Use of biomimetic diversity-oriented synthesis to discover galanthamine-like molecules with biological properties beyond those of the natural product [3]

Full text of DOI:10.1021/ja016093h

6740
J. Am. Chem. Soc. 2001, 123, 6740-6741
Use of Biomimetic Diversity-Oriented Synthesis to
Discover Galanthamine-Like Molecules with
Biological Properties beyond Those of the Natural
Product
Henry E. Pelish,1,2 Nicholas J. Westwood,1,2 Yan Feng,2
Tomas Kirchhausen,2,3 and Matthew D. Shair*,1,2
Department of Chemistry and Chemical Biology
HarVard UniVersity, Cambridge Massachusetts 02138
Institute of Chemistry and Cell Biology
Department of Cell Biology, and
Center for Blood Research
HarVard Medical School, Boston, Massachusetts 02115
ReceiVed April 26, 2001
ReVised Manuscript ReceiVed May 31, 2001
Natural products are central to biology and medicine, serving
as pharmaceutical leads, drugs,4 and powerful reagents for
studying cell biology.5 To date, libraries based upon natural
products have been synthesized primarily for the purpose of
improving the known biological and pharmacokinetic properties
of the parent natural products.6,7 In contrast, we have used
diversity-oriented synthesis to construct a library based on a
natural product, galanthamine (2, Figure 1), with the goal of
discovering molecules that exhibit biological effects beyond those
previously associated with the natural product. Although 2 is a
potent acetylcholinesterase inhibitor,8 our aim was not to improve
this activity. Galanthamine was selected because it offered a range
of functionality for diversity-generating reactions, it presented a
rigid polycyclic core that might lower the potential entropy penalty
associated with protein binding, and it allowed for the use of
powerful biomimetic reactions in the synthesis.9 In this com-
munication, we report a biomimetic solid-phase synthesis of 2527
molecules based on the alkaloid natural product galanthamine and
the identification of a molecule from the library that perturbs the
secretory pathway in mammalian cells10sa process unrelated to
the acetylcholinesterase inhibitory activity of 2.
Figure 1. Biomimetic diversity-oriented synthesis parallels the biosyn-
thesis of galanthamine.
rene beads through a Si-O bond to generate 5 upon deprotection
(Scheme 1).13 Reductive amination14 and protecting-group adjust-
ments produced 7. Exposure of 7 to PhI(OAc)215 afforded 8 which
was then converted to 9 via Pd-mediated deprotection and spon-
taneous cyclization. For the library synthesis, building blocks were
selected that reacted in >80% yield and as a group possessed
diverse physical characteristics. (Figure 2). The first diversity step
was accomplished by coupling the phenol of 9 with five primary
alcohols to afford 10 (Scheme 1).16 Treatment of 10 with thiols
n
in the presence of BuLi afforded 11 as a single diastereomer.17
The nitrogen of 11 was either acylated or alkylated, providing
compounds that would be neutral or positively charged, respec-
tively, at physiological pH. The last diversification step involved
treatment of 12 with hydrazines and hydroxylamines, generating
13.18
The library was prepared as a single copy (1 bead per library
member), arrayed in 384-well plates (1 bead per well), and
detached from the solid-support with HF-pyridine (13 f 14).
Following completion of the synthesis, the presence of 2527
out of 2946 (86%) potential compounds was confirmed by mass
spectrometry.19 Evaporation of the cleavage reaction solution and
resuspension in 7 µL of DMSO afforded 2527 stock solutions
for biological screening.
Our library synthesis strategy took advantage of efficient bio-
mimetic reactions (3 f 4),11 paralleling the biosynthesis of the
natural product (1 f 2).12 Following biomimetic solid-phase syn-
thesis of the core structure 4, four diversity-generating reactions
were performed to complete the library synthesis (Figure 1).
The library synthesis commenced with attachment of a tyrosine
derivative to 500-600µm high capacity (1.43 mmol/g) polysty-
* Towhomcorrespondenceshouldbeaddressed.Email: shair@chemistry.harvard.edu.
(1) Department of Chemistry and Chemical Biology, Harvard University.
