Activity of lycorine analogues against the fish bacterial pathogen Flavobacterium columnare

Article abstract of DOI:10.1021/jf200452z

In a continuing effort to discover natural products and natural product-based compounds for the control of columnaris disease in channel catfish (Ictalurus punctatus), 17 lycorine analogues were synthesized, including new benzoyl analogues 6-16, and evaluated for antibacterial activity against two isolates (ALM-00-173 and BioMed) of Flavobacterium columnare using a rapid bioassay. Two of the lycorine analogues had greater antibacterial activity than 1-O-acetyllycorine, an analogue of lycorine evaluated previously that is highly active against both isolates. Carbamate analogue 18 (1S,2S,3a ¹S,12bS)-2,3a¹,4,5,7,12b-hexahydro-1H-[1,3]dioxolo[4,5-j] pyrrolo[3,2,1-de]phenanthridin-1,2-diylbis(o-tolylcarbamate) had the strongest antibacterial activity toward both F. columnare isolates ALM-00-173 and BioMed, with 24-h IC₅₀ values of 3.0 ± 1.3 and 3.9 ± 2.2 mg/L, respectively, and a MIC of 5.5 ± 0 mg/L for both isolates. Compound 18 appears to be the most promising lycorine analogue for future efficacy studies to determine its potential for use as an alternative to the currently used compounds to control columnaris disease in channel catfish.

Full text of DOI:10.1021/jf200452z

ARTICLE
pubs.acs.org/JAFC
Activity of Lycorine Analogues against the Fish Bacterial Pathogen
Flavobacterium columnare
Cheng-Xia Tan,†,§ Kevin K. Schrader,† Cassia S. Mizuno,† and Agnes M. Rimando*,†
†Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 8048, University,
Mississippi 38677, United States
§College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310014, China
ABSTRACT: In a continuing effort to discover natural products and natural product-based compounds for the control of
columnaris disease in channel catfish (Ictalurus punctatus), 17 lycorine analogues were synthesized, including new benzoyl
analogues 6ꢀ16, and evaluated for antibacterial activity against two isolates (ALM-00-173 and BioMed) of Flavobacterium
columnare using a rapid bioassay. Two of the lycorine analogues had greater antibacterial activity than 1-O-acetyllycorine, an
analogue of lycorine evaluated previously that is highly active against both isolates. Carbamate analogue 18 (1S,2S,3a1S,12bS)-2,
3a1,4,5,7,12b-hexahydro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-1,2-diylbis(o-tolylcarbamate) had the strongest
antibacterial activity toward both F. columnare isolates ALM-00-173 and BioMed, with 24-h IC50 values of 3.0 ( 1.3 and 3.9 (
2.2 mg/L, respectively, and a MIC of 5.5 ( 0 mg/L for both isolates. Compound 18 appears to be the most promising lycorine
analogue for future efficacy studies to determine its potential for use as an alternative to the currently used compounds to control
columnaris disease in channel catfish.
KEYWORDS: antibacterial activity, channel catfish, columnaris disease, Flavobacterium columnare, lycorine
’ INTRODUCTION
of F. columnare, BioMed (genomovar I) and ALM-00-173
(genomovar II).
Columnaris disease is a worldwide bacterial fish disease that
results in heavy economic losses to the aquaculture industry.1
The bacterial pathogen Flavobacterium columnare is the cause of
columnaris disease, and this Gram-negative rod-shaped bacter-
ium is in the family Flavobacteriaceae.2 Columnaris disease may
cause severe gill-rotting and possible skin ulceration in catfish.
Epidemics of columnaris disease can occur in populations of
pond-raised channel catfish (Ictalurus punctatus) in the south-
eastern United States, and, therefore, this common disease is a
major concern for the channel catfish aquaculture industry.
Catfish producers currently have several available management
approaches including the application of medicated feeds, attenu-
ated vaccines,3 and nonantibiotic therapeutic agents such as 35%
Perox-Aid for external columnaris. However, Perox-Aid is not
recommended for use in earthen ponds without water exchange.
Although additional agents such as copper sulfate pentahydrate
Lycorine is a pyrrolo[de]phenanthridine ring-type alkaloid
extracted from various genera of plants in the Amaryllidaceae,
and its structure was first elucidated by Nagakawa et al.9 The
various biological properties of lycorine include inhibition of the
following: (1) ascorbic acid (AA) biosynthesis;10,11 (2) growth
and cell division in higher plants, algae, and yeasts;12 (3) cyanide-
insensitive respiration;13 (4) Trichomonas vaginalis nucleoside
triphosphate diphosphohydrolase and ecto-50-nucleotidase
activities;14 and (5) apoptosis-resistant cancer cells.15 In addi-
tion, lycorine is antiplasmodial16 and has significant cytostatic
activity toward cancer cells.17 These interesting properties make
lycorine a valuable tool for studying a number of important
physiological processes.
Because of our interest in improving the activity of lycorine and
discovering promising compounds with the potential to control
columnaris disease, we pursued the synthesis of various lycorine
analogues. Two genomovars of F. columnare were included in the
bioassay to identify potential selective toxicity of the lycorine
analogues. Here we report the synthesis and antibacterial activity
of 17 analogues, many of which are novel and, therefore, have not
been previously evaluated against the two F. columnare genomovars.
(CuSO4 5H2O) and potassium permanganate (KMnO4) have
3
been mentioned as potential treatment options for columnaris,4
the efficacy of these therapeutants can be adversely affected by
certain water quality variables. In addition, these compounds must
be applied carefully due to their broad-spectrum toxicity toward
nontarget organisms (e.g., channel catfish).5
Previously, a rapid 96-well microplate bioassay developed by
Schrader and Harries6 has been utilized to evaluate numerous
natural products and natural product-based compounds for
antibacterial activity toward F. columnare. Subsequently, promis-
ing compounds have been identified; for example, tannic acid is
highly toxic toward an isolate of F. columnare obtained from an
infected catfish in Mississippi.7 A more recent study by Schrader
et al.8 discovered that lycorine (1) (Figure 1) and two lycorine
derivatives, 1,2-O,O0-diacetyllycorine (2) and 1-O-acetyllycorine
(4), possess strong antibacterial activity toward two genomovars
’ MATERIALS AND METHODS
General Methods. General laboratory chemicals and solvents were
purchased from commercial sources and used without purification.
Received: February 1, 2011
Revised:
April 19, 2011
Accepted: April 25, 2011
Published: April 25, 2011
r
2011 American Chemical Society
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|

Journal of Agricultural and Food Chemistry
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Figure 1. Chemical structures of lycorine analogues under study.
Reactions were performed in a round-bottom flask, under N2 atmo-
sphere, and were monitored by thin-layer chromatography (TLC). 1H
and 13C NMR spectra were recorded at 400 MHz on an AVANCEIII
Bruker spectrometer (Bruker Daltonics Inc., Billerica, MA). ESI
MS spectra were recorded on a LC-MS system, that is, an Agilent
1100 HPLC (Agilent Technologies Inc., Santa Clara, CA) coupled to a
JEOL AccuTOF (JMS-T100LC) mass spectrometer (JEOL USA, Inc.,
Peabody, MA). Analytical and preparative TLCs were performed on
silica gel GF plates (Analtech uniplate, scored 10 ꢁ 20 cm, 250 μm, and
20 ꢁ 20 cm, 250 μm, respectively); the spots were visualized under
UV light.
Antibacterial Bioassay. Two isolates of F. columnare [BioMed
(genomovar I) and ALM-00-173 (genomovar II)] were obtained from
Dr. Covadonga Arias (Department of Fisheries and Allied Aquacultures,
Auburn University, Alabama). To ensure purity, cultures of both strains
were maintained separately on modified Shieh agar plates (pH
7.2ꢀ7.4).18 Prior to the bioassay, single colonies of the test cultures
were used to prepare the assay culture materials by separately culturing
each genomovar isolate in 75 mL of modified Shieh broth (18 h for
BioMed and 24 h for ALM-00-173) at 29 ( 1 °C at 150 rpm on a rotary
shaker (model C24KC; New Brunswick Scientific, Edison, NJ).
