whereas upon expo-
thesis.
sure to LiAlH4 the silyl ether protection was lost and exten-
sive racemization occurred (data not shown). A different
synthetic approach was therefore conceived (Scheme 1),
which was based on the reduction of diazide 9 prepared
from the Staudinger reaction[17] of ditosylate 8. In principle,
8 appeared to be available by tosylation of diol 7, which in
turn was readily achievable from the reduction of O-TIPS
protected (S)-dimethylmalate 6 with DIBAL-H by a pro-
cedure outlined by Goti et al.[18]
The reduction of 6 gave diol 7 in good yields (86%); how-
ever, its subsequent bistosylation was marred by the co-oc-
curring intramolecular SN2 reaction of the intermediate
monotosylate, which led to the TIPS derivative of 3-hy-
droxytetrahydrofuran. Under optimized experimental con-
ditions, specifically, excess TsCl and pyridine as the base
instead of NEt3 at 5 °C, the yield of 8 could be raised to
about 42%. In the following conversion of ditosylate 8 into
diazide 9,[17,19] the solvent proved to exert a major role, as
the azidation was slow and largely incomplete after 16 h in
95% aqueous MeCN at 70 °C, whereas it went to comple-
tion in 45 min in DMF at 110 °C. Subsequently, diazide 9
was immediately treated with PPh3 to afford diamine (S)-
Table 2. 13C NMR (100 MHz, CD3OD) chemical shifts (in ppm)
of compounds 4 and 5.
Carbon
4
5
1
2
3
4
46.7 (t)
69.2 (d)
35.4 (t)
37.4 (t)
169.0 (s), 168.8 (s)
121.8 (d), 121.1 (d)
141.9 (d), 141.7 (d)
136.2 (2ϫd)
43.1 (t)
72.2 (d)
32.4 (t)
36.8 (t)
168.7 (s), 168.4 (s)
121.6 (d), 121.5 (d)
141.9 (d), 141.5 (d)
136.1 (d), 136.0 (d)
129.7 (4ϫd)
130.7 (2ϫd), 130.6 (2ϫd)
128.7 (d), 128.6 (d)
172.3 (s)
1Ј, 1ЈЈ
2Ј, 2ЈЈ
3Ј, 3ЈЈ
4Ј, 4ЈЈ
5Ј, 9Ј, 5ЈЈ, 9ЈЈ 129.9 (4ϫd)
6Ј, 8Ј, 6ЈЈ, 8ЈЈ 130.8 (4ϫd)
7Ј, 7ЈЈ
1ЈЈЈ
2ЈЈЈ
3ЈЈЈ
4ЈЈЈ
5ЈЈЈ
6ЈЈЈ
129.9 (2ϫd)
–
–
–
–
–
–
46.6 (t)
70.9 (s)
46.8 (t)
175.2[a] (s)
27.9 (q)
[a] Low intensity.
Both (S)- and (R)-hydroxyputrescine were previously (+)-10 (91% overall yield from 8), which was then acylated
synthesized from commercial (S)-and (R)-dimethylmal- with freshly prepared cinnamoyl chloride. Exposure of di-
ate[16] by diborane reduction of the corresponding hydroxy amide 11 to aqueous TFA at room temperature resulted in
unprotected primary diamides. In contrast, the direct re- smooth cleavage of the hydroxy protecting group to yield
duction of O-TBDMS protected (S)-hydroxydisuccinamide (S)-hydroxyputrescine-1,4-dicinnamamide (12). The syn-
to the corresponding diamine, envisaged as an advanced thetic sample was found to be identical to natural com-
Scheme 1. Synthetic route to (S)-2-hydroxyputrescine 1,4-dicinnamamide (12) from dimethyl (S)-malate. Yields are in brackets.
Eur. J. Org. Chem. 2007, 5551–5559
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M. Clericuzio et al.
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pound 4 [1H and 13C NMR spectra, low- and high-resolu- all the HMGA moieties occurring as esters in different fun-
tion MS (ESI+), TLC mobility under different chromato- gal metabolites show this same absolute configuration, irre-
graphic conditions] except for the sign of the optical rota- spective of the structures of the alcoholic counterparts. This
tion, which was negative for (S)-12; as a consequence, natu- observation seems to indicate that the enzyme(s) catalyzing
ral product 4 was assigned the (R) configuration.
the esterification step are rather substrate-aspecific, but
In nature, (+)-2-hydroxyputrescine has been isolated completely enantioselective as to the recognition of the two
from Pseudomonas bacteria,[20] and it was assigned the (S) enantiotopic carboxylic groups of HMGA. Nothing is
configuration as the corresponding (+)-dihydrochloride.[16] known so far on the biochemistry of these HMGA deriva-
Biosynthetically, it derives from putrescine.[20] Two HCA tives.
derivatives of 2-hydroxyputrescine are reported in the litera-
ture,[21] in which the amino moiety is derived from the (S)-
enantiomer. However, the unique polyamine-derived amino
acid hypusine, contained in the eukaryotic translation initi-
ation factor 5A (eIF5A), is derived from the (R) enantiomer
of 2-hydroxyputrescine.[22]
The last dicinnamamide to elute was 5 and it showed a
more complex 13C NMR spectrum than those of 3 and 4,
and two additional C=O resonances were found at δ =
172.3 and 175.2 ppm in CD3OD. The MS (ESI) spectrum
of 5 gave a clear pseudomolecular ion at m/z = 507 in the
negative ionization mode, which corresponds to MW = 508.
A typical IR broad band around 3000 cm–1 suggested that
one of the additional carbonyl groups belonged to a car-
boxylic acid functionality, whereas the second carbonyl
group was assigned to an ester moiety on the basis of the
observed downfield acylation shift of the secondary alcohol
1
resonance from δ = 3.8 ppm in the H NMR spectrum of
dicinnamamide 4 to δ = 5.1 ppm in the spectrum of 5. This
proton correlated, in the HSQC spectrum, with a methine
oxygenated carbon at δ = 72.2 ppm. The additional pres-
Scheme 2. Absolute stereochemistry determination of the HMGA
moiety of pholiotic acid (5).
ence of a quaternary carbon resonance at δ = 70.9 ppm
along with a singlet at δ = 1.4 ppm for the methyl group
and two ABq signals at δ = 2.5 and 2.7 ppm in the 1H NMR
spectrum firmly established that 5 corresponded to the ester
of 4 with 3-hydroxy-3-methyl glutaric acid (HMGA). This
acidic moiety was found in a small group of biosynthetically
Antitumour Activity
unrelated fungal metabolites, among which the most repre-
The polyamines putrescine, spermidine and spermine are
sentative are a few lanostane triterpenes, like fasciculol E, organic cations found in all mammalian cells and are re-
previously isolated by us from the same mushroom (Ph. quired for cell proliferation. Polyamine analogues inhibit
spumosa)[1] or the hebelomic acids A, B, E and F isolated cell growth and kill cancer cells both in tissue culture as
from Hebeloma senescens.[23] We gave compound 5 the triv- well as in experimental animal models.[25–29] Polyamines are
ial name pholiotic acid.
important for prostate cancer cell function.