(2) Institute of Chemistry and Cell Biology (ICCB), Harvard Medical
School.
(3) Department of Cell Biology, and Center for Blood Research, Harvard
Medical School.
(4) Newman, D. J.; Cragg, G. M.; Snader, K. M. Nat. Prod. Rep. 2000,
17, 215-234.
(5) Schreiber, S. L. Chem. Eng. News 1992 (October 26), 22-32.
(6) Hall, D. G.; Manku, S.; Wang, F. J. Comb. Chem. 2001, 3(2), 125-
150.
(7) (a) Nicolaou, K. C.; Vourloumis, D.; Li, T.; Pastor, J.; Winssinger, N.;
He, Y.; Ninkovis, S.; Sarabia, F.; Vallberg, H.; Roschanger, F.; King, N. P.;
Finlay, R. V.; Giannakakou, P.; Verdier-Pinard, P.; Hamel, E. Angew. Chem.,
Int. Ed. Engl. 1997, 36, 2097-2103. (b) Nicolaou, K. C.; Winssinger, D.;
Vourloumis, D.; Ohshima, T.; Kim, S.; Pfefferkorn, J.; Xu, J.-Y.; Li, T. J.
Am. Chem. Soc. 1998, 120, 10814-10826. (c) Lee, K. J.; Angulo, A.; Ghazal,
P.; Janda, K. D. Org. Lett. 1999, 1, 1859-1862. (d) Xu, R.; Greiveldinger,
G.; Marenus, L. E.; Cooper, A.; Ellman, J. A. J. Am. Chem. Soc. 1999, 121,
4898-4899. (e) Wipf, P.; Reeves, J. T.; Balachandran, R.; Giuliano, K. A.;
Hamel, E.; Day, B. W. J. Am. Chem. Soc. 2000, 122, 9391-9395. (f) Boger,
D. L.; Fink, B. E.; Hedrick, M. P. J. Am. Chem. Soc. 2000, 122, 6382-6394.
(g) Nicolaou, K. C.; Pfefferkorn, J. A.; Barluenga, S.; Mitchell, H. J.; Roecker,
A. J.; Cao, G.-Q. J. Am. Chem. Soc. 2000, 122, 9968-9976 and references
therein.
(11) Biomimetic syntheses of galanthamine: (a) Barton, D. H. R.; Kirby,
G. W. J. Chem. Soc. 1962, 806-817. (b) Shimizu, K.; Tomioka, K.; Yamada,
S.; Koga, K. Chem. Pharm. Bull. 1978, 26, 3765-3771. (c) Kita, Y.; Arisawa,
M.; Gyoten, M.; Nakajima, M.; Hamada, R.; Tohma, H.; Takada, T. J. Org.
Chem. 1998, 63, 6625-6633.
(12) Barton, D. H. R.; Cohen, T. Festschrift A. Stoll; Birkhauser: Basel,
1957.
(13) Tallarico, J. A.; Depew, K. M.; Pelish, H. E.; Westwood, N. J.;
Lindsley, C. W.; Shair, M. D.; Schreiber, S. L.; Foley, M. A. J. Comb. Chem.,
2001, 3, 312-318.
(14) Look, G. C.; Murphy, M. M.; Campbell, D. A.; Gallop, M. A.
Tetrahedron Lett. 1995, 36, 2937-2940.
(15) For the use of hypervalent iodine(III) reagents in similar oxidations,
see Krihna, K. V. R.; Sujatha, K.; Kapil, R. S. Tetrahedron Lett. 1990, 31,
1351-1352. Kita, Y.; Takada, T.; Gyoten, M.; Tohma, H.; Zenk, M. H.;
Eichhorn, J. J. Org. Chem. 1996, 61, 5857-5864 and references therein.
(16) “Skip codon” (Figure 2) refers to the absence of a building block which
renders the core structure functionality a diversity element.
(17) Observed by NMR and correlated with molecular modeling. See
Supporting Information.
(8) Coyle, J.; Kershaw, P. Biol. Psychiatry 2001, 49(3), 289-299.
(9) Lindsley, C. W.; