Synthesis and biological activity of 10-aza-narciclasine

Article abstract of DOI:10.1002/adsc.201400993

The first active aza analogue of narciclasine was synthesized from a pentasubstituted derivative of nicotinic acid. The key features of the synthesis include a halogen dance of bromopyridine and an intramolecular Heck reaction with a conduramine derived chemoenzymatically from bromobenzene. 10-Aza-narciclasine was found to have reasonable activity against several cancer cell lines.

Full text of DOI:10.1002/adsc.201400993

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DOI: 10.1002/adsc.201400993
Synthesis and Biological Activity of 10-Aza-narciclasine
Sergey Vshyvenko,a Zemane W’Giorgis,a Audrey Weber,a Natalia Neverova,b
Bradley Hedberg,b and Tomas Hudlickya,*
a
Department of Chemistry and Centre for Biotechnology, Brock University, St. Catharines, ON L2S 3 A1, Canada
E-mail: thudlicky@brocku.ca
The Centre for Drug Research and Development 2405 Wesbrook Mall, Fourth Floor Vancouver, BC V6T 1Z3
b
Received: October 20, 2014; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201400993.
Abstract: The first active aza analogue of narcicla-
sine was synthesized from a pentasubstituted deriv-
ative of nicotinic acid. The key features of the syn-
thesis include a halogen dance of bromopyridine
and an intramolecular Heck reaction with a condur-
amine derived chemoenzymatically from bromo-
benzene. 10-Aza-narciclasine was found to have
reasonable activity against several cancer cell lines.
C-1 benzoate of pancratistatin, prepared and evaluat-
ed by Pettit.[4] From the collaborations of our group
with Pettit,[3g,j,k,m] and from the work of McNulty,[3c,e,l,p]
Chapleur,[3a] Fessner,[3i] Kornienko,[3o,q,r] Alonso,[3v,w]
Banwell,[3b] Pandey,[3h] DeShong,[3n] and Kim,[3t] it
became evident that very few variations may be made
either in stereochemistry or in the functionality on
the periphery of these compounds.
The relatively unexplored space for alterations of
the structure in Amaryllidaceae constituents proved
to be the aromatic nucleus as well as the C-1 hydroxyl.
We have reported the synthesis and biological activity
of C-1 homologues of pancratistatin and 7-deoxypan-
cratisatin and found that the C-1 benzoylmethyl de-
rivative was more potent than narciclasine.[3s,u] C-
Keywords: alkaloids; antitumor agents; asymmetric
synthesis; drug design; heterocycles
Narciclasine (1), a member of the Amaryllidaceae 1 amino derivatives have also been reported and
family of alkaloids, is a highly cytotoxic compound found active.[5] Turning to the investigating of changes
with nanomolar activity against a variety of cancer in the aromatic core of narciclasine, we prepared the
cell lines.[1] Along with other Amaryllidaceae conge- truncated version 2, lacking the methylene dioxole
ners such as pancratistatin, lycoricidine, and 7-deoxy moiety, Figure 1.[6] That this compound as well as its
pancratistatin, it attracted the attention of organic N-oxide were inactive, confirmed that the 7-hydroxy
chemists as an attractive target for total synthesis.[2] functionality is essential for maintaining activity. We
The initial studies of the cytotoxic activities of these therefore turned to the synthesis of 10-aza-derivative
natural products were hampered not only by the lack 3, which would maintain all other functionality and
of availability but also by the low solubility of the features of narciclasine (1). Herein we report the syn-
compounds. For these reasons the attention of the thesis of 3 and its biological evaluation.
chemical community shifted from total synthesis of
these alkaloids to the preparation of unnatural and/or
truncated analogues. Many such derivatives have
been prepared and tested by our group and others.[3]
One of the most potent unnatural derivatives was the
Figure 1. Narciclasine and its aza-analogues.
Scheme 1.
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Sergey Vshyvenko et al.
Scheme 2.
