A β-carboline-1-one mimic of the anticancer Amaryllidaceae constituent pancratistatin: Synthesis and biological evaluation

Article abstract of DOI:10.1002/anie.200460218

New drug design possibilities are suggested by the results of a biological evaluation of the indole derivative 1 of pancratistatin, a known anticancer agent, in a panel of cancer cell lines. Enzymatic dihydroxylation was one of the key steps in the short,

Full text of DOI:10.1002/anie.200460218

Communications
Synthetic Methods
A b-Carboline-1-one Mimic of the Anticancer
Amaryllidaceae Constituent Pancratistatin:
Synthesis and Biological Evaluation**
Uwe Rinner, Tomas Hudlicky,* Heather Gordon, and
George R. Pettit
Pancratistatin (1) and narciclasine (3; Scheme 1), well-known
constituents[1] of the Amaryllidaceae species, have been the
[2]
subject of intense study, both in the realm of total synthesis
and with respect to their anticancer activities.[3] Both com-
pounds contain a free phenolic hydroxy group that is part of
the enolized b-ketoamide function. It is this functional group
that accounts for the greater (10-fold or more) activityof
Scheme 1. Amaryllidaceae constituents containing the enolized b-keto-
amide motif and their b-carboline-1-one analogue.
[*] Dr. U. Rinner, Prof. Dr. T. Hudlicky, Prof. Dr. H. Gordon
Department of Chemistry
Brock University
500 Glenridge Avenue, St. Catharines, Ontario L2S 3A1 (Canada)
Fax: (+1)905-984-4841
E-mail: thudlicky@brocku.ca
Prof. Dr. G. R. Pettit
Department of Chemistry & Biochemistry and
the Cancer Research Institute
Arizona State University
Tempe, Arizona 85287 (USA)
[**] The authors are grateful to the following agencies for their support
of this work: the National Science and Engineering Research
Council, the Canadian Foundation for Innovation, the Ontario
Innovation Trust, Brock University, the donors of the Petroleum
Research Fund (administered by the American Chemical Society;
Grant no. PRF-38075-AC), TDC Research Inc., and TDC Research
Foundation. G.R.P. also thanks the Divison of Cancer Research
Treatment and Diagnosis, National Cancer Institute, Department of
Health and Human Services (Grant no. R01-CA90441-03), and the
Arizona Disease Control Research Commission for financial
support and is also grateful for the assistance provided by Dr. Jean-
Charles Chapuis and Dr. Jean M. Schmidt.
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DOI: 10.1002/anie.200460218
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Chemie
these compounds compared to that of their congeners 7-
deoxypancratistatin (2) and lycoricidine (4), which lack the
phenol group.[4]
Several research groups have focused their efforts on
identifying the pharmacophore of these plant constituents.[5]
To this end, a series of derivatives of 1 have been prepared in
which functionalities in the aminoinositol moietyhave been
deleted or changed,[6] or in which the skeleton has been
truncated to produce smaller derivatives.[5c] No modification
of the aminoinositol ring has led to an increase in the activity
of such derivatives. Truncated derivatives that retain the
phenanthridone moietyshow decreased activities compared
to that of the parent compound. An account of the synthesis
of a lactone analogue of 1 containing a carbohydrate motif
appeared recentlybut no biological evaluation of the com-
pound was reported.[7]
Pancratistatin has been proved active in antiviral
screens[3f] and is highlyactive against various cancer cell
lines in vitro and in vivo. However, it has poor bioavailability
and a major effort has been made to develop more soluble
analogues or prodrugs.[5a,8] We speculated that the potencyof
1 and 3 mayin part be due to the hydrogen-bonding donor–
acceptor pairing of the b-ketoamide motifs present in these
compounds but absent from the 7-deoxycongeners. We
therefore decided to test the b-carboline-1-one analogue of 1,
compound 5, in which such donor–acceptor pairing is
extended through the vinyl indole ring, as shown in
Scheme 2. The steric and, to some degree, the electronic
Figure 1. Comparison of the electrostatic potential energy surfaces of
minimum energy conformations of pancratistatin (1; left) and b-carbo-
line-1-one analogue 5 (right). The isodensity surfaces are color-coded
according to the electrostatic potential: red À0.05, yellow 0.00, green
0.05, light blue 0.10, blue 0.15 e.
Scheme 2. Donor–acceptor pairing for hydrogen bonding in pancratis-
tatin (1) and b-carboline-1-one analogue 5.