To assign the absolute configuration of the HMGA moi-
Prostate cancer, a disease still globally widespread, usu-
ety in 5, we followed our general procedure first used for ally progresses from an androgen-dependent to an indepen-
hebelomic acid A.[23] To this purpose, pholiotic acid (5) was dent stage, which makes antiandrogen therapy ineffective
condensed with (S)-1-(1-naphthyl)ethylamine according to and leads to an increase in metastatic potential and incur-
the Kaminsky protocol[24] to yield triamide 13, which was able malignancy.[30] Cytotoxic chemotherapy is used to con-
transesterified with NaOMe to afford 2-hydroxyputrescine trol and treat prostate cancer at this stage, but it remains
1,4-dicinnamamide and glutaramide 14 (Scheme 2). The relatively unselective and highly toxic to normal tissues.
former was found to be identical to natural product 4, in- Therefore, there has been increasing interest in the use of
cluding the sign and magnitude of the optical rotation. This structural polyamine analogues as prostate cancer chemo-
indicated that the absolute configuration at C-2 in pholiotic therapeutic agents, as a variety of analogues have been syn-
acid (5) is (R), as in dicinnamamide 4, and in agreement thesized and shown to be active against several prostate
with straightforward biosynthetic considerations. Moreover, cancer cells.[31,32] Our previous study demonstrated that pu-
compound 14 was identical in all respects to an authentic trescine-1,4-dicinnamamide (1), isolated from the same fun-
sample prepared from hebelomic acid A, including the op- gus (Pholiota spumosa),[1] inhibits the cell growth of DU-
tical rotation;[23] the stereocentre of HMGA in 5 was there- 145 cancer cells,[12] as it was also found for some synthetic
fore assigned the (S) absolute configuration. Noteworthy, polyamine analogues.[31,32]
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Non-Phenolic Dicinnamamides From Pholiota Spumosa
We have now investigated the responses of the human Table 3. Lactate dehydrogenase (LDH) release in LNCaP and DU-
145 cells: untreated and treated with compounds 3 and 5 at dif-
prostate cancer cell lines LNCaP and DU-145 to maytenine
(3) and pholiotic acid (5). These cell lines were chosen be-
ferent concentrations for 72 h. The stock solution of the com-
pounds was prepared in DMSO and the final concentration of this
cause they represent a spectrum of androgen-dependent
solvent was kept constant at 0.25%. Control cultures received
and androgen-independent prostate cancers, respectively.
The results, summarized in Figure 1, show that compounds
3 and 5, used at nontoxic concentrations in normal cells
(data not shown), exhibited a significant (pϽ0.001) and a
dose-dependent inhibitory effect only on DU-145 cell
growth. In fact, the vitality was 87 and 91% of LNCaP cells
exposed to 50 µ of compounds 3 and 5, respectively. In
contrast, other studies have evidenced that DU-145, a well-
characterized androgen-independent human prostate cancer
cell line, was more sensitive to cell growth inhibition in-
duced by polyamine analogues than androgen-sensitive
LNCaP prostate cancer cells, and that the latter cancer cells
maintained the largest total polyamine content.[31]
DMSO alone. The results are expressed as percentage of LDH re-
leased into the cell medium with respect to total LDH. Each value
represents the meanϮSD of three experiments, performed in qua-
druplicate.
Treatments
% LDH released
LNCaP cells
Control
3
12.5 µ
25 µ
50 µ
5
6.9Ϯ0.7
7.3Ϯ0.1
6.4Ϯ0.9
7.5Ϯ0.8
12.5 µ
25 µ
50 µ
4.9Ϯ0.9
6.3Ϯ0.7
7.5Ϯ0.6
DU-145 cells
Control
3
12.5 µ
25 µ
50 µ
5
4.6Ϯ0.3
4.4Ϯ0.5
5.4Ϯ0.5
5.7Ϯ0.9
12.5 µ
25 µ
50 µ
3.4Ϯ0.6
4.4Ϯ0.5
5.8Ϯ0.9
Table 4. Comet assay of genomic DNA in LNCaP and DU-145
cells: untreated and treated with compounds 3 and 5 at different
concentrations for 72 h. The stock solution of the compounds was
prepared in DMSO and the final concentration of this solvent was
kept constant at 0.25%. Control cultures received DMSO alone.
Reported values are the meanϮSD of three experiments performed
in quadruplicate.
Treatments
TDNA[a]
TMOM[b]
LNCaP cells
Control
3
12.5 µ
25 µ
50 µ
5
Figure 1. Effect of compounds 3 and 5, at different concentrations
for 72 h, on LNCaP and DU-145 cell growth. The stock solution
of the compounds was prepared in DMSO and the final concentra-
tion of this solvent was kept constant at 0.25%. Control cultures
received DMSO alone. Reported values are the meanϮSD of three
experiments performed in quadruplicate.
15Ϯ3.5
97Ϯ5.7
17Ϯ5.3
17Ϯ3.4
18Ϯ2.3
105Ϯ16
99Ϯ7
103Ϯ13
12.5 µ
25 µ
50 µ
16Ϯ7.3
14Ϯ4.1
13Ϯ2.7
95Ϯ11
111Ϯ9
100Ϯ14
Necrosis results in a disruption of cytoplasmic mem-
brane and the necrotic cells release cytoplasmic LDH and
other cytotoxic substances into the medium. We therefore
examined the membrane permeability of the treated cells
and the existence of LDH in their culture medium. No sta-
tistically significant increase in LDH release was observed
in DU-145 cells treated with compounds 3 and 5 even at
the highest concentration, specifically, 50 µ (Table 3). The
tested compounds were also inactive on LNCaP cells in this
assay (Table 4).
