Two new pyridine monoterpene alkaloids by chemical conversion of a commercial extract of Harpagophytum procumbens

Article abstract of DOI:10.1021/np980149c

The treatment of harpagide (1), harpagoside (2), or 8-O-p- coumaroylharpagide (3), the main iridoids of Harpagophytum procumbens and Harpagophytum zeyheri, with NH3 and HCl led to aucubinine B(4), a pyridine monoterpene alkaloid (PMTA). A similar procedure applied to a commercial extract of H. procumbens yielded 4 and two new PMTAs named beatrine A (5) and beatrine B (6). The structures of these new PMTAs were established using ESIMS and 2D NMR. Their semisynthesis was analyzed in terms of reaction mechanisms.

Full text of DOI:10.1021/np980149c

J . Nat. Prod. 1999, 62, 211-213
211
Tw o New P yr id in e Mon oter p en e Alk a loid s by Ch em ica l Con ver sion of a
Com m er cia l Extr a ct of Ha r pa goph ytu m pr ocu m ben s
B. Baghdikian,† E. Ollivier,*,† R. Faure,‡ L. Debrauwer,§ P. Rathelot,† and G. Balansard†
Laboratory of Pharmacognosy and Laboratory of Organic Chemistry, Faculty of Pharmacy, 27 Boulevard J ean Moulin,
University Aix-Marseille II, 13385 Marseille Cedex 5, France, ESA 6009, University Aix-Marseille III, Avenue Escadrille
Normandie Niemen, 13397 Marseille Cedex 20, France, and INRA, Center of Research of Toulouse, Laboratory of Xenobiotics,
180 Chemin de Tournefeuille, BP 3, 31931 Toulouse, France
Received April 16, 1998
The treatment of harpagide (1), harpagoside (2), or 8-O-p-coumaroylharpagide (3), the main iridoids of
Harpagophytum procumbens and Harpagophytum zeyheri, with NH3 and HCl led to aucubinine B(4), a
pyridine monoterpene alkaloid (PMTA). A similar procedure applied to a commercial extract of H.
procumbens yielded 4 and two new PMTAs named beatrine A (5) and beatrine B (6). The structures of
these new PMTAs were established using ESIMS and 2D NMR. Their semisynthesis was analyzed in
terms of reaction mechanisms.
Sch em e 1. Proposed Mode of Formation of Aucubinine B (4)
from Harpagoside (2).
The formation of pyridine monoterpene alkaloids (PM-
TAs) from iridoids such as antirrhinoside,1 and from
secoiridoids such as gentiopicroside2 and swertiamarine,2
by treatment with ammonia and acid has been reported.
The conversion of iridoids into PMTAs has also been
reported using human intestinal bacteria3,4 or by enzymatic
reaction with â-glucosidase on aucubin,3 geniposide,5,6
gardenoside,4 8-epiloganin,6 cornin,6 and antirrhinoside.6
In this paper, we report the transformation of harpagide
(1), harpagoside (2), or 8-O-p-coumaroylharpagide (3), the
main iridoids of the genus Harpagophytum, to PMTAs
using ammonia and hydrochloric acid. H. procumbens
(Pedaliaceae) is an herbaceous plant growing in the Kala-
hari desert and in the Namibian steppes. The secondary
tuberized roots of this plant, which contain iridoids, are
used for their antiinflammatory and analgesic effects.7
Conversion of harpagoside (2) into a PMTA could account
for the observed discrepancies between pharmacological
data obtained with harpagoside and with H. procumbens
preparations.8
mixture that was purified by preparative liquid chroma-
tography on Sephadex LH-20 to give aucubinine B (4) as
the primary product. The ESIMS of 4 showed [MH]+ at m/z
148, corresponding to a molecular formula of C9H9ON, and
other prominent fragmentation ions at m/z 133 [MH -
CH3]+, 130 [MH - H2O]+, 120 [MH - CO]+, and 106 [MH
- CH2CO]+. The 1H 400 MHz NMR spectrum of 4 had
signals characteristic of a disubstituted pyridine 3,4-ring9
at δ 8.94 (sbr), 8.67 (d, J ) 5.0 Hz), and 7.52 (dd, J ) 4.9,
1.0 Hz). Close examination of the remaining resonances
indicated a partial structure -CH(CH3)CH2- (see the
Experimental Section for data). The 13C NMR spectrum
confirmed these results and further indicated the presence
of nine carbons including a carbonyl group and five
aromatic carbons. At this point, 4 was identified as aucu-
binine B on the basis of the above arguments and general
1
Resu lts a n d Discu ssion
similarity of its H NMR spectrum with that of previously
reported aucubinine B.3 Complete assignments of the 13C
NMR signals of 4 was realized from the concerted applica-
tion of HMQC10 and HMBC11 experiments.
