Biological evaluation of ent-narciclasine, ent-lycoricidine, and certain enantiomerically-related congeners

Article abstract of DOI:10.1002/cbdv.200800319

The non-natural enantiomeric forms of narciclasine and lycoricidine ((-)-1 and (-)-2, respectively), as well as congeners 3-6 are available through chemoenzymatic synthesis. Accordingly, they have now been tested for their cytotoxic effects in a 13-member

Full text of DOI:10.1002/cbdv.200800319

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
685
Biological Evaluation of ent-Narciclasine, ent-Lycoricidine, and Certain
Enantiomerically-Related Congeners
by Maria Matveenkoa), Martin G. Banwell*a), Max Joffeb), Soosan Wanb), and Emmanuelle Fantinob)
a) Research School of Chemistry, Institute of Advanced Studies, The Australian National University,
Canberra, ACT 0200, Australia
(phone: þ61-2-6125-8202; fax: þ61-2-6125-8114; e-mail: mgb@rsc.anu.edu.au)
b) Cytopia Pty Ltd, 576 Swan Street, Richmond, VIC 3121, Australia
The non-natural enantiomeric forms of narciclasine and lycoricidine ((ꢀ)-1 and (ꢀ)-2, respectively),
as well as congeners 3–6 are available through chemoenzymatic synthesis. Accordingly, they have now
been tested for their cytotoxic effects in a 13-member human cancer cell-line panel and found to be only
weakly active. In contrast, an authentic sample of the natural enantiomeric form of narciclasine ((þ)-1)
was found to be highly active in the same screens.
1. Introduction. – While it has been estimated that only 15% of the worldꢀs plants
have been screened for their therapeutic potential [1], the daffodil (a.k.a. narcissus) has
been the subject of extensive study and proven to be a rich source of biologically active
compounds1). In particular, and partly as a result of ethnobotanical studies [2], the
isocarbostyril-type natural product narciclasine ((þ)-1) was isolated from a variety of
Narcissus bulbs [3], as well as Lycoris radiata [4], and shown to possess potent
antimitotic properties [3]. Its structure was established by single-crystal X-ray analysis
[5]. The 7-deoxy analogue (þ)-2, also known as lycoricidine, was isolated at around the
same time from L. radiata [4] and L. sanguinea [6], as well as from Pancratium littorale
[7] and Haemanthus kalbreyeri [8]. Like congener (þ)-1, lycoricidine ((þ)-2) exhibits
nanomolar growth-inhibitory potencies against a range of human cancer cell lines [9].
As a consequence, an extraordinary effort has been devoted to the development of
syntheses of compounds (þ)-1 and (þ)-2 as well as a range of congeners [2]2).
1
)
)
For an excellent review of this topic, see [2] and refs. cit. therein.
For relevant reviews, see [10]. For recent synthetic efforts in this area that have not been covered by
the aforementioned reviews, see [11].
2
ꢁ 2009 Verlag Helvetica Chimica Acta AG, Zꢂrich

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As part of our continuing interest in developing new approaches to this fascinating
class of compounds3), we have recently reported efficient, chemoenzymatic total
syntheses of the non-natural enantiomeric forms of narciclasine [13] and lycoricidine
[14] ((ꢀ)-1 and (ꢀ)-2, resp.) as well as congeners 3–6 [14]. These vary in terms of the
nature and degree of substitution on the A- and C-rings as well as in configuration of
certain of the substituents. A constant feature within this series is that each member
possesses the non-natural configuration at C(2) and C(4a). Such work provided the
first total synthesis of (ꢀ)-narciclasine ((ꢀ)-1) and only the second synthesis of (ꢀ)-
lycoricidine ((ꢀ)-2)4). Given that nothing has been reported about the cyctotoxic
profiles of the non-natural enantiomeric forms of narciclasine and lycoricidine, we have
subjected compounds (ꢀ)-1, (ꢀ)-2, and 3–6, as well as (þ)-1, to relevant screening
regimes and now report the outcomes of such studies herein.
2. Results and Discussion. – For the sake of completeness, and before detailing the
outcomes of the screening of the abovementioned compounds, it is appropriate to
provide a short commentary on the synthetic approach used in obtaining compounds
(ꢀ)-1 and (ꢀ)-2 [13][14]. This is presented immediately below and is followed by a
brief section detailing the physical and spectroscopic similarities between compound
(ꢀ)-1 and an authentic sample of naturally occurring narciclasine, viz. (þ)-1.
2.1. Synthetic Studies. A convergent strategy was employed in obtaining targets (ꢀ)-
1 and (ꢀ)-2 (Scheme) [13][14]. Specifically, the progenitor, 8, to the C-ring of each of
these compounds was prepared by elaboration of the enantiomerically pure and
enzymatically-derived cis-1,2-dihydrocatechol 75) using relatively conventional chem-
ical transformations and an Overman rearrangement process6) to introduce the pivotal
3
)
)
)
)
For a key earlier publication arising from our efforts in this area, see [12].
The first synthesis of (ꢀ)-lycoricidine was reported by Keck et al. [15].
4
5
6
For reviews on the production and general synthetic utility of these types of metabolites, see [16].
For a discussion of the application of this type of process in this context, see [17].