DU-145 cells
Control
3
12.5 µ
25 µ
50 µ
5
12.5 µ
25 µ
50 µ
21Ϯ5
96Ϯ7.7
86Ϯ15[c]
101Ϯ13[c]
198Ϯ19[c]
996Ϯ15[c]
1112Ϯ39[c]
2009Ϯ36[c]
88Ϯ14[c]
111Ϯ13[c]
176Ϯ17[c]
989Ϯ18[c]
1123Ϯ27[c]
1889Ϯ36[c]
Nuclear DNA fragmentation was analyzed by using the
COMET assay, a sensitive method for the detection of
DNA strand breaks in individual cells; this is a versatile
tool that is highly effective in human biomonitoring of nat-
[a] TDNA is the percentage of the fragmented DNA. [b] TMOM
is the tail moment expressed as the product of TD (distance be-
tween head and tail) and TDNA. [c] Significant vs. control un-
ural compounds. Quantification of the COMET data is re- treated cells (pϽ0.001).
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M. Clericuzio et al.
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separated by MPLC (flow 7 mLmin–1, pressure 100 psi, λ =
245 nm) on an RP-18 reverse-phase column (particle size: 15–
35 µm) by using a H2O/MeOH gradient (from 7:3 to 1:3). Nineteen
fractions were collected. The most polar fractions (A1–3) contained
a mixture of styryl–pyrones, whereas fasciculol E[1] was eluted in
A4 and A5. Fractions A6 and A7 contained maytenine (3), which
after a second separation with Sephadex LH-20 (MeOH/EtOAc,
7:3) was obtained in pure form (40 mg). (R)-2-Hydroxyputrescine
1,4-dicinnamamide (4) was eluted in fractions A8–13, together with
pholiotic acid (5) and putrescine 1,4-dicinnamamide (1), which was
ported as TDNA and TMOM values in Table 4. The results
clearly evidence DNA damage only in DU-145 cells ex-
posed to compounds 3 and 5 for 72 h and drastic increases
in both the TDNA and TMOM values at 25 and 50 µ
concentrations are also observed. These results seem to
confirm an apoptotic cell death, as recent literature data[33]
indicate that only comets with high values of TMOM (tail
moments) and TDNA (distance between head and tail of
the comet) can be related to apoptosis.
In summary, these data suggest that compounds 3 (may- obtained pure (70 mg) in fractions A14 and A15. Fractions A8–13
were subsequently purified by gel-permeation chromatography on
a Sephadex LH-20 column, eluted with MeOH, which allowed sep-
aration of 4 from acid 5. Coeluting dicinnamamides 4 and 1
(20 mg) were finally purified by an RP-18 column (particle
size:12 µm), eluted with a gradient mixture of H2O/MeOH/MeCN
(3:2:1 to 1:1:1 + 0.5% HCOOH). This allowed alcohol 4 to be
obtained in an almost pure form (6 mg). The LH-20 fractions con-
taining pholiotic acid (5; 60 mg) were submitted to a final purifica-
tion with an RP-18 column (particle size: 12 µ) and eluted with a
mixture of H2O/MeOH/MeCN (1:1:1 to 1:2:1 + 0.5% HCOOH);
28 mg of 5 were obtained in this way. In fractions A16 and A17,
dicinnamamide 1 and small amounts of (E)-2,3-dehydroputrescine
1,4-dicinnamamide (2) coeluted.
tenine) and 5 (pholiotic acid), which are fungal metabolites
belonging to the class of polyamine conjugates, may have
chemotherapeutic potential in the case where prostatic can-
cer has become androgen independent, which justifies fur-
ther investigations in order to discover new biological
mechanisms.
Experimental Section
General Conditions: Optical rotations were determined with a Per-
kin–Elmer 241 polarimeter. Melting points were determined with
a Stuart Scientific SMP3 apparatus. IR spectra were recorded with
an FTIR Perkin–Elmer BX spectrometer. The NMR experiments
were performed with a Bruker Avance 400 NMR spectrometer; the
1H and 13C chemical shifts are reported relative to residual solvent
signals: CD3OD: 3.31 (1H), 49.0 (13C); CDCl3: 7.26 (1H), 77.10
(13C); DMSO: 2.50 (1H), 39.5 (13C); THF: 3.74, 1.88 (1H), 68.7,
25.7 (13C); CD3CN: 1.94 (1H), 118.2, 1.3 (13C). The abbreviation s
= singlet, d = doublet, t = triplet, q = quartet, m = multiplet and
br. = broad are used throughout. MS (ESI) experiments were car-
ried out both in positive and in negative ion mode by using an
ion-trap Thermo Finnigan LCQ Duo Thermoquest spectrometer
equipped with the Xcalibur software. High-resolution ESI mass
spectra were determined with an FT-ICR Apex II Mass Spectrome-
ter of Bruker Daltonics. UV spectra were recorded with a UN-
ICAM 8700 UV/Vis Spectrophotometer, and CD spectra were re-
N1,N8-Dicinnamoylspermidine (Maytenine; 3): Yellow waxy solid.
