Isolation, Synthesis, and Semisynthesis of Amaryllidaceae Constituents from Narcissus and Galanthus sp.: De Novo Total Synthesis of 2-epi-Narciclasine

Article abstract of DOI:10.1021/acs.jnatprod.8b00218

An efficient protocol for the isolation of narciclasine from common Amaryllidaceae bulbs, separation from haemanthamine, and the occurrence of a trace alkaloid, 2-epi-narciclasine, are reported. Attempts to convert natural narciclasine to its C-2 epimer by Mitsunobu inversion or oxidation/reduction sequences were compromised by rearrangement and aromatization processes, through which a synthesis of the alkaloid narciprimine was achieved. The methylation of the 7-hydroxy group of natural narciclasine followed by protection of the 3,4-diol function and oxidation/reduction sequence provided the target C-2 epimer. A de novo chemoenzymatic synthesis of 2-epi-narciclasine from m-dibromobenzene is also described. Haemanthamine and narciprimine were readily detected in the crude extracts of Narcissus and Galanthus bulbs containing narciclasine, and the occurrence of 2-epi-narciclasine as a trace natural product in Galanthus sp. is reported for the first time.

Full text of DOI:10.1021/acs.jnatprod.8b00218

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Isolation, Synthesis, and Semisynthesis of Amaryllidaceae
Constituents from Narcissus and Galanthus sp.: De Novo Total
Synthesis of 2-epi-Narciclasine
Suresh Borra,† Ringaile Lapinskaite,‡ Christine Kempthorne,§ David Liscombe,§ James McNulty,*,†
and Tomas Hudlicky*,‡
†Department of Chemistry & Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4M1, Canada
‡Department of Chemistry and Centre for Biotechnology, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, ON L2S 3A1,
Canada
§Vineland Research and Innovation Centre, 4890 Victoria Avenue North, Box 4000, Vineland Station, ON L0R 2E0, Canada
S
* Supporting Information
ABSTRACT: An efficient protocol for the isolation of narciclasine from common Amaryllidaceae bulbs, separation from
haemanthamine, and the occurrence of a trace alkaloid, 2-epi-narciclasine, are reported. Attempts to convert natural narciclasine
to its C-2 epimer by Mitsunobu inversion or oxidation/reduction sequences were compromised by rearrangement and
aromatization processes, through which a synthesis of the alkaloid narciprimine was achieved. The methylation of the 7-hydroxy
group of natural narciclasine followed by protection of the 3,4-diol function and oxidation/reduction sequence provided the
target C-2 epimer. A de novo chemoenzymatic synthesis of 2-epi-narciclasine from m-dibromobenzene is also described.
Haemanthamine and narciprimine were readily detected in the crude extracts of Narcissus and Galanthus bulbs containing
narciclasine, and the occurrence of 2-epi-narciclasine as a trace natural product in Galanthus sp. is reported for the first time.
arciclasine (1), isolated in 1967,1 is one of the potent
biologically active Amaryllidaceae constituents shown in
has been developed to separate narciclasine from related
constituents. Attempts to invert the C-2 configuration employ-
ing Mitsunobu chemistry, which has instead led to a synthesis
of the natural product narciprimine (7) via aromatization of the
C-ring, are also discussed. We also report the identification of
2-epi-narciclasine as a natural product for the first time in an
extract of Galanthus sp., the occurrence of narciprimine (7) in
both Narcissus and Galanthus, and the conversion of
narciclasine to its C-2 epimer as well as a de novo
chemoenzymatic total synthesis of this compound.
N
Figure 1. Originally described as antimitotic,2 narciclasine
induces apoptosis by activating death receptors FAS and DR4
and mitochondrial pathways in tumor cells but not in normal
cells.3 The targeting of eEF1A protein by narciclasine has been
reported.4 The potent anticancer activity led to the intense
interest of the organic chemistry community in synthetic
approaches to narciclasine, which is slightly more abundant
(30−140 mg/kg wet weight)5 than pancratistatin (2) (∼19
mg/kg), isolated by Pettit in 1984.6
Many total syntheses of narciclasine have been accomplished
to date since the first three reports by Rigby in 19977 and Keck8
and Hudlicky in 1999.9 Other syntheses followed,10,11 and the
interest in this Amaryllidaceae consitutent continues unabated,
in view of the medicinal chemistry potential of the natural
product and its derivatives.10a,b,e−h In this paper an optimized
procedure for isolation of narciclasine from Narcissus
pseudonarcissus bulbs is reported. A solvent partition sequence
RESULTS AND DISCUSSION
■
Optimized Isolation Protocol. At least five different
extraction protocols for the isolation of narciclasine from
Narcissus bulbs have been reported.10e The narciclasine content
Received: March 16, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b00218
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Figure 1. A selection of Amaryllidaceae constituents.
a
Scheme 1. Attempted Mitsunobu Reaction of Narciclasine-3,4-acetonide
a
Reagents: (i) 2,2-DMP, p-TSA (cat), DCM, rt (65−80%); (ii) PBu3, BzOH, DEAD, THF, rt (30%).
a
Scheme 2. Second Attempt at a Mitsunobu Inversion at C-2 in Narciclasine-3,4-acetonide 8
a
Reagents: (i) Ph3P, DIAD, p-NO2BzOH, THF; (ii) TFA/H2O, 2:1, THF (35%, over 2 steps).
in the bulbs and leaves varies considerably with season,
reaching a maximum content as the plant approaches full
flowering.5a Plant material is normally processed with MeOH
or EtOH, and narciclasine is separated through solvent
partitioning and chromatographic purification. In our hands,
narciclasine has been isolated over several years following
literature procedures in quantities approaching 50 mg/kg wet
bulbs. We have now developed an optimized, alternative
isolation protocol in which extraction of the plant material into
MeOH (48 h), dilution with 250 mL of H2O, and
concentration yield an aqueous extract from which the less
polar materials are removed by extraction into dichloromethane
(DCM). The optimized process involves extraction of the
remaining aqueous residue with a mixture of EtOAc and
MeOH (9:1). This process allows clean extraction of
narciclasine and a trace contaminant into the EtOAc phase
from the polar aqueous phase. A single chromatographic
purification then allows isolation of narciclasine in pure form.
The initial DCM extraction process completely removes
other compounds such as galanthamine (6) and a contaminant
that has a similar Rf to narciclasine. This compound was
1
identified as the crinane alkaloid haemanthamine (5). The H
and 13C NMR data of haemanthamine proved identical to the
reported data.12,13 However, analysis of the 1H−1H and
1H−13C 2D spectra showed that the C-6 and C-12 methylene
signals have been previously misassigned. The C-12 methylene
1
(13C: 63.4 ppm; H: 2.9, 3.3 ppm) correlates to the C-11
1
signals (13C: 79.8 ppm; H: 3.8 ppm), in contrast to the
isolated resonances observed for the C-6 methylene (13C: 60.4
1
ppm; H: 4.2, 3.6 ppm). The assignment of these carbon and
proton signals should therefore be reversed.12,13
Analysis of the EtOAc content from Narcissus by TLC
showed essentially single-spot separation; however, the 1H
NMR data show the presence of narciclasine and a trace of an
unidentified contaminant. The NMR data of narciclasine and
the trace contaminant did not permit identification by
comparison with known co-metabolites.5a The presence of
B
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a
Scheme 3. Oxidation/Reduction of Protected Narciclasine (Free Phenol at C-7)
a
Reagents: (i) MnO2, THF; (ii) CeCl3·7H2O, NaBH4, MeOH (30% over 2 steps).