Compounds were evaluated for antibacterial activity using a rapid 96-
well microplate bioassay following the procedures of Schrader and
Harries.6 Florfenicol and oxytetracycline HCl were included as positive
controls for each assay. Also, control wells (no test compound added)
and wells with 1-O-acetyllycorine were included in each assay. The
analogue 1-O-acetyllycorine was included in the bioassay for comparison
because it was more active than lycorine against both genomovars of
F. columnare,8 and it was more readily available in sufficient quantities for
testing. To determine the 24-h 50% inhibition concentration (IC50) and
minimum inhibition concentration (MIC), sterile 96-well polystyrene
microplates (type Costar; Corning Corp., Acton, MA) with flat-bottom
wells were used to conduct the bioassay. Technical grade methanol was
used to dissolve the test compounds. Final concentrations of test
compounds, 1-O-acetyllycorine, and drug controls in the microplate
wells were 0.01, 0.1, 1.0, 10.0, and 100.0 μM. Dissolved test compounds
and drug controls were added separately to appropriate microplate wells
(10 μL/well), and only methanol was added to control wells (10 μL/
well). Solvent was allowed to completely evaporate before 0.5 MacFar-
land bacterial culture (see ref 6) was added to the microplate wells
(200 μL/well). The0.5 McFarland standard provideda culture suspension
of actively growing cells of F. columnare at 108 colony forming units
(CFU)/mL. Three replications were used for each dilution of each test
compound and controls; each bioassay was repeated. Microplates were
incubated at 29 °C (VWR model 2005 incubator; Sheldon Manufac-
turing, Inc., Cornelius, OR). A Packard model SpectraCount micro-
plate photometer (Packard Instrument Co., Meriden, CT) was used to
measure the absorbance (630 nm) of the microplate wells at times 0
and 24 h. Final results were converted to units of milligrams per liter to
allow comparison with previous studies.
The means and standard deviations of absorbance measurements
were calculated, graphed, and compared to controls to help determine
the 24-h IC50 and MIC for each test compound.6 The 24-h IC50 and
MIC results for each compound tested were divided by the respective
24-h IC50 and MIC results obtained for the positive controls florfe-
nicol and oxytetracycline to determine the relative to drug control
florfenicol (RDCF) and relative to drug control oxytetracycline
(RDCO) values.
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Journal of Agricultural and Food Chemistry
ARTICLE
Scheme 1a
a Conditions: DMAP, pyridine; lycorine/substituted benzoylchloride (1:1.05).
Preparation and Synthesis of Lycorine Analogues 2ꢀ5.
Compounds 2 and 3 were prepared from 1 (Sigma-Aldrich Co., St. Louis,
MO), whereas 4 and 5 were prepared from 2 by following published
procedures.17,19ꢀ21
(1H, br s, ꢀOCH2Oꢀ), 5.60 (1H, br s, H-3), 5.58 (1H, br s, H-2), 4.67
(1H, br s, H-1), 4.16 (1H, d, J = 14.2 Hz, H-7b), 3.58 (1H, d,
J = 14.1 Hz, H-7a), 3.40 (1H, m, H-5b), 2.92 (1H, d, J = 10.4 Hz, H-11b),
2.82 (1H, d, J = 10.4 Hz, H-11c), 2.70 (2H, m, H2ꢀ4), 2.61 (3H, s,
PhꢀCH3), 2.45 (1H, ddd, J = 8.1, 8.1, 8.1 Hz, H-5a); 13C NMR
(100 MHz, CDCl3) δ 166.9 (C-100), 146.7 (C-3a), 146.4 and 145.9
(C-10, C-9), 140.4 (PhꢀC-2), 132.2 (PhꢀC-4), 131.8 (PhꢀC-3),
130.9 (PhꢀC-1), 129.8 (PhꢀC-6), 127.2 (PhꢀC-5), 129.2 (C-7a),
125.8 (C-11a), 113.9 (C-3), 107.7 (C-8), 104.7 (C-11), 101.0
(ꢀOCH2Oꢀ), 73.9 (C-1), 69.2 (C-2), 64.4 (C-11c), 56.9 (C-7),
53.7 (C-5), 41.8 (C-11b), 28.8 (C-4), 22.0 (PhꢀCH3). HRMS: calcd
for C24H24NO5 [M þ H], 406.1655; found, 406.1634.
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl-diacetate (2) and (1S,
2S,3a1S,12bS)-1-Hydroxy-2,3a1,4,5,7,12b-hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridin-2-yl-acetate (3). Synthesis of
2 and 3 was according to published procedures.22 A mixture of 1 (30 mg,
0.10 mmol), pyridine (3 mL), and acetic anhydride (5 mL) in a sealed
flask was stirred at room temperature for 30 h, and then the reaction
mixture was quenched with cold water and extracted with chloroform
(3 ꢁ 10 mL). The combined organic layers were washed with brine and
dried over anhydrous magnesium sulfate. After removal of the volatiles,
the crude product was purified by SiO2 open column chromatography
(EtOAc/CHCl3/MeOH, 40:40:20) to give 2 (14.5 mg, 38% yield) and 3
(7.0 mg, 21% yield), both as white needles.
(1S,2S,3a1S,12bS)-1-Hydroxy-2,3a1,4,5,7,12b-hexahydro-1H-[1,
3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-2-yl-4-methylbenzoate
(7). The reaction of 1 (25 mg, 0.085 mmol) and 4-methylbenzoyl
chloride (14 mg, 0.09 mmol) afforded 7 (17 mg, 49% yield) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 7.93 (2H, d, J = 8.2 Hz, Ph2ꢀH-
2,6), 7.23 (2H, d, J = 8.2 Hz, Ph2ꢀH-3,5), 6.82 (1H, s, H-11), 6.61
(1H, s, H-8), 5.93 (1H, d, J = 1.3 Hz, ꢀOCH2Oꢀ), 5.92 (1H, d,
J = 1.3 Hz, ꢀOCH2Oꢀ), 5.60 (2H, brs, H-2, 3), 4.67 (1H, br s, H-1), 4.19
(1H, d, J = 14.1 Hz, H-7b), 3.58 (1H, d, J = 14.1 Hz, H-7a), 3.42
(1H, m, H-5b), 3.14 (1H, d, J = 10.4 Hz, H-11b), 2.88 (1H, d,
J = 10.4 Hz, H-11c), 2.70 (2H, m, H2ꢀ4), 2.45 (1H, ddd, J = 8.2, 8.2,
8.2 Hz, H-5a), 2.41 (3H, s, PhꢀCH3); 13C NMR (100 MHz, CDCl3)
δ 164.3 (C-100), 146.5 (C-3a), 146.4 and 146.2 (C-10, C-9), 139.3
(Ph2ꢀC-4), 131.1 (Ph2ꢀC-2,6), 129.2 (C-7a), 128.5 (Ph2ꢀC-3,5),
128.3 (Ph2ꢀC-1), 126.0 (C-11a), 113.3 (C-3), 107.2 (C-8), 105.3
(C-11), 100.0 (ꢀOCH2Oꢀ), 71.2 (C-1), 70.0 (C-2), 61.2 (C-11c), 56.5
(C-7), 53.4 (C-5), 41.4 (C-11b), 28.4 (C-4), 22.1 (PhꢀCH3). HRMS:
calcd for C24H24NO5 [M þ H], 406.1655; found, 406.1614.
(1S,2S,3a1S,12bS)-2-Hydroxy-2,3a1,4,5,7,12b-hexahydro-1H-[1,
3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-1-yl acetate (4). A
solution of 2 (18 mg, 0.05 mmol) in methanol (3 mL) and 35%
hydrochloric acid (0.5 mL) was heated on an oil bath for 1 h.23 After
cooling, the mixture was cautiously basified (pH 8) with aqueous
ammonia solution, diluted with water, and then extracted with chloro-
form (3 ꢁ 10 mL). The combined organic layers were washed with
brine and dried over anhydrous magnesium sulfate. After evaporation,
the residue was chromatographed over silica gel (EtOAc/CHCl3/
MeOH, 40:40:20) to give 4 (9 mg, 55% yield) as a colorless solid.
