648
Vol. 51, No. 6
useful information regarding the metabolism of drugs in HR-ESI-MS m/z [MϩH]ϩ: 199.0865 (Calcd for C12H10N2OϩH: 199.0866).
Compound 7 was amorphous; Rf 0.23; UV (MeOH) lmax(log e): 256 (4.44),
mammalian systems.
316 (4.28), 344 (3.68), 358 (3.54) nm; IR nmax (KBr) cmϪ1: 3353, 3195,
1
1618, 1455, 1382, 1204, 1171, 1094, 762; H-NMR (500 MHz, CDCl3): d
Experimental
8.07 (1H, d, Jϭ6.4, H-3), 7.93 (1H, d, Jϭ6.7, H-5), 7.78 (1H, d, Jϭ6.4, H-
4), 7.49 (1H, dd, Jϭ7.2, 7.5, H-7), 7.45 (1H, d, 7.5, H-8), 7.21 (1H, dd,
Jϭ6.7, 7.2, H-6), 2.71 (3H, s, Me-1). 13C-NMR: d 142.54 (C-8a), 135.96
(C-8b), 134.60 (C-1), 130.58 (C-3), 128.30 (C-7), 122.77 (C-4a), 121.27 (C-
4b), 121.14 (C-5), 120.69 (C-6), 114.22 (C-4), 111.80 (C-8), 11.71 (Me-1)
HR-ESI-MS m/z [MϩH]ϩ: 199.0876 (Calcd for C12H10N2OϩH: 199.0866).
Preparation of Harman-2-oxide 7 A mixture of harman (5) (100 mg)
and m-chloroperbenzoic acid (100 mg) in CHCl3 was allowed to stand at
room temperature for 24 h. It was washed with 5% NaHCO3 solution. The
organic layer was evaporated and the residue was chromatographed over Si
gel to yield harman-2-oxide (7) as a white solid (18.60 mg, 17.2%) with
physical data identical to those of the product from microbial transforma-
tion.
General Experimental Procedure Melting points were determined on
a Thomas-Hoover apparatus and are uncorrected. The IR spectra were run in
CHCl3 using an ATI Mattson Genesis Series FTIR Spectrophotometer. The
1H- and 13C-NMR were recorded in CDCl3 on a Bruker Avance DRX-500
FT spectrometer. High resolution electrospray ionization mass spectroscopy
(HR-ESI-MS) data were obtained using a Bruker GioApex 3.0 apparatus.
Substrates Harman, harmaline and harmalol were from Aldrich (Mil-
waukee, U.S.A.). The authenticity of the alkaloids was established by physi-
cal methods including NMR and HPLC.
Organisms and Metabolism Thirty-seven microorganisms obtained
from the National Center for Natural Products Research, University of Mis-
sissippi were used for screening. All the fermentation experiments were car-
ried out in medium a, consisting of dextrose, 20 g; NaCl, 5 g; K2HPO4, 5 g;
bacto-peptone (Difco Labs), 5 g and yeast extract (Difco Labs, Detroit, MI,
U.S.A.), 5 g per liter of distilled water. Initial fermentations were conducted
in 125 ml Erlenmeyer flasks containing 25 ml medium a. A two-stage fer-
mentation procedure was adopted in all experiments.14) Each substrate was
added in dimethylformamide (0.5 mg/ml) to 24 h old stage II cultures. They
were incubated at room temperature on a rotary shaker (New Brunswick
Model G10-21) at 100 rpm for a period 14 d. Sampling and TLC monitoring
were carried out at 7 d intervals. Precoated Si gel 60 F254 plates (E. Merck)
were used with CHCl3 : (CH3)2CO : Et2NH (5 : 4 : 1) as the solvent system.
UV light (254 nm) and p-anisaldehyde spray reagent were used to visualize
the spots. Scale-up fermentations were performed under the same conditions
with six 2 l flasks, containing 300 ml of medium and 50 mg of substrate,
each. Extraction of the culture filtrates and residues was carried out with
EtOAc. The solvent was evaporated in vacuo at 40 °C to obtain the residues.