Scheme 4. Conversion of Narciclasine (1) to 2-epi-Narciclasine 19
a
Reagents: (i) CH2N2, EtOH, Et2O; (ii) 2,2-DMP, p-TSA, DCM: (iii) DMP, DCM; (iv) L-Selectride, DCM, THF (50% over two steps); (v) HCl
(2 M), DCM, THF (30%); vi) TMSCl, KI, MeCN (51%).
minor peaks for a methylenedioxy −CH2− and an olefinic
proton (1H NMR: 6.06−6.12 ppm) led to a hypothesis that this
material may be a minor epimer of narciclasine. Purification of
the EtOAc-soluble portion from Narcissus by chromatography
on silica yields off-white narciclasine in high yield (120 mg/kg).
The disappearance of the minor metabolite upon a single silica
gel column led to the assumption that the compound may be
the C-2-allylic alcohol epimer. Thus, 2-epi-narciclasine became
a target for semisynthesis from narciclasine and also for a de
novo total synthesis (vide infra). This work and a direct
comparison provided proof that the minor isomer from
Narcissus is not the C-2-epimer, and this unstable minor
compound remains unidentified. Access to authentic 2-epi-
narciclasine obtained by semisynthesis (vide infra) and also by
de novo total synthesis (Scheme 4) and comparison with
extracts of Narcissus by LC-MS confirmed the absence of this
metabolite in Narcissus. However, surprisingly, an identical
analysis of an extract of Galanthus by LC-MS allowed
confirmation of the presence of the 2-epimer by comparison
of MS and LC-MS data.
alkoxyphosphonium intermediate, and yielded the mixture of
dienamine 9 and benzoic acid (1:1) in 30% yield (Scheme 1).
The Mitsunobu reaction was next attempted on compound 8
under alternative conditions using triphenylphosphine and
diisopropyl diazodicarboxylate (DIAD) (Scheme 2). These
conditions resulted in the formation of the target product 10
(16%) and the product of allylic alcohol oxidation, followed by
olefin migration to give the stable conjugated enamine 11
(84%). This rearrangement of the double bond into the lactam
moiety has previously been reported.14 In the present case, the
reaction produced an inseparable mixture of the products 10
and 11. Most interestingly, treatment of the mixture with
trifluoroacetic acid (TFA), in an attempt to remove the
acetonide, yielded a major product in 35% isolated yield from
acetonide 8. This product proved to be identical with the minor
Amaryllidaceae constituent narciprimine (7), a compound that
exhibits acetylcholinesterase inhibitory activity.2,15
B. Oxidation/Reduction Sequence. Because of the failure of
the direct C-2-inversion reactions, an alternative attempt at the
conversion of narciclasine to its C-2 epimer was investigated
involving the oxidation of the C-2 allylic alcohol group and
stereoselective reduction. This sequence also proved problem-
atic: Oxidation of compound 8 was unsuccessful under Swern
as well as Dess-Martin conditions. Enone 12 was prepared by
oxidation of narciclasine with MnO2, but its isolation was
arduous because of its instability. As noted in the original
publication by Krohn, enone 12 readily rearranges to its
Conversion of Narciclasine to 2-epi-Narciclasine.
A. Mitsunobu Inversion. In an attempt to convert narciclasine
to its C-2 epimer Mitsunobu reaction conditions with
tributylphosphine and benzoic acid were first applied to the
3,4-acetonide-protected allylic alcohol 8. Although starting
material was consumed, none of the target product was isolated.
Instead, elimination took place, most likely from the C-2-
C
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Figure 2. Extracted ion chromatograms (m/z 306.0614) for narciclasine (A, C) and 2-epi-narciclasine (B, D) from N. pseudonarcissus extracts (A, B)
compared to synthesized standards (C, D).
a
Scheme 5. Synthesis of 2-epi-Narciclasine (19)
a
Reagents: (i) toluene dioxygenase in E. coli JM109(pDTG601A) (0.5−0.8 g/L); (ii) DMP, p-TsOH, then MocNHOH, NaIO4, MeOH, H2O (45−
70%); (iii) Pd(PPh3)4, Na2CO3, PhH, H2O, EtOH, then Mo(CO)6, MeCN, H2O (60−72%); (iv) CeCl3·7H2O, NaBH4, MeOH (68−90%); (v) HCl
(2 M), DCM, THF (95%); (vi) Ac2O, Et3N, DMAP, DCM (84%); (vii) Tf2O, DMAP, DCM (31%); (viii) TMSCl, Kl, MeOH (40−60%); (ix)
K2CO3, MeOH (60−95%).
deconjugated isomer 11 (Scheme 3).14 Attempts to perform a
one-pot oxidation/reduction without isolation of enone 12
proved irreproducible, and only traces of allylic alcohol 13 were
observed in reaction mixtures.
We suspected that the free phenolic moiety might be
contributing to the problem encountered in oxidations of the
C-2 allylic alcohol moiety and to the instability of enone 12.
Thus, narciclasine was treated with diazomethane to produce
the methyl ether 14, whose protection as an acetonide yielded
compound 15, Scheme 4. The allylic alcohol was oxidized
under Dess-Martin conditions to enone 16, followed by in situ
reduction. The Luche reduction of 16 yielded a mixture of both
diastereomers 15 and 17 in 45% yield and a 6:1 ratio. The
reduction with a bulkier reagent, L-selectride, provided only the
allylic alcohol 17 in 50% yield. Acetonide deprotection and O-
demethylation of 18 furnished 2-epi-narciclasine (19). UPLC
analysis of the crude extract from Narcissus bulbs revealed the
absence of 2-epi-narciclasine, as evidenced by co-injection
comparison with the synthetic sample (Figures 2B and 2D).
De Novo Synthesis to 2-epi-Narciclasine. A total
synthesis of 2-epi-narciclasine was accomplished from 1,3-
dibromobenzene via enzymatic dihydroxylation to diol 20,
D
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Figure 3. Extracted ion chromatograms (m/z 306.0614) for narciclasine (A, C) and 2-epi-narciclasine (B, D) from Galanthus sp. extracts (A, B)
compared to synthesized standards (C, D).
which was converted in five steps to the allylic alcohol 23
[acetonide protection, nitroso Diels−Alder reaction, Suzuki
coupling, Mo(CO)6 and Luche reductions] (Scheme 5). Details
of these transformations were recently published [up to the
allylic alcohol 23].16 The allylic alcohol 23 was deprotected
with 2 M HCl, and the resulting triol 24 was reprotected as
triacetate 25. Formation of ring B of the narciclasine backbone
was achieved employing the Banwell-modified Bishler−
Napieralski reaction and yielded phenantridone 26.17 Depro-
tection of 26 can be accomplished in two ways: (a) O-
demethylation (yielding compound 27), followed by hydrolysis
of acetoxy groups, or (b) hydrolysis of acetoxy groups (yielding
compound 18), followed by O-demethylation. The initial
pathway was combined in one pot and furnished 2-epi-
narciclasine (19) in 50% yield over the two steps. As stated
above, 2-epi-narciclasine was not detected in the crude extract
from Narcissus (Figures 2B and 2D).
The literature contains a few misleading references to this
epimer, including the structure as represented by 19.18 This
arose from initial misassignment of the structure of narciclasine
(1), which was corrected by Krohn and Mondon in 1972.19
They synthesized oxygenated derivatives and compared their
1H NMR data with those of the isomerized narciclasine
derivative. In this manner the correct C-2 configuration was
reassigned for the natural product, as shown in Figure 1.