Several efficient syntheses of narciclasine and lycor-
Scheme 3.
icidine have utilized some form of cross-coupling to
establish the C-10a/C-10b connectivity. Our approach
to 3 was therefore based on the Heck reaction of an
appropriately functionalized halopyridine 14 with the ed well and yielded the same lithium carboxylate 14,
known conduramine 8, which is derived from cyclo- which was, without isolation, coupled with condura-
hexadiene diol 4 (Scheme 1), the well-known metabo- mine 8, as shown in Scheme 3.
lite[7] from the enzymatic oxidation of bromobenzene
Amide 15 was then subjected to the conditions ap-
with toluene dioxygenase overexpressed in E.coli plied in the synthesis of the truncated 7-aza analogue.
JM109 (pDTG601 A).[8]
Protection of the amide as the Boc derivative resulted
For the synthesis of the more synthetically challeng- in imide 16, which underwent an intramolecular Heck
ing aromatic fragment, namely penta-substituted nico- reaction to give azaphenanthridone 17 in 58% yield
tinic acid 14, we decided to utilize a halogen dance (30% for the sequence starting with 12), Scheme 4. 17
strategy.[9] This approach has been applied several was then deprotected in the same way as in our syn-
times to the synthesis of polyoxygenated pyridine- thesis of pancratistatin analogues.[3s,u] Demethylation
containing natural products such as atpenins[10] and with anhydrous lithium chloride was followed by desi-
piericidin.[11]
Halopyridine 11, which represents the core of the desired 10-aza-narciclasine (3).
lylation and acid-catalyzed deprotection to furnish the
desired compound, was produced from furfural in two
Biological evaluation of 10-aza-narciclasine 3 in
steps by a known procedure.[12] Despite the low yields several cancer cell lines showed reasonable activity, as
of some of the steps in this sequence, its simplicity al- shown in Table 1. That it was active (unlike the 7-aza
lowed the production of multigram quantities of inter- analogue 2) confirms the hypothesis that the 7-hy-
mediate 11. The bromopyridine was then submitted droxy functionality is an essential part of the pharma-
to a directed ortho-metallation/borylation/oxidation cophore for active Amaryllidaceae constituents. Fur-
sequence followed by direct methylation with diazo-
methane to furnish bromopyridine 12, Scheme 2.
The next goal was to study the key transformation
– the halogen dance reaction of halopyridine 12. At-
tempts to follow a procedure described for similar
systems[10,11] did not lead to the desired product. Slow
addition of the substrate to a solution of lithium tetra-
methylpiperidide (LTMP) at low temperature (be-
tween À908C and À808C) led to simple deprotona-
tion at the a-position with no halogen dance, and at
higher temperatures (between À758C and À708C) de-
composition was observed. However, reverse slow ad-
dition of LTMP at the lower temperature (between
À908C and À808C) followed by methanol quench led
to the product of halogen dance, isomeric halopyri-
dine 13. In order to prove this observation we decided
to match the products of carbon dioxide quench, ob-
tained by halogen dance reaction of 12, with the prod-
uct of the direct addition of base to 2-bromopyridine
13. To our satisfaction both of these reaction proceed- Scheme 4.
2
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10-Aza-narciclasine
Table 1. Results of biological testing (EC50, nM) and assessment of potency of cytotoxicity in cancer cell lines.
Compound
HCC1954
IC50
Jurkat
PANC-1
95% confidence inter- IC50
95% confidence inter- IC50
95% confidence inter-
val (nM)
(nM)
val (nM)
(nM)
val (nM)
(nM)
10 aza-narcicla-
sine (3)
Narciclasine (1)
193.7
173.2
132.8 to 282.5
82.85
71.89 to 95.48
1644
1346 to 2010
229.3 to 322.3
134.0 to 223.8
36.89
33.13 to 41.07
271.9
ther studies on water-soluble derivatives of 3 with the pancreatic carcinoma cell line PANC-1 (Figure 2C).
functionalities of the more active C-1 homologues of Compound concentrations capable of 50% growth in-
pancratistatin may produce compounds with higher hibition compared to untreated control cells are pre-
activity.
sented along with the corresponding 95% confidence
Table 1 summarizes the cytotoxic activity of 10-aza- interval of those values. Independent of the cell line
narciclasine when tested using the human mammary tested, 10-aza narciclasine was less potent or similarly
carcinoma cell line HCC1954 (Figure 2A) human T- potent compared to narciclasine. The data presented
cell leukemia cell line Jurkat (Figure 2B), or human are representative of two independent experiments.
