A new pyrrole alkaloid from seeds of Castanea sativa

Article abstract of DOI:10.1016/S0367-326X(01)00344-6

A new pyrrole alkaloid, methyl-(5-formyl-1H-pyrrole-2-yl)-4-hydroxybutyrate (1), was isolated from sweet chestnut seeds and its structure elucidated on the basis of data from NMR spectroscopy and by comparison with synthetic analogues.

Full text of DOI:10.1016/S0367-326X(01)00344-6

Fitoterapia 73 Ž2002. 22᎐27
A new pyrrole alkaloid from seeds of
Castanea sati¨a
a,U
a
Alois Hiermann , Samir Kedwani ,
b
b
Hans Wolfgang Schramm , Christoph Seger
a
Institute of Pharmacognosy, Karl Franzens Uni¨ersity, Uni¨ersit ¨a tsplatz 4, A-8010 Graz, Austria
b
Institute of Pharmaceutical Chemistry and Pharmaceutical Technology, Karl Franzens Uni¨ersity,
Uni¨ersit ¨a tsplatz 1, A-8010 Graz, Austria
Received 9 January 2001; accepted in revised form 7 July 2001
Abstract
A new pyrrole alkaloid, methyl-Ž5-formyl-1H-pyrrole-2-yl.-4-hydroxybutyrate (1), was
isolated from sweet chestnut seeds and its structure elucidated on the basis of data from
NMR spectroscopy and by comparison with synthetic analogues. ᮊ 2002 Elsevier Science
B.V. All rights reserved.
Keywords: Castanea sati¨a; Alkaloids; Methyl-Ž5-formyl-1H-pyrrole-2-yl.-4-hydroxybutyrate
1
. Introduction
Seeds of Castanea sati¨a Mill. ŽFagaceae., a deciduous tree growing in Southern
Europe Žespecially in the Mediterranean region and the Balkans., are currently
used in paediatrics for treatment of gastroenteritis and as a gluten-free diet in
cases of coeliac disease w1,2x. Only few reports have concerned the phytochemistry
of these seeds w3᎐6x. In the Middle Ages the raw seeds were disclosed by the
German herbalist Hildegard von Bingen as useful in the treatment of heart
disorders w7x. As part of our ongoing phytochemical studies on the sweet chestnut
seeds we isolated a new pyrrole alkaloid 1. According to our knowledge, no pyrrole
U
Corresponding author. Tel.: q43-316-3805525; fax: q43-316-3809860.
E-mail address: alois.hiermann@kfunigraz.ac.at ŽA. Hiermann..
0
367-326Xr02r$ - see front matter ᮊ 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 6 7 - 3 2 6 X Ž 0 1 . 0 0 3 4 4 - 6

A. Hiermann et al. rFitoterapia 73 (2002) 22᎐27
23
derivative of a similar structure has been isolated from plants before. In this paper,
we report on the isolation and structure elucidation of this compound.
2
. Experimental
2
.1. General
NMR spectra were recorded with a Bruker AMX 500 spectrometer equipped
with a 5-mm inverse probe head at 300 K. TMS was used as internal standard.
Before NOE experiments were performed, dissolved oxygen was removed by
bubbling Ar through the solution. LSI-MS spectra were measured on a Mat 8500
ŽFinnigan., matrix glycerol, 4.5 kV Cs beam, negative and positive ion mode.
UVrVIS spectra were recorded on a Shimazu UV160A and the IR measurements
were performed on a Perkin-Elmer 1720-X. Elementary analysis was performed at
the Microanalytical Laboratory, Institute of Physical Chemistry, University Vienna,
Austria.
2
.2. Plant material
Dried seeds Ž400 g. of C. sati¨a were collected from Grubberg, near Graz,
Austria, in October 1992. The plant material was authenticated by macro- and
microscopy. A voucher specimen ŽHIKED 1r92. is deposited at the Institute of
Pharmacognosy at the University of Graz.
2
.3. Extraction and isolation
Powdered seeds were Soxhlet extracted successively with petrol and MeOH. The
MeOH extract was separated on a Sephadex LH 20 column Žlength, 80 cm; i.d., 5
cm. eluting with MeOH. Fractions of 100 ml were collected and monitored by
TLC. Fractions 8᎐12 were combined, concentrated to dryness and the residue
suspended in 40 ml H O. The aqueous phase was then subjected to CC on
2
Polyamide Žlength, 40 cm; i.d., 3 cm. using a H O᎐MeOH step gradient ŽH O 1000
2
2
ml, 50% MeOH 2000 ml, MeOH 1000 ml.. Each fraction was analysed by TLC
ŽSi-gel 60 F254, CHCl rMeOHrH O, 36:25:5, detection by spraying with 2,4-di-
3
2
nitrophenylhydrazine and potassium ferricyanide.. Fractions of the 50% MeOH
eluates containing compound 1 were pooled and further purified by preparative
HPLC on Lichrosorb RP-18 Ž250 = 20 mm. 7 ␮m, MeOHrH O 15:85, UV 296
2
nm. The total isolation procedure was repeated =4 to afford 4 mg of the oily
compound 1.
Treatment of 1 with excess diazomethane afforded the N-methyl derivative 2.
Methyl-(5-formyl-1H-pyrrole-2-yl)-4-hydroxybutyrate (1). TLC:
R
0.66
f
1
ŽCHCl rMeOHrH O 36:25:5.; UV max ŽMeOH.: 291, 255 sh nm; H and
3
2
1
3
C-NMR: see Tables 1 and 2, respectively; LSI-MS negative ion mode mrz: 210

