Synthesis and evaluation of a fluorescent ritterazine-cephalostatin hybrid

Article abstract of DOI:10.1021/ol202139z

The cephalostatin and ritterazine natural products comprise a potent family of bis-steroidal pyrazines that display potent single-digit nanomolar inhibition of tumor cell growth. An active fluorescent ritterazine-cephalostatin hybrid probe was developed u

Full text of DOI:10.1021/ol202139z

ORGANIC
LETTERS
2011
Vol. 13, No. 19
5334–5337
Synthesis and Evaluation of a Fluorescent
RitterazineꢀCephalostatin Hybrid
Kanduluru Ananda Kumar,† James J. La Clair,*,‡ and Philip L. Fuchs*,†
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907,
United States, and Xenobe Research Institute, P.O. Box 3052, San Diego,
California 92163-1052, United States
i@xenobe.org; pfuchs@purdue.edu
Received August 13, 2011
ABSTRACT
The cephalostatin and ritterazine natural products comprise a potent family of bis-steroidal pyrazines that display potent single-digit nanomolar
inhibition of tumor cell growth. An active fluorescent ritterazineꢀcephalostatin hybrid probe was developed using detailed SAR data derived
through total synthetic efforts. A combination of time course and confocal imaging studies indicate that this natural product family is rapidly taken
up in tumor cells and localizes subcellularly within ER and surrounding the nuclearꢀER interface.
Ritterazine B (1) is a member of a large family of natural
products (over 45 cephalostatin and ritterazine congeners)
that were isolated from marine organisms by the labora-
tories of Pettit1a and Fusetani.1b The unique structure of
these bis-steroidal compounds (see Figure 1) along with
their potent single-digit nanomolar inhibition of tumor cell
growth is highlighted by an average GI50 value of 1.2 nM
for cephalostatin 1 (2) in the NCI-60 cell line screen. This
potent activity along with their unique cell selectivity and
apoptotic response has drawn attention to their potential
as clinical leads for cancer.2
Soon after their isolation, synthetic programs were
launched, with the first total synthesis reported in 1995.3
While these synthetic tools now provide access to ana-
logues with improved activity and ease of access,4 a detailed
understanding of the mechanisms by which the ritterazines
and cephalostatins induce tumor cell apoptosis remains
unclear.5 Recent studies from a team led by Shair suggest
that 1 and 2 target an oxysterol binding protein (OSBP);
† Purdue University.
‡ Xenobe Research Institute.
(1) (a) Pettit, G. R.; Inoue, M.; Kamano, Y.; Herald, D. L.; Arm, C.;
Dufresne, C.; Christie, N. D.; Schmidt, J. M.; Doubek, D. L.; Krupa,
T. S. J. Am. Chem. Soc. 1988, 110, 2006–2007. (b) Fukuzawa, S.;
Matsunaga, S.; Fusetani, N. J. Org. Chem. 1994, 59, 6164–6166.
(2) (a) Moser, B. R. J. Nat. Prod. 2008, 71, 487–491. (b) Flessner, T.;
Jautelat, R.; Scholz, U.; Winterfeldt, E. Fortschr. Chem. Org. Naturst.
2004, 87, 1–80. (c) Lee, S.; LaCour, T. G.; Fuchs, P. L. Chem. Rev. 2009,
109, 2275–2314.
(3) (a) Jeong, J. U.; Sutton, S. C.; Kim, S.; Fuchs, P. L. J. Am. Chem.
Soc. 1995, 117, 10157–10158. (b) Fortner, K. C.; Kato, D.; Tanaka, Y.;
Shair, M. D. J. Am. Chem. Soc. 2010, 132, 275–280.
(4) (a) Lee, S.; LaCour, T. G.; Fuchs, P. L. Chem. Rev. 2009, 109,
2275–314. (b) Phillips, S. T.; Shair, M. D. J. Am. Chem. Soc. 2007, 129,
6589–6598.
r
10.1021/ol202139z
Published on Web 09/14/2011
2011 American Chemical Society

however, a link between this target and the apoptotic
activity of 1 and 2 has yet to be established.6
R-aminomethoxime.4a Since both steroidal precursors
pass through the R-azidoketone stage, there are two coupl-
ing modes to the same product. The bottom line is that this
unsymmetrical pyrazine synthesis provides dependable
late-stage coupling of an exceptionally valuable pair of
3-ketosteroids with the expectation of excellent overall
yield and preservation of acid-labile spiroketal stereo-
chemistry. The average yield for the initial 59 cases
examined was 72%.