In summary, the first active aza derivative of narci-
clasine, 10-aza analogue 3, was synthesized by a che-
moenzymatic strategy. The biological evaluation
against several cancer cell lines indicated activity of
a similar order of magnitude as those of the natural
product. This finding bodes well for the development
of further aza-analogues with focus on increased solu-
bility of their salts. By extension, we may pursue 10-
aza derivatives of the active C-1 analogues of pancra-
tisatin that were reported earlier.[3s,u] These results
will be reported in due course.
Experimental Section
The key steps are reported here: the halogen dance reaction
of 12 to 13, the formation of 15 from 13, and the intramolec-
ular Heck of 16 to 17. Experimental details of all reactions,
spectral data, and the biological assays are included in the
supporting information.
5-Bromo-7-methoxy[1,3]dioxolo[4,5-b]pyridine (13):
To a solution of 2,2,6,6-tetramethylpiperidine (0.320 mL,
1.86 mmol) in THF (5 mL) at À508C nBuLi (0.770 mL,
1.82 mmol) was added dropwise. The reaction mixture was
stirred for 15 min at À308C, then cooled down to À808C
and added dropwise to a solution of 12 (0.206 g, 0.89 mmol)
in THF (5 mL) while maintaining a temperature of À808C.
The reaction mixture was stirred for 20 min at this tempera-
ture, followed by consecutive quenching with methanol
(1 mL) and solution of NH4Cl (sat. aq., 2 mL). The reaction
mixture was extracted with extracted with EtOAc (3ꢁ
30 mL). The organic layer was dried over Na2SO4, evaporat-
ed, and subjected to flash column chromatography (silica
gel, hexane/EtOAc 4:1) to yield 13 as a white crystalline
compound (0.110 g, 54%).
Figure 2. Cytotoxicity Assay non-linear regression plots.
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(5 mL) and HBTU (0.178 g, 0.47 mmol) was added, followed
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to 2:1) to yield 15 as a white crystalline compound (0.114 g,
52% based on 13).
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tert-Butyl (3aS,3bR,12S,12aS)-12-{[tert-
butyl(dimethyl)silyl]oxy}-6-methoxy-2,2-dimethyl-5-
oxo-3a,5,12,12a-tetrahydro[1,3]benzodioxolo[4,5 h]
[1,3]dioxolo[4,5-b]-1,6-naphthyridine-4(3bH)-
carboxylate (17)
To a solution of 16 (0.30 g, 0.457 mmol) in toluene (35 mL)
was added Pd(OAc)2 (0.025 g, 0.113 mmol) and Ag3PO4
(0.140 g, 0.33 mmol). The reaction mixture was degassed by
passing argon through it for 5 min. After addition of 1,2-bis-
(diphenylphosphino)ethane (0.036 g, 0.091 mmol), the reac-
tion mixture was stirred at 958C for 20 h. The product was
isolated by flash column chromatography (hexanes/EtOAc
2:1 to 1:1) affording 17 as a white solid (0.152 g, 58%, 69%
based on recovered starting material) and 0.05 g of starting
material.
Acknowledgements
The authors are grateful to the following agencies for finan-
cial support of this work: Natural Sciences and Engineering
Research Council of Canada (NSERC) (Idea to Innovation
and Discovery Grants), Canada Research Chair Program,
Canada Foundation for Innovation (CFI), TDC Research,
Inc., TDC Research Foundation, the Ontario Partnership for
Innovation and Commercialization (OPIC), and The Ad-
vanced Biomanufacturing Centre (Brock University). We
also appreciate the skillful assistance of Razvan Simionescu
and Tim Jones for their help with NMR and mass spectrome-
try analysis.
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6 Synthesis and Biological Activity of 10-Aza-narciclasine
Adv. Synth. Catal. 2014, 356, 1 – 6
Sergey Vshyvenko, Zemane W’Giorgis, Audrey Weber,
Natalia Neverova, Bradley Hedberg, Tomas Hudlicky*
6
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