2
4
A. Hiermann et al. rFitoterapia 73 (2002) 22᎐27
Table 1
1
H-NMR spectral data of compounds 1 ŽCD OD., 3, 5, 7 and 9 ŽDMSO-d6.
3
H
3
1
5
7
9
2
3
4
5
Ј
4.49 Žs.
6.22 Žd.
6.93 Žd.
9.42 Žs.
4.64 Žs.
6.25 Žd.
6.97 Žd.
9.41 Žs.
5.11 Žs.
6.31 Žd.
6.94 Žd.
9.41 Žs.
5.11 Žs.
6.30 Žd.
6.92 Žd.
9.40 Žs.
11.62 Žs br.
2.49 Žt.
5.05 Žs.
6.27 Žd.
6.90 Žd.
9.38 Žs.
11.56 Žs br.
2.35 Žt.
1.85 Žm.
3.42 Žt.
4.38 Žs.
a
Ј
b
NH
11.93 Žs br.
nd
nd
2
3
4
5
6
7
Љ
2.24 Žt.
1.98 Žm.
4.37 Žt.
2.43 Žt.
1.95 Žm.
4.06 Žt.
Љ
2.08 Žm.
3.41 Žt.
Љ
Љ
Љ
1.99 Žs.
Љ ᎐9Љ
7.25 Žm.
a
J3,4 f 4 Hz, J
f 7 Hz, J3Љ ,4Љ f 7 Hz for all cases.
2Љ ,3Љ
b
nd, not detected.
q
q
q
ŽM y H .; LSI-MS positive ion mode mrz: 212 ŽMH ., 194 ŽMH y H O., 166
2
q
ŽMH y C H O.. Calculated: C H NO .
2
6
10
13
4
1
Methyl-(5-formyl-1-methyl-1H-pyrrole-2-yl)-4-hydroxybutyrate (2). H-NMR ŽC D .:
6
6
␦
1.50 Ž1H, m, H-3Љ ., 1.95 Ž1H, t, J 7.3 Hz, H-2Љ ., 3.20 Ž3H, s, N-CH ., 4.15 Ž1H, t,
3
J 7.6 Hz, H-4Љ ., 4.20 Ž1H, s, H-5Ј., 5.90 Ž1H, d, J 4.0 Hz, H-4., 6.50 Ž1H, d, J 4.0
q
Hz, H-3., 9.45 Ž1H, s, CHO.; LSI-MS negative ion mode mrz: 224 ŽM y H ..
Calculated: C H NO .
1
1
15
4
Table 2
1
3
C-NMR spectral data of compounds 1, 3, 5, 7 and 9
a
b
a
a
a
b
C
3
1
5
7
9
9
2
2
3
4
5
5
1
2
3
4
5
6
7
8
9
Ј
56.11
56.38
61.44
61.60
61.37
59.12
132.09
108.81
120.89
142.44
178.61
136.51
111.41
126.09
136.53
114.44
124.35
138.90
182.87
176.05
33.22
26.87
66.45
174.71
22.64
136.47
114.61
124.57
138.89
183.03
175.79
35.16
136.47
114.49
124.48
139.02
182.88
176.55
33.70
27.95
72.05
75.68
136.64
112.21
122.24
134.10
182.77
174.31
31.59
25.75
69.99
73.31
c
nd
Ј
Љ
Љ
Љ
Љ
Љ
Љ
Љ
Љ
Љ
180.81
175.85
32.05
28.56
61.12
30.87
35.45
141.54
130.67
131.26
130.53
139.28
128.97
128.30
128.20
a
b
c
Solvent: DMSO-d6.
Solvent: CD OD.
3
nd, not detected.