(1S,2S,3a1S,12bS)-1,2-Diacetoxy-1,2,3a1,4,5,6,7,12b-octahydro-[1,
3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-6-ium chloride (5).
To a solution of 2 (18 mg, 0.05 mmol) in ethanol (3 mL) was added
hydrogen chloride (2 mL, 10%), and the reaction mixture was stirred at
room temperature for 10 h. Then, the mixture was cautiously basified
(pH 7.5) with aqueous NaHCO3 solution, diluted with water, and
extracted with chloroform (3 ꢁ 10 mL). After removal of the volatiles,
the crude product was purified by SiO2 open column chromatography
(CHCl3/MeOH, 60:40) to give 5 (17 mg, 82% yield) as a white solid.
General Procedure for the Synthesis of Compounds 6ꢀ8.
To a solution of 1 (25 mg, 0.085 mmol) in dry pyridine (2 mL) was
added substituted benzoyl chloride and 4-dimethylaminopyridine
(DMAP) (1.05 mg, 0.01 mmol) (Scheme 1). The reaction mixture
was stirred at room temperature for 12 h and then carefully poured into
ice water (30 mL), causing precipitation. The precipitate was filtered,
washed with brine, dried, and purified by flash column chromatography
(SiO2, EtOAc/hexane = 60:40).
(1S,2S,3a1S,12bS)-1-Hydroxy-2,3a1,4,5,7,12b-hexahydro-1H-[1,
3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-2-yl-4-chlorobenzoate
(8). The reaction of 1 (25 mg, 0.085 mmol) and 4-chlorobenzoyl chloride
(16 mg, 0.09 mmol) afforded 8 (24 mg, 66% yield) as a white solid: 1H
NMR (400 MHz, CDCl3) δ 8.00 (2H, d, J = 8.6 Hz, Ph-H-2,6), 7.42 (2H,
d, J = 8.6 Hz, Ph-H-3,5), 6.82 (1H, s, H-11), 6.62 (1H, s, H-8), 5.94 (1H,
d, J = 1.3 Hz, ꢀOCH2Oꢀ), 5.93 (1H, d, J = 1.3 Hz, ꢀOCH2Oꢀ), 5.92
(1H, br s, H-3), 5.60 (1H, br s, H-2), 4.67 (1H, br s, H-1), 4.18 (1H, d,
J= 14.1 Hz, H-7b), 3.65 (1H, d, J= 14.1 Hz, H-7a), 3.41 (1H, m, H-5b), 3.14
(1H, d, J = 10.4 Hz, H-11b), 3.00 (1H, d, J = 10.4 Hz, H-11c), 2.73
(2H, m, H-4), 2.52 (1H, ddd, J = 8.3, 8.3, 8.3 Hz, H-5a); 13C NMR
(100 MHz, CDCl3) δ 164.5 (C-100), 146.3 (C-3a), 146.6 and 146.4
(C-10, C-9), 139.5 (PhꢀC-4), 131.2 (PhꢀC-2,6), 128.6 (PhꢀC-3,5),
128.2 (PhꢀC-1), 129.1 (C-7a), 126.0 (C-11a), 113.5 (C-3), 107.3 (C-8),
105.1 (C-11), 100.1 (ꢀOCH2Oꢀ), 71.3 (C-1), 70.1 (C-2), 61.3 (C-11c),
56.6 (C-7), 53.6 (C-5), 41.2 (C-11b), 28.6 (C-4). HRMS: calcd for
C23H22ClNO5 [M þ H], 426.1108; found, 426.1117.
(1S,2S,3a1S,12bS)-1-Hydroxy-2,3a1,4,5,7,12b-hexahydro-1H-[1,
3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-2-yl 2-methylbenzoate
(6). The reaction of 1 (25 mg, 0.085 mmol) and 2-methylbenzoyl
chloride (14 mg, 0.09 mmol) afforded 6 (15 mg, 43% yield) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 7.90 (1H, d, J = 7.7 Hz, Ph-H-2),
7.40 (1H, t, J = 7.6, 7.4 Hz, Ph-H-40), 7.23 (2H, m, Ph-H-3, 5), 6.81
(1H, s, H-11), 6.60 (1H, s, H-8), 5.91 (1H, br s, ꢀOCH2Oꢀ), 5.90
General Procedure for the Synthesis of Compounds
9ꢀ16. Substituted benzoyl chloride (0.18 mmol) and DMAP (1.05 mg,
0.01 mmol) were added to a solution of 1 (25 mg, 0.085 mmol) in dry
pyridine (2 mL) (Scheme 2). The reaction mixture was stirred at
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Scheme 2a
a Conditions: DMAP, pyridine; lycorine/substituted benzoyl chloride (1:2.1), 24 h, 30ꢀ80% yield.
room temperature for 24 h and then carefully poured into ice water
(30 mL). The precipitate was filtered and washed with brine. Purification
was by flash column chromatography (SiO2, EtOAc/hexane = 52: 48).
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl-bis(2-methylbenzoate)
(9). The reaction of 1 (25 mg, 0.085 mmol) and 2-methylbenzoyl
chloride (28 mg, 0.18 mmol) afforded 9 (24 mg, 54% yield) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 7.95 (1H, d, J = 7.6 Hz, Ph2ꢀH-
2), 7.67 (1H, d, J = 7.6 Hz, Ph1ꢀH-2), 7.35ꢀ7.43 (2H, m, Ph2ꢀH-4,
Ph1ꢀH-4), 7.16ꢀ7.25 (4H, m, Ph2ꢀH-3, 5, Ph1ꢀH-3, 5), 6.88 (1H, s,
H-11), 6.60 (1H, s, H-8), 6.15 (1H, br s, H-1), 5.90 (1H, d,
J = 1.2 Hz, ꢀOCH2Oꢀ), 5.88 (1H, d, J = 1.2 Hz, ꢀOCH2Oꢀ), 5.74
(1H, br s, H-3), 5.69 (1H, br s, H-2), 4.23 (1H, d, J = 14.2 Hz, H-7b),
3.64 (1H, d, J = 14.1 Hz, H-7a), 3.48 (1H, m, H-5b), 3.19 (1H, d, J = 9.3
Hz, H-11b), 3.07 (1H, d, J = 9.3 Hz, H-11c), 2.75 (2H, m, H2ꢀ4), 2.65
(3H, s, Ph2ꢀCH3), 2.58 (1H, ddd, J = 8.1, 8.1, 8.1 Hz, H-5a), 2.47 (3H,
The reaction of 1 (25 mg, 0.085 mmol) and 4-methylbenzoyl chloride
(28 mg, 0.18 mmol) afforded 11 (23 mg, 52% yield) as a white solid:
1H NMR (400 MHz, CDCl3) δ 7.94 (2H, d, J = 8.2 Hz, Ph2ꢀH-2,6), 7.80
(2H, d, J = 8.1 Hz, Ph1ꢀH-2,6), 7.23 (2H, d, J = 8.1 Hz, Ph2ꢀH-3,5), 7.18
(2H, d, J = 8.1 Hz, Ph1ꢀH-3,5), 6.87 (1H, s, H-11), 6.57 (1H, s, H-8), 6.12
(1H, br s, H-1), 5.89 (1H, d, J = 1.3 Hz, ꢀOCH2Oꢀ), 5.85 (1H, d,
J = 1.3 Hz, ꢀOCH2Oꢀ), 5.68 (1H, br s, H-3), 5.67 (1H, br s, H-2),
4.22 (1H, d, J = 14.1 Hz, H-7b), 3.62 (1H, d, J = 14.1 Hz, H-7a), 3.44
(1H, m, H-5b), 3.14 (1H, d, J = 10.4 Hz, H-11b), 3.03 (1H, d,
J = 10.4 Hz, H-11c), 2.73 (2H, m, H2ꢀ4), 2.50 (1H, ddd, J = 8.3, 8.3,
8.3 Hz, H-5a), 2.42 (3H, s, Ph2ꢀCH3), 2.38 (3H, s, Ph1ꢀCH3); 13
C
NMR (100 MHz, CDCl3) δ 164.4 and 164.2 (C-10, C-100), 146.4
(C-3a), 146.5 and 146.3 (C-10, C-9), 139.6 and 139.5 (Ph1ꢀC-4,
Ph2ꢀC-4), 131.2 and 131.1 (Ph1ꢀC-2,6, Ph2ꢀC-2,6), 129.8 (C-7a),
128.6 and 128.5 (Ph1ꢀC-3,5, Ph2ꢀC-3,5), 128.3 and 128.1 (Ph1ꢀC-1,
Ph2ꢀC-1), 126.0 (C-11a), 113.5 (C-3), 107.3 (C-8), 105.1 (C-11),
100.0 (ꢀOCH2Oꢀ), 71.3 (C-1), 70.1 (C-2), 61.5 (C-11c), 56.6 (C-7),
53.5 (C-5), 41.0 (C-11b), 28.2 (C-4), 21.3 and 21.2 (Ph1ꢀCH3,
Ph2ꢀCH3). HRMS: calcd for C32H30NO6 [M þ H], 524.2073; found,
524.2095.