Using SAR studies as a guide,9 the C-25 position was
targeted, as it has shown tolerance to modification. Our
studies focused on use of an immunoaffinity fluorescent
(IAF) label,10 as this label has been shown to provide
effective probes without phenotypic modification and
modest loss in cell viability and activity.
Scheme 1. Synthesis of IAF-Labeled Fluorescent Probe 7
Figure 1. Structures of ritterazine B (1), cephalostatin 1 (2), 230-
deoxycephalostatin 1 (3), ritterostatin GN1N (4), and 25-epi-
ritterostatin hybrid GN1N (5).
Our ability to synthesize cephalostatin, ritterazine, and
hybrid analogues has been used to supply sufficient quan-
titiesofmaterialsfordetailedbiologicaland invivostudies.
For instance, the first-generation synthesis of 110 mg of
230-deoxycephalostatin 1 (3) was used to characterize their
apoptotic activity and evaluate the accompanying mito-
chondrial damage and cytosolic Ca2þ increase.7 These
studies have also shown that 3 engenders a 50ꢀ60% increase
in mouse lifespan in U87MG brain cancer xenografts.8 Syn-
thetic samples (from 245 mg total) of 25-epi-ritterostatin
GN1N (5) were further screened for in vitro activity against
multiple cancer cells lines of different tissue origins.3a,4a
Given our synthetic access, application of these materi-
als to prepare and evaluate fluorescent probes was a logical
next step. Our unsymmetrical pyrazine synthetic route
operates through a coupling of a R-azidoketone and a
Glycine IAF label 6a11 was coupled with 25-epi-ritterosta-
tin GN1N (5) at the C-25 position to deliver probe 7(Scheme1).
The same probe 7 was also obtained through a stepwise pro-
cedure involving an Fmoc-protected intermediate 8.
Using the MTT assay, probe 7 demonstrated an activity
of IC50 value of 79 ( 4 nM against HCT-116 cells.12 With
ꢀ
ꢀ
(5) (a) Rudy, A.; Lopez-Anton, N.; Dirsch, V. M.; Vollmar, A. M. J.
Nat. Prod. 2008, 71, 482–486. (b) Komiya, T.; Fusetani., T.; Matsunaga,
S.; Kubo, A.; Kaye, F. J.; Kelley, J. M.; Tamura, K.; Yoshida, M.;
Fukuoka, M.; Nakagawa, K. Cancer Chemother. Pharmacol. 2003, 51,
202–208.
(9) (a) LaCour, T. G.; Guo, C.; Ma, S.; Jeong, J. U.; Boyd, M. R.;
Matsunaga, S.; Fusetani, N.; Fuchs, P. L. Bioorg. Med. Chem. Lett.
1999, 9, 2587–2592. (b) Guo, C.; LaCour, T. G.; Fuchs, P. L. Bioorg.
Med. Chem. Lett. 1999, 9, 419–424.
(10) Yu, W. L.; Guizzunti, G.; Foley, T. L.; Burkart, M. D.; La Clair,
J. J. J. Nat. Prod. 2010, 73, 1659–1666.
(11) (a) Hughes, C. C.; MacMillan, J. B.; Gaudencio, S. P.; Fenical,
(6) Burgett, A. W.; Poulsen, T. B.; Wangkanont, K.; Anderson,
D. R.; Kikuchi, C.; Shimada, K.; Okubo, S.; Fortner, K. C.; Mimaki,
Y.; Kuroda, M.; Murphy, J. P.; Schwalb, D. J.; Petrella, E. C.; Cornella-
Taracido, I.; Schirle, M.; Tallarico, J. A.; Shair, M. D. Nat. Chem. Biol.
2011, 7, 639–647.