1H NMR (400 MHz, CD3OD): δ = 7.59–7.48 (m, 6 H, Ar), 7.46
(d, J = 16.0 Hz, 2 H, 3Ј-H and 3ЈЈ-H), 7.34–7.27 (m, 4 H, Ar), 6.58
(d, J = 16.0 Hz, 2 H, 2Ј-H and 2ЈЈ-H), 3.41 (m, 2 H, 1-H), 3.30 (m,
2 H, 7-H), 3.02 (m, 4 H, 3-H and 4-H), 1.94 (m, 2 H, 2-H), 1.71
(m, 2 H, 5-H), 1.48 (m, 2 H, 6-H) ppm. 13C NMR (100 MHz,
CD3OD): δ = 169.5 (s, C-1Ј or C-1ЈЈ), 168.8 (s, C-1ЈЈ or C-1Ј), 142.3
(d, C-3Ј or C-3ЈЈ), 141.8 (d, C-3ЈЈ or 3Ј), 136.1 (s, C-4Ј or C-4ЈЈ),
136.0 (s, C-4ЈЈ or C-4Ј), 130.9 (d, 2 C, C-7Ј and C-7ЈЈ), 130.0 (d, 4
C, C-6Ј, C-6ЈЈ, C-8Ј, C-8ЈЈ), 128.9 (d, 2 C, C-5Ј and C-5ЈЈ or C-9Ј
and C-9ЈЈ), 128.8 (d, 2 C, C-9Ј and C-9ЈЈ or C-5Ј and C-5ЈЈ), 121.7
(d, C-2Ј or C-2ЈЈ), 121.3 (d, C-2ЈЈ or C-2Ј), 48.1 (t, C-4), 46.2 (t,
C-3), 39.6 (t, C-7), 37.1 (t, C-1), 27.7 (t, C-2), 27.5 (t, C-6), 24.6 (t,
C-5) ppm. 1H NMR (400 MHz, [D8]THF + 5% D2O): δ = 8.21
(m, 2 H, amidic N-H), 7.34–7.32 (m, 4 H, Ar), 7.30 (d, J = 15.0 Hz,
1 H, 3Ј-H or 3ЈЈ-H), 7.28 (d, J = 15.0 Hz, 1 H, 3ЈЈ-H or 3Ј-H),
7.12–7.05 (m, 4 H, Ar), 6.48 (d, J = 15.0 Hz, 1 H, 2Ј-H or 2ЈЈ-H),
6.46 (d, J = 15.0 Hz, 1 H, 2ЈЈ-H or 2Ј-H), 3.34 (br. t, 2 H, 1-H),
3.26 (t, J = 6.4 Hz, 2 H, 7-H), 2.99 (m, 4 H, 3-H and 4-H), 1.99
(m, 2 H, 2-H), 1.79 (m, 2 H, 5-H), 1.59 (m, 2 H, 6-H) ppm. 13C
NMR (100 MHz, [D8]THF + 5% D2O): δ = 168.2 (s, C-1Ј or C-
1ЈЈ), 167.7 (s, C-1ЈЈ or C-1Ј), 141.2 (d, C-3Ј or C-3ЈЈ), 140.8 (d, C-
3ЈЈ or C-3Ј), 136.8 (s, C-4Ј or C-4ЈЈ), 136.6 (s, C-4ЈЈ or C-4Ј), 130.5
(d, Ar), 130.4 (d, Ar), 129.9 (d, Ar), 129.8 (d, Ar), 129.1 (d, Ar),
129.0 (d, Ar), 121.9 (d, C-2Ј or C-2ЈЈ), 121.5 (d, C-2ЈЈ or C-2Ј),
48.5 (t, C-4), 46.5 (t, C-3), 39.9 (t, C-7), 37.7 (t, C-1), 27.6 (t, 2 C,
corded with
a
Jasco J-710 spectropolarimeter. Thin-layer
chromatography was performed on silica gel F254 sheets (direct
phase TLC) or RP-18 F254 sheets (reverse-phase TLC). The com-
pounds were visualized under UV light (254 and 366 nm) and by
spraying with sulfoanisaldehyde, phosphomolybdic acid or ninhyd-
rin solutions, followed by heating. Medium-pressure liquid
chromatography (MPLC) was performed with Fluka silica gel 100
C-18 (15–35 µm, fully endcapped) or Merck-VWR LiChrospher
RP-18 (12 µm). Lipophilic Sephadex LH-20 (25–100 µm), pur-
chased from Sigma–Aldrich, was employed for gel-filtration liquid
chromatography. Gas chromatographic analysis was performed
with a Perkin–Elmer Autosystem apparatus (Agilent Technologies)
equipped with a dimethylpolysiloxane capillary column (25 m long,
0.20 mm i.d., 0.33 µm film thickness). All glassware used in the
synthesis of compounds 12 and 14 were dried in an oven at 100 °C
overnight and assembled under a stream of dry N2.
C-2 and C-6), 24.8 (t, C-5) ppm. IR (thin film): ν = 3300, 3273,
˜
2930, 2891, 1670, 1657, 1614, 1515, 1224, 979 cm–1. MS (ESI+):
m/z = 406 [M + H]+, 833 [2M + Na]+. MS (ESI–, NEt3 added):
+
m/z = 404.45 [M – H]–. HRMS (ESI+): calcd. for C25H32N3O2
[M + H]+ 406.24890; found 406.24864.
Fungal Material, Extraction and Isolation: Fruiting bodies of Ph.
spumosa (1.2 kg) were collected in the Reserva Nacional Forestal
Lago Peñuelas (Valparaiso, Chile) and identified by one of us
(M. C.). A voucher specimen was deposited at the University F. S.
Maria of Valparaiso. Immediately after collection, the mushrooms
were frozen at –20 °C; eventually they were minced whilst frozen
and then extracted with EtOH, and the solution was allowed to
reach r.t. The solution thus obtained was filtered and then concen-
trated under reduced pressure at 35 °C; the solid residue (5 g) was
(R)-(+)-2-Hydroxyputrescine 1,4-Dicinnamamide (4): Microcrystal-
line powder, [α]2D2 = +3.0 (c = 0.5, MeOH). 1H and 13C NMR
(CD3OD), see Tables 1 and 2, respectively. UV (MeOH): λ (ε,
Lmol–1 cm–1) = 275.0 (10600), 204.5 (9200) nm. CD (MeOH): λ
(∆ε) = 285.0 (+0.26), 256.0 (–0.33) nm. MS (ESI+): m/z = 365.2
[M
+ +
H]+, 387.2 [M Na]+. HRMS (ESI+): calcd. for
C22H24N2O3Na+ 387.16791; found 387.16807.
5556
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 5551–5559

Non-Phenolic Dicinnamamides From Pholiota Spumosa
Pholiotic Acid {(2R)-2-[(S)-3-Hydroxy-3-methylglutaryloxy]putres-
cine Dicinnamamide; 5}: Waxy solid. [α]2D2 = +18.5 (c = 0.4, MeOH).