13
s, Ph1ꢀCH3); C NMR (100 MHz, CDCl3) δ 166.5 (C-100), 166.1
(C-10), 146.5 (C-3a), 146.4 and 145.9 (C-10, C-9), 140.4 (Ph2ꢀC-2),
139.4 (Ph1ꢀC-2), 132.2 (Ph2ꢀC-4), 132.1 (Ph1ꢀC-4), 131.7
(Ph2ꢀC-3), 131.6 (Ph1ꢀC-3), 131.0 (Ph2ꢀC-1), 130.9 (Ph1ꢀC-1),
129.4 (C-7a), 129.3 (Ph2ꢀC-6), 129.1 (Ph1ꢀC-6), 127.2 (Ph2ꢀC-5),
127.0 (Ph1ꢀC-5), 125.7 (C-11a), 113.2 (C-3), 107.4 (C-8), 105.0
(C-11), 100.0 (ꢀOCH2Oꢀ), 70.9 (C-1), 68.9 (C-2), 61.6 (C-11c),
56.6 (C-7), 53.6 (C-5), 40.5 (C-11b), 28.1 (C-4), 22.0 (Ph2ꢀCH3),
21.6 (Ph1ꢀCH3). HRMS: calcd for C32H30NO6 [M þ H], 524.2073;
found, 524.2090.
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl-bis(2,3-dimethylbenzoate)
(12). The reaction of 1 (25 mg, 0.085 mmol) and 2,3-dimethylbenzoyl
chloride (30 mg, 0.18 mmol) afforded 12 (15 mg, 32% yield) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 7.66 (1H, d, J = 7.3 Hz, Ph2ꢀH-6),
7.35 (1H, d, J = 8.2 Hz, Ph2ꢀH-4), 7.30 (1H, d, J = 7.5 Hz, Ph1ꢀH-6),
7.24 (1H, d, J = 7.4 Hz, Ph1ꢀH-4), 7.13 (1H, t, J = 7.8, 7.6 Hz, Ph2ꢀH-5),
7.06 (1H, t, J = 7.8, 7.6 Hz, Ph1ꢀH-5), 6.88 (1H, s, H-11), 6.58
(1H, s, H-8), 6.13 (1H, br s, H-1), 5.91 (1H, s, ꢀOCH2Oꢀ), 5.89 (1H,
s, ꢀOCH2Oꢀ), 5.73 (1H, br s, H-3), 5.66 (1H, br s, H-2), 4.21 (1H, d,
J = 13.3 Hz, H-7b), 3.56 (1H, d, J = 13.3 Hz, H-7a), 3.43 (1H, m, H-5b),
3.12 (1H, d, J = 10.4 Hz, H-11b), 2.97 (1H, br s, H-11c), 2.72 (2H, m,
H2ꢀ4), 2.52 (3H, s, Ph2ꢀ3-CH3), 2.46 (1H, ddd, J = 8.1, 8.1, 8.1 Hz,
H-5a), 2.34 (3H, s, Ph1ꢀ3-CH3), 2.30 (3H, s, Ph2ꢀ2-CH3), 2.27 (3H,
s, Ph1ꢀ2-CH3); 13C NMR (100 MHz, CDCl3) δ 165.9 and 165.7
(C-10, C-100), 149.7 and 149.3 (C-10, C-9), 146.2 (C-3a), 141.7 and
141.6 (Ph1ꢀC-2, Ph2ꢀC-2), 140.5 and 140.3 (Ph1ꢀC-3, Ph2ꢀC-3),
137.4 and 137.0 (Ph1ꢀC-6, Ph2ꢀC-6), 132.0 and 131.8 (Ph1ꢀC-4,
Ph2ꢀC-4), 131.3 and 130.9 (Ph1ꢀC-1,5, Ph2ꢀC-1,5), 129.3 (C-7a),
126.6 (C-11a), 113.4 (C-3), 107.0 (C-8), 104.9 (C-11), 101.0
(ꢀOCH2Oꢀ), 70.6 (C-1), 69.3 (C-2), 61.5 (C-11c), 56.4 (C-7),
53.3 (C-5), 41.2 (C-11b), 28.0 (C-4), 21.6, 21.3, 21.0, and 20.9
(Ph1ꢀ2,3-CH3, Ph2ꢀ2,3-CH3). HRMS: calcd for C34H34NO6 [M þ
H], 552.2308; found, 552.2317.
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl-bis(3-methylbenzoate)
(10). The reaction of 1 (25 mg, 0.085 mmol) and 3-methylbenzoyl
chloride (28 mg, 0.18 mmol) afforded 10 (26 mg, 59% yield) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 7.88 (2H, br s, Ph2ꢀH-2,6), 7.74
(1H, s, Ph1ꢀH-2), 7.69 (1H, d, J = 7.6 Hz, Ph1ꢀH-6), 7.39ꢀ7.24 (4H,
m, Ph2ꢀH-4,5, Ph1ꢀH-4,5), 6.89 (1H, s, H-11), 6.58 (1H, s, H-8),
6.16 (1H, br s, H-1), 5.89 (1H, d, J = 1.0 Hz, ꢀOCH2Oꢀ), 5.85 (1H, d,
J = 1.0 Hz, ꢀOCH2Oꢀ), 5.70 (2H, br s, H-2, 3), 4.22 (1H, d,
J = 14.1 Hz, H-7b), 3.62 (1H, d, J = 14.1 Hz, H-7a), 3.45 (1H, m, H-5b),
3.18 (1H, d, J = 10.4 Hz, H-11b), 3.04 (1H, d, J = 10.4 Hz, H-11c), 2.75
(2H, m, H2ꢀ4), 2.52 (1H, ddd, J = 8.2, 8.2, 8.2 Hz, H-5a), 2.39 (3H, s,
Ph2ꢀCH3), 2.36 (3H, s, Ph1ꢀCH3); 13C NMR (100 MHz, CDCl3)
δ 165.5 and 165.4 (C-10, C-100), 146.5 and 146.4 (C-10, C-9), 146.2
(C-3a), 138.2 and 138.1 (Ph1ꢀC-3, Ph2ꢀC-3), 133.9 and 133.8 (Ph1ꢀC-4,
Ph2ꢀC-4), 130.4 and 130.3 (Ph1ꢀC-1, Ph2ꢀC-1), 129.9 and 129.6
(Ph1ꢀC-2, Ph2ꢀC-2), 129.3 (C-7a), 128.3 and 128.2 (Ph1ꢀC-5,
Ph2ꢀC-5), 127.0 and 126.9 (Ph1ꢀC-6, Ph2ꢀC-6), 126.5 (C-11a),
114.1 (C-3), 107.3 (C-8), 105.2 (C-11), 100.9 (ꢀOCH2Oꢀ), 71.0
(C-1), 69.5 (C-2), 61.6 (C-11c), 56.9 (C-7), 53.9 (C-5), 41.1 (C-11b),
28.8 (C-4), 21.3 and 21.2 (Ph1ꢀCH3, Ph2ꢀCH3). HRMS: calcd for
C32H30NO6 [M þ H], 524.2073; found, 524.2097.