Detection of 2-epi-Narciclasine in Amaryllidaceae
Crude Extracts. Narciclasine was detected in extracts of
both N. pseudonarcissus and Galanthus sp. (Figures 2A and 3A,
respectively) as compared to an authentic standard (Figures 2C
and 3C). However, as indicated by coelution with synthesized
2-epi-narciclasine (Figures 2D and 3D), this compound was
only detected in extracts of Galanthus sp. (Figure 3B), and not
extracts of N. pseudonarcissus (Figure 2B). 2-epi-Narciclasine has
not been reported as a natural product. Narciclasine,
haemanthamine, and narciprimine were readily detectable in
LC-MS chromatograms of the crude extract from both N.
pseudonarcissus and Galanthus sp. and alignment of retention
time and mass spectrum with standard compounds (see HRMS
data in Figure S2-1 in the Supporting Information). Analysis of
the relative content of 2-epi-narciclasine in Galanthus sp.
showed it to be approximately 100-fold less abundant than
narciclasine (Figure S2-2 in the Supporting Information),
indicating its presence at sub-ppm level (<1.0 mg/kg) in
Galanthus bulbs. The compound would have been identified
only with much difficulty through standard isolation protocols
but was readily matched with a sample of synthesized 2-epi-
narciclasine. On the other hand, significant amounts of
narciprimine (Figure S2-3, Supporting Information) and
haemanthamine (Figure S2-4, Supporting Information) were
detected in the extracts of both species.
An efficient solvent extraction and single-column protocol for
the isolation of narciclasine from bulbs of N. pseudonarcissus is
reported in high (>100 mg/kg wet weight) yield. The crinane
alkaloid haemanthamine was also isolated, and several
resonances in the NMR spectrum of 5 were reassigned on
the basis of 2D NMR correlations. Clean separation of
narciclasine from haemanthamine is achieved during this
solvent-extraction process. A trace contaminant co-occurring
with narciclasine in the EtOAc extract of N. pseudonarcissus was
hypothesized to be 2-epi-narciclasine, leading to both the semi-
and total syntheses of this 2-epimer. This compound proved not
to be identical to a minor metabolite, whose constitution is as-
yet unidentified. Nonetheless, through an interesting twist of
fate, 2-epi-narciclasine was identified as a trace metabolite (<1.0
ppm) in an extract of Galanthus sp. and is here reported as a
natural product for the first time. Attempted Mitsunobu
inversions of the allylic alcohol functionality in narciclasine as
an entry to the C-2 epimer were complicated by side reactions;
however this work resulted in a serendipitous synthetic entry to
the natural product narciprimine. Analysis of the crude extracts
of N. pseudonarcissus and Galanthus sp. using LC-MS and
synthesized standards confirmed the presence of narciclasine,
haemanthamine, and narciprimine in both extracts and the trace
occurrence of 2-epi-narciclasine in only the Galanthus sample.
Given the low natural abundance of 2-epi-narciclasine, the
optical rotation data or a detailed analysis on a chiral support
E
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Narciclasine (1): Specific rotation and other physical data were in
accord with the literature. Rf = 0.3 [DCM/MeOH (9:1)]; mp = 236−
240 °C (dec > 245 °C), lit.11c 246 °C; [α]2D0 +111 (c 0.4, MeOH), lit.8
and comparison with the racemate could not be obtained. The
overall absolute configuration is assumed to be in accord with
related lycoranes such as narciclasine and pancratistatin. Of
special note is the rare fact that this total synthesis of 2-epi-
narciclasine preceded its identification as a natural product
1
[α]2D0 +112 (c 0.6, MeOH); H NMR (700 MHz, DMSO-d6) δ 13.26
(s, 1H), 7.89 (s, 1H), 6.86 (s, 1H), 6.17−6.14 (m, 1H), 6.08 (d, J = 2.4
Hz, 2H), 5.18 (dd, J = 16.0, 5.8 Hz, 2H), 5.02 (d, J = 3.8 Hz, 1H), 4.19
(d, J = 8.5 Hz, 1H), 4.02 (s, 1H), 3.81−3.77 (m, 1H), 3.70 (d, J = 2.3
Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 168.9, 152.4, 144.8,
133.4, 132.1, 129.3, 124.8, 105.6, 102.1, 95.8, 72.4, 69.1, 68.8, 52.9;
HRMS (ESI) (M+Na)+ calcd for C14H13NO7Na 330.0590; found
330.0581.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All solvents were dried and
distilled before usage. Reactions were done in an inert atmosphere (Ar
or N2). All reagents were obtained from commercial sources. Before
using, all glassware was oven- or flame-dried. NMR analysis was carried
out on 400, 600, and Bruker AV-III 700 mHz spectrometers running
Topspin 2.1 and 3.5 software. Probes were equipped with gradients
and variable-temperature accessories. Chemical shifts are given in δ,
and coupling constants J are given in Hz. Melting points were
determined using a capillary melting point apparatus. HRMS data were
recorded using an LTQ Orbitrap XL or double focusing sector mass
spectroscopy, and the mass ion was determined by electrospray
ionization, fast atom bombardment, or electron ionization. Infrared
spectra were recorded on an FT-IR spectrophotometer as CHCl3
solutions or neat and are reported in wave numbers (cm−1). Optical
rotations were determined on an Autopol IV automatic polarimeter
and a PerkinElmer 241-MC polarimeter. Flash grade 60 silica gel was
used for column chromatography unless otherwise noted. Deactivated
silica was prepared by stirring it with 10% H2O for 5 h. TLC was
performed on silica gel 60 F254-coated aluminum sheets and visualized
with UV and CAM or KMnO4 solutions. UPLC-ESIMS analysis of
crude extracts obtained from TEVA and synthesized standards was
performed on an Acquity UPLC Class I equipped with an Acquity
BEH C18 1.7 μm column coupled to a Waters Xevo-G2XS QTOF.
Leucine enkephalin (200 pg/μL in acidified 50:50 MeCN/H2O) was
used as a reference calibrant with a Waters LockSpray ion source for
exact mass measurement. Narciprimine, narciclasine, and 2-epi-
narciclasine standards were diluted in acidified 5% MeCN/H2O
(0.1% formic acid) and hemeeanthamine standard diluted in DCM for
UPLC-MS analysis. Crude extracts obtained from TEVA were
prepared in MeOH at 2 mg/mL and diluted (1:10) in acidified 5%
MeCN/H2O (0.1% formic acid) or DCM.
(3aS,3bR,12S,12aR)-6,12-Dihydroxy-2,2-dimethyl-3b,4,12,12a-
tetrahydrobis[1,3]dioxolo[4,5-c:4′,5′-j]phenanthridin-5(3aH)-one
(8). Protocol A: Narciclasine (1) (10 mg, 0.032 mmol) was suspended
in DCM (3 mL), and 2,2-DMP (0.3 mL) added followed by addition
of p-toluenesulfonic acid (p-TSA, cat.). The mixture was stirred at
room temperature for 2 h. The reaction mixture was adsorbed on
deactivated silica and purified by flash column chromatography
(EtOAc/hexanes, 1:2 to 1:1). The product was isolated as a colorless,
oily solid, 8 mg, 71%.