^
W.; La Clair, J. J. Angew. Chem., Int. Ed. 2009, 48, 728–732. (b)
Alexander, M. D.; Burkart, M. D.; Leonard, M. S.; Portonovo, P.;
ꢀ
Liang, B.; Ding, X.; Joullie, M. M.; Gulledge, B. M.; Aggen, J. B.;
(7) Unpublished results with Prof. Peng Huang (M. D. Anderson).
(8) (a) Unpublished studies with Dr. John Beutler (National Cancer
Institute). (b) Synthesis of 25-epi-ritterostatin GN1N will be reported in
due course.
Chamberlin, A. R.; Sandler, J.; Fenical, W.; Cui, J.; Gharpure, S. J.;
Polosukhin, A.; Zhang, H. R.; Evans, P. A.; Richardson, A. D.; Harper,
M. K.; Ireland, C. M.; Vong, B. G.; Brady, T. P.; Theodorakis, E. A.; La
Clair, J. J. ChemBioChem 2006, 7, 409–416.
Org. Lett., Vol. 13, No. 19, 2011
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Figure 2. Time-course imaging depicting the uptake and subcelluar localization of probe 7 in live HCT-116 cells. Images were collected
using a custom built fiberoptic microscope (see the Supporting Information) that was mounted on a computer-driven XYZ micrometer
adjustable stage and was equipped to excite with an LED at λex = 380 nm and collect emission at λem = 448 ( 20 nm. Images were
collected at 30 min intervals over a 24 h period using identical optical parameters. A concentration gradient of each probe was
conducted and compared to cells treated with dragmacidin D (9), an ER stain, and IAF-labeled nogalamycin (10), a nuclear stain and
an IAF control, compound 6b. Images are provided at (a) 1 h, (b) 6 h, (c) 12 h, and (d) 24 h. Bar denotes 10 μm.
active probe and access to panel of IAF-labeled natural
products and controls,11b our studies shifted to develop a
detailed description of the uptake and subcellular localiza-
tion of 7.
Using inexpensive components, a live cell imaging sys-
tem was built containing a polycarbonate CO2 incubator
mounted on the head of computer-driven fiberoptic micro-
scope (see the Supporting Information). The instrument
was designed to rapidly capture images from 24 experi-
ments in parallel at 30 min intervals over a 24 h period.
In this system, the uptake of three concentrations of probe
7 was measured in triplicate along the respective positive
and negative controls (Figure 2).
An identical outcome was observed after multiple of
repetitions. After 6 h, probe 7 appeared in the nuclear
membrane and extending into the endoplasmic reticulum
(ER). The intensity of this stain increased until 12 h where
it remained consistent thereafter (imaging was conducted
upto24h). Thislocalization wasconfirmedbycomparison
with a complementary blue-fluorescent nuclear stain, IAF-
labeled nogalamycin (10),8b and blue-fluorescent ER stain
dragmacidin D (9) (structures of 9 and 10 are provided in
the Supporting Information).13 Further controls indicated
that the label alone did not stain cells under identical
conditions.14 Even at concentrations up to 250 μM, the
lack of fluorescence from controls such as 6b (Scheme 1) in
live cells indicated that the uptake and localization was
indeed due to the natural product’s activity.
While time course imaging served for initial analyses,
confocal microscopy provided improved resolution. Using
the data from the time course studies, HCT-116 cells were
treated with 0.1 μM 7 for 12 h, washed with media, and
imaged. These studies provided further support for the
localization in the endoplasmic reticulum (er, Figure 3a)
and around the nuclear membrane (nm, Figure 3a). How-
ever, probe 7 was also observed on chromatin (label c,
Figure 3a) within the nucleus of HCT-116 cells. By eval-
uating other cell lines, this observation was linked to a
unique selectivity in HCT-116 cells. For example, probe 7
was not localized on chromatin in HeLa cells (label c,
Figure 3b).
While control experiments indicated the label did not
alter the subcellular localization, further studies were con-
ducted to confirm the localization of probe 7 was compar-
able to ritterazine B (1) or celphalostatin 1 (2). Both 1 and
2 were shown to effectively block the uptake of probe 7
(as shown in Figure 3c,d).