The purification of metabolites was carried out by column (Si gel 230—400
mesh: E. Merck) and preparative layer (Si gel 60 F254) chromatography. Cul-
ture and substrate controls were run simultaneously with the above experi-
ments.18)
Microbial Transformation of Harmaline (1) by R. rubra The incuba-
tion mixtures of harmaline (1) (300 mg) were pooled and filtered. The filtrate
(1.8 l) was extracted exhaustively with EtOAc and a brownish solid (941 mg)
was obtained on evaporation of the solvent. It was column chromatographed
over silica gel with CHCl3 gradually enriched with MeOH as the eluting sol-
vent. 2-Acetyl-3-(2-acetamidoethyl)-7-methoxyindole, 3 was crystallized
from CHCl3, to yield while needles (110 mg, 28.6% yield), mp 162 °C (lit.5)
mp 160—162 °C; Rf 0.64; UV (MeOH) lmax (log e): 218 (3.88), 258 (3.38),
336 (3.86) nm; IR nmax (CHCl3) cmϪ1: 3287, 3092, 2933, 1640, 1630, 1573,
1529, 1428, 1161; 1H- and 13C-NMR: see Table 2; HR-ESI-MS m/z
[MϩNa]ϩ: 297.1196 (Calcd for C15H18N2O3ϩNa: 297.12092).
Microbial Transformation of Harmalol (2) by R. rubra ATCC 20129
The procedures for fermentation of harmalol 2 (300 mg), extraction and sep-
aration of the new tryptamine, 2-acetyl-3-(2-acetamidoethyl)-7-hydroxyin-
dole (4) were as described for harmaline. The EtOAc extract of the filtrate
on separation gave a white solid (30 mg, 7.6%); Rf 0.79; UV (MeOH) lmax
(log e): 220 (3.75), 258 (3.34), 336 (3.85) nm; IR nmax (CHCl3) cmϪ1: 3290,
2933, 1624, 1572, 1530, 1430; 1H- and 13C-NMR: see Table 2; HR-ESI-MS
m/z [MϩNa]ϩ: 283.1053 (Calcd for C14H16N2O3ϩNa: 283.1061).
Acknowledgements The authors thank Dr. Chuck Dunbar for conduct-
ing HR-ESI-MS analysis and Mr. Frank Wiggers for assistance in obtaining
2D NMR spectra. This work was supported, in part, by the United States De-
partment of Agriculture, Agricultural Research Specific Cooperative Agree-
ment No. 58-6408-2-0009.
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Microbial Transformation of Harman (5) by C. echinulata NRRL
3665 The EtOAc extract (773.2 mg) of the culture medium (300 mg of 5)
when subjected to column chromatography with CHCl3 enriched with
MeOH yielded 6-hydroxyharman (6) (21.4 mg, 6.5%) and harman-2-oxide
(7) (8.7 mg, 2.7%). Compound (6) gave yellow needles from MeOH, mp 319
320 °C; Rf 0.31; UV (MeOH) lmax (log e): 234 (4.35), 246 (4.28), 258
(4.15), 288 (3.98), 296 (4.16) 360 (3.60) nm; IR nmax (KBr) cmϪ1: 3207,
1
1577, 1458, 1199, 1027, 817; H-NMR (500 MHz, CD3OD): d 8.07 (1H, d,
Jϭ5.4, H-3), 7.76 (1H, d, Jϭ5.4, H-4), 7.48 (1H, d, Jϭ2.2, H-5), 7.42 (1H,
Jϭ8.7, H-8), 7.12 (1H, dd, Jϭ8.7, 2.2, H-7), 2.76 (3H, s, Me-1). 13C-NMR:
d 151.2 (C-6), 142.0 (C-1), 136.0 (C-3), 135.9 (C-4b), 128.3 (C-4a), 122.3
(H-8a), 118.4 (C-7), 113.0 (C-4), 112.5 (C-8), 105.7 (H-5), 18.6 (Me-1);