360 J ournal of Natural Products, 2004, Vol. 67, No. 3
Pungitore et al.
stirred vigorously under hydrogen (1 atm). When the con-
sumption of H2 had subsided, the mixture was filtered through
a thin layer of Si gel over Celite and concentrated to yield 4
(170 mg, 85%) as an amorphous yellow solid identical in all
respects with previously reported data.1
The impossibility of the aforementioned additional rec-
ognition factor, as well as the steric hindrance produced
by the introduced bulky groups in compounds 5 and 6,
should explain their inactivity, although their HOMOs
were similar to those of compounds 1-3. For compound 7,
the lack of activity could be attributed to both steric
hindrance and the dramatic change in the HOMO proper-
ties due to the aromatic rings.
In conclusion, these iridoids are allelochemicals to the
generalist insect T. castaneum. Taking into account the
observed taq DNA polymerase inhibition, it is possible to
propose a disruption of DNA synthesis as the basis of the
observed allelochemical effect. The experimental results are
in agreement with the theoretical basis of recognition, and
it is possible to propose that the molecular orbital proper-
ties of the assayed iridoids are important factors in the
recognition process. To our knowledge, it is the first time
that iridoids have been reported as DNA polymerase
inhibitors.
P er -O-a cetylca ta lp ol (5). Compound 1 (150 mg, 0.41
mmol) was dissolved in dry pyridine (3 mL), acetic anhydride
(3 mL) was added, and the mixture was stirred at room
temperature for 2 days. The reaction mixture was partitioned
between cold water and Et2O. The organic layer was washed
with aqueous CuSO4 (5%) to remove pyridine followed by a
solution of NaHCO3 (5%) and water. After drying over Na2-
SO4, the residue was purified by Si gel column chromatography
using mixtures of CHCl3-MeOH in increasing polarities to
yield compound 5 (120 mg, 80%) as yellow crystals: [R]D25
-54.4 (c 0.45, CHCl3); IR (KBr) νmax 2942, 1754, 1369, 1224,
1043, 1014 cm-1; 1H NMR (CDCl3, 200 MHz) δ 6.30 (1H, dd, J
) 6.0, 1.9 Hz, H-3), 5.20 (1H, dd, J ) 11.0, 7.3 Hz, H-4′), 5.05
(1 H, dd, J ) 6.0, 4.0 Hz, H-4), 4.97 (1 H, br s, H-1′), 4.95 (1H,
br s, H-2′), 4.82 (1H, d, J ) 12.8 Hz, H-10b), 4.75 (1H, d, J )
9.1 Hz, H-1), 4.36 (1H, dd, J ) 12.8, 3.65 Hz, H-6′a), 4.11(1H,
dd, J ) 12.8, 3.65 Hz, H-6b), 3.92 (1H, d, J ) 12.8 Hz, H-10
a), 3.69 (1H, m, H-5′), 3.58 (1 H, br s, H-6), 3.56 (1H, br s,
H-7), 2.51 (1H, dd, J ) 9.1, 7.1, H-9), 2.42 (1H, m, H-5), 2.15-
1.95 (18H, OAc); 13C NMR (CDCl3, 50.3 MHz) δ 171.0-169.0
(C, OAc), 140.5 (CH, C-3), 103.3 (CH, C-4), 96.4 (CH, C-1′),
94.2 (CH, C-1), 86.9 (CH, C-6), 72.5 (CH, C-3′), 72.1 (CH, C-5′),
70.5 (CH, C-2′), 68.2 (CH, C-4′), 62.8 (CH2, C-10), 62.0 (C, C-8),
61.0 (CH2, C-6′), 57.8 (CH, C-7), 41.4 (CH, C-9), 35.6 (CH, C-5);
anal. C 52.73%, H 5.70%, calcd for C27H34O16, C 52.70%, H
5.54%.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Combustion analy-
ses were performed on an Eager 200 instrument at Instituto
Universitario de Bioorga´nica Antonio Gonza´lez, La Laguna
University, Tenerife, Spain. Optical rotations were obtained
on a Perkin-Elmer 341 polarimeter. The 1H NMR spectra were
recorded at 200.13 MHz on a Bruker AC 200 with TMS as
internal standard. The 13C NMR spectra were obtained with
the same instruments at 50.23 MHz. 2D experiments were
obtained using standard Bruker microprograms. IR spectra
were recorded on an FT-IR Nicolet Prote´ge´ 460 spectrometer.
PCR experiments were done on a Perkin-Elmer GeneAmp
2400. Column chromatography was performed on Si gel G 70-
230 mesh and Kieselgel 60 H; TLC was carried out on Si gel
60 F254 (0.2 mm thick plates) using CHCl3-MeOH (70:30) as
solvent. The iridoid-containing fractions were detected by
anisaldehyde-AcOH-H2SO4 (0.5:50:1) spray reagent.
