Human cytochrome P450 liability studies of trans-dihydronarciclasine: A readily available, potent, and selective cancer cell growth inhibitor

Article abstract of DOI:10.1021/np100657w

The cytochrome P45O activities of the naturally occurring Amaryllidaceae alkaloid narciclasine (3), isolated from Narcissus pseudonarcissus, and synthetic derivative trans-dihydronarciclasine (5) are reported. While narciclasine was found to possess potent inhibitory activity to human CYP3A4, its dihydro analogue was inactive. This study revealed that the C1-C10b double bond is required for inhibition of this crucial metabolizing enzyme. Compound 5 also demonstrated no inhibition of the related human cytochromes CYP19 and CYP1A1. This study elevates the status of trans-dihydronarciclasine (5) as a highly privileged, readily available molecule, with potent and selective anticancer activity.

Full text of DOI:10.1021/np100657w

106
J. Nat. Prod. 2011, 74, 106–108
Human Cytochrome P450 Liability Studies of trans-Dihydronarciclasine: A Readily Available,
Potent, and Selective Cancer Cell Growth Inhibitor
James McNulty,*,† Amol Thorat,† Nesrin Vurgun,† Jerald J. Nair,† Emilija Makaji,‡ Denis J. Crankshaw,‡ Alison C. Holloway,‡
and Siyaram Pandey§
Department of Chemistry and Chemical Biology, McMaster UniVersity, 1280 Main Street West, Hamilton, Ontario, Canada, L8S 4M1,
Department of Obstetrics and Gynecology, McMaster UniVersity, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5, and
Department of Chemistry and Biochemistry, UniVersity of Windsor, Windsor, Ontario, Canada, N9B 3P4
ReceiVed September 21, 2010
The cytochrome P45O activities of the naturally occurring Amaryllidaceae alkaloid narciclasine (3), isolated from
Narcissus pseudonarcissus, and synthetic derivative trans-dihydronarciclasine (5) are reported. While narciclasine was
found to possess potent inhibitory activity to human CYP3A4, its dihydro analogue was inactive. This study revealed
that the C1-C10b double bond is required for inhibition of this crucial metabolizing enzyme. Compound 5 also
demonstrated no inhibition of the related human cytochromes CYP19 and CYP1A1. This study elevates the status of
trans-dihydronarciclasine (5) as a highly privileged, readily available molecule, with potent and selective anticancer
activity.
The Amaryllidaceae alkaloids have attracted significant attention
by virtue of their diverse structures1 coupled with important
biological activities that have been documented for members of
this family. The lycorane derivatives pancratistatin2 (1) and
narciclasine (3), in particular, have attracted considerable interest
over the last two decades due to the potent, selective anticancer
activity demonstrated, as well as their unknown mechanism of
action.2d,3 Recent studies showed that pancratistatin induces apo-
ptosis, or programmed cell death, selectively in cancer cells, with
minimal effect on normal cells, and that the mitochondria in
cancerous cells are a site of action.3,4b Pancratistatin was shown to
have a greater specificity than etoposide (VP-16) or paclitaxel in
selectively inducing apoptosis. Other recent studies have shown
that narciclasine disrupts organization of the actin skeleton in cancer
cells at low (30-90 nM) concentrations.3e Narciclasine (3) has also
been shown to increase survival in preclinical models of human
glioblastoma multiforme and clearly has much potential.3f
(3) over the past few years.3e,f,7,9 Narciclasine along with pancrat-
istatin and the naturally occurring derivatives 7-deoxypancratistatin
(2), 7-deoxynarciclasine (4), trans-dihydronarciclasine (5) (also
known as 1-deoxypancratistatin), and 7-deoxy-trans-dihydronarci-
clasine (6) all demonstrate potent anticancer activity. These
derivatives share the same profile and hence elusive mechanism of
action as determined through COMPARE pattern-recognition
analysis.2d While trans-dihydronarciclasine (5) has potent anticancer
activity,2d,8 cis-dihydronarciclasine (10b epimer of 5) was inactive,2d
demonstrating the well-known fine subtleties of this anticancer
pharmacophore. Narciclasine (3) is available in relatively large
quantities from many of the Amaryllidaceae, including common
varieties of daffodil (Narcissus pseudonarcissus).7c The synthesis
and biological assessment of a large number of narciclasine
derivatives, including water-soluble pro-drugs, was recently reported
by Kiss and co-workers3e and further semisynthetic interconversions
by Pettit et al.8 The third issue facing the development of a viable
clinical derivative in the series is the determination of their potential
toxicity, particularly their liability in terms of interaction with human
cytochrome P450s. Surprisingly, this issue has been overlooked,
and our group recently documented the selective interaction of
certain of these alkaloids with human cytochromes, in particular
CYP3A4.9 The CYP3A subfamily comprises about 30% of the total
liver cytochrome P450 enzyme pool in humans, and the isoenzyme
CYP3A4 accounts for approximately 60% of drugs metabolized.10c
Many drug development programs have failed or are dose-
compromised due to interactions with this most predominant
enzyme.10 Inhibition of CYP3A4 can lead to drug accumulation,
contribute significantly to negative drug-drug interactions, and may
lead to hepatic toxicity. In our recent study,9 narciclasine (3) was
shown to be a potent inhibitor of human CYP3A4 (Ki ) 0.63 µM),
raising serious concerns for the future development of a narciclasine
derivative. This study also revealed considerable selectivity, most
importantly showing that pancratistatin was inactive to CYP3A4.9
These results immediately draw attention to the double bond in
narciclasine as a likely site of interaction with CYP3A4. In the
present work, we report the isolation and conversion of natural
narciclasine (3) to the known anticancer derivative trans-dihydr-
onarciclasine (5) (1-deoxypancratistatin)2d,11 and demonstrate that
this derivative, like native pancratistatin, exhibits no inhibition of
CYP3A4 and other human cytochrome P450s.
Three major issues have been of concern in the development of
a clinically relevant schedule involving pancratistatin or a derivative.
The first issue is its low aqueous solubility (53 µg/mL), a problem
that has been tackled through the synthesis of phosphate derivatives4
including conversion of natural pancratistatin to the biologically
active 7-phosphate pro-drug.4b A second issue is the low natural
abundance of pancratistatin2a,b and hence access to a reliable supply.
Two solutions to this problem are the semisynthetic conversion of
the readily available derivative narciclasine (3) to pancratistatin (1)
(10 steps, 3.6% overall yield)5 or total synthesis.6 These two issues
have served to refocus attention on the relatively abundant alkaloid
* To whom correspondence should be addressed. E-mail: jmcnult@
mcmaster.ca.
† Department of Chemistry and Chemical Biology, McMaster University.
‡ Department of Obstetrics and Gynecology, McMaster University.
§ University of Windsor.
10.1021/np100657w  2011 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/24/2010