1
8: Rf = 0.6 [EtOAc/hexanes (2:1)]; H NMR (600 MHz, DMSO-
d6) δ 13.76 (s, 1H), 8.83 (s, 1H), 7.03 (s, 1H), 6.49 (t, J = 3.0 Hz,
1H), 6.07 (dd, J = 5.1, 0.8 Hz, 2H), 5.82 (s, 1H), 4.19−4.14 (m, 1H),
4.14−4.09 (m, 1H), 4.07 (t, J = 7.8 Hz, 1H), 3.97 (dd, J = 7.9, 6.2 Hz,
1H), 1.46 (s, 3H), 1.32 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ
167.7, 152.6, 145.2, 133.4, 128.9, 128.3, 126.0, 109.8, 104.3, 102.1,
94.3, 79.0, 78.5, 71.0, 54.6, 27.1, 24.8; HRMS (EI) calcd for
C17H17O7N 347.1005; found 347.1004.
Protocol B: To a stirred solution of narciclasine (1) (10 mg, 0.032
mmol) and 2,2-DMP (0.3 mL) in DMF (0.3 mL) was added
pyridinium p-toluene sulfonate (0.8 mg, 0.0032 mmol) at 0 °C. The
reaction mixture was allowed to warm to room temperature and stirred
for 8 h. The reaction mixture was poured into H2O (5 mL) and
extracted with EtOAc (3 × 5 mL). The combined organic phase was
washed with cold H2O (1 × 10 mL) and brine (1 × 10 mL), dried
over anhydrous Na2SO4, filtered, and concentrated in vacuo. The
resulting mixture was purified by silica gel column (DCM/MeOH,
250:1 to 165:1). Acetonide 8 was obtained as a white solid (9 mg,
82%).
8: Rf = 0.6 [DCM/MeOH (19:1)]; mp = 270−274 °C, lit. 270−
2735b and 275−276 °C;14 [α]D20 −11.4 (c 1.2, CHCl3), lit.14 [α]2D0 −33
(c 0.35, THF) and lit.8 [α]D20 −24 (c 0.35, THF); 1H NMR (600 MHz,
DMSO-d6) δ 13.73 (s, 1H), 8.79 (s, 1H), 7.00 (s, 1H), 6.46 (t, J = 2.9
Hz, 1H), 6.05 (d, J = 5.0 Hz, 2H), 5.79 (d, J = 5.7 Hz, 1H), 4.17−4.12
(m, 1H), 4.11−4.08 (m, 1H), 4.07−4.02 (m, 1H), 3.95 (dd, J = 7.8,
6.3 Hz, 1H), 1.44 (s, 3H), 1.30 (s, 3H); 13C NMR (150 MHz, DMSO-
d6) δ 167.6, 152.5, 145.2, 133.3, 128.9, 128.3, 125.9, 109.8, 104.2,
102.0, 94.2, 79.0, 78.4, 71.0, 54.6, 27.0, 24.8; HRMS (ESI) (M+H)+
calcd for C17H18NO7 348.1083; found 348.1075.
(3aS,12aR)-6-Hydroxy-2,2-dimethyl-4,12a-dihydrobis[1,3]-
dioxolo[4,5-c:4′,5′-j]phenanthridin-5(3aH)-one mixture with ben-
zoic acid (9): Allylic alcohol 8 (15 mg, 0.043 mmol), PBu3 (17.5 mg,
0.086 mmol, 21.3 μL), and benzoic acid (10.6 mg, 0.086 mmol) were
suspended in tetrahydrofuran (THF) (1 mL). It was followed by slow
dropwise addition of diethyl azodicarboxylate (DEAD) (15 mg, 0.086
mmol, 13.7 μL). The reaction mixture was stirred at room temperature
for 1.5 h (full consumption of starting material). The reaction mixture
was adsorbed on deactivated silica and purified by column
chromatography (EtOAc/hexanes, 1:4 to 2:3). The product was
isolated as a 1:1 mixture with benzoic acid, 6 mg, 30%.
Extraction and Purification of Narciclasine (1) and Hae-
manthamine (5) from Narcissus pseudonarcissus Bulbs. Finely
chopped bulbs of Narcissus pseudonarcissus (1.0 kg wet weight) were
soaked in MeOH (3.0 L) for 2 days at ambient temperature.
Considerable darkening of the material and decomposition is observed
if bulbs are allowed to soak for longer periods. The resulting solution
and suspended plant parts were filtered through a bed of Celite and
washed with MeOH (3 × 250 mL). The combined filtrate was
concentrated at 45 °C under reduced pressure to give approximately
250 mL of an aqueous residue, which was further diluted with water
(250 mL). The aqueous phase was extracted with DCM (3 × 250
mL). The combined DCM phases were concentrated in vacuo, giving a
residue (12.8 g) that was purified by column chromatography (DCM/
MeOH, 24:1 to 19:1). Haemanthamine (5) was obtained as a pale
yellow solid (11 mg, 0.0011%).
Haemanthamine (5): Rf = 0.3 [DCM/MeOH (9:1)]; mp = 200−
204 °C, lit.20 199−202 °C; [α]D20 +41 (c 0.2, CHCl3); lit.20 [α]2D4 +42
(c 0.3, CHCl3); 1H NMR (600 MHz, DMSO-d6) δ 6.96 (s, 1H), 6.59
(s, 1H), 6.46 (d, J = 10.1 Hz, 1H), 6.11 (dd, J = 10.0, 5.0 Hz, 1H), 5.92
(s, 2H), 5.75 (s, 1H), 5.03 (s, 1H), 4.17 (d, J = 16.8 Hz, 1H), 3.82−
3.78 (m, 1H), 3.77−3.74 (m, 1H), 3.65−3.60 (m, 1H), 3.37−3.28 (m,
2H), 3.22 (s, 3H), 3.07 (dd, J = 13.4, 4.0 Hz, 1H), 2.97−2.93 (m, 1H),
2.01 (td, J = 13.3, 4.2 Hz, 1H), 1.76 (d, J = 9.8 Hz, 1H); 13C NMR
(150 MHz, DMSO-d6) δ 145.9, 145.4, 136.2, 129.2, 128.8, 106.8,
103.3, 100.5, 79.8, 72.2, 63.4, 62.5, 60.4, 55.6, 54.9, 49.9, 28.0; HRMS
(ESI) (M + H)+ calcd for C17H20NO4 302.1392; found 302.1387.
The aqueous phase from the DCM extraction was extracted with
EtOAc/MeOH (9:1, 3 × 250 mL). The combined organic phase was
concentrated in vacuo to give a residue (1.53 g), which was further
purified by column chromatography (DCM/MeOH, 19:1 to 13:1).
Narciclasine (1) was isolated as an off-white solid (0.12 g, 0.012%).
9: Rf = 0.4 [EtOAc/hexanes (1:2)]; [α]2D3 −109 (c 0.05, CHCl3); 1H
NMR (600 MHz, CDCl3) δ 6.81−6.73 (m, 2H), 6.12 (d, J = 1.0 Hz,
2H), 6.05 (dd, J = 9.9, 4.5 Hz, 1H), 5.09 (d, J = 7.7 Hz, 1H), 4.82 (dd,
J = 7.6, 4.5 Hz, 1H), 1.51 (s, 3H), 1.36 (s, 3H); 13C NMR (150 MHz,
CDCl3) δ 171.2, 165.8, 154.5, 144.6, 133.8, 132.8, 132.4, 131.6, 130.3,
129.6, 128.6, 122.9, 120.9, 108.6, 108.5, 102.6, 92.8, 71.3, 70.8, 27.2,
25.6; LRMS (ESI+) calcd for C17H15O6N+Na 352.1; found 352.0.