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl-bis(2,4-dimethylbenzoate)
(13). The reaction of 1 (25 mg, 0.085 mmol) and 2,4-dimethylbenzoyl
chloride (30 mg, 0.18 mmol) afforded 13 (14 mg, 30% yield) as a white solid:
1H NMR (400 MHz, CDCl3) δ 7.85 (1H, d, J = 7.9 Hz, Ph2ꢀH-6), 7.59
(1H, d, J =7.9 Hz, Ph1ꢀH-6), 6.97ꢀ7.06 (4H, m, Ph2ꢀH-3,5, Ph1ꢀH-3,5),
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl-bis(4-methylbenzoate) (11).
5980
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Journal of Agricultural and Food Chemistry
ARTICLE
Scheme 3a
a Conditions: Pyridine, lycorine/isocyanate (1:1.1), room temperature, 20 h, 31% yield.
6.87 (1H, s, H-11), 6.58 (1H, s, H-8), 6.12 (1H, br s, H-1), 5.90 (1H, d,
J = 1.3 Hz, ꢀOCH2Oꢀ), 5.87 (1H, d, J = 1.3 Hz, ꢀOCH2Oꢀ), 5.71 (1H,
br s, H-3), 5.65 (1H, br s, H-2), 4.21 (1H, d, J = 13.3 Hz, H-7b), 3.58 (1H, d,
J = 13.3 Hz, H-7a), 3.43 (1H, m, H-5b), 3.14 (1H, d, J = 10.4 Hz, H-11b),
3.00 (1H, br s, H-11c), 2.72 (2H, m, H2ꢀ4), 2.61(3H, s, Ph2ꢀ1-CH3), 2.55
(1H, ddd, J = 8.0, 8.0, 8.0 Hz, H-5a), 2.44 (3H, s, Ph1ꢀ1-CH3), 2.36 (3H, s,
Ph2ꢀ4-CH3), 2.31 (3H, s, Ph1ꢀ4-CH3); 13C NMR (100 MHz, CDCl3) δ
166.4 and 166.1 (C-10, C-100), 150.4 and 149.5 (C-10, C-9), 146.4 (C-3a),
142.7 and 142.6 (Ph1ꢀC-4, Ph2ꢀC-4), 140.5 and 140.3 (Ph1ꢀC-2, Ph2ꢀ
C-2), 132.5 and 132.3 (Ph1ꢀC-3,6, Ph2ꢀC-3,6), 131.1 and 130.8 (Ph1ꢀ
C-1,5, Ph2ꢀC-1,5), 129.4 (C-7a), 126.4 (C-11a), 113.8 (C-3), 107.3 (C-8),
105.2 (C-11), 100.9 (ꢀOCH2Oꢀ), 70.9 (C-1), 69.6 (C-2), 61.8 (C-11c),
56.9 (C-7), 53.7 (C-5), 41.0 (C-11b), 28.2 (C-4), 21.9, 21.6, 21.3, and 21.2
(Ph1ꢀ2,4-CH3, Ph2ꢀ2,4-CH3). HRMS: calcd for C34H34NO6 [M þ H],
552.2308; found, 552.2320.
(100 MHz, CDCl3) δ 164.3 and 164.0 (C-10, C-100), 146.5 (C-3a),
146.8 and 146.5 (C-10, C-9), 134.6 and 134.5 (Ph1ꢀC-3, Ph2ꢀC-3),
133.3 and 133.2 (Ph1ꢀC-4, Ph2ꢀC-4), 131.6 and 131.3 (Ph1ꢀC-1,
Ph2ꢀC-1), 129.9 and 129.8 (Ph1ꢀC-2, Ph2ꢀC-2), 129.7 and 129.6
(Ph1ꢀC-5, Ph2ꢀC-5), 129.3 (C-7a), 128.1 and 128.0 (Ph1ꢀC-6,
Ph2ꢀC-6), 126.1 (C-11a), 113.6 (C-3), 107.4 (C-8), 105.0 (C-11),
101.0 (ꢀOCH2Oꢀ), 71.4 (C-1), 70.0 (C-2), 61.5 (C-11c), 56.8
(C-7), 53.6 (C-5), 41.0 (C-11b), 28.8 (C-4). HRMS: calcd for
C30H24Cl2NO6 [M þ H], 564.0981; found, 564.0989.
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl-bis(4-chlorobenzoate)
(16). The reaction of 1 (25 mg, 0.085 mmol) and 4-chlorobenzoyl
chloride (32 mg, 0.18 mmol) afforded 16 (37 mg, 77% yield) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 8.00 (2H, d, J = 8.6 Hz, Ph2ꢀH-
2,6), 7.82 (2H, d, J = 8.6 Hz, Ph1ꢀH-2,6), 7.42 (2H, d, J = 8.6 Hz,
Ph2ꢀH-3,5), 7.35 (2H, d, J = 8.6 Hz, Ph1ꢀH-3,5), 6.83 (1H, s,
H-11), 6.58 (1H, s, H-8), 6.11 (1H, br s, H-1), 5.89 (1H, d, J =
1.3 Hz, ꢀOCH2Oꢀ), 5.86 (1H, d, J = 1.3 Hz, ꢀOCH2Oꢀ), 5.67 (2H,
br s, H-2,3), 4.22 (1H, d, J = 14.1 Hz, H-7b), 3.62 (1H, d, J = 14.1 Hz,
H-7a), 3.44 (1H, m, H-5b), 3.14 (1H, d, J = 10.4 Hz, H-11b), 3.01
(1H, d, J = 10.4 Hz, H-11c), 2.75 (2H, m, H2ꢀ4), 2.53 (1H, ddd,
J = 8.4, 8.4, 8.4 Hz, H-5a); 13C NMR (100 MHz, CDCl3) δ 164.6 and
164.4 (C-10, C-100), 146.5 (C-3a), 146.6 and 146.4 (C-10, C-9), 139.7
and 139.6 (Ph1ꢀC-4, Ph2ꢀC-4), 131.3 and 131.2 (Ph1ꢀC-2,6, Ph2ꢀC-
2,6), 128.7 and 128.6 (Ph1ꢀC-3,5, Ph2ꢀC-3,5), 128.3 and 128.0 (Ph1ꢀC-
1, Ph2ꢀC-1), 129.2 (C-7a), 126.1 (C-11a), 113.6 (C-3), 107.4 (C-8),
105.0 (C-11), 100.0 (ꢀOCH2Oꢀ), 71.2 (C-1), 70.0 (C-2), 61.4 (C-11c),
56.7 (C-7), 53.7 (C-5), 41.1 (C-11b), 28.7 (C-4). HRMS: calcd for
C30H24Cl2NO6 [M þ H], 564.0981; found 564.0973.