Figure 2. Direct overlap of the structures of pancratistatin (1, top) and
b-carboline-1-one analogue 5 (bottom).
properties of 1 and 5 are similar. We expected the b-carboline-
1-one analogue to interfere with RNA transcription bythe
mechanism proposed for the activityof narciclasine ( 3), the
onlymember of the Amaryllidaceae familyfor which data on
the possible mode of action are available.[3c,d] In addition, b-
carboline-1-ones and stericallyconstrained tryptamines have
were obtained bycarrying out a concerted dihedral search
using the CHARMm molecular mechanics forcefield.[11] Not
surprisingly, the lowest energy conformation of the phenolic
hydroxy group under these conditions is that required to form
an intramolecular hydrogen bond with the amide carbonyl
oxygen atom.
The isodensitysurfaces displayed in Figure 1 were con-
structed with the program Molden[12] and are color-coded
according to the electrostatic potential. The geometry-opti-
mized structures were superimposed based on the positions of
the atoms of the shared aminoinositol moiety. Figure 1 clearly
illustrates the difference between the electrostatic potential
of the putative hydrogen-bond donor–acceptor pair of the b-
ketoamide motif of 1 and that of the analogous portion of 5.
The hydrogen-bonding b-ketoamide motif has three distinct
regions of negative electrostatic potential, while the equiv-
[9]
been found to have serotonin-regulating activity.
Molecular models of these two compounds, pancratistatin
(1) and its carboline-1-one mimic 5, show interesting spatial
similarities (Figures 1 and 2). Except for the electron density
associated with the methylenedioxy bridge of 1, the com-
pounds seem to occupyalmost identical space, as confirmed in
Figure 2, which shows that the functionalities of the two
compounds directlyoverlap.
Geometryoptimizations of both pancratistatin ( 1) and its
b-carboline-1-one analogue 5 were performed at the HF/6-
31G* level of theorybyusing the Gaussian 03 package. [10] We
calculated the optimal geometries of the compounds in vacuo.
The initial orientations of the aminoinisitol hydroxy groups
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alent part of 5 has negative electrostatic energyonlyaround
the carbonyl oxygen atom.
Herein we report a concise synthesis of 5 that features a
number of interesting transformations (Scheme 3), along with
to open aziridines under InCl3 catalysis[16] and condensed
smoothlywith 6 under silica gel catalysis. However, we did not
expect ester 8 to react well with 6 because of the decreased
nucleophilicityof the 3-position of the indole as a result of
conjugation to the vinyl urethane function. Lewis acid
catalysis provided low yields of 9 compared to those obtained
from the reactions on the silica surface.[17–19]
The methyl ester group of 9 was hydrolyzed (LiOH/H2O,
12 h, RT) and the free acid 10 subjected to iodolactonization
to produce lactone 11 as a single stereoisomer in 71% yield.[20]
This protocol allowed full control of the stereochemistry
while avoiding the use of oxidizing agents or other epoxida-
tion procedures that would have been detrimental to the fate
of the indole core. Exposure of lactone 11 to LiOMe/MeOH
gave epoxide 12 cleanly. This transformation could also be
achieved byusing a two-step procedure (LiOH/H 2O; CH2N2;
85% yield). Detosylation of compounds of this type has been
shown to proceed more smoothlywhen the tosylamide is first
converted into an imide.[2e,5b] We converted 12 into the bis-
Boc-protected material 13 (88% yield), which was easily
detosylated in 71% yield by treatment with sodium naph-
thalide at À658C.
The final transformation of 13 into the b-carboline-1-one
analogue 5 was accomplished in a one-flask sequence during
which four separate events took place. A sample of 13 was
dissolved in acetone and adsorbed on silica. The drypowder
was suspended in H2O, placed in a thick-walled pressure tube,
and heated at 1708C. After one hour the material had been
quantitativelytransformed into the free amide 14 bya
thermal retro-ene reaction of the Boc-carbamate and internal
amidation of the methyl ester. Thermolysis on wet silica gel is
superior to the previouslyreported technique, in which the
starting material was treated with 5% aqueous sodium
benzoate under reflux.[21] Separation of polar products from
benzoic acid is often problematic on a small scale. Continued
heating of 14 for an additional 16 h at 1608C resulted in its
conversion into 5, which was obtained as a single stereoisomer
through transdiaxial opening of the epoxide and thermolytic
cleavage of the acetonide. Pure carboline-1-one derivative 5,
a sparinglysoluble compound, was isolated bychromatog-
raphy. The crude product was converted into its pentaacetate
15 for use in detailed NMR studies because 15 is more easily
purified than 5.