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
687
amino group. Subjection of the amino-conduritol 8 to Suzuki–Miyaura cross-coupling
[18] with either the aryl boronate ester 9 [13] or its deoxy counterpart 10 [14] then gave
the fully MOM-protected derivative of (ꢀ)-narciclasine ((ꢀ)-1) and (ꢀ)-lycoricidine
((ꢀ)-2) in 63 and 83% yield, respectively. The precise order of events involved in these
conversions has not been confirmed, but it seems likely that the Suzuki–Miyaura cross-
coupling process precedes a lactamization reaction leading to the formation of the B-
ring of compounds 11 and 12. Exhaustive removal of the MOM groups associated with
each of isocarbostyrils 11 and 12 could be accomplished using TMS-Br to give the
corresponding targets (ꢀ)-1 and (ꢀ)-2 in 48 and 62% yield, respectively.
Scheme
The structures of compounds (ꢀ)-1 and (ꢀ)-2 were initially established through the
acquisition of the usual sets of spectral data and comparisons of these with those
reported in the literature7). In addition, the triacetate derivative, 3, of (ꢀ)-lycoricidine
was prepared by established procedures and then subjected to single-crystal X-ray
analysis [14].
After the publication of the details of our synthesis of (ꢀ)-narciclasine [13], we
were kindly provided with an authentic sample of its natural enantiomer by Dr. Hank
Fales of the National Institutes of Health in the USA. Accordingly, we have been able to
make, for the first time, direct spectroscopic comparisons of the two samples. A
summary of such comparisons is provided in the following section.
2.2. Spectroscopic Characterization and Comparison of (þ)- and (ꢀ)-Narciclasine.
The 13C- and 1H-NMR spectra of naturally derived (þ)-narciclasine ((þ)-1) and
synthetically derived (ꢀ)-narciclasine ((ꢀ)-1) were recorded under essentially identical
conditions in (D6)DMSO, and the outcomes of such experiments are presented in
Table 1. As is readily discerned from inspection of the tabulated chemical shifts, the two
sets of data are identical within the limits of error of the NMR experiments.
7
)
In the case of (ꢀ)-narciclasine ((ꢀ)-1), the initial comparisons were with those data reported for
(þ)-narciclasine ((þ)-1); see [15] and [19].