Narciprimine 4,7-Dihydroxy-[1,3]dioxolo[4,5-j]phenanthridin-
6(5H)-one (7): Ph3P (60 mg, 0.23 mmol) and p-nitrobenzoic acid
(38 mg, 0.23 mmol) were added to a stirred solution of the acetonide
F
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8 (20.0 mg, 0.057 mmol) in THF (2 mL), followed by the dropwise
addition of DIAD (0.051 mL, 0.25 mmol) at 0 °C. The reaction
mixture was warmed to rt and stirred for 16 h. The reaction mixture
was then cooled to 0 °C and quenched with slow addition of 1 M HCl
(0.2 mL) and H2O (5.0 mL). The mixture was extracted with EtOAc
(3 × 5 mL). The combined organic extract was washed with H2O (1 ×
10 mL) and brine (1 × 10 mL), dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. The resulting crude mixture was purified
by silica gel column (EtOAc/hexanes, 1:5 to 1:4) and afforded an
inseparable mixture of 10 and 1116 (1:6, 10 mg), which was
(2R,3R,4S,4aR)-2,3,4-Trihydroxy-7-methoxy-3,4,4a,5-tetrahydro-
3,4-[a][2,2-dimethyl-1,3-dioxolanyl]-[1,3]dioxolo[4,5-j]-
phenanthridin-6(2H)-one (17): A solution of allylic alcohol 15 (15
mg, 0.042 mmol) in DCM (2 mL) was cooled to 0 °C, and Dess-
Martin periodinane (20 mg, 0.045 mmol) added. The mixture was left
to warm to room temperature for 1.5 h. To the cooled reaction
mixture (0 °C) was added L-selectride (1 M solution in THF, 0.2 mL)
in a dropwise manner. The mixture was left to warm to room
temperature. After consumption of the intermediate enone 16 [TLC,
DCM/MeOH (50:1)], the reaction mixture was quenched with a
saturated NH4Cl solution (1 mL) and filtered through a plug of Celite.
Compound 17 was purified by column chromatography [DCM/
MeOH (100:1 to 50:1)] (6 mg, 40%, yellow oil).
1
characterized by H NMR and COSY data. Rf = 0.70 [DCM/MeOH
(19:1)]. The mixture of 10 and 11 was used directly in the next step.
To a solution of the above mixture (10.0 mg) in THF (2.0 mL) was
added TFA/H2O (2:1, 0.9 mL) at 0 °C. The reaction mixture was
allowed to warm to rt and stirred for 2 h. The solvent was evaporated
by flushing with a stream of nitrogen. The crude product was purified
by silica gel column (DCM/MeOH, 199:1 to 140:1). Narciprimine
(7) was obtained as a white solid (5 mg, 35% over 2 steps), the
physical data of which were fully consistent with the literature.15
7: Rf = 0.45 [DCM/MeOH (19:1)]; mp = 309−313 °C, lit.17 310−
315 °C; 1H NMR (600 MHz, DMSO-d6) δ 13.78 (s, 1H), 7.75 (d, J =
8.2 Hz, 1H), 7.57 (s, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.97 (d, J = 7.8 Hz,
1H), 6.20 (s, 2H); 13C NMR (175 MHz, DMSO-d6) δ 165.0, 153.6,
144.8, 144.4, 132.3, 131.9, 123.9, 123.2, 119.4, 113.8, 113.5, 107.1,
102.3, 93.7; HRMS (ESI) (M + H)+ calcd for C14H10NO5 272.0559;
found 272.0555.
(2S,3R,4S,4aR)-2,3,4-Trihydroxy-7-methoxy-3,4,4a,5-tetrahydro-
[1,3]dioxolo[4,5-j]phenanthridin-6(2H)-one (14): N-Nitroso-N-meth-
ylurea (1.3 g, 13 mmol) was added portionwise to a cold (0 °C)
mixture of aqueous KOH solution (8 M, 4 mL) and Et2O (14 mL).
The organic layer containing freshly prepared diazomethane solution
was decanted onto 2 g of cold KOH pellets and added to the
suspension of narciclasine (1) (100 mg, 0.325 mmol) in EtOH (17
mL) at 0 °C. The reaction flask was covered with a loose rubber
septum and left to warm to room temperature, and the mixture stirred
for 16 h. After full consumption of starting material (TLC, DCM/
MeOH, 10:1), the mixture was stirred for another 16 h under
atmospheric conditions and then quenched with the addition of glacial
acetic acid (1 mL). The reaction mixture was concentrated and used
for acetonide formation without further purification. The crude
product was purified by column chromatography (DCM/MeOH, 10:1
to 5:1) to afford 14 (83 mg, 79%).
17: Rf = 0.3 [DCM/MeOH (50:1)]; [α]2D4 +119 (c 0.1, CHCl3/
1
MeOH, 92:8); IR (neat) 3306, 2924, 2854, 1659, 1478 cm−1; H
NMR (400 MHz, CDCl3) δ 6.93 (s, 1H), 6.47 (dd, J = 5.9, 3.5 Hz,
1H), 6.04 (dd, J = 8.1, 1.3 Hz, 2H), 5.97 (s, 1H), 4.79 (ddd, J = 7.0,
3.5, 1.2 Hz, 1H), 4.68 (dd, J = 6.4, 4.6 Hz, 1H), 4.31 (dd, J = 8.3, 4.5
Hz, 1H), 4.27−4.19 (m, 1H), 4.03 (s, 3H), 2.70 (s, 1H), 1.57 (s, 3H),
1.42 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 161.6, 152.3, 145.2,
140.2, 134.8, 130.7, 120.1, 114.3, 112.1, 102.2, 98.3, 80.6, 73.4, 63.6
61.2, 54.9, 26.8, 24.8; HRMS (EI) calcd for C18H19O7N 361.1162;
found 361.1153.
2-epi-7-Methoxynaciclasine [(2R,3R,4S,4aR)-2,3,4-Trihydroxy-7-
methoxy-3,4,4a,5-tetrahydro-[1,3]dioxolo[4,5-j]phenanthridin-
6(2H)-one] (18): Procedure from Synthesized Intermediate 17:
Acetonide 17 (5 mg, 0.014 mmol) was dissolved in a mixture of DCM
and THF (1:2, 0.5 mL), and 2 M HCl (0.3 mL) was added in a
dropwise manner at 0 °C. After stirring for 30 min, the reaction
mixture was quenched with a saturated solution of NaHCO3 (3 mL)
and extracted with DCM (3 × 4 mL). The organic phase was dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
triol was purified by preparative TLC [DCM/MeOH (8:1)]. The
reaction yielded 2 mg (44%) of compound 18 as a white, crystalline
product.
Procedure from Synthesized Intermediate 26. A round-bottom
flask was charged with triacetate 26 (25 mg, 0.056 mmol), K2CO3 (23
mg, 0.17 mmol), and MeOH (3 mL). After stirring for 5 min, the
reagents dissolved and the mixture became homogeneous. TLC
analysis showed complete consumption of the starting material. The
reaction mixture was concentrated under reduced pressure, and the
product was isolated by preparative TLC [DCM/MeOH (8:1)] to
yield 18 as a white, crystalline solid (17 mg, 95%).