1H and 13C NMR (CD3OD), see Tables 1 and 2, respectively. 1H
NMR (400 MHz, [D6]DMSO): δ = 8.40 (m, 1 H, NH), 8.11 (br. t,
1 H, NH), 7.55–7.51 (m, 4 H, Ar), 7.49–7.40 (m, 8 H, Ar, 3Ј-H and
3ЈЈ-H), 6.67 (d, J = 14.6 Hz, 1 H, 2Ј-H or 2ЈЈ-H), 6.58 (d, J =
14.6 Hz, 1 H, 2ЈЈ-H or 2Ј-H), 4.89 (m, 1 H, 2-H), 3.45 (m, 2 H, 1-
(S)-2-(Triisopropylsilyloxy)butane-1,4-diol (7):
A
solution of
DIBAL-H (1  in CH2Cl2, 31.2 mL, 5.2 equiv.) was added drop-
wise to compound 6 (1.9 g, 6.0 mmol) in CH2Cl2 (5 mL) under a
N2 flow at –15 °C, and the disappearance of 6 was monitored by
GC. After 3 h, the reaction mixture was quenched by adding 1-
propanol (6 mL) and H2O (3 mL) and then partitioned between
an aqueous saturated solution of NH4Cl (10 mL) and 2-butanone
H), 3.21 (m, 2 H, 4-H), 2.57 (ABq, 2 H, 2ЈЈЈ-H or 4ЈЈЈ-H), 2.41 (20 mL). The organic layer was washed with H2O (5 mL) and dried
(ABq, 2 H, 4ЈЈЈ-H or 2ЈЈЈ-H), 1.73 (m, 2 H, 3-H), 1.27 (s, 3 H, 6ЈЈЈ- with anhydrous Na2SO4; the solvent was evaporated under vacuum
H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 173.0 (s, C-5ЈЈЈ),
170.4 (s, C-1ЈЈЈ), 165.3 (s, C-1Ј or C-1ЈЈ), 165.0 (s, C-1ЈЈ or C-1Ј),
to afford pure diol 7 (1.34 g, 86%) as a viscous liquid. [α]2D2 = –7.3
(c = 4.2, MeOH). 1H NMR (400 MHz, CD3CN): δ = 4.12 (m, 1
139.0 (d, C-3Ј or C-3ЈЈ), 138.6 (d, C-3ЈЈ or C-3Ј), 135.0 (s, 2 C, C- H, 2-H), 3.80 (m, 2 H, 1-H), 3.70 (m, 2 H, 4-H), 1.93 (m, 2 H, 3-
4Ј and C-4ЈЈ), 129.5 (d, 4 C, Ar), 129.0 (d, 2 C, Ar), 127.6 (d, 4 C, H), 1.30 [m, 3 H, 3ϫCH(CH3)2], 1.16 [br., 18 H, 3ϫCH(CH3)2]
Ar), 122.3 (d, C-2Ј or C-2ЈЈ), 122.1 (d, C-2ЈЈ or C-2Ј), 70.8 (d, C- ppm. 13C NMR (100 MHz, CD3CN): δ = 72.0 (t, C-1), 66.5 (d, C-
2), 69.2 (s, C-3ЈЈЈ), 46.0 (t, 2 C, C-2ЈЈЈ and C-4ЈЈЈ), 41.3 (t, C-1),
2), 58.9 (t, C-4), 38.2 (t, C-3), 18.5 [q, 6 C, 3ϫCH(CH3)2], 13.2 [d,
35.3 (t, C-4), 31.3 (t, C-3), 27.6 (q, C-6ЈЈЈ) ppm. IR (thin film): ν
3 C, 3ϫCH(CH3)2] ppm. MS (ESI+): m/z = 285.2 [M + Na]+.
˜
= 3700–2400 (br., COOH stretching), 3308, 3077, 2924, 2851, 1723,
1714, 1659, 1620, 1614, 1556, 1449, 1343, 1226, 1079, 1028,
978 cm–1. UV (MeOH): λ (ε, Lmol–1 cm–1): 274.5 (9000), 216.5
(6800) nm. CD (MeOH): λ (∆ε) = 331.0 (+0.06), 268.5 (+0.29),
220.0 (–0.47) nm. MS (ESI–): m/z = 507.4 [M – H]–. MS (ESI+,
HCOOH added): m/z = 509.6 [M + H]+, 531.2 [M + Na]+. MS
(EI): m/z (%) = 448 (1) [M – CH3COOH]+, 407 (4), 406 (5), 405
(3), 259 (10), 199 (65), 131 (100), 103 (71), 77 (46). HRMS (ESI+):
(S)-2-(Triisopropylsilyloxy)butane-1,4-diol Ditosylate (8): Com-
pound 7 (1.34 g, 5.1 mmol) in dry MeCN (3 mL) was added drop-
wise to p-toluenesulfonyl chloride (5 g, 26 mmol) in CH2Cl2 (6 mL)
to which dry pyridine (1.5 mL) had been previously added. The
reaction mixture was stirred for 1 h at 5 °C, followed by an ad-
ditional hour at r.t., and then quenched by adding H2O (10 mL).
The aqueous phase was extracted with CH2Cl2 (2ϫ10 mL). The
organic layers were collected, dried with anhydrous Na2SO4 and
the solvents evaporated under vacuum. The resulting residue
(2.15 g) was chromatographed on a silica gel column deactivated
with Et3N (petroleum ether/CH2Cl2, 8.5:1.5 to 3:7) to afford 8
(1.2 g, 42%) as a colourless viscous liquid. [α]2D2 = –8.6 (c = 1.1,
MeOH). 1H NMR (400 MHz, CDCl3): δ = 7.74 (m, 4 H, Ar), 7.36–
7.32 (m, 4 H, Ar), 4.08 (m, 3 H, 1a-H, 2-H and 4a-H), 3.87 (m, 1
H, 4b-H), 3.82 (m, 1 H, 1b-H), 2.45 (s, 3 H, Ar-CH3), 2.44 (s, 3 H,
Ar-CH3), 1.83 (m, 2 H, 3-H), 0.92 [m, 3 H, 3ϫCH(CH3)2], 0.88
[br., 18 H, 3ϫCH(CH3)2] ppm. 13C NMR (100 MHz, CDCl3): δ =
145.2 (s, Ar), 145.0 (s, Ar), 132.9 (s, Ar), 132.7 (s, Ar), 130.0 (d, 2
C, Ar), 129.9 (d, 2 C, Ar), 128.1 (d, 2 C, Ar), 128.0 (d, 2 C, Ar),
72.1 (t, C-1), 66.9 (d, C-2), 66.3 (t, C-4), 33.5 (t, C-3), 27.7 (q, 2 C,
calcd. for C28H31N2O7 [M – H]– 507.21367; found 507.21325.
–
Methyl Pholiotate (5a): An ethereal solution of CH2N2 (0.4 mL)
was added dropwise to pholiotic acid (5; 8 mg) in MeOH (0.5 mL).
The resulting solution was evaporated under vacuum, and the resi-
due was chromatographed on a silica gel column (benzene/EtOAc,
2:1) to afford methyl pholiotate (5a; 4 mg) as a colourless oil.