Notes
Journal of Natural Products, 2011, Vol. 74, No. 1 107
Scheme 1. Synthesis of cis- and trans-Dihydronarciclasine
correlation of the structure with its NMR data proved difficult. Since
isomerization of the olefinic double bond is known to occur in this
series under hydrogenation conditions,14a other isomers are possible.
We were able to crystallize a sample of compound (7) and
characterize the molecule via single-crystal X-ray structural analysis
for the first time, confirming the structure and absolute configuration
as shown (Figure 1).
The interaction of trans-dihydronarciclasine (5) with human
CYP3A4 was investigated under standard conditions, via kinetic
monitoring of the conversion of 7-benzyloxyquinoline to 7-hy-
droxyquinoline by fluorometric measurement of emission at 538
Results and Discussion
Although trans-dihydronarciclasine (5) is a natural product,2d it
is not readily available, and we elected to pursue a semisynthetic
approach. Narciclasine (3) was isolated from freshly harvested bulbs
of N. pseudonarcissus according to a literature protocol.12
The tetraacetate derivative (8) was prepared under standard
acetylation conditions as outlined in Scheme 1. Stereoselective
hydrogenation of the double bond proved capricious and was highly
dependent on the solvent employed. Pettit and co-workers studied
this hydrogenation reaction (1 atm H2, 25 °C) in a variety of solvents
and found that the highest stereoselectivity favoring the trans-
dihydro diastereomer (9) was obtained in a 1:1 ratio of EtOH/
CH2Cl2 (trans:cis 65:25).8 The diastereomer ratio also varied
considerably upon the nature of the Pd catalyst employed. In our
hands, initial hydrogenation of (8) in EtOH/CH2Cl2, 1:1, over 10%
Pd/C (4 h, rt) favored formation of the cis-diastereomer (10), which
was isolated in 60% yield. In this reaction, hydrogen was allowed
to bubble through the reaction medium at room temperature, which,
it was subsequently realized, resulted in partial loss of volatile
CH2Cl2, altering the facial selectivity of the hydrogenation. In
separate experiments, the catalyst was first purged with hydrogen
in the presence of the tetraacetate (8), followed by addition of
solvent (EtOH/CH2Cl2, 1:1). This protocol resulted in formation
of the trans-dihydro diastereomer (9) (isolated in 50% yield),
confirming the important role of CH2Cl2 for selective hydrogenation.
Removal of the acetate protecting groups independently from (9)
and (10) then provided a sample of trans-dihydronarciclasine (5),
identical to that reported, and cis-dihydronarciclasine (7).
Although cis-dihydronarciclasine has been reported several times
in the literature,2d,8,14a no X-ray data are available, and the direct
Figure 1. X-ray structure of cis-dihydronarciclasine 7.

108 Journal of Natural Products, 2011, Vol. 74, No. 1
Notes
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nm after excitation at 410 nm, using ketoconazole as a positive
control. While 3 proved to be a potent inhibitor of CYP3A4 (Ki )
0.63 µM), 5 was found to be inactive, similar to the data described
for pancratistatin.9 Dihydronarciclasine (5) also proved inactive to
the related human cytochromes P450s CYP1A1 and CYP19 at 10
µM. This study confirms the critical role of the C1-C10b double
bond in derivatives such as narciclasine for CYP3A4 interaction.
These P450 liability studies also now confer a privileged status to
trans-dihydronarciclasine (5),2d a compound that has attracted a
recent flurry of synthetic activity,15 toward the development of a
readily available, potent, selective anticancer agent.
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Acknowledgment. Financial support of this work by the Lotte and
John Hecht Memorial Foundation, NSERC, and McMaster University
is gratefully acknowledged. We thank Dr. D. Hughes for high-field
NMR and Dr. J. Britten for the X-ray structural elucidation.
Supporting Information Available: Isolation of native narciclasine,
synthetic procedures, 1H and 13C NMR spectra of all compounds, and
crystallographic data for compound 7 (tables of selected bond lengths
and angles) are available free of charge via the Internet at http://
pubs.acs.org. Full details have been deposited at the Cambridge
Crystallographic Data Centre under deposition number CCDC 786216
(www.ccdc.cam.ac.uk).
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NP100657W