14: Rf = 0.1 [DCM/MeOH (10:1)]; mp = 208 °C, lit.2 206 °C;
[α]D23 +256 (c 0.7, CHCl3/MeOH, 1:1), lit.11b [α]2D6 +204 (c 0.3,
DMSO); IR (neat) 3379, 2980, 2912, 1359, 1650, 1611, 1478 cm−1;
1H NMR (600 MHz, DMSO-d6) δ 7.15 (s, 1H), 7.00 (s, 1H), 6.13 (d,
J = 0.9 Hz, 1H), 6.11−6.09 (m, 1H), 6.07 (d, J = 0.9 Hz, 1H), 5.19 (d,
J = 5.8 Hz, 1H), 5.15 (d, J = 5.5 Hz, 1H), 4.96 (d, J = 3.8 Hz, 1H),
4.04−3.99 (m, 2H), 3.84 (s, 3H), 3.77 (ddd, J = 7.8, 5.5, 2.0 Hz, 1H),
3.69−3.65 (m, 1H); 13C NMR (150 MHz, DMSO-d6) δ 162.1, 151.4,
143.5, 139.2, 133.5, 131.3, 123.6, 115.3, 102.0, 99.5, 72.5, 69.2, 69.2,
60.5, 52.6; HRMS (EI) calcd for C15H15O7N 321.0849; found
321.0847.
(2S,3R,4S,4aR)-2,3,4-Trihydroxy-7-methoxy-3,4,4a,5-tetrahydro-
3,4-[a][2,2-dimethyl-1,3-dioxolanyl]-[1,3]dioxolo[4,5-j]-
phenanthridin-6(2H)-one (15):16 7-Methoxynarciclasine (14) (22
mg, 0.07 mmol) was suspended in DCM (2 mL), and 2,2-DMP (0.2
mL) was added, followed by the addition of a catalytic amount of p-
TSA. The reaction mixture was stirred at room temperature for 2 h,
adsorbed on deactivated silica, and purified by gravity column
chromatography [DCM/MeOH (100:1 to 30:1)] to afford 15 as a
colorless, oily solid, 17 mg, 68%.
26b: Rf = 0.4 [DCM/MeOH (5:1)]; [α]2D4 +9 (c 0.1, CHCl3/
1
MeOH, 1:1); IR (neat) 3342, 2923, 2852, 1651 cm−1; H NMR (600
MHz, acetone-d6) δ 6.87 (s, 1H), 6.11 (d, J = 0.9 Hz, 1H), 6.06 (d, J =
0.9 Hz, 1H), 6.00 (d, J = 1.4 Hz, 1H), 4.43 (d, J = 2.7 Hz, 1H), 4.33−
4.27 (m, 1H), 4.11 (s, 1H), 3.91 (s, 3H), 3.73 (dd, J = 8.7, 1.7 Hz,
1H); 13C NMR (150 MHz, acetone-d6) δ 163.6, 153.0, 145.2, 140.4,
134.3, 130.7, 126.2, 116.3, 103.1, 99.9, 74.1, 72.8, 68.8, 61.1, 52.9;
HRMS (EI) calcd for C15H15O7N 321.0849; found 321.0849.
(2R,3R,4S,4aR)-2,3,4,7-Tetrahydroxy-3,4,4a,5-tetrahydro-[1,3]-
dioxolo[4,5-j]phenanthridin-6(2H)-one (19): Procedure from Syn-
thesized Intermediate 18: Triol 18 (20 mg, 0.062 mmol) was
dissolved in MeCN (2 mL), and a solution of trimethylsilyl chloride
(TMSCl) in MeCN (0.5 M, 0.16 mL) and KI (11 mg, 0.068 mmol)
were added. The reaction mixture was heated at 60 °C for 1 h, cooled
to 0 °C, and quenched with H2O (2 mL). The product was extracted
with EtOAc (3 × 5 mL), and the organic phase was dried over
Na2SO4, filtered, and concentrated under reduced pressure.
Purification by flash column chromatography [DCM/MeOH (30:1
to10:1)] yielded 11 mg (60%) of 2-epi-narciclasine (19).
15: Rf = 0.3 [DCM/MeOH (50:1)]; [α]2D4 +47.9 (c 0.5, CHCl3); IR
(neat) 3338, 2988, 2923, 2853, 1655, 1611, 1478 cm−1; 1H NMR (600
MHz, CDCl3) δ 6.85 (s, 1H), 6.31−6.27 (m, 1H), 6.04−6.02 (m, 3H),
4.37 (m, 1H), 4.16−4.05 (m, 3H), 4.02 (s, 3H), 2.77 (d, J = 4.5 Hz,
1H), 1.51 (s, 3H), 1.38 (s, 3H); 13C NMR (150 MHz, CDCl3) δ
161.8, 152.5, 145.3, 139.8, 130.4, 128.2, 125.0, 113.7, 111.4, 102.1,
97.7, 79.8, 79.0, 72.9, 61.2, 55.6, 27.2, 25.0; HRMS (EI) calcd for
C18H19O7N 361.1162; found 361.1165.
Procedure from Synthesized Intermediate 27: Triacetate 27 (15
mg, 0.035 mmol) was dissolved in MeOH (3 mL), and K2CO3 (20
mg, 0.14 mmol) was added at rt. The reaction mixture was stirred for
10 min until full consumption of starting material (TLC, DCM/
MeOH, 10:1). The reaction mixture was quenched with HCl (2 M, 0.2
mL) and diluted with H2O(10 mL), and the product extracted with
DCM (20 mL) over 16 h. The organic layer was dried over Na2SO4,
filtered, and concentrated on deactivated silica, and the product was
G
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purified by flash column chromatography [DCM/MeOH (30:1 to
10:1)], to yield 7 mg (66%) of 19 as a white precipitate, 66%.
19: Rf = 0.2 [DCM/MeOH (10:1)]; [α]2D1 +69 (c 0.1, i-PrOH); IR
(neat) 3356, 2922, 2852, 1660, 1632 cm−1; 1H NMR (600 MHz,
DMSO-d6) δ 13.25 (s, 1H), 7.92 (s, 1H), 6.73 (s, 1H), 6.08 (d, J = 5.2
Hz, 2H), 5.98 (s, 1H), 5.31 (s, 1H), 4.95 (s, 1H), 4.89 (d, J = 7.4 Hz,
1H), 4.29 (d, J = 2.1 Hz, 1H), 4.22 (dt, J = 8.5, 3.1 Hz, 1H), 3.89 (s,
1H), 3.64 (d, J = 8.6 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ
169.1, 152.4, 144.8, 133.3, 131.9, 127.8, 126.9, 105.6, 102.1, 95.5, 72.0,
71.7, 67.6, 53.2; HRMS (EI) calcd for C14H13O7N 307.0692; found
307.0691.
Methyl (1R,4R,5R,6S)-4,5,6-Trihydroxy-2-(7-methoxybenzo[d]-
[1,3]dioxol-5-yl)cyclohex-2-enylcarbamate (24): Allylic alcohol 23
(61 mg, 0.16 mmol) was dissolved in a mixture of DCM and THF
(1:2, 3 mL), and 6 M HCl (0.5 mL) was added dropwise at room
temperature. The reaction mixture was stirred for 15 min, quenched
with a saturated solution of NaHCO3 (5 mL), and extracted with
DCM (3 × 10 mL). The organic phase was dried over Na2SO4,
filtered, and concentrated under reduced pressure. The triol was
purified by column chromatography [DCM/MeOH (100:1 to 10:1)],
and compound 24 was isolated by precipitation from CHCl3 as a white
solid [52 mg (95%)].
column chromatography [EtOAc/hexanes (1:3 to 1:1)] to yield 31 mg
(48%) of triacetate 26 as a colorless oil.