1
[α]2D2 = +4.0 (c = 0.2, MeOH). H NMR (400 MHz, CD3OD): δ =
7.60–7.48 (m, 4 H, Ar), 7.41–7.34 (m, 8 H, Ar, 3Ј-H and 3ЈЈ-H),
6.61 (d, J = 15.8 Hz, 2 H, 2Ј-H and 2ЈЈ-H), 5.14 (m, 1 H, 2-H),
3.68 (m, 1 H, 1a-H), 3.64 (s, 3 H, COOCH3), 3.45 (m, 1 H, 1b-H),
3.39 (m, 2 H, 4-H), 2.74 (ABq, 2 H, 2ЈЈЈ-H or 4ЈЈЈ-H), 2.70 (ABq,
2 H, 4ЈЈЈ-H or 2ЈЈЈ-H), 1.89 (m, 2 H, 3-H), 1.41 (s, 3 H, 6ЈЈЈ-H)
ppm. 13C NMR (100 MHz, CD3OD): δ = 173.2 (s, C-5ЈЈЈ), 172.4
(s, C-1ЈЈЈ), 169.1 (s, C-1Ј or C-1ЈЈ), 168.9 (s, C-1ЈЈ or C-1Ј), 142.3
(d, C-3Ј or C-3ЈЈ), 142.1 (d, C-3ЈЈ or C-3Ј), 136.3 (d, 2 C, C-4Ј and
C-4ЈЈ), 130.1 (d, 4 C, Ar), 129.0 (d, 2 C, Ar), 122.4 (d, 4 C, Ar),
121.7 (d, C-2Ј or C-2ЈЈ), 121.6 (d, C-2ЈЈ or C-2Ј), 72.3 (d, C-2), 71.1
(s, C-3ЈЈЈ), 52.1 (q, COOCH3), 46.6 (t, C-2ЈЈЈ or C-4ЈЈЈ), 46.3 (t, C-
4ЈЈЈ or C-2ЈЈЈ), 43.4 (t, C-1), 37.1 (t, C-4), 32.5 (t, C-3), 26.1 (q, C-
6ЈЈЈ) ppm. MS (ESI+): m/z = 523 [M + H]+, 545 [M + Na]+.
2ϫAr-CH3), 18.0 [q,
6 C, 3ϫCH(CH3)2], 12.4 [br., 3 C,
3ϫCH(CH3)2] ppm. MS (ESI+): m/z = 593.16 [M + Na]+, 571.26
[M + H]+.
(S)-(1,4-Diazidobutyl-2-oxy)triisopropylsilane (9): Solid NaN3
(1.40 g, 21.6 mmol) was added portionwise to compound 8 (1.2 g,
2.16 mmol) in DMF (6 mL), and the reaction mixture was stirred
at 110 °C for 45 min. H2O (10 mL) was added, and the mixture
was extracted with EtOAc (2ϫ10 mL). The collected organic layers
were dried with anhydrous Na2SO4, and the solvent was evaporated
under vacuum at r.t. to yield diazide 9 (0.64 g, 95%), which was
immediately used in the next reaction.
Dimethyl (S)-(–)-2-(Triisopropylsilyloxy)butandioate (6): Triisopro-
pylsilyl chloride (3.86 g, 20 mmol) and imidazole (0.64 g, 9.5 mmol)
were added to dimethyl (S)-malate (1.5 g, 9.2 mmol) in DMF
(10 mL). The reaction mixture was stirred at r.t. for 27 h under a
N2 atmosphere. At the end, H2O (10 mL) was added, and the mix-
ture was extracted with CH2Cl2 (2ϫ10 mL). The organic layer was
dried with anhydrous Na2SO4, and the solvent was removed under
vacuum. The residue (5.6 g) was chromatographed on a silica gel
column deactivated with Et3N (toluene/CH2Cl2, 3:1) to afford the
protected dimethyl malate 6 (2.28 g, 78%) as a colourless viscous
liquid. [α]2D2 = –12.8 (c = 1.9, MeOH). 1H NMR (400 MHz,
(S)-(+)-2-(Triisopropylsilyloxy)butane-1,4-diamine
(10):
PPh3
(1.47 g, 5.61 mmol) dissolved in MeCN (5 mL) was added to a
solution of freshly prepared diazide 9 (0.64 g, 2.04 mmol) in MeCN
(5 mL) whilst stirring, and the resulting mixture was kept at 60 °C
for 1 h. Subsequently, H2O (3 mL) and 30% NH4OH (7 mL) were
added and stirring was continued at 60 °C for an additional hour.
The reaction mixture was cooled to r.t. and then was extracted with
EtOAc; the organic layer was dried with Na2SO4, and the solvent
CDCl3): δ = 4.72 (t, J = 6.2 Hz, 1 H, 2-H), 3.71 (s, 3 H, OCH3), was evaporated under vacuum. The residue was chromatographed
3.65 (s, 3 H, OCH3), 2.77 (d, J = 6.0 Hz, 1 H, 3a-H), 2.74 (d, J =
on an RP-18 column (H2O/MeOH, 2:3 to 1:9 + 1% acetic acid) to
6.0 Hz, 1 H, 3b-H), 1.13 [m, 3 H, 3ϫCH(CH3)2], 1.06 [br., 18 H, yield 10 (0.48 g, 91%) as a viscous liquid. [α]2D2 = +1.7 (c = 0.33,
3ϫCH(CH3)2] ppm. 13C NMR (100 MHz, CDCl3): δ = 173.3 (s, MeOH). 1H NMR (400 MHz, CD3OD): δ = 4.15 (m, 1 H, 2-H),
C-1), 171.3 (s, C-4), 70.1 (d, C-2), 52.6 (q, OCH3), 52.3 (q, OCH3), 2.91 (m, 1 H, 1a-H), 2.87 (m, 1 H, 1b-H), 2.82 (m, 1 H, 4a-H),
41.1 (t, C-3), 18.5 [q, 6 C, 3ϫCH(CH3)2], 13.2 [br., 3 C,
2.79 (m, 1 H, 4b-H), 1.86 (m, 1 H, 3a-H), 1.80 (m, 1 H, 3b-H), 1.15
[m, 3 H, 3ϫCH(CH3)2], 1.10 [d, J = 6.6 Hz, 18 H, 3ϫCH(CH3)2]
3ϫCH(CH3)2] ppm. MS (ESI+): m/z = 341.2 [M + Na]+.