A. Hiermann et al. rFitoterapia 73 (2002) 22᎐27
25
2
.4. General procedure for synthesis of 5, 7 and 9
One of the O-substituted 4-hydroxybutyric acids 4, 6 and 7 Žsee Fig. 1. Ž0.02 mol.
and 0.02 mol of N,NЈ-carbonyldiimidazole was dissolved in 60 g anhydrous DMF
and the mixture was stirred at room temperature for 1 h, 2.5 g of 5-hydroxymethyl-
pyrrole-2-carbaldehyde (3) were suspended with 60 g of anhydrous DMF and
catalytic amounts of NaH in another flask and the mixture was stirred at 40ЊC for 1
h. Then, both mixtures were pooled and kept under stirring at 40ЊC overnight. The
separated imidazole was filtered off and the filtrate was evaporated in high
vacuum. The residue was dissolved in a mixture of Et O and water. The aqueous
2
layer was extracted with Et O and the organic layer was washed several times with
2
water and sodium hydrogen carbonate. The organic layer was dried over anhydrous
sodium sulfate and the solvent was evaporated to yield the reaction product Ž5, 7
and 9, respectively; yield: 32, 19 and 34%, respectively..
Methyl-(5-formyl-1H-pyrrol-2-yl)-4-acetoxybutyrate (5). Yellow-brown oil; TLC: Rf
0
.81 ŽCHCl rEtOAc 4:1.; UV max ŽMeOH.: 290, 204 nm; IR bands ŽKBr.: 3236
3
y1
1 13
Žs., 3132 Žw., 2968 Žw., 1734 Žs., 1640 Žs. cm ; H and C-NMR: see Tables 1 and
, respectively. ŽFound: C, 56.94; H, 5.92; N, 5.48. Calculated for C H NO : C,
2
12 15 5
5
6.91; H, 5.97; N, 5.53..
Methyl-(5-formyl-1H-pyrrol-2-yl)-4-bromobutyrate (7). Yellow oil; TLC: Rf 0.88
ŽCHCl rEtOAc 4:1.; IR bands ŽKBr.: 3236 Žs., 3132 Žw., 2968 Žw., 1734 Žs., 1640
3
y1
1
13
Žs. cm ; H and C-NMR: see Tables 1 and 2, respectively. ŽFound: C, 48.75; H,
.47; N, 5.09. Calculated for C H BrNO : C, 48.82; H, 4.41; N, 5.11..
4
1
0
12
3
Methyl-(5-formyl-1H-pyrrol-2-yl)-4-benzyloxybutyrate (9). Yellow-brown oil; TLC:
R 0.71 ŽCHCl rEtOAc 4:1.; UV max ŽMeOH.: 291, 207 nm; IR bands ŽKBr.:
f
3
y1
1
3
264 Žs., 3140, 3065, 3040 Žw., 2940, 2860 Žw., 1738 Žs., 1646 Žs. cm ; H and
C-NMR: see Tables 1 and 2, respectively. ŽFound: C, 67.71; H, 6.39; N, 4.72.
1
3
Calculated for C H NO : C, 67.76; H, 6.35; N, 4.65..
1
7
19
4
3
. Results and discussion
A methanolic extract of defatted C. sati¨a seeds was worked up by CC on
Sephadex LH-20 and polyamide as well as preparative RP-HPLC to give the
pyrrole alkaloid 1, which is present in very low concentration. The structure of 1
1
13
was determined by H and C-NMR as well as by comparison with synthetically
obtained derivatives.
1
The H-NMR spectrum of 1 showed a pair of coupled olefinic protons at 6.25
and 6.97 ppm. Their assignment as H-4 and H-3, respectively, was made possible by
the analysis of the HMBC spectrum of compound 9 as well as by a NOE
experiment performed with the title compound. This experiment resulted in the
signal enhancement of H-3 upon irritation of the aldehyde proton. The vicinity of
the aldehyde group and the NH of the pyrrol ring was proved by NOE contacts
between CHO and NCH in the N-methylated derivative 2. The position of the
3

2
6
A. Hiermann et al. rFitoterapia 73 (2002) 22᎐27

A. Hiermann et al. rFitoterapia 73 (2002) 22᎐27
27
attachment of the CH O side chain to the pyrrol ring was proved by a HMBC
2
contact from H-2Ј to C-2 of the core structure w11,12x.
To confirm the structure of 1 independently, we tried to prepare it or a related
compound by total synthesis ŽFig. 1.. The starting material 5-hydroxymethyl-pyr-
role-2-carbaldehyde (3), which can be obtained in a well documented four step
synthesis w8,9x, was reacted in a one-pot reaction with ␻-protected 4-hydroxybutyric
acids 4, 6, 8 and N,NЈ-carbonyl-diimidazole w10x in the presence of catalytic
amounts of sodium hydride in DMF. Thus, the synthesis of methyl-Ž5-formyl-1H-
pyrrole-2-yl.-4-acetoxybutyrate (5), methyl-Ž5-formyl-1H-pyrrole-2-yl.-4-bromo-
butyrate (7) and methyl-Ž5-formyl-1H-pyrrole-2-yl.-4-benzyloxybutyrate (9) was
achieved. Hydrolysis from 5 and 7, carried out by using various methods, did not
result in the expected methyl-Ž5-formyl-1H-pyrrole-2-yl.-4-hydroxybutyrate (1), only
5
-hydroxymethyl-pyrrole-2-carbaldehyde (3) being recovered. Hydrogenation of 9
with palladium on charcoal under different conditions yielded exclusively 5-methyl-
pyrrole-2-carbaldehyde (10).
The chemical shifts in the H- ŽTable 1. and in the 13C-NMR spectra ŽTable 2.
1
of the starting material 3, the isolated compound 1 and the protected synthetic
1
3
analogues 5, 7 and 9 were very similar. The assignment of the C shift values was
supported by the CH correlation analysis ŽHMQC, HMBC. of compound 9.
Acknowledgements
The authors want to thank Doris Reiter for doing most of the preparative work
and Manfred Schubert-Zsilavecz for measuring the mass spectra.
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