Triple hybrids of steroids, spiroketals, and oligopeptides as new biomolecular chimeras

Article abstract of DOI:10.1021/ol802247m

An oxidative enol ether rearrangement was the key methodology in the construction of steroid-spiroketal-RGD peptides. Biological studies demonstrated potent integrin CD11b/CD18 antagonistic effects.

Full text of DOI:10.1021/ol802247m

ORGANIC
LETTERS
2009
Vol. 11, No. 1
65-68
Triple Hybrids of Steroids, Spiroketals,
and Oligopeptides as New Biomolecular
Chimeras
Abhisek Banerjee,† Eduard Sergienko,‡ Stefan Vasile,‡ Vineet Gupta,§
Kristiina Vuori,‡ and Peter Wipf*,†
Department of Chemistry and Center for Chemical Methodologies & Library
DeVelopment, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15260, Burnham
Center for Chemical Genomics, Burnham Institute for Medical Research, 10901 North
Torrey Pines Rd., La Jolla, California 92037, and DiVision of Nephrology and
Hypertension, UniVersity of Miami, 1600 NW 10th AVenue, Miami, Florida 33136
pwipf@pitt.edu
Received October 11, 2008
ABSTRACT
An oxidative enol ether rearrangement was the key methodology in the construction of steroid-spiroketal-RGD peptides. Biological studies
demonstrated potent integrin CD11b/CD18 antagonistic effects.
Steroids are often conjugated to other building blocks that
modulate their biological activity.1 For example, carbohy-
drates are attached to the steroid A-ring hydroxyl groups and
D-ring side chains, and the resulting hybrid molecules have
improved solubility and modified physicochemical properties
as well as distinct biological functions. Synthetic sugar-
steroid conjugates have been shown to target phospholipid
membranes.2 Peptide-steroid conjugates have been applied
as artificial proteolytic enzymes,3 mimics of cationic anti-
biotics,4 and synthetic receptors for oligopeptides (Figure
1).5 The steroid skeleton is rearranged into a spiroketal
moiety in hippurin-1,6 cephalostatins, and ritterazine M,7
which show potent reversal of multidrug resistance and
anticancer activities. A library approach toward the synthesis
of peptidomimetic spirostane hybrids took advantage of the
four-component Ugi reaction,8 and macrocyclic hybrid
structures were assembled with the goal to construct chiral
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P. B. Org. Lett. 2004, 6, 3433–3436.
† University of Pittsburgh.
‡ Burnham Institute for Medical Research.
§ University of Miami.
(5) (a) Cheng, Y.; Suenaga, T.; Still, W. C. J. Am. Chem. Soc. 1996,
118, 1813–1814. (b) Boyce, R.; Li, G.; Nestler, H. P.; Suenaga, T.; Still,
W. C. J. Am. Chem. Soc. 1994, 116, 7955–7956.
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10.1021/ol802247m CCC: $40.75
Published on Web 12/09/2008
2009 American Chemical Society