b-Carboline-1-one 5 and each of the keyintermediates in
this nine-step sequence were tested against a small panel of
cancer cell lines. The results are shown in Table 1 as GI50
values (amount of substrate in mgmLÀ1 cell solution necessary
to stop the growth of 50% of the cancer cells in 1 mL cell
solution). The cutoff for activityin human cancer cell lines is
considered to be 10 mgmLÀ1. Some of the indole modifica-
tions produced compounds that meet this criterion for activity
against certain cancer cell lines. Biological evaluation of 5
confirmed borderline activity in the murine P388 lymphocytic
leukemia assay. Surprisingly, iodolactone 11 showed promis-
ing activities against pancreas and breast adenocarcinoma
(GI50 = 1.9 and 4.3 mgmLÀ1). It is possible that the mechanism
of action of the iodolactone is different from that of
pancratistatin and it maybe beneficial to examine 11 as a
completelydifferent scaffold on which the design of further
Scheme 3. Synthesis of b-carboline-1-one analogue 5. Boc=tert-butoxycar-
bonyl, DMAP=4-dimethylaminopyridine, pyr=pyridine, Ts=tosyl.
the biological evaluation of the product in a series of cancer
cell lines as a prelude to a more focused drug discoveryeffort
aimed at the heterocyclic variants of 1. We found that the
synthesis of 5 is easier than the preparation or extraction of
the natural alkaloids.
The synthesis began with vinylaziridine 6,[2e,13] which was
prepared in three steps from enzymatically derived diene-diol
7,[14] and methyl indole-2-carboxylate (8), prepared from
[15]
commerciallyavailable indole-2-carboxylic acid.
The two
compounds were adsorbed on silica and heated at 708C for
48 h to provide tosylamide 9 cleanlyin 68% iyeld. This
transformation was surprising. Indole itself has been reported
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Table 1: (Continued)
Table 1: Evaluation of the activities of b-carboline-1-one and intermedi-
ates in the synthesis, as well as pancratistatin and 7-deoxypancratistatin,
against Murine P388 lymphocytic leukemia and human cancer cell lines.
GI50-values in [mgmLÀ1][a].
Compound
P388[b]
BXPC-3[c] MCF-7[d] KM20L2[e]
Compound
P388[b]
BXPC-3[c] MCF-7[d] KM20L2[e]
11.7
1.9
4.3
>10
0.02
0.03
–
–
0.045
[a] No significant activity against human SF268 (CNS glioblastoma),
NCI-H460 (lung large cell), or DU-145 (prostate carcinoma) was
detected. [b] P388, lymphocytic leukemia. [c] BXPC-3, pancreas adeno-
carcinoma (human). [d] MCF-7, breast adenocarcinoma (human).
[e] KM20L2, colon adenocarcinoma (human).
0.44
–
0.22
derivatives could be based. bis-Boc derivative 13 and
unsaturated analogues 17 and 18 are also active against
murine P388 lymphocytic leukemia, with GI50 values (12.8,
11.8, and 4.6 mgmLÀ1, respectively) one order of magnitude
smaller than that of 7-deoxypancratistatin (GI50 =
0.44 mgmLÀ1). These exciting results suggest new possibilities
that should be examined in the next series of analogues.
Some guidelines for the design of new analogues emerged
as a result of this particular study: 1) The presence of both the
oxygen atoms in the methylenedioxy bridge of pancratistatin
seems essential for high activity. We recently showed that
deletion of the C8 methoxygroup from the dimethoxy
derivative of 1 leads to GI50 values 10- to 20-fold higher than
that of the natural product.[5d] 2) The activityof compounds
such as lactone 11 opens up the possibilitythat analogues can
be structured around completelydifferent scaffolds in future
because such compounds can be further functionalized at a
number of positions. 3) The synthesis of 5 is, with nine steps,
the shortest existing preparation of pancratistatin analogues
containing the aminoinositol motif. Some of the transforma-
tions that were employed will no doubt find further applica-
tion in the design of heterocyclic analogues of pancratistatin.
4) The vinylaziridine 6 proved useful as a scaffold for the
generation of diversityand will be further exploited in the
design of new derivatives of the title compounds.
22.8
>10
>10
>10
>10
12.8
>10
>10
11.8
4.6
8.6
10.5
>10
>10
>10
>10
>10
>10
>100
3.5
Future endeavors in this area will focus on heteroatom
alterations in the aromatic portion of pancratistatin as it has
alreadybeen shown that the aminoinositol moietymust
remain intact for these compounds to retain activity. We will
report our findings in due course.