26: Rf = 0.7 [EtOAc/hexanes (1:1)], [α]2D4 +7 (c 0.2, CHCl3); IR
(neat) 2956, 2926, 2856, 1750, 1669, 1239 cm−1; 1H NMR (600 MHz,
CDCl3) δ 6.78 (s, 1H), 6.06 (d, J = 1.1 Hz, 1H), 6.03 (d, J = 1.1 Hz,
1H), 5.94 (s, 1H), 5.90 (s, 1H), 5.80 (dd, J = 6.1, 3.4 Hz, 1H), 5.75−
5.72 (m, 1H), 5.08 (dd, J = 9.1, 1.8 Hz, 1H), 4.58 (dt, J = 9.0, 2.8 Hz,
1H), 4.06 (s, 3H), 2.13 (s, 3H), 2.11 (s, 3H), 2.09 (s, 3H); 13C NMR
(150 MHz, CDCl3) δ 170.6, 170.3, 169.8, 163.0, 152.5, 144.9, 139.6,
132.3, 132.1, 120.1, 114.7, 102.2, 99.5, 72.8, 68.1, 67.8, 61.2, 50.7, 21.0,
20.9 × 2; HRMS (EI) calcd for C21H21O10N 447.1165; found
447.1156.
(2R,3R,4S,4aR)-7-Hydroxy-6-oxo-2,3,4,4a,5,6-hexahydro-[1,3]-
dioxolo[4,5-j]phenanthridine-2,3,4-triyl triacetate (27): Phenantri-
done 26 (30 mg, 0.067 mmol) was dissolved in MeCN (3 mL), and a
solution of TMSCl in MeCN (0.5 M, 0.17 mL) and KI (12 mg, 0.072
mmol) were added. The reaction mixture was heated at 60 °C for 1 h,
cooled to 0 °C, and quenched with H2O (3 mL). The product was
extracted with EtOAc (3 × 5 mL), and the organic phase was dried
over Na2SO4, filtered, and concentrated under reduced pressure.
Purification by flash column chromatography [DCM/MeOH (gradient
100:1 to 10:1)] yielded 15 mg (50%) of phenol 27.
27: Rf = 0.3 [DCM/MeOH (20:1)]; [α]2D1 −28 (c 0.2, CHCl3); IR
(neat) 3308, 2923, 1749, 1675 cm−1; 1H NMR (600 MHz, DMSO-d6)
δ 13.19 (s, 1H), 8.91 (s, 1H), 6.94 (s, 1H), 6.14−6.09 (m, 3H), 5.88−
5.85 (m, 1H), 5.59−5.55 (m, 1H), 5.17 (dd, J = 9.4, 1.7 Hz, 1H), 4.61
(dt, J = 9.1, 2.8 Hz, 1H), 2.07 (s, 3H), 2.03−2.00 (m, 6H); 13C NMR
(150 MHz, DMSO-d6) δ 170.2, 170.1, 169.4, 169.1, 152.5, 144.8,
133.9, 130.5, 129.3, 121.6, 105.8, 102.3, 96.1, 71.5, 67.5, 67.4, 50.0,
21.0, 20.6, 20.5; HRMS (EI) calcd for C20H19O10N 433.1009; found
433.1003.
24: Rf = 0.4 [DCM/MeOH (10:1)]; mp = 184−185 °C (MeOH);
[α]2D3 −145 (c 0.3, MeCN); IR (neat) 3366, 2938, 2904, 2851, 1697,
1
1626 cm−1; H NMR (600 MHz, DMSO-d6) δ 7.01 (d, J = 9.8 Hz,
1H), 6.59 (d, J = 1.4 Hz, 1H), 6.56 (d, J = 1.5 Hz, 1H), 5.96 (s, 2H),
5.79−5.76 (m, 1H), 4.89 (d, J = 5.9 Hz, 1H), 4.73 (d, J = 4.4 Hz, 1H),
4.65 (d, J = 8.0 Hz, 1H), 4.63−4.58 (m, 1H), 4.14 (dt, J = 6.3, 2.9 Hz,
1H), 3.84 (t, J = 3.9 Hz, 1H), 3.79 (s, 3H), 3.63−3.57 (m, 1H), 3.45
(s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 156.9, 148.2, 142.7, 136.7,
134.1, 133.9, 128.8, 105.7, 101.1, 100.2, 72.8, 71.1, 67.7, 56.0, 52.3,
51.2; HRMS (EI) calcd for C16H19O8N 353.1111; found 353.1102.
(1S,2R,3R,6R)-5-(7-Methoxybenzo[d][1,3]dioxol-5-yl)-6-
(methoxycarbonylamino)cyclohex-4-ene-1,2,3-triyl triacetate (25):
Triol 24 (362 mg, 1.03 mmol) was dissolved in DCM (12 mL), and
the solution cooled to 0 °C. Et3N (570 μL) and Ac2O (390 μL) were
added dropwise. After addition of DMAP (cat., 4.2 mg), the reaction
flask was removed from the ice bath. After 10 min TLC (DCM/
MeOH, 10:1) showed full consumption of starting material. The
reaction mixture was quenched with H2O (15 mL), the organic layer
was separated, and the aqueous phase was washed with DCM (4 × 10
mL). The combined organic phases were washed with saturated NaCl
solution (5 mL), dried over Na2SO4, filtered, and concentrated. The
crude product was purified by flash column chromatography [EtOAc/
hexanes (gradient 1:2 to 1:1.5)] to yield 412 mg (84%) of triacetate 25
as a white foamy oil.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00218.
NMR data and LC-MS results (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
*Tel: +1-905-525-9140, ext. 27393. E-mail: jmcnult@
mcmaster.ca (J. McNulty).
25: Rf = 0.5 [EtOAc/hexanes (1:1)]; [α]2D2 −210 (c 1, CHCl3); IR
*Tel: +1-905-688-5550, ext. 44956. Fax: +1-905-984-4841. E-
mail: thudlicky@brocku.ca (T. Hudlicky).
1
(CHCl3) 3333, 2941, 2849, 1744, 1722 cm−1; H NMR (600 MHz,
CDCl3) δ 6.54 (s, 1H), 6.51 (s, 1H), 5.95 (dd, J = 7.0, 1.4 Hz, 2H),
5.74 (dd, J = 3.5, 2.0 Hz, 1H), 5.70 (dd, J = 6.4, 2.8 Hz, 1H), 5.63−
5.58 (m, 1H), 5.20 (dd, J = 8.5, 1.2 Hz, 1H), 5.12 (t, J = 9.0 Hz, 1H),
4.66 (d, J = 10.0 Hz, 1H), 3.87 (s, 3H), 3.57 (s, 3H), 2.15 (s, 3H), 2.05
(s, 3H), 2.02 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 170.6, 170.5,
170.1, 157.0, 148.9, 143.5, 140.2, 135.5, 132.0, 123.8, 106.2, 101.8,
101.1, 77.2, 71.8, 69.1, 68.0, 56.6, 52.5, 50.6, 21.0, 20.9 × 2; HRMS
(EI) calcd for C22H25O11N 479.1422; found 479.1427; anal. calcd for
C22H25O11N: C, 55.11; H, 5.26; found C, 55.15; H, 5.34.