Treatment of individual pure compounds 1, 2, or 3 first
with ammonia and then hydrochloric acid yielded a product
Aucubinine B (4) was thus obtained for the first time
from these iridoids by a controlled chemical transformation.
Scheme 1 gives a plausible mechanism for the conversion
of harpagoside (2) into aucubinine B (4).
* To whom correspondence should be addressed. Phone/fax: 04 91 83 55
93. E-mail: Evelyne.Ollivier@ pharmacie.univ-mrs.fr.
† University Aix-Marseille II.
‡ University Aix-Marseille III.
§ INRA, Center of Research of Toulouse.
10.1021/np980149c CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/18/1998

212 J ournal of Natural Products, 1999, Vol. 62, No. 2
Baghdikian et al.
Studies of the conversion of these iridoids by an enzymatic
pathway, â-glucosidase, and by microbiological pathways
with intestinal bacteria are in progress in order to deter-
mine if these compounds may be produced in vivo.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. NMR spectra were
recorded in CDCl3 on a Bruker AMX400 spectrometer using
tetramethylsilane (TMS) as internal reference in 1H and13
C
F igu r e 1. Proton-carbon connectivities found in the HMBC spectrum
of beatrine A (5).
measurements and standard Bruker pulse sequences for two-
dimensional experiments. ESIMS were obtained using a
Nermag R-10-10 H mass spectrometer. Melting points were
determined on a Bu¨chi apparatus and are uncorrected. UV
spectra were recorded with a Kontron 930 spectrophotometer
and IR spectra (film) with a Perkin-Elmer 1600 spectrometer.
Optical rotations [R] were measured on a Perkin-Elmer 341
Orot polarimeter. Elemental analyses were performed with a
Technicon auto-analyzer. Column chromatography was carried
out on Sephadex LH-20 (Pharmacia) and Lichroprep C18
(Merck).
P la n t Ma ter ia ls. H. procumbens and H. zeyheri were
collected, identified, and provided by G. J . R. Betti in Namibia.
Voucher specimens (H. procumbens 4-93 and H. zeyheri 4-93)
are kept in the department of Pharmacognosy, Faculty of
Pharmacy, Marseilles, France. Harpagide (1) and harpagoside
(2) were purified from H. procumbens roots by preparative
liquid chromatography (J obin-Yvon, France), on RP-18 (Li-
chroprep C18, Merck, 200 g), elution with aqueous MeOH
solutions. 8-O-p-Coumaroylharpagide (3) was purified from H.
zeyheri roots as previously described.8 The commercial extract
of H. procumbens was from Indena (Milano, Italy). This
aqueous extract contained 2.5% of 1, 10.2% of 2, and 4.3% of
3.8
Compounds 1-3 are the main iridoids present in H.
procumbens and H. zeyheri.8 A commercial extract of H.
procumbens treated first with ammonia and then with
hydrochloric acid gave, in low yield (near 1.0%), a brown
viscous residue. From this residue, three PMTAs, aucubi-
nine B (4), beatrine A (5), and beatrine B (6), were isolated
by preparative liquid chromatography. The ESIMS of
beatrine A (5) showed the molecular ion [MH]+ at m/z 256
in agreement with a molecular formula of C15H13O3N.