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Table 1. Comparison of the 13C- and 1H-NMR Data Recorded in (D6)DMSO for Naturally Derived (þ)-
Narciclasine ((þ)-1) and Synthetically Derived (ꢀ)-Narciclasine ((ꢀ)-1)a). d in ppm, J in Hz.
d(C)b)
d(H)c)
(þ)-1
(ꢀ)-1
(þ)-1
(ꢀ)-1
169.0
152.4
144.8
133.5
132.2
129.3
124.8
105.6
102.1
95.9
169.0
152.4
144.8
133.5
132.2
129.3
124.8
105.6
102.2
95.9
13.26 (s, 1 H)
7.91 (s, 1 H)
6.86 (s, 1 H)
13.26 (s, 1 H)
7.92 (s, 1 H)
6.86 (s, 1 H)
6.18–6.13 (m, 1 H)
6.10–6.07 (m, 2 H)
5.24–5.15 (m, 2 OH)
5.06–5.00 (m, OH)
4.19 (d, J¼8.0, 1 H)
4.04–3.98 (m, 1 H)
3.82–3.76 (m, 1 H)
3.72–3.67 (m, 1 H)
6.17–6.14 (m, 1 H)
6.10–6.07 (m, 2 H)
5.23–5.17 (m, 2 OH)
5.04 (d, J¼3.0, OH)
4.19 (d, J¼8.0, 1 H)
4.04–3.98 (m, 1 H)
3.82–3.76 (m, 1 H)
3.72–3.68 (m, 1 H)
72.4
72.4
69.2
69.2
68.8
68.8
52.9
52.9
a) The central peak (d 39.5) of the signals due to (D6)DMSO was used as the internal reference for
13C-NMR spectra, while the signal (d 2.50) due to residual protio-DMSO was used as the internal
reference for 1H-NMR spectra. b) At 75 MHz. c) At 500 MHz.
The ESI mass spectra of (þ)- and (ꢀ)-narciclasine were each recorded in positive-
ionization mode, and they proved to be superimposable as did the corresponding IR
spectra. In keeping with expectations, the specific rotation of the naturally occurring
enantiomer ([a]D þ116 (c¼0.19, MeOH)) was of the same magnitude but opposite
sign to that of ent-narciclasine ([a]D ꢀ116 (c¼0.21, MeOH)). The enantiomeric
relationship between the two compounds was reinforced through the acquisition of the
CD spectra for the two samples. These are shown together in the Figure which reveals
their near-complementary relationship. The slight distortion in the CD spectrum of
(ꢀ)-narciclasine ((ꢀ)-1) observed at ca. 250 nm is attributed to the presence of trace
amounts of an unidentified impurity displaying an intense absorption at this wave-
length.
The naturally occurring (þ)-enantiomer had no sharp melting point but began to
color intensely at 2058 and decomposed slowly above that temperature. Synthetic (ꢀ)-
narciclasine, on the other hand, colored intensely from 2148 and decomposed slowly
above this temperature. It changed from white to pale-yellow at 1808. Such a change
was not observed for the naturally occurring sample as it was a tan-colored material to
begin with.
2.3. Biological Screening of Compounds (þ)-1, (ꢀ)-1, (ꢀ)-2, and 3–6 against
Certain Human Cancer Cell Lines. Each of the synthetically derived compounds (ꢀ)-1,
(ꢀ)-2, and 3–6 was screened, at 2 mm concentrations, against a panel of 13 human
cancer cell lines as listed in Table 2 (details of the protocols used are presented in the
Exper. Part). An authentic sample of (þ)-narciclasine ((þ)-1) was also tested against
the same panel. As a consequence, it became clear that the synthetic compounds, which
possess the non-natural configuration at C(2) and C(4a), are all one or two orders of

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
689
Figure. Comparison of the CD spectral data of naturally derived (þ)-narciclasine ((þ)-1; red trace) and
synthetically derived (ꢀ)-narciclasine ((ꢀ)-1; blue trace). Spectra were recorded on 1 mg samples of (þ)-
or (ꢀ)-1 dissolved in 2.0 ml of HPLC-grade EtOH using an Applied Photophysic Chirascan instrument
operating at 188.
Table 2. Inhibition of Cell Growth [%]a) Caused by 2 mm Samples of Compounds (þ)-1, (ꢀ)-1, (ꢀ)-2,
and 3–6 in Cytotoxicity Screens Using 13 Human Cancer Cell Linesb)
Cell linec)
(þ)-1
(ꢀ)-1
(ꢀ)-2
3
4
5
6
A-375
A-431
HCT-15
K-562
MDA MB-231
A-549
103
100
105
102
118
105
107
135
151
116
155
135
104
2
1
1
ꢀ2
3
2
12
ꢀ4
11
2
4
0
5
5
6
3
5
6
ꢀ1
14
5
6
6
9
7
12
6
ꢀ6
ꢀ7
18
17
14
1
5
ꢀ15
ꢀ3
3
8
ꢀ2
ꢀ1
ꢀ21
4
12
15
ꢀ6
ꢀ2
ꢀ21
8
ꢀ6
PA-1
ꢀ4
ꢀ5
ꢀ2
HT-1376
HTB-178
MES-SA
HepG2
MCF-7
DU-145
ꢀ10
14
ꢀ10
7
ꢀ14
15
1
6
3
13
17
12
ꢀ20
16
8
5
ꢀ10
5
15
10
ꢀ9
8
ꢀ8
15
ꢀ3
ꢀ20
a) Percentage-inhibition values were calculated by comparison with vehicle-treated cells. The numbers
shown are as calculated. Since cell growth has some inherent variability, the values shown are
approximate measurements of inhibition of cellular proliferation, and those identified as >100% and
<0% simply represent complete inhibition and no inhibition of cell growth, respectively. b) DMSO was
used as solvent in these screens. c) See Exper. Part for details.