(12) Activities were measured using the protocols described in: Kang,
M.; Jones, B. D.; Mandel, A. L.; Hammons, J. C.; DiPasquale, A. G.;
Rheingold, A. L.; La Clair, J. J.; Burkart, M. D. J. Org. Chem. 2009, 74,
9054–9061.
(13) Forsyth, C. J.; Ying, L.; Chen, J.; La Clair, J. J. J. Am. Chem.
Soc. 2006, 128, 3858–3859.
This observation suggests two key points. First, ritter-
azine B (1) as well as probe 7 bind to their target in a strong
and irreversible manner (if this was not the case treatment
of cells exposed to 1 with probe 7, as in Figure 3c or d,
would have led to comparable localization as in Figure 3a).
(14) See examples: La Clair, J. J. Nat. Prod. Rep. 2010, 27, 969–995.
5336
Org. Lett., Vol. 13, No. 19, 2011

Further insight into the cellular targeting was obtained
from examining mitotic cells treated with 7 (m, Figure 3b).
While localizing on chromatin in mature cells, probe 7 was
not observed binding to the chromosomes during mitosis.
As illustrated in Figure 3b, fluorescence from 7 was
observed in the cytosolic space prophase to telophase. This
observation was consistent with the transitioning of the
nuclear envelope and ER membranes during mitosis.16
In summary, this study describes the preparation of a
fluorescent probe based on a ritterazineꢀcephalostatin
hybrid. Time course and confocal microscopy indicate
that a ritterazineꢀcephalostatin hybrid probe shares a
common uptake and subcellular localization in the ER,
nuclear membrane, and chromatin with ritterazine B (1)
and cephalostain 1 (2). The observed uptake and subcel-
lular localization was consistent with the transitioning
during mitosis.13
The study further illustrates how scarce natural materi-
als made available through complex chemical synthesis
play a pivotal role in evaluating natural product activity.
Our program is now focused on applying these materials to
identify the molecular targets and pathways modulated by
this unique class of bis-steroidal alkaloid, with the goal of
identify the pathways regulated by probe 7 during cell
growth and division.
Figure 3. Confocal fluorescent images detailing the subcellular
localization of probe 7 and corresponding natural products
ritterazine B (1) or celphalostatin 1 (2): (a) live HCT-116 cells
that were incubated with probe 7 for 12 h; (b) comparable
uptake arising from treating HeLa cells with probes 7 for 12 h;
(c) HCT-116 cells that were treated with ritterazine B (1) for 12 h
followed by probe 7 for an additional 12 h; (d) HCT-116 cells
that were treated with cephalostatin 1 (2) for 12 h followed
by probe 7 for an additional 12 h. Labels denote chromatin
(c), endoplasmic reticulum (er), nuclear membrane (nm), and
mitotic cell (m). Images were collected with excitation at λex
=
405 nm and emission at λem = 448 ( 20 nm. Bars denote 10 μm.
Acknowledgment. This work was supported by funding
from the NIH (5U01CA060548-17 and CA 60548) and
internal support from XRI. We sincerely thank Dr. Dou-
glas Lantrip for laboratory assistance and Dr. Karl Wood
and Arlene Rothwell of Purdue University for assistance
with collection of the mass spectral data.
Second, and perhaps most importantly, probe 7 shares
a common target with ritterazine 1 and cephalostatin 2.
While suggested by related hybrid natural products,15 this
study provides direct evidence that the cephalostatins,
ritterazines, and hybrids of both natural products share a
common mode of action.
Supporting Information Available. Synthetic proce-
dures, copies of NMR spectra on the probes, as well
as detailed procedures for the cellular imaging studies
and time course imaging system are available. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(15) (a) Suzuki, K. Chem. Rec. 2010, 10, 291–307. (b) Meunier, B.
Acc. Chem. Res. 2008, 41, 69–77.
(16) Ellenberg, J.; Siggia, E. D.; Moreira, J. E.; Smith, C. L.; Presley,
J. F.; Worman, H. J.; Lippincott-Schwartz, J. J. Cell. Biol. 1997, 138,
1193–1206.
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