integrins, including RVꢀ3 (Kd ≈ 10-9 M).12 Several integrin
antagonists based on the RGD sequence have been designed
for applications in the control of angiogenesis, tumor cell
metastasis, osteoporosis, and other diseases.13 However, the
development of selective integrin antagonists still constitutes
a major challenge in RGD mimetic design.
Scheme 2. Steroid Spiroketal R-Alkoxy Ester Synthesis
Figure 1. Selected natural and designed steroid-peptide, steroid-
amino acid, and steroid-spiroketal hybrid structures.2-7
host molecules.9 However, conjugates of spiroketal modified
steroids to peptides or peptide mimetics have not yet been
explored; we hypothesizd that these chimeras could dem-
onstrate interesting membrane affinities and receptor recogni-
tion properties.
Scheme 1. Oxidative Rearrangement of Alkyl Enol Ethers
Our synthesis of the steroid spiroketal carboxylic acid 5
began with commercially available epi-androsterone (1).
Treatment of 1 with 5-lithio-2,3-dihydrofuran followed by
the acidic Dowex 50X initiated a pinacol-type14 rearrange-
ment (Scheme 2). Enolization and O-methylation of ketone
2 was performed with KHMDS and Me2SO4 in a 4:1 mixture
of THF and DMF. Other conditions led to significant
amounts of C-methylation. The reaction of the enol ether 3
with m-CPBA buffered with Na2HPO4 produced the penta-
cyclic spiroketal ester 4 as a single diastereomer after basic
workup.15 Saponification of 4 with KOH provided carboxylic
acid 5, and the X-ray structure of amide 6 confirmed the
structural assignment of the spiroketal portion of 5.
We have recently demonstrated an oxidative rearrangement
of enol ethers to lactones and spiroketal esters.10 Our
methodology allows for a rapid formation of these common
structural subunits (Scheme 1). We now report an extension
of this methodology for the construction of a small library
of steroid-spiroketal-peptide triple hybrid structures that were
designed to anchor the RGD motif in the cell membrane.
The tripeptide sequence Arg-Gly-Asp (RGD)11 is widely
used for cell adhesion studies, as it is recognized by many
A microwave Fmoc-SPPS protocol16 was selected for the
preparation of the chimera. However, when Wang resin was
used, cleavage of the peptide from the solid support by
aminolysis with benzylamine13 did not provide the desired
(9) Wessjohann, L. A.; Voigt, B.; Rivera, D. G. Angew. Chem., Int. Ed.
2005, 44, 4785–4790.
(13) Gentilucci, L.; Cardillo, G.; Squassabia, F.; Tolomelli, A.; Spam-
pinato, S.; Sparta, A.; Baiula, M. Bioorg. Med. Chem. Lett. 2007, 17, 2329–
2333.
(10) Waller, D. L.; Stephenson, C. R. J.; Wipf, P. Org. Biomol. Chem.
2007, 5, 58–60.
(14) Paquette, L. A.; Andrews, J. F. P.; Vanucci, C.; Lawhorn, D. E.;
Negri, J. T.; Rogers, R. D. J. Org. Chem. 1992, 57, 3956–3965.
(15) Inorganic bases such as Na2CO3, NaHCO3, Cs2CO3, and NaH2PO4/
Na2HPO4 (1:1) gave no improvement in yield.
(11) Ruoslahti, E.; Pierschbacher, M. D. Science 1987, 238, 491–497.
(12) (a) Andronati, S. A.; Karaseva, T. L.; Krysko, A. A. Curr. Med.
Chem. 2004, 11, 1183–1211. (b) Cacciari, B.; Spalluto, G. Curr. Med. Chem.
2005, 12, 51–70. (c) Carlson, C. B.; Mowery, P.; Owen, R. M.; Dykhuizen,
E. C.; Kiessling, L. L. ACS Chem. Biol. 2007, 2, 119–127.
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143–148.
66
Org. Lett., Vol. 11, No. 1, 2009

Scheme 3
.
Solid-Phase Synthesis of Steroid-Peptide Chimeras
Table 1. Structures of Variable Amino Acid Segments and
Steroid-Spiroketal-Tripeptide (R-X-D) Products
product. Alternative aminolysis protocols, including benzyl-
amine-catalytic sodium cyanide,17 lithium aluminum ben-
zylamide,18 and benzylamine-dimethylaluminum complex,19
were also unproductive. In contrast, the preparation of
Scheme 4. Solid-Phase R-X-D Triple Hybrid Library Synthesis
a All products were synthesized in a manner analogous to the procedure
shown in Scheme 3. b Isolated yield after RP-HPLC. c SPPS was conducted
on 0.05 mmol of resin as opposed to 0.1 mmol for all other entries.
chimera 13 succeded in good overall yield on FMPB-AM
Rink amide resin (Scheme 3).20,21
Reductive amination of aldehyde 7 with benzylamine was
followed by iterative coupling of 8 under microwave
conditions22 to suitably N- and side chain protected amino
acid building blocks. After conjugation of tripeptide chain
11 to acid 5, the resin was cleaved under global deprotection
conditions to provide the chimera sequence 13. This product
was purified by RP-HPLC and submitted to a cell-based
(17) Hoegberg, T.; Stroem, P.; Ebner, M.; Raemsby, S. J. Org. Chem.
1987, 52, 2033–2036.
(18) Solladie-Cavallo, A.; Bencheqroun, M. J. Org. Chem. 1992, 57,
5831–5834.
Org. Lett., Vol. 11, No. 1, 2009
67