(2R,3R,4S,4aR)-7-Methoxy-6-oxo-2,3,4,4a,5,6-hexahydro-[1,3]-
dioxolo[4,5-j]phenanthridine-2,3,4-triyl triacetate (26): Triacetate
25 (21 mg, 0.044 mmol) was dissolved in DCM (1 mL), and DMAP
(16 mg, 0.132 mmol, 3 equiv) was added. The reaction mixture was
cooled to 0 °C, and Tf2O (37 μL, 0.219 mmol, 5 equiv) was added
dropwise. The mixture was stirred for 18 h at 3 °C, poured into a
saturated solution of NaHCO3 (5 mL), and extracted with EtOAc (3 ×
10 mL). The organic layer was concentrated under reduced pressure.
The crude mixture was redissolved in THF (2 mL), 2 M HCl (0.2
mL) was added, and the mixture was stirred at room temperature for 3
h. The reaction mixture was poured into a saturated solution of
NaHCO3 (5 mL), extracted with EtOAc (3 × 10 mL), dried over
Na2SO4, filtered, and concentrated. The crude product was purified by
ORCID
James McNulty: 0000-0002-7123-7588
Tomas Hudlicky: 0000-0003-3239-6815
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the following agencies for financial support
of this work: Stanley Medical Research Institute (Grant No.
13R-002), Natural Sciences and Engineering Research Council
of Canada (NSERC) (Grant No. RGPIN-2018-03723), Canada
Research Chair Program, Canada Foundation for Innovation
(CFI), TDC Research, Inc., TDC Research Foundation, the
Ontario Partnership for Innovation and Commercialization
(OPIC), The Advanced Biomanufacturing Centre (Brock
University), and Growing Forward 2. We are thankful to H.
Endoma (Brock University) for her skillful assistance in the
fermentations. We thank R. Simionescu and Dr. L. Qui for their
help with NMR and mass spectrometry analyses. We would like
H
DOI: 10.1021/acs.jnatprod.8b00218
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
to thank Dr. L. Cvak from TEVA [Opava, Czech Republic] for
a generous gift of narciclasine and Galanthus sp. crude extracts.
REFERENCES
(1) Ceriotti, G. Nature 1967, 213, 595−596.
■
(2) Piozzi, F.; Fuganti, C.; Mondelli, R.; Ceriotti, G. Tetrahedron
1968, 24, 1119−1131.
(3) Dumont, P.; Ingrassia, L.; Rouzeau, S.; Ribaucour, F.; Thomas, S.;
Roland, I.; Darro, F.; Lefrancy, F.; Kiss, R. Neoplasia 2007, 9, 766−
776.
(4) Van Goietsenoven, G.; Hutton, J.; Becker, J. P.; Lallemand, B.;
Robert, F.; Lefranc, F.; Pirker, Ch.; Vandenbussche, G.; Van
Antwerpen, P.; Evidente, A.; Berger, W.; Prevost, M.; Pelletier, J.;
Kiss, R. FASEB J. 2010, 24, 4575−4584.
(5) (a) Lubbe, A.; Gude, H.; Verpoorte, R.; Choi, Y. H.
Phytochemistry 2013, 88, 43−53. (b) Pettit, G. R.; Melody, N.;
Herald, D. L. J. Org. Chem. 2001, 66, 2583−2587.
(6) Pettit, G. R.; Gaddamidi, V.; Cragg, G. M. J. Nat. Prod. 1984, 47,
1018−1020.
(7) Rigby, J. H.; Mateo, M. E. J. Am. Chem. Soc. 1997, 119, 12655−
12656.
(8) Keck, G. E.; Wager, T. T.; Rodriquez, J. F. D. J. Am. Chem. Soc.
1999, 121, 5176−5190.
(9) Gonzalez, D.; Martinot, T.; Hudlicky, T. Tetrahedron Lett. 1999,
40, 3077−3080.
(10) Reviews: (a) Martin, S. F. In The Alkaloids, Vol. 30; Brossi, A.,
Ed.; Academic Press: New York, 1987; pp 251−376. (b) Hoshino, O.
In The Alkaloids, Vol. 51; Cordell, G. A., Ed.; Academic Press: New
York, 1998; pp 323−424. (c) Rinner, U.; Hudlicky, T. Synlett 2005,
365−387. (d) Chapleur, Y.; Chrtien, F.; Ahmed, S. I.; Khaldi, M. Curr.
Org. Synth. 2006, 3, 341−378. (e) Kornienko, A.; Evidente, A. Chem.
Rev. 2008, 108, 1982−2014. (f) Jin, Z. Nat. Prod. Rep. 2009, 26, 363−
381. (g) Ghavre, M.; Froese, J.; Pour, M.; Hudlicky, T. Angew. Chem.,
Int. Ed. 2016, 55, 5642−5691. (h) Furst, R. Planta Med. 2016, 82,
̈
1389−1394. (i) Zepeda-Velazquez, C.; McNulty, J. Studies in Natural
Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier, 2017; Vol. 53, pp
85−108.
(11) Total syntheses since 1999: (a) Rigby, J. H.; Maharoof, U. S. M.;
Mateo, M. E. J. Am. Chem. Soc. 2000, 122, 6624−6628. (b) Hudlicky,
T.; Rinner, U.; Gonzalez, D.; Akgun, H.; Schilling, S.; Siengalewicz, P.;
Martinot, T. A.; Pettit, G. R. J. Org. Chem. 2002, 67, 8726−8743.
(c) Elango, S.; Yan, T.-H. J. Org. Chem. 2002, 67, 6954−6959.
(d) Matveenko, M.; Banwell, M. G.; Willis, A. C. Tetrahedron 2008,
64, 4817−4826. (e) Maji, B.; Yamamoto, H. J. Am. Chem. Soc. 2015,
137, 15957−15963. (f) Southgate, E. H.; Holycross, D. R.; Sarlah, D.
Angew. Chem., Int. Ed. 2017, 56, 15049−15052.
(12) Frahm, A. W.; Ali, A. A.; Ramadan, M. A. Magn. Reson. Chem.
1985, 23, 804−808.
(13) Bastida, J.; Viladomat, F.; Llabres, J.; Codina, C.; Feliz, M.;
Rubiralta, M. Phytochemistry 1987, 26, 1519−1524.
(14) Mondon, A.; Krohn, K. Chem. Ber. 1975, 108, 445−463.
(15) Nair, J. J.; Aremu, A. O.; Staden, J. V. J. Ethnopharmacol. 2011,
137, 1102−1106.
(16) Lapinskaite, R.; Ghavre, M.; Rintelmann, C. L.; Bedard, K.; Dela
Paz, H. E.; Hudlicky, T. Synlett 2017, 28, 2896−2900.
(17) Banwell, M. G.; Bissett, B.; Cowden, C. J.; Hockless, D. C. R.;
Holman, J. W.; Read, R. W.; Wu, A. W. J. Chem. Soc., Chem. Commun.
1995, 2551−2553.
(18) (a) Mondon, A.; Krohn, K. Tetrahedron Lett. 1970, 24, 2123−
2126. (b) Numata, A.; Takemura, T.; Ohbayashi, H.; Katsuno, T.;
Yamamoto, K.; Sato, K.; Kobayashi, S. Chem. Pharm. Bull. 1983, 31
(6), 2146−2149.
(19) Mondon, A.; Krohn, K. Tetrahedron Lett. 1972, 21, 2085−2088.
(20) Bohno, M.; Sugie, K.; Imase, H.; Yusof, Y. B.; Oishi, T.; Chida,
N. Tetrahedron 2007, 63, 6977−6989.
I
DOI: 10.1021/acs.jnatprod.8b00218
J. Nat. Prod. XXXX, XXX, XXX−XXX