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl-bis(2-chlorobenzoate)
(14). The reaction of 1 (25 mg, 0.085 mmol) and 2-chlorobenzoyl
chloride (32 mg, 0.18 mmol) afforded 14 (35 mg, 74% yield) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 7.88 (1H, d, J = 7.6 Hz, Ph2ꢀH-6),
7.62 (1H, d, J = 7.6 Hz, Ph1ꢀH-6), 7.47ꢀ7.41 (2H, m, Ph2ꢀH-3,
Ph1ꢀH-3), 7.35ꢀ7.39 (2H, m, Ph2ꢀH-4, Ph1ꢀH-4), 7.30ꢀ7.34 (1H,
m, Ph2ꢀH-5), 7.22ꢀ7.27 (1H, m, Ph1ꢀH-5), 6.88 (1H, s, H-11), 6.58
(1H, s, H-8), 6.19 (1H, br s, H-1), 5.90 (1H, d, J = 1.2 Hz,
ꢀOCH2Oꢀ), 5.88 (1H, d, J = 1.2 Hz, ꢀOCH2Oꢀ), 5.72 (2H, br
s, H-2, 3), 4.19 (1H, d, J = 14.2 Hz, H-7b), 3.57 (1H, d, J = 14.1 Hz,
H-7a), 3.41 (1H, m, H-5b), 3.15 (1H, d, J = 9.3 Hz, H-11b), 3.02
(1H, d, J = 9.0 Hz, H-11c), 2.71 (2H, m, H2ꢀ4), 2.45 (1H, d,
J = 8.3 Hz, H-5a); 13C NMR (100 MHz, CDCl3) δ 164.9 (C-100), 164.4
(C-10), 146.7 (C-3a), 146.6 and 146.4 (C-10, C-9), 134.0 (Ph2ꢀC-4),
133.7 (Ph1ꢀC-4), 132.8 (Ph2ꢀC-2), 132.7 (Ph1ꢀC-2), 131.8 (Ph2ꢀC-1),
131.6 (Ph1ꢀC-1), 131.2 (Ph2ꢀC-6), 131.0 (Ph1ꢀC-6), 130.9
(Ph2ꢀC-1), 129.8 (Ph2ꢀC-3), 129.7 (Ph1ꢀC-3), 129.3 (C-7a),
126.6 (Ph2ꢀC-5), 126.5 (Ph1ꢀC-5), 126.4 (C-11a), 113.5 (C-3),
107.3 (C-8), 105.1 (C-11), 101.0 (ꢀOCH2Oꢀ), 71.9 (C-1), 70.5
(C-2), 61.6 (C-11c), 56.9 (C-7), 53.7 (C-5), 40.8 (C-11b), 28.8 (C-4).
HRMS: calcd for C30H24Cl2NO6 [M þ H], 564.0981; found, 564.0992.
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl-bis(3-chlorobenzoate)
(15). The reaction of 1 (25 mg, 0.085 mmol) and 3-chlorobenzoyl
chloride (32 mg, 0.18 mmol) afforded 15 (33 mg, 70% yield) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 8.03 (1H, s, Ph2ꢀH-2), 7.95(1H,
d, J = 7.8 Hz, Ph2ꢀH-6), 7.85 (1H, s, Ph1ꢀH-2), 7.79 (1H, d, J = 7.8 Hz,
Ph1ꢀH-6), 7.54 (1H, d, J = 8.0 Hz, Ph2ꢀH-4), 7.49 (1H, d, J = 8.0 Hz,
Ph1ꢀH-4), 7.39 (1H, t, J = 7.9 Hz, Ph2ꢀH-5), 7.33 (1H, t,
J = 7.9 Hz, Ph1ꢀH-5), 6.84 (1H, s, H-11), 6.59 (1H, s, H-8), 6.13
(1H, br s, H-1), 5.89 (1H, d, J = 1.3 Hz, ꢀOCH2Oꢀ), 5.86 (1H, d, J =
1.3 Hz, ꢀOCH2Oꢀ), 5.67 (2H, br s, H-2, 3), 4.22 (1H, d, J = 14.2 Hz,
H-7b), 3.62 (1H, d, J = 14 Hz, H-7a), 3.44 (1H, m, H-5b), 3.16 (1H, d,
J = 10.4 Hz, H-11b), 3.01 (1H, d, J = 10.4 Hz, H-11c), 2.74 (2H, m,
H2ꢀ4), 2.54 (1H, ddd, J = 8.2, 8.2, 8.2 Hz, H-5a); 13C NMR
(1S,2S,3a1S,12bS)-1-Hydroxy-2,3a1,4,5,7,12b-hexahydro-1H-[1,
3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-2-yl-o-tolylcarbamate
(17). 2-Methylphenyl isocyanate (14 mg, 0.11 mmol) was added to a
solution of 1 (30 mg, 0.10 mmol) in dry pyridine (2 mL) (Scheme 3).
The reaction mixture was stirred at room temperature for 20 h and then
carefully poured into ice water (30 mL), causing precipitation. The
precipitate was filtered, washed with brine, and purified by flash
column chromatography (SiO2, EtOAc/hexane = 70:30) to afford
17 (13 mg, 31% yield) as a white solid: 1H NMR (400 MHz, CDCl3)
δ 7.82 (1H, br s, NH), 7.23ꢀ7.15 (2H, m, PhꢀH-5,6), 7.04 (1H, t,
J = 7.4, 7.4 Hz, PhꢀH-3), 6.81 (1H, s, H-11), 6.62 (1H, s, H-8), 6.59
(1H, m, PhꢀH-4), 5.94 (1H, d, J = 1.4 Hz, ꢀOCH2Oꢀ), 5.92 (1H, d,
J = 1.4 Hz, ꢀOCH2Oꢀ), 5.58 (1H, br s, H-3), 5.33 (1H, br s, H-2),
4.60 (1H, br s, H-1), 4.16 (1H, d, J = 14.2 Hz, H-7b), 3.56 (1H, d, J = 1
4.1 Hz, H-7a), 3.38 (1H, m, H-5b), 2.90 (1H, d, J = 10.4 Hz, H-11b),
2.74 (1H, d, J = 10.4 Hz, H-11c), 2.67 (2H, m, H2ꢀ4), 2.45 (1H, ddd,
J = 8.1, 8.1, 8.1 Hz, H-5a), 2.25 (3H, s, PhꢀCH3); 13C NMR (100 MHz,
CDCl3) δ 153.3 (C-100), 146.7 (C-3a), 146.4 and 145.9 (C-10, C-9), 135.7
(PhꢀC-1), 130.4 (PhꢀC-2), 130.0 (C-7a), 129.8 (PhꢀC-3), 127.4
(PhꢀC-5), 126.9 (PhꢀC-4), 125.3 (C-11a), 124.3 (PhꢀC-6), 114.0
(C-3), 107.6 (C-8), 104.8 (C-11), 101.0 (ꢀOCH2Oꢀ), 74.6
(C-1), 69.3 (C-2), 60.7 (C-11c), 56.8 (C-7), 53.6 (C-5), 41.6 (C-11b),
5981
dx.doi.org/10.1021/jf200452z |J. Agric. Food Chem. 2011, 59, 5977–5985

Journal of Agricultural and Food Chemistry
ARTICLE
Scheme 4a
a Conditions: Pyridine, lycorine/isocyanate (1:2.2) room temperature, 40 h, 29% yield.
Scheme 5a
a Conditions: (i) DMAP, pyridine, lycorine/substituted benzoyl chloride (1:2); (ii) CH3I, CH3CN, 24 h, 40 °C.
28.7 (C-4), 17.6 (PhꢀCH3). HRMS: calcd for C24H23N2O5 [M ꢀ H],
419.16070; found, 419.16072.
reaction mixture was concentrated, and the residue was purified by SiO2 open
column chromatography (CHCl3/MeOH, 80:20), affording 19 as a white
1
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]dioxolo[4,
5-j]pyrrolo[3,2,1-de]phenanthridin-1,2-diyl-bis(o-tolylcarbamate)
(18). 2-Methylphenyl isocyanate (28 mg, 0.22 mmol) was added to a
solution of 1 (30 mg, 0.10 mmol) in dry pyridine (2 mL) (Scheme 4).