1
The H NMR spectrum of 5 revealed signals at δ 8.98,
8.72 (d, J ) 4.4 Hz) and 7.65 (d, J ) 4.8 Hz), which
characterized the disubstituted pyridine ring. Further
analysis of the 1H NMR data indicated a secondary methyl
group at δ 1.58 (d, J ) 7.0 Hz), a methylene bearing an
oxygen at δ 4.74, and a deshielded resonance at δ 7.43 (d,
J ) 1.7 Hz) showing long-range allylic coupling between
the methine signal and the methyl group. Finally, the
presence of a 2′,5′-disubstituted furan ring in 5 was
suggested by a pair of low-field doublets at δ 6.81 (d, J )
3.4 Hz) and 6.49 (d, J ) 3.4 Hz).
Con ver sion of Ha r p a gid e (1), Ha r p a gosid e (2), or 8-O-
p-Cou m a r oylh a r p a gid e (3) to Au cu bin in e B (4). Each
individual iridoid (1.0 g) was dissolved in 200 mL 25% aqueous
NH4OH-MeOH (50:50) and stirred for 1 h at room tempera-
ture. The reaction mixture was concentrated to 100 mL under
vacuum, and 50 mL of 8 N HCl was added. After reflux for 15
min, the mixture was basified with NH4OH (pH ) 8-9) and
extracted with CHCl3. The organic layer was extracted with 3
N HCl, and then the aqueous layer was rebasified and
extracted with CHCl3. Chromatography of the residue on
Sephadex LH-20 (MeOH) yielded 5 mg of 4: mp 110-113 °C;
[R] -10°; UV (MeOH) λmax 225, 280 nm; IR (MeOH) νmax 1720
The occurrence of a conjugated carbonyl function was
supported by the signal at δ 193.6 in the 13C NMR
spectrum of 5. Multiplicities of the individual 13C peaks
were determined using the DEPT pulse sequence12 and the
results suggested a tricyclic structure for 5.
1
The molecular framework and the complete H and 13C
chemical shift assignments of beatrine A (5) were deduced
from the combination of both direct (HMQC) and long-
range (HMBC) correlation experiments. Consideration of
the various connectivities (Figure 1) in conjunction with
the inferences drawn from the 1D NMR data establish that
5 is 6-[(5′-hydroxymethylfuran-2′-yl)methylene]-7-methyl-
6,7-dihydro[2]pyrindin-5-one.
1
cm-1; H NMR (CDCl3, 400 MHz) δ 8.94 (1H, sbr, H-1), 8.67
(1H, d, J ) 5.0 Hz, H-3), 7.52 (1H, dd, J ) 1.0, 4.9 Hz, H-4),
3.55 (1H, m, H-7), 2.30 (1H, dd, J ) 3.5, 19.4 Hz, H-8a), 2.95
(1H, dd, J ) 7.5, 19.4 Hz, H-8b), 1.45 (3H, d, J ) 7.1 Hz, H-10);
13C NMR (CDCl3, 100 MHz) δ 149.22 (C-1), 148.56 (C-3), 116.24
(C-4), 142.39 (C-5), 152.61 (C-6), 31.63 (C-7), 45.32 (C-8),
206.07 (C-9), 21.29 (C-10); ESIMS m/z 148 [MH]+.
The stereochemistry at C-10 was unequivocally estab-
lished using a phase-sensitive NOESY experiment13 in
which H-12 NOE cross-peaks were observed with H-16 and
H-7. The molecular formula for beatrine B (6) was estab-
lished as C15H13O2N (ESIMS [MH]+ m/z 240). The struc-
tural similarity between beatrine A (5) and beatrine B (6)
was shown by the close similarity of the 1H and 13C
chemical shifts, the striking difference being the presence
of a methyl group instead of the hydroxymethyl function
at C-14. Beatrine A and B (5 and 6) are new compounds.
The furan ring in 5 and 6 may be due to carbohydrates
present in the H. procumbens extract. To confirm this
hypothesis, we have applied the experimental procedure
previously described to 2 in the presence of added sugars
(stachyose, fructose, galactose, or glucose); these com-
pounds are the main sugars of H. procumbens. Under these
conditions the reaction yielded 5, especially in the presence
of fructose. Aucubinine B (4) may undergo a keto-enol
tautomerism, and under acidic conditions, the enol form
can react with the aldehydic group of 5-(hydroxymethyl)-
furfural obtained by dehydration of the corresponding
carbohydrate, yielding 5. The formation of 6 could be
explained by the presence of 6-deoxyhexose in the extract.