Eur. J. Org. Chem. 2007, 5551–5559
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5557

M. Clericuzio et al.
FULL PAPER
ppm. 13C NMR (100 MHz, CD3OD): δ = 71.4 (d, C-2), 51.9 (t, C-
and the residue was suspended in EtOAc (15 mL). This suspension
1), 40.1 (t, C-3), 40.0 (t, C-4), 18.5 [q, 6 C, 3ϫCH(CH3)2], 13.4 [d, was washed successively with H2O (5 mL), a 10% citric acid solu-
3 C, 3ϫCH(CH3)2] ppm. MS (ESI+): m/z = 261 [M + H]+. HRMS
tion (5 mL), H2O (5 mL), a saturated NaHCO3 solution (5 mL)
and finally H2O (5 mL). The organic layer was dried (Na2SO4), and
the solvent was evaporated. The residue was chromatographed on
a silica gel column (toluene/EtOAc, 2.5:1 to 1:1) to afford pure
triamide 13 (15 mg, 61%) as a waxy solid [α]2D2 = +5.0 (c = 0.9,
MeOH). 1H NMR (400 MHz, CD3OD): δ = 7.85 (m, 1 H, Ar),
7.76 (d, J = 8.0 Hz, 1 H, Ar), 7.51–7.46 (m, 7 H, Ar, 3Ј-H and 3ЈЈ-
H), 7.44 (m, 1 H, Ar), 7.42 (m, 1 H, Ar), 7.38–7.34 (m, 8 H, Ar),
6.60 (d, J = 15.8 Hz, 1 H, 2Ј-H or 2ЈЈ-H), 6.58 (d, J = 15.8 Hz, 1
H, 2ЈЈ-H or 2Ј-H), 5.85 (q, J = 6.9 Hz, 1 H, 1ЈЈЈЈ-H), 5.09 (m, 1 H,
2-H), 3.89 (dd, J = 8.8 and 3.8 Hz, 1 H, 1a-H), 3.65 (m, 1 H, 1b-
H), 3.59 (m, 1 H, 4a-H), 3.43 (m, 1 H, 4b-H), 2.60 (m, 2 H, 2ЈЈЈor-
H 4ЈЈЈ-H), 2.59 (m, 2 H, 4ЈЈЈ-H or 2ЈЈЈ-H), 1.84 (m, 2 H, 3-H), 1.58
(d, J = 6.9 Hz, 3 H, 2ЈЈЈЈ-H), 1.37 (s, 3 H, 6ЈЈЈ-H) ppm. 13C NMR
(100 MHz, CD3OD): δ = 172.5 and 172.4 (s, 2 C, C-1ЈЈЈ and C-
5ЈЈЈ), 168.9 and 168.6 (s, 2 C, C-1Ј and C-1ЈЈ), 142.1 and 141.8 (d,
2 C, C-3Ј and C-3ЈЈ), 140.2 (s, Ar), 136.3 (s, Ar), 136.2 (s, Ar),
135.4 (s, Ar), 132.2 (s, Ar), 130.9 (d, Ar), 130.8 (d, Ar), 130.0 (d,
Ar), 129.9 (d, Ar), 129.0 (d, Ar), 128.9 (d, Ar), 128.8 (d, Ar), 127.3
(d, Ar), 126.7 (d, Ar), 126.4 (d, Ar), 124.2 (d, Ar), 123.6 (d, Ar),
121.8 and 121.6 (d, 2 C, C-2Ј and C-2ЈЈ), 72.3 (d, C-2), 71.6 (s, C-
3ЈЈ), 47.7 (t, C-2ЈЈЈ or C-4ЈЈЈ), 46.8 (t, C-1), 46.0 (d, C-1ЈЈЈЈ), 43.3
(t, C-4ЈЈЈ or C-2ЈЈЈ), 36.9 (t, C-4), 32.5 (t, C-3), 28.2 (q, C-6ЈЈЈ),
21.6 (q, C-2ЈЈЈЈ) ppm. MS (ESI+): m/z = 684.4 [M + Na]+, 662.4
[M + H]+.
(ESI+): calcd. for C13H33N2OSi+ 261.23567; found 261.23545.
Cinnamoyl Chloride: To a solution of cinnamic acid (1.3 g, 9 mmol)
in CH2Cl2 (5 mL) was added oxalyl chloride (3.4 g, 27 mmol). The
reaction mixture was stirred at r.t. for 3 h; subsequently, the solvent
and excess oxalyl chloride were removed under vacuum. The cinnam-
oyl chloride thus obtained was employed in the next reaction step
with no further purification.
(S)-(–)-2-(Triisopropylsilyloxy)putrescine 1,4-Dicinnamamide (11):
To freshly prepared cinnamoyl chloride was added dropwise a solu-
tion of 10 (0.45 g, 1.73 mmol) in CH2Cl2 (5 mL) followed by dry
pyridine (1 mL). Whilst stirring, the reaction was kept at 30 °C for
1 h. Subsequently, the solvent was evaporated under vacuum, and
the residue was chromatographed on a silica gel column deactivated
with Et3N (toluene/EtOAc, 3:2) to yield dicinnamamide 11 (0.75 g,
73%) as a waxy solid. [α]2D2 = –4.6 (c = 0.4, MeOH). 1H NMR
(400 MHz, CDCl3): δ = 7.82 (d, J = 15.6 Hz, 1 H, 3Ј-H or 3ЈЈ-H),
7.75 (d, J = 15.6 Hz, 1 H, 3ЈЈ-H or 3Ј-H), 7.64–7.60 (m, 6 H, Ar),
7.53–7.39 (m, 4 H, Ar), 7.22 (m, 2 H, 2ϫN-H), 6.68 (d, J =
15.6 Hz, 1 H, 2Ј-H or 2ЈЈ-H), 6.60 (d, J = 15.6 Hz, 1 H, 2ЈЈ-H or
2Ј-H), 4.24 (m, 1 H, 2-H), 3.90 (m, 1 H, 1a-H), 3.75 (m, 1 H, 1b-
H), 3.63 (m, 1 H, 4a-H), 3.51 (m, 1 H, 4b-H), 2.03 (m, 2 H, 3-H),
1.39 [m, 3 H, 3ϫCH(CH3)2], 1.18 [br., 18 H, 3ϫCH(CH3)2] ppm.