The reaction mixture was stirred at room temperature for 40 h and then
carefully poured into ice water (30 mL). The precipitate that formed
was filtered and washed with brine. Purification by flash column
chromatography (SiO2, EtOAc/hexane = 60:35) afforded 18 (16 mg,
29% yield) as a white solid: 1H NMR (400 MHz, CDCl3) δ 7.84 (1H,
br s, 20-NH), 7.67 (1H, br s, 10-NH), 7.23ꢀ7.11 (4H, m, Ph1ꢀH-5,6,
Ph2ꢀH-5,6), 7.05ꢀ6.94 (2H, m, Ph1ꢀH-3, Ph2ꢀH-3), 6.81 (1H, s,
H-11), 6.60 (1H, s, H-8), 6.47 (2H, m, Ph1ꢀH-4, Ph2ꢀH-4), 6.28 (1H,
br s, H-1), 5.93 (1H, d, J = 1.4 Hz, ꢀOCH2Oꢀ), 5.91 (1H, d,
J = 1.4 Hz, ꢀOCH2Oꢀ), 5.71 (1H, br s, H-3), 5.43 (1H, br s, H-2),
4.19 (1H, d, J = 14.2 Hz, H-7b), 3.57 (1H, d, J = 14.1 Hz, H-7a), 3.40
(1H, m, H-5b), 2.95 (1H, d, J = 10.4 Hz, H-11b), 2.85 (1H, d, J = 10.4
Hz, H-11c), 2.69 (2H, m, H2ꢀ4), 2.45 (1H, ddd, J = 8.1, 8.1, 8.1 Hz,
H-5a), 2.27 (3H, s, Ph2ꢀCH3), 2.16 (3H, s, Ph1ꢀCH3); 13C NMR
(100 MHz, CDCl3) δ 152.8 (C-100), 152.5 (C-10), 146.6 (C-3a), 146.4
and 145.9 (C-10, C-9), 135.7 (Ph2ꢀC-1), 135.4 (Ph1ꢀC-1), 130.4
(Ph2ꢀC-2, Ph1ꢀC-2), 130.3 (Ph2ꢀC-3, Ph1ꢀC-3), 129.3 (C-7a),
126.9 (Ph2ꢀC-5, Ph1ꢀC-5), 126.8 (C-11a), 126.7 (Ph2ꢀC-4, Ph1ꢀ
C-4), 124.5 (Ph2ꢀC-6), 124.2 (Ph1ꢀC-6), 114.4 (C-3), 107.3 (C-8),
105.3 (C-11), 101.0 (ꢀOCH2Oꢀ), 71.8 (C-1), 68.8 (C-2), 61.2 (C-
11c), 56.8 (C-7), 53.7 (C-5), 40.5 (C-11b), 28.7 (C-4), 17.7
(Ph2ꢀCH3), 17.6 (Ph1ꢀCH3). HRMS: calcd for C32H30N3O6 [M ꢀ H],
552.21020; found, 552.21073.
solid (13 mg, 52% yield): H NMR (400 MHz, CDCl3) δ 8.02 (1H, s,
Ph2ꢀH-2), 7.93 (1H, d, J = 7.6 Hz, Ph2ꢀH-6), 7.90 (3H, s, NꢀCH3), 7.83
(1H, s, Ph1ꢀH-2), 7.80 (1H, d, J = 7.6 Hz, Ph1ꢀH-6), 7.52 (1H, d, J = 8.1
Hz, Ph2ꢀH-4), 7.47 (1H, d, J = 8.1 Hz, Ph1ꢀH-4), 7.40 (1H, t, J = 7.7 Hz,
Ph2ꢀH-5), 7.32 (1H, t, J = 7.7 Hz, Ph1ꢀH-5), 6.82 (1H, s, H-11), 6.60 (1H,
s, H-8), 6.12 (1H, br s, H-1), 5.90 (1H, d, J = 1.2 Hz, ꢀOCH2Oꢀ), 5.88
(1H,d, J=1.2Hz, ꢀOCH2Oꢀ), 5.68 (2H, br s, H-2, 3), 4.43 (1H, d, J= 14.0
Hz, H-7b), 3.84 (1H, d, J = 14.0 Hz, H-7a), 3.54 (1H, m, H-5b), 3.25 (1H, d,
J = 10.2 Hz, H-11b), 3.14 (1H, d, J = 10.2 Hz, H-11c), 2.88 (2H, m, H2ꢀ4),
2.64 (1H, ddd, J = 8.0, 8.0, 8.0 Hz, H-5a); 13C NMR (100 MHz, CDCl3)
δ 164.4 and 164.1 (C-10, C-100), 146.5 and 146.3 (C-10, C-9), 135.5 (C-3a),
134.7 and 134.4 (Ph1ꢀC-3, Ph2ꢀC-3), 133.6 and 133.3 (Ph1ꢀC-4, Ph2ꢀ
C-4), 131.7 and 131.4 (Ph1ꢀC-1, Ph2ꢀC-1), 129.7 and 129.5 (Ph1ꢀC-2,
Ph2ꢀC-2), 129.3 and 129.0 (Ph1ꢀC-5, Ph2ꢀC-5), 128.9 (C-7a), 128.3 and
128.1 (Ph1ꢀC-6, Ph2ꢀC-6), 127.1 (C-11a), 121.6 (C-3), 110.3 (C-8), 108.1
(C-11), 100.9 (ꢀOCH2Oꢀ), 82.5 (C-11c), 72.4 (C-1), 71.0 (C-2), 64.8
(C-7), 61.7 (C-5), 47.6 (NꢀCH3), 32.0 (C-11b), 23.8 (C-4). HRMS: calcd
for C31H26Cl2NO6 [Mþ þ H], 578.1137; found, 578.1127.
’ RESULTS AND DISCUSSION
It was previously shown that 1-O-acetyllycorine (4) is more
active than lycorine against both isolates of F. columnare, ALM-
00-173 and BioMed.8 Therefore, in this study, 4 was selected as a
standard to gauge the activities of the lycorine analogues
synthesized for evaluation against both genomovars of F. columnare.
The IC50, MIC, RDCF, and RDCO values obtained for 4 in this
study were very similar to those of previous studies;8 therefore,
results from this study can be compared with those from the
previous study with confidence.
(1S,2S,3a1S,12bS)-1,2-bis((2-chlorobenzoyl)oxy)-6-methyl-1,2,3a1,4,
5,6,7,12b-octahydro-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-
6-ium iodide (19). Iodomethane (0.5 mL, excess) via cannula was
added to a solution of 14 (20 mg, 0.035 mmol) in acetonitrile (2 mL,
anhydrous) (Scheme 5). The reaction mixture was stirred under N2
and heating at 40 °C for 24 h. After completion of the reaction, the
Analogues of lycorine were prepared by introducing diversity
at the C1-O- and C2-O-positions while the [1,3]dioxolo-pyrrolo-
[de]phenanthridine ring was kept intact. Acetylation at the
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Table 1. Results of the Bioassay Evaluation of Lycorine Analogues for Toxicity toward Flavobacterium columnare ALM-00-173a
MICc
b
24-h IC50
b
compd
24-h IC50
2
MICc
RDCFd
RDCOe
RDCFd
RDCOe
2f
3
4
3
3
10
11
17.7 (8.8)
7.3 (1.1)
12.2 (0)
16.6 (16.3)
3.3 (0)
27.8 (13.8)
10.4 (1.8)
15.5 (0)
26.4 (13.1)
9.9 (1.7)
14.8 (0)
50.5 (49.5)
10.0 (0)
54.3 (53.2)
10.8 (0)
4
5
40.7 (0)
46.4 (5.9)
4.7 (0.6)
42.6 (0)
4.1 (0)
100.0 (0)
100.0 (0)
10.0 (0)
107.5 (0)
107.5 (0)
10.8 (0)
6
>52.3
>51.8
>49.3
7
14.6 (1.6)
13.0 (0.2)
16.6 (7.7)
16.4 (0.3)
15.8 (0.3)
21.3 (9.9)
15.6 (0.3)
15.1 (0.3)
20.2 (9.4)
8
100.0 (0)
10.0 (0)
107.5 (0)
10.8 (0)
9
10
11
12
13
14
15
16
17
18
19
>52.3
5.2 (0)
>51.8
>49.3
10.0 (0)
10.8 (0)
13.8 (2.9)
13.8 (0)
46.4 (5.9)
30.4 (24.9)
5.5 (0)
15.3 (1.3)
13.0 (0)
14.6 (1.3)
12.3 (0)
100.0 (0)
55.0 (45.0)
10.0 (0)
107.5 (0)
59.2 (48.4)
10.8 (0)
15.8 (0.9)
9.4 (5.8)
14.8 (0.8)
8.7 (5.4)
14.1 (0.8)
8.3 (5.1)
31.0 (25.4)
5.6 (0)
55.0 (45.0)
10.0 (0)
59.2 (48.4)
10.8 (0)
13.6 (4.0)
21.8 (11.6)
11.2 (1.5)
3.0 (1.3)
12.5 (3.7)
20.0 (10.7)
13.7 (1.8)
2.8 (1.2)
11.9 (3.5)
19.0 (10.1)
13.1 (1.7)
2.6 (1.1)
31.0 (25.4)
23.2 (19.0)
5.5 (0)
55.0 (45.0)
55.0 (45.0)
10.0 (0)
59.2 (48.4)
59.2 (48.4)
10.8 (0)
>70.6
>70.6
>51.8
>49.3
>100.0
>107.5
a Numbers in parentheses are the standard error of the mean. b 24-h IC50 = 50% inhibition concentration in mg/L. c Minimum inhibition concentration
in mg/L. d Relative to drug control florfenicol; numbers closer to “1” indicate greater activity because a value of “1” indicates the same activity compared
to drug control. e Relative to drug control oxytetracycline; numbers closer to “1” indicate greater activity because a value of “1” indicates the same activity
compared to drug control. f Values are from ref 10.