10,6′-Di-O-oleylca ta lp ol (6). To 1 (100 mg, 0.28 mmol) in
CH2Cl2 (10 mL) were added oleic acid (160 µL, 0.50 mmol),
N,N-dicyclohexylcarbodiimide (284 mg, 1.38 mmol), and 4-(dim-
ethylamino)pyridine (170 mg, 1.39 mmol).30 The reaction
mixture was stirred at room temperature for 1 day. The
mixture was filtered to remove the precipitated dicyclohexy-
lurea. The filtrate was concentrated, and the residue was
purified by Si gel column chromatography using mixtures of
n-hexane-EtOAc in increasing polarities to yield 20 mg (20%)
of compound 6 as a colorless oil: [R]2D5 -44.7 (c 0.15, CHCl3);
IR (KBr) νmax 2927, 2856, 1741, 1459, 1247, 1166, 1093 cm-1
;
P la n t Ma ter ia l. Aerial parts of Buddleja cordobensis
Griseb. were collected during J une 2001, in Cerro de La Cruz,
Departamento La Capital, San Luis, Argentina, and a voucher
specimen is deposited at the Herbarium of the Universidad
Nacional de San Luis: L.A. Del Vitto & E.M. Petenatti-4868
(UNSL).
1H NMR (CDCl3, 200 MHz) δ 6.25 (1H, dd, J ) 6.0, 1.9 Hz,
H-3), 5.32 (4 H, H-9′′, H-9′′′, H-10′′, H-10′′′), 1.30 (56 H, -CH2-
oleyl); 13C NMR (CDCl3, 50.3 MHz) δ 174.3 (C, C-1′′or C-1′′′),
173.4 (C, C-1′′or C-1′′′), 140.2 (CH, C-3), 130.0 (CH, C-9′′ and
C-9′′′), 129.6 (CH, C-10′′ and C-10′′′), 103.8 (CH, C-4), 96.0 (CH,
C-1′), 93.7 (CH, C-1), 87.0 (CH, C-6), 74.7 (CH, C-3′), 74.1 (CH,
C-5′), 70.2 (CH, C-2′), 68.9 (CH, C-4′), 64.8 (C, C-8), 61.8 (CH2,
C-6′), 57.6 (CH2, C-10), 42.0 (CH, C-9), 35.7 (CH, C-5), 34.2-
22.6 (28 carbons, -CH2-oleyl moiety), 14.1 (CH3, C-18′′ and
C-18′′′); anal. C 69.06%, H 10.02%, calcd for C51H82O12, C
69.07%, H 9.25%.
Extr a ction a n d Isola tion . The dry aerial parts (3.2 kg)
were chopped and macerated twice for 10 day periods with
MeOH. The solvent was evaporated under reduced pressure
at low temperature, and the residue (177.5 g) taken up in
CHCl3 and partitioned against H2O. The aqueous layer was
subjected to lyophylization, and the brown amorphous residue
(80.0 g) was purified by Si gel column chromatography using
mixtures of CHCl3-MeOH in increasing polarities. After
several purifications, compound 1 (3.8 g) was obtained. The
structure of 1 was confirmed by comparison of both spectro-
scopical and physical data with previously published values.1,29
Compound 2 was kindly supply by Prof. J osep Coll Toledano,
Departamento de Qu´ımica Orga´nica Biolo´gica, Institut d’Investi-
gacions Quimiques i Ambientals “J osep Pascual Vila”, Barce-
lona, Spain. This iridioid was obtained from Ajuga reptans L.13
Ha r p a gid e (3). To 2 (30 mg, 0.082 mmol) in MeOH (20 mL)
was added NaH (oil dispersion 57-63%). The reaction mixture
was stirred at room temperature for 20 min, the volume was
reduced to 8 mL in vacuo, and the reaction mixture was
washed with n-hexane in order to remove the oil. The MeOH
layer was concentrated, and the residue was purified by Si
gel column cromatography to yield 3 (25 mg, 83%) as colorless
crystals. The structure of 3 was confirmed by comparison of
both spectral and physical data with previously published
values.1
6,2′,3′,4′-Tetr a -O-a cetyl-10,6′-d i-O-(ter t-bu tyld ip h en yl-
silyl)ca ta lp ol (7). To 1 (100 mg, 0.28 mmol) dissolved in CH2-
Cl2 (25 mL) were added tert-butylchlorodiphenylsilane (200 µL,
0.54 mmmol) and imidazole (200 mg, 2.9 mmol). The mixture
was stirred overnight at room temperature and then was
partitioned between H2O and Et2O. The organic layer was
dried and concentrated, and the residue was purified by Si
gel column chromatography using mixtures of n-hexane-
EtOAc in increasing polarities to yield 10,6′-di-O-(tert-butyl-
diphenylsilyl)catalpol (65 mg, 65%) as a colorless oil. The
silylderivative was peracetylated by standard techniques to
yield compound 7 (35 mg, 54%) as an amorphous yellow solid:
[R]2D5 -22.3 (c 0.20, CHCl3); IR (KBr) νmax 3072, 2931, 2858,
1
1760, 1652, 1473, 1429, 1218, 1114, 823, 701 cm-1; H NMR
(CDCl3, 200 MHz) δ 7.8-6.9 (20H, aromatic protons), 6.32 (1H,
dd, J ) 6.0, 1.9 Hz, H-3), 5.15-4.65 (6H, m, H-4′, H-4, H-1′,
H-2′, H-10b, H-1), 4.30-4.05 (3H, m, H-6a, H-6b, and H-10a),
3.65-3.30 (3H, m, H-5′, H-6, and H-7), 2.60-2.45 (2H, m, H-9,
and H-5), 2.10-1.80 (12 H, s, OAc), 1.15-1.10 (18H, tert-butyl
protons); anal. C 67,49%, H: 6,88%, calcd for C55H66O14Si2, C
67.48%, H 6.74%.
Dih yd r oca ta lp ol (4). To 1 (200 mg, 0.55 mmol) in MeOH
(50 mL) was added Pt/C (5%) (20 mg). The suspension was