Con ver sion of a Com m er cia l Extr a ct of Ha r pa goph y-
tu m pr ocu m ben s (5 a n d 6). The procedure previously
described for the pure compounds was applied to 10 g of
commercially dried extract. A portion of the residue obtained
(200 mg) was purified on a column of Sephadex LH-20 (elution
with CH2Cl2) yielding 4 mg of 4 and 10 mg of beatrine A (5):
mp 95-98 °C; [R] -36° (MeOH); UV (MeOH) λmax 250, 380
nm; IR (MeOH) νmax 1750, 1615, 1570 cm-1; 1H NMR (CDCl3,
400 MHz) δ 8.98 (1H, sbr, H-1), 8.72 (1H, dbr, J ) 4.4 Hz,
H-3), 7.65 (1H, dbr, J ) 4.8 Hz, H-4), 4.42 (1H, dq, J ) 1.7,
7.0 Hz, H-7), 7.43 (1H, d, J ) 1.7 Hz, H-8), 6.81 (1H, d, J )
3.4 Hz, H-3′), 6.49 (1H, d, J ) 3.4 Hz, H-4′), 4.74 (2H, s, CH2-
OH), 1.58 (3H, d, J ) 7 Hz, CH3); 13C NMR (CDCl3, 100 MHz)
δ 148.9 (C-1), 148.7 (C-3), 116.7 (C-4), 149.1 (C-4a), 193.6 (C-
5), 137.1 (C-6), 36.3 (C-7), 148.8 (C-7a), 20.6 (CH3), 121.7 (C-
8), 151.0 (C-2′), 120.0 (C-3′), 111.0 (C-4′), 158.3 (C-5′), 57.9
(CH2OH); ESIMS m/z 256 [MH]+; anal. C 70.26%, H 5.80%, N
5.37%, calcd for C15H13O3N, C 70.58%, H 5.13%, N 5.49%.
The remaining residue (300 mg) was separated by chroma-
tography on Lichroprep C18 (50% MeOH f MeOH) leading
to 6 mg of beatrine B (6): mp 80-82 °C; [R] -51° (MeOH);

Pyridine Monoterpene Alkaloids from Iridoids Harpagophytum
J ournal of Natural Products, 1999, Vol. 62, No. 2 213
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UV (MeOH) λmax 252, 386 nm; IR (MeOH) νmax 1750, 1615,
1
1570 cm-1; H NMR (CDCl3, 400 MHz) δ 8.96 (1H, sbr, H-1),
8.70 (1H, dbr, J ) 4.9 Hz, H-3), 7.64 (1H, dbr, J ) 4.9 Hz,
H-4), 4.38 (1H, dq, J ) 1.3, 7.0 Hz, H-7), 7.39 (1H, d, J ) 1.3
Hz, H-8), 6.77 (1H, d, J ) 3.3 Hz, H-3′), 6.20 (1H, d, J ) 2.8
Hz, H-4′), 2.44 (3H, s, H- CH3), 1.57 (3H, d, J ) 7.0 Hz, CH3);
13C NMR (CDCl3, 100 MHz) δ 148.7 (C-1), 148.5 (C-3), 116.7
(C-4), 143.4 (C-4a), 193.7 (C-5), 135.4 (C-6), 36.3 (C-7), 148.9
(C-7a), 20.4 (CH3), 122.0 (C-8), 149.8 (C-2′), 121.3 (C-3′), 109.9
(C-4′), 157.6 (C-5′), 14.3 (CH3); ESIMS m/z 240 [MH]+; anal.
C 74.98%, H 5.15%, N 5.64%, calcd for C15H13O2N, C 75.31%,
H 5.44%, N 5.86%.
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Ack n ow led gm en t. We thank G. Boudon for his technical
collaboration.
Refer en ces a n d Notes
(1) Franzyk, H.; Frederiksen, S. M.; J ensen, S. R. J . Nat. Prod. 1997,
60, 1012-1016.
(2) Popov, S. S.; Marekov, N. L.; Do, T. N. J . Nat. Prod. 1988, 51, 765-
768.
NP980149C