>100
>10
>10
3.8
Received: April 5, 2004
Revised: June 21, 2004
Keywords: antitumor agents · asymmetric synthesis ·
.
natural products · pancratistatin · synthetic methods
18.3
>10
>10
>10
[1] The pancratistatin, narciclasine, and lycoricidine group of
natural products has been collectivelyreferred to as “ Amarylli-
daceae alkaloids” bymost, if not all, synthetic chemists (Martin,
Hudlicky, Keck, Polt, and others). Such nomenclature is not
accurate as these compounds do not contain basic nitrogen
atoms. Another term used in the literature is “isocarbostyril”
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Communications
(Pettit), a term which is correct but not generallyknown in the
[9] S. Elz, H. Zimmermann, K. Rehse, Arch. Pharm. 1993, 326, 893 –
899; b) K. Rehse, H. Zimmermann, Arch. Pharm. 1994, 327, 67 –
75.
synthetic community. To avoid further inaccuracies we will avoid
both names in future publications and simplyrefer to these
compounds as “constituents”.
[10] Gaussian03, RevisionB.03, M. J. Frisch, G. W. Trucks, H. B.
Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A.
Montgomery, Jr., T. Vreven, K. N. Kudin, J. C. Burant, J. M.
Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M.
Cossi, G. Scalami, N. Rega, G. A. Petersson, H. Nakatsuji, M.
Hada, M. Ehara, K. Toyota, R. Fukudy, J. Hasegawa, M. Ishida,
T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E.
Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J. Jaramillo, R.
Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi,
C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A.
Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S.
Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick,
A. D. Rabuck, K. Raghgvachari, J. B. Foresman, J. V. Ortiz, Q.
Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G.
Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J.
Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M.
Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong,
C. Gonzalez, J. A. Pople, Gaussian, Inc., Pittsburgh, PA, 2003.
[11] a) CHARMm, Accelrys, Inc., 2001; b) B. R. Brooks, R. E.
Bruccoleri, B. D. Olafson, D. J. States, S. Swaminathan, M.
Karplus, J. Comput. Chem. 1983, 4, 187 – 217.
[12] G. Schaftenaar, J. H. Noordik, J. Comput.-Aided Mol. Des. 2000,
14, 123 – 134.
[13] T. Hudlicky, X. Tian, K. Königsberger, J. Rouden, J. Org. Chem.
1994, 59, 4037 – 4039.
[14] T. Hudlicky, H. Luna, H. F. Olivo, C. Andersen, T. Nugent, J. D.
Price, J. Chem. Soc. Perkin Trans. 1 1991, 2907 – 2917.
[15] T. Martin, C. J. Moody, J. Chem. Soc. Perkin Trans. 1 1998, 235 –
240.
[16] J. S. Yadav, B. V. S. Reddy, S. Abraham, G. Sabitha, Tetrahedron
Lett. 2002, 43, 1565 – 1567.
[2] For reviews, see: a) S. F. Martin in The Alkaloids, Vol. 40 (Ed.:
A. R. Brossi), Academic Press, New York, 1987, pp. 251 – 376;
b) R. Polt in Organic Synthesis: Theory and Applications, Vol. 3
(Ed.: T. Hudlicky), JAI, Greenwich, CT, 1998, pp. 109 – 148;
c) O. Hoshino in The Alkaloids, Vol. 51 (Ed.: G. A. Cordell),
Academic Press, New York, 1998, pp. 323 – 424; for total
syntheses of pancratistatin, see: d) S. Danishefsky, J. Y. Lee, J.
Am. Chem. Soc. 1989, 111, 4829 – 4837; e) X. R. Tian, T.
Hudlicky, K. Königsberger, J. Am. Chem. Soc. 1995, 117,
3643 – 3644; f) B. M. Trost, S. R. Pulley, J. Am. Chem. Soc.
1995, 117, 10143 – 10144; g) G. E. Keck, S. F. McHardy, J. A.
Murry, J. Am. Chem. Soc. 1995, 117, 7289 – 7290; h) T. Hudlicky,
X. R. Tian, K. Königsberger, R. Maurya, J. Rouden, B. Fan, J.
Am. Chem. Soc. 1996, 118, 10752 – 10765; i) N. Chida, M.
Jitsuoka, Y. Yamamoto, M. Ohtsuka, S. Ogawa, Heterocycles
1996, 43, 1385 – 1390; j) T. J. Doyle, M. Hendrix, D. VanDerveer,
S. Javanmard, J. Haseltine, Tetrahedron 1997, 53, 11153 – 11170;
k) P. Magnus, I. K. Sebhat, Tetrahedron 1998, 54, 15509 – 15524;
l) P. Magnus, I. K. Sebhat, J. Am. Chem. Soc. 1998, 120, 5341 –
5342; m) J. H. Rigby, U. S. M. Maharoof, M. E. Mateo, J. Am.