13C NMR (100 MHz, CDCl3): δ = 167.6 (s, C-1Јor C-1ЈЈ), 167.3 (s,
C-1ЈЈ or C-1Ј), 142.4 (d, C-3Ј or C-3ЈЈ), 141.5 (d, C-3ЈЈ or C-3Ј),
135.8 (s, C-4Ј or C-4ЈЈ), 135.4 (s, C-4ЈЈ or C-4Ј), 130.3 (d, 5 C, Ar),
129.5 (d, 5 C, Ar), 121.6 (d, C-2Ј or 2ЈЈ), 121.1 (d, C-2ЈЈ or C-2Ј),
71.0 (d, C-2), 45.1 (t, C-1), 37.0 (t, C-4), 34.2 (t, C-3), 18.9 [q, 6 C,
3ϫCH(CH3)2], 13.1 [d, 3 C, 3ϫCH(CH3)2] ppm. MS (ESI+): m/z
= 543.4 [M + Na]+, 521.3 [M + H]+. MS (ESI–): m/z = 519.1 [M –
H]–.
Methyl (S)-4-[(S)-1-(1-Naphthyl)ethylcarbamoyl]-3-hydroxy-3-meth-
ylbutanoate (14): Amide 13 (0.015 g, 22.7 µmol) was dissolved in
MeOH (2 mL) and a freshly prepared 10% solution of MeONa in
MeOH (0.5 mL) was added. The mixture was stirred at 30 °C for
30 min and then quenched with 5% aqueous HCl. Volatiles were
evaporated under reduced pressure, and the residue was chromato-
graphed on a silica gel column (toluene/EtOAc, 7:3 to EtOAc/
CH3OH, 9:1) to afford dicinnamide 4 (7.2 mg, 87%), which was
identical to the natural sample, and ester 14 (6.5 mg) as a waxy
solid. [α]2D2 = –6.5 (c = 0.25, MeOH).[34] The 1H and 13C NMR
spectra (CD3OD) of compound 14 matched those reported in the
literature.[23] MS (ESI+): m/z = 330.1 [M + H]+, 352.2 [M + Na]+,
680.8 [2M + Na]+.
(S)-(–)-2-Hydroxyputrescine 1,4-Dicinnamamide (12): To dicinnam-
amide 11 (0.75 g, 1.44 mmol) was added THF (3 mL), H2O (1 mL)
and CF3COOH (1 mL) whilst stirring at r.t. The reaction was mon-
itored by silica gel TLC (toluene/EtOAc, 3:7), and it went to com-
pletion in 6 h. After neutralization with a saturated aqueous solu-
tion of NaHCO3, the volatiles were removed under vacuum, and
the residue was chromatographed on a silica gel column (toluene/
EtOAc, 1:1 to EtOAc/MeOH, 9:1) to afford compound 12.
Recrystallization from MeOH gave a white microcrystalline powder
(0.51 g, 98%). M.p. Ͼ190 °C (decomp.). [α]2D2 = –2.5 (c = 1.6,
Studies on Human Tumour Cell Lines
Cell Culture and Treatments: Androgen-responsive LNCaP cells
and androgen-insensitive prostate cancer cells DU-145 cells were
obtained from American Type Culture Collection (ATCC, Rock-
ville, MD, USA). LNCaP cells were grown in RPMI-1640 medium
supplemented with 10% fetal calf serum, 100 UmL–1 penicillin,
100 µgmL–1 streptomycin and 1 m glutamine. Androgen-nonre-
sponsive DU-145 human prostate cancer cells were grown in Dul-
becco’s modified Eagle’s medium (DMEM) containing 10% FCS,
100 UmL–1 penicillin, 100 µgmL–1 streptomycin, 1 m glutamine
and 1% nonessential amino acids. The cells were plated at a con-
stant density to obtain identical experimental conditions in the dif-
ferent tests to thus achieve a high accuracy of the measurements.
After 24 h incubation at 37 °C under a humidified 5% carbon diox-
ide environment to allow cell attachment, the cells were treated
with different concentrations of maytenine (3) and pholiotic acid
(5) and incubated for 72 h under the same conditions. Stock solu-
tions of natural compounds were prepared in DMSO. Control cul-
tures received DMSO alone.
1
MeOH). The H and 13C NMR spectra in CD3OD were identical
to those of natural product 4 (see Tables 1 and 2, respectively). CD
(MeOH): λ (∆ε) = 288.0 (–0.28), 258.0 (+0.29) nm. MS (ESI+): m/z
= 365.2 [M + H]+, 387.2 [M + Na]+. HRMS (ESI+): calcd. for
C22H24N2O3Na [M + Na]+ 387.16791; found 387.16764. HRMS
(ESI+): calcd. for C44H48N4O6Na [2M + Na]+ 751.34661; found
751.34725.
(R)-2-{(S)-4-[(S)-1-(1-Naphthalen-1-yl)ethylacarbamoyl]-3-hydroxy-
3-methylbutanoyloxy}putrescine 1,4-Dicinnamamide (13): To
a
stirred solution of pholiotic acid (5; 20 mg, 39.3 µmol) and 2-
chloro-4,6-dimethoxy-1,3,5-triazine (CDMT; 7 mg, 40 µmol) in
MeCN (2 mL), was added dropwise N-methylmorpholine (4.4 µL,
40 µmol) whilst the temperature of the solution was kept at about
–5 °C. Stirring was continued at 0 °C for 4 h, and the reaction was
monitored by silica gel TLC (EtOAc/MeOH, 9:1). Subsequently, to
the solution was added a mixture of N-methylmorpholine (4.4 µL,
40 µmol) and (S)-1-(1-naphthyl)ethylamine (7 mg, 40 µmol) in
THF (2 mL). The reaction was stirred for an additional 2 h at 0 °C
and for 14 h at r.t. The volatiles were evaporated under vacuum,
MTT Bioassay: Cellular growth was determined by using the MTT
assay on 96-well microplates as described previously.[12] The optical
density of each sample well was measured with a microplate spec-
5558
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Eur. J. Org. Chem. 2007, 5551–5559

Non-Phenolic Dicinnamamides From Pholiota Spumosa
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stained with 100 µL of ethidium bromide (2 µgmL–1) for 10 min
and scored by using a fluorescence microscope (Leica, Wetzlar,
Germany) interfaced with a computer. Software (Leica-QWIN) was
used for the analysis and quantification of DNA damage by meas-
uring: (1) tail length (TL), intensity (TI) and area (TA); (2) head
length (HL), intensity (HI) and area (HA). These parameters are
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Received: June 19, 2007
Published Online: September 25, 2007
Eur. J. Org. Chem. 2007, 5551–5559
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5559