Table 2. Results of the Bioassay Evaluation of Lycorine Analogues for Toxicity toward Flavobacterium columnare BioMeda
MICc
b
24-h IC50
b
compd
24-h IC50
10
MICc
37
RDCFd
RDCOe
RDCFd
100
RDCOe
108
2f
3
11
15
6.1 (0.8)
9.4 (1.2)
12.8 (0.2)
3.3 (0)
3.3 (0)
7.8 (1.1)
12.1 (1.5)
13.4 (0.2)
10.9 (1.5)
16.9 (2.1)
18.5 (0.3)
10.0 (0)
10.0 (0)
10.8 (0)
10.8 (0)
4
5
40.7 (0)
22.9 (17.7)
22.9 (17.7)
42.6 (0)
4.1 (0)
100.0 (0)
55.0 (45.0)
55.0 (45.0)
100.0 (0)
10.0 (0)
107.5 (0)
59.2 (48.4)
59.2 (48.4)
107.5 (0)
10.8 (0)
6
>52.3
>42.4
>58.8
7
14.8 (1.0)
14.5 (1.3)
9.3 (0.8)
13.6 (0.9)
14.4 (1.3)
9.8 (0.9)
18.9 (1.2)
20.0 (1.8)
13.6 (1.2)
8
9
10
11
12
13
14
15
16
17
18
19
>52.3
28.8 (23.6)
22.9 (17.7)
55.2 (0)
30.4 (24.9)
5.6 (0)
>42.4
>58.8
55.0 (45.0)
100.0 (0)
100.0 (0)
55.0 (45.0)
10.0 (0)
59.2 (48.4)
107.5 (0)
107.5 (0)
59.2 (48.4)
10.8 (0)
5.8 (4.3)
14.6 (0.3)
14.9 (1.7)
8.5 (6.2)
11.7 (1.1)
11.2 (0.2)
11.4 (1.3)
6.4 (4.7)
16.2 (1.5)
15.6 (0.3)
15.9 (1.8)
8.9 (6.5)
13.6 (2.3)
15.0 (4.3)
15.0 (1.1)
3.9 (2.2)
5.6 (0)
10.2 (1.7)
11.3 (3.2)
15.0 (1.1)
3.0 (1.7)
14.2 (2.4)
15.6 (4.4)
20.9 (1.5)
4.1 (2.4)
10.0 (0)
10.8 (0)
5.6 (0)
10.0 (0)
10.8 (0)
42.1 (0)
5.5 (0)
100.0 (0)
10.0 (0)
107.5 (0)
10.8 (0)
>70.6
>70.6
>42.4
>58.8
>100.0
>107.5
a Numbers in parentheses are the standard error of the mean. b 24-h IC50 = 50% inhibition concentration in mg/L. c Minimum inhibition concentration
in mg/L. d Relative to drug control florfenicol; numbers closer to “1” indicate greater activity because a value of “1” indicates the same activity compared
to drug control. e Relative to drug control oxytetracycline; numbers closer to “1” indicate greater activity because a value of “1” indicates the same activity
compared to drug control. f Values are from ref 10.
C2-O-position was performed initially, yielding 3, to compare the
antibacterial effect of the two monoacetylated analogues 3 and 4.
Acetylation at the C2-O-position resulted in weaker activity
against F. columnare ALM-00-173 (Table 1); the activity against
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F. columnare BioMed was similar for both compounds (Table 2).
The lycorine diacetate 2 is more active against genomovar ALM-
00-173 than monoacetate 3 (Table 1). It must be noted that
F. columnare ALM-00-173 is considered to be potentially more
problematic for catfish producers because genomovar II isolates
are more pathogenic for channel catfish.24 To improve the
solubility of 1,2-O,O0-diacetyllycorine, its hydrochloride salt
(5) was synthesized. However, this approach did not result in
increased antibacterial activity (see Tables 1 and 2).
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (662) 915-1037. Fax: (662) 915-1035. Email: agnes.
rimando@ars.usda.gov.
Funding Sources
We are grateful for the financial support to C.-X.T. from the
Chinese Scholarship Council.
On the basis of the preceding discussions, esterification at
C1 and C2 was performed. Esterification reaction between
benzoyl chloride and lycorine yielded novel compounds 6ꢀ16.
Of these analogues, 9 and 14 showed activities similar to those
of 3 and 4 against F. columnare BioMed (see IC50, MIC, RDFC,
RDCO values in Table 2). Noticeably, 9 and 14 are distinct
from the other benzoyl chloride ester analogues in having a
single substituent at the C2-position of the benzoyl ring. This
structural feature, however, did not seem to be of significance
for activity against F. columnare ALM-00-173 as the IC50, MIC,
RDFC, and RDCO values were not consistently lower than
those of 4 (see Table 1). Compound 9, which is substituted at
both the C1-O- and C2-O-positions, is more active than the
monosubstituted analogue 6. This is similar to results observed
with the acetylated analogues. To improve water solubility, an
iodo-salt of 14, the most active compound among the benzoyl
chloride analogues, was prepared, yielding 19. However, im-
proved solubility did not correspond to improved antibacterial
activity.
Carbamates 17 and 18 were synthesized from lycorine and the
appropriate isocyanate, via a Curtius reaction mechanism, con-
verting the isocyanate to an amine by nucleophilic addition on
the isocyanate. Compound 18 demonstrated high activity against
both F. columnare isolates, and 18 appears to have stronger
antibacterial activity than 4 (Tables 1 and 2). Consistent with the
observation with the acetylated analogues, the monosubstituted
analogue 17 was less active (Tables 1 and 2). Interestingly,
18 differs from 9 only in having nitrogen between the carbonyl
and the toluene group. It appears that the addition of a “spacer
unit” between these two moieties might be responsible for
improved activity. Notably, 18 inhibited the growth of
F. columnare ALM-00-173 and F. columnare BioMed.
In conclusion, the lycorine analogues synthesized having
substitution at both the C1-O- and C2-O-positions have better
antibacterial activity compared to having only one substitution at
either carbon. Salt preparations of the disubstituted analogues
have better solubility but no enhanced antibacterial activity. The
carbamate is more effective than the benzoyl chloride analogues.
1,2-O,O0-Diacetyllycorine is highly active and selective toward
the genomovar II isolate ALM-00-173; however, we have suc-
ceeded in preparing a lycorine analogue, 18, which had high
antibacterial activity against both isolates and considerably better
than that of 4.
Efficacy studies still need to be conducted to determine the
potential of 18 for use as an alternative to the antibiotic
florfenicol, currently under U.S. Food and Drug Administration
conditional approval, or other management approaches (e.g., use
of 35% Perox-Aid) for controlling columnaris disease in channel
catfish. In addition, palatability studies would need to be con-
ducted to determine the potential for incorporation of 18 into
catfish feed. However, results from our in vitro study certainly
indicate 18 to be the most promising of the lycorine analogues
evaluated in this study.
’ ACKNOWLEDGMENT
We thank Marcuslene Harries and Phaedra Page for technical
assistance.
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