Chem. Soc. 2000, 122, 6624 – 6628; n) G. R. Pettit, N. Melody,
D. L. Herald, J. Org. Chem. 2001, 66, 2583 – 2587; o) S. Kim, H.
Ko, E. Kim, D. Kim, Org. Lett. 2002, 4, 1343 – 1345; p) H. J. Kim,
E. Kim, J. E. Park, D. Kim, S. Kim, J. Org. Chem. 2004, 69, 112;
for total syntheses of narciclasine, see: q) J. H. Rigby, M. E.
Mateo, J. Am. Chem. Soc. 1997, 119, 12655 – 12656; r) G. E.
Keck, T. T. Wager, J. F. D. Rodriquez, J. Am. Chem. Soc. 1999,
121, 5176 – 5190; s) D. Gonzalez, T. Martinot, T. Hudicky,
Tetrahedron Lett. 1999, 40, 3081 – 3084; t) S. Elango, T.-H. Yan,
J. Org. Chem. 2002, 67, 6954 – 6959.
[3] For studies on the biological activityof Amaryllidaceae constit-
uents, see: a) D. B. Fitzgerald, J. L. Hartwell, J. Leiter, J. Natl.
Cancer Inst. 1958, 20, 763 – 764; b) G. Ceriotti, Nature 1967, 213,
595 – 596; c) L. Carrasco, M. Fresno, D. Vazquez, FEBS Lett.
1975, 52, 236 – 239; d) A. Jimenez, L. Sanchez, D. Vazquez,
FEBS Lett. 1975, 55, 53 – 56; e) A. Jimenez, A. Santos, G.
Alonso, D. Vazquez, Biochim. Biophys. Acta 1976, 425, 342 –
348; f) G. R. Pettit, V. Gaddamidi, D. L. Herald, S. B. Singh,
G. M. Cragg, J. M. Schmidt, F. E. Boettner, M. Williams, Y.
Sagawa, J. Nat. Prod. 1986, 49, 995 – 1002; g) B. Gabrielsen, T. P.
Monath, J. W. Huggins, D. F. Kefauver, G. R. Pettit, G. Groszek,
M. Hollingshead, J. J. Kirsi, W. M. Shannon, E. M. Schubert, J.
Dare, B. Ugarkar, M. A. Ussery, M. J. Phelan,J. Nat. Prod. 1992,
55, 1569 – 1581.
[17] M. Hudlicky, J. Org. Chem. 1974, 39, 3460 – 3461.
[18] T. Hudlicky, T. Srnak, Tetrahedron Lett. 1981, 22, 3351 – 3354.
[19] A. K. Banerjee, M. S. L. Mimo, W. J. V. Vegas, Usp. Khim. 2001,
70, 1094 – 1115.
[20] M. Jung, J. Ham, J. Song, Org. Lett. 2002, 4, 2763 – 2765.
[21] M. Mandel, T. Hudlicky, L. D. Kwart, G. M. Whited, J. Org.
Chem. 1993, 58, 2331 – 2333.
[4] G. R. Pettit, N. Melody, D. L. Herald, J. M. Schmidt, R. K. Pettit,
J.-C. Chapuis, Heterocycles 2002, 56, 139 – 155.
[5] a) G. R. Pettit, S. Freeman, M. J. Simpson, M. A. Thompson,
M. R. Boyd, M. D. Williams, G. R. Pettit III, D. L. Doubek, Anti-
Cancer Drug Des. 1995, 10, 243 – 250; b) J. McNulty, R. Mao, S.
Wolf, G. R. Pettit, D. L. Herald, M. R. Boyd, Bioorg. Med.
Chem. Lett. 2001, 11, 169 – 172; c) T. Hudlicky, U. Rinner, D.
Gonzalez, H. Akgun, S. Schilling, P. Siengalewicz, T. A. Marti-
not, G. R. Pettit, J. Org. Chem. 2002, 67, 8726 – 8743; d) U.
Rinner, H. Hillebrenner, D. R. Adams, T. Hudlicky, G. R. Pettit,
Bioorg. Med. Chem. Lett. 2004, in press.
[6] S. Schilling, U. Rinner, C. Chan, I. Ghiviriga, T. Hudlicky,Can. J.
Chem. 2001, 79, 1659 – 1667.
[7] A. N. Phung, M. T. Zannetti, G. Whited, W. D. Fessner, Angew.
Chem. 2003, 115, 4970 – 4972; Angew. Chem. Int. Ed. 2003, 42,
4821 – 4824.
[8] G. R. Pettit, B. Orr, S. Ducki, Anti-Cancer Drug Des. 2000, 15,
389 – 395.
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