adhesion assay for high-throughput screening of regulators
of the leukocyte-specific integrin CD11b/CD18, a validated
therapeutic target for inflammatory diseases.23,24
6.5 µM, respectively. These assay data validated our
hypothesis and provide the basis for planned expansions of
the chimera motif for integrin antagonist design.
In conclusion, we have successfully extended our oxidative
enol ether rearrangement methodology toward the construc-
tion of steroidal RGD mimics. These chimeras contain
structural elements from three major classes of natural
products, i.e., steroids, spiroketals, and peptides. The biologi-
cal studies provided evidence for potent integrin CD11b/
CD18 antagonistic effects for the glycine-containing 13 and
its corresponding N-debenzylated primary amide analog.
Further work will mainly focus on determining selectivity
and binding sites for these and related antagonists.
In addition to 13, additional RGD encoded triple hybrid
chimeras were prepared for biological evaluations as well
as for exploring the scope of our synthetic strategy (Scheme
4, Table 1). Coupling steps were conducted in the microwave
at 40 W for 5 min at 70 °C. The coupling with Fmoc-
Asp(OtBu)-OH was repeated with 2.5 equiv of acid to ensure
complete loading. For other acids, a single acylation with
3.5 equiv of Fmoc-amino acid was used, and 2.0 equiv of 5
were employed in the final amide bond formation. The
cleavage of the Fmoc groups with 20% piperidine in DMF
also took advantage of microwave heating (50 W, 3 min, 50
°C). Products were released from the resin by treatment with
a TFA cleavage cocktail at room temperature for 2 h.
In the design of our chimera, we hypothesized that the
steroid scaffold would anchor the peptide in the membrane,
the spiroketal linker would rigidly project the peptide strand
and provide selectivity, and the replacement of the glycine
residue in the RGD sequence would induce features such as
conformational preorganization, rigidity, ꢀ-turn stabilization,
and resistance toward proteolytic degradation. Thus, we
synthesized both L- and D-proline containing scaffolds 15
and 17. Similarly, the Fmoc-protected enantiomeric γ-amino-
R,ꢀ-cyclopropyl acids 18 and 20 were used to introduce turn
structures.25 ꢀ-Amino acids also have a profound effect on
secondary sturctures and are finding increasing applications
in peptide mimicry.26 The ꢀ-amino acids 22 and 2427 were
readily inserted into the triple hybrid scaffold. Finally, for
cellular localization studies, we prepared the fluorescein
labeled derivative 27 by inserting the modified Fmoc-lysine
residue 2628 into the R-X-D tripeptide sequence.
Acknowledgment. This work was supported by the
NIGMS CMLD program (GM067082). We thank Ms.
Stephanie Nicolay, Mr. David Turner, and Mr. Pete Cham-
bers at the Pittsburgh CMLD and Mr. Michael Hedrick and
Mr. Brock T. Brown at the Burnham Center for Chemical
Genomics for their valuable contributions. The x-ray structure
of 6 was solved by Dr. Steven Geib (University of Pitts-
burgh).
Supporting Information Available: Experimental pro-
cedures and spectral data for all new compounds, including
copies of 1H and 13C NMR spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL802247M
(22) A CEM monomode microwave peptide synthesizer was used, with
individual coupling times of 3-8 min per residue. For other recent
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Lett. 2007, 17, 2329–2333.
The biological evaluation24 of all triple hybrid RGD
mimics revealed no agonist activities. However, two com-
pounds, 13 and the corresponding C-terminal NH2amide,
displayed potent antagonistic effects and IC50’s of 10.9 and
(24) The assay was an adaptation of Park, J. Y.; Arnaout, M. A.; Gupta,
V. J. Biomol. Screen. 2007, 12, 406–417.
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