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magnitude less cytotoxic than the parent compound and can thus be regarded as
essentially inactive materials. Such results seem consistent with the observation that
ent-7-deoxypancratistatin (an ꢃalkene hydratedꢀ form of (ꢀ)-lycoricidine) exhibits
cytotoxic potencies that are tenfold lower than those of the corresponding natural
enantiomer [20].
Conclusions. – The differences in the cytotoxic properties of naturally derived (þ)-
narciclasine ((þ)-1) and its synthetically derived optical antipode (ꢀ)-1 are particularly
noteworthy. These results are consistent with earlier findings [2][20] that high levels of
carcinostatic activity are only observed in compounds possessing the full structural
framework of the lycorine alkaloids and incorporating the natural configurational
relationships of functional groups in the C-ring.
We thank the Australian Research Council and The Institute of Advanced Studies at the Australian
National University for generous financial support. Dr. Hank Fales (National Institutes of Health,
Bethesda, Maryland USA) is warmly acknowledged for providing an authentic sample of (þ)-
narciclasine ((þ)-1). We are also grateful to Mr. Paul Gugger (Research School of Chemistry, ANU) for
assisting with the acquisition of the CD spectra of compounds (þ)-1 and (ꢀ)-1. Dr. Chris Burns (Cytopia
Pty Ltd.) is thanked for facilitating the biological testing described herein.
Experimental Part
General. M.p.: Reichert hot-stage microscope apparatus; uncorrected. Optical rotations: at 188 with
a Perkin-Elmer 241 polarimeter at the sodium-D line (589 nm) and the concentrations (c) (g/100 ml)
indicated using spectroscopic grade solvents; measurements carried out in a cell with a path length (l) of
1 dm. CD Spectra: Applied Photophysics Spectroscan instrument. IR Spectra (˜nmax): Perkin-Elmer 1800
1
Series FTIR Spectrometer; as KBr disks. H- and 13C-NMR spectra: at 188 in (D6)DMSO on a Varian
Inova 500 or 300 spectrometer operating at 500 MHz for 1H and 75 MHz for 13C nuclei. Low- and high-
resolution (LR and HR, resp.) electrospray (ESI) MS: VG Quattro II triple quadrupole MS instrument
operating in positive-ionization mode.
Sources of Compounds and Spectral Properties. Samples of compounds (ꢀ)-1, (ꢀ)-2, and 3–6
required for biological testing were prepared according to previously described procedures [13][14].
An authentic sample of (þ)-narciclasine ((þ)-1) (308.0768 ([MþH]þ , C14H13NO7þ ; calc. 308.0770)
was kindly provided by Dr. Hank Fales of the National Institutes of Health, Bethesda, Maryland, USA.
This material was subjected, as obtained, to 1H- and 13C-NMR, as well as CD spectral analyses, the results
of which are presented in Table 1 and the Figure, resp. IR (KBr): 3437, 3323, 1674, 1652, 1640, 1477, 1437,
1408, 1377, 1281, 1229, 1097, 1088, 1034, 1019, 803. ESI-MS: 330 (50, [MþNa]þ ), 308 (100, [MþH]þ ).
Cytotoxicity Testing. Stock solns. of the test compounds were prepared at 20 mm in neat DMSO and
diluted in tissue culture medium to a concentration of 20 mm. Compounds were screened against ATCC
cell lines at a final concentration of 2 mm in flat-bottom TC plates. The cells were incubated for 72 h at 378
in a 5% CO2 incubator. Inhibition of cell proliferation by the test compounds was determined by
measuring conversion of Alamar-Blue fluorescent dye at excitation/emission 544/590 nm with a BMG
FluoStar plate reader.
The ATCC cell lines used were: A-375 (CRL-1619), human malignant melanoma; A-431 (CRL-
1555), human epidermoid carcinoma; HCT-15 (CCL-225), human colon adenocarcinoma; K-562 (CCL-
243), human chronic myelogenous leukaemia; MDA-MB231 (HTB-26), human breast adenocarcinoma;
A-549 (CCL-185), human lung carcinoma; PA-1 (CRL-1572), human ovarian teratocarcinoma; HT-1376
(CRL-1472), human bladder carcinoma; HTB-178 (NCI-H82), human small-cell lung carcinoma; MES-
SA (CRL-1976), human uterine sarcoma; HepG2/C3A (CRL-10741), human hepatoblastoma; MCF-7
(HTB-22), human breast adenocarcinoma; and DU-145, human prostate carcinoma.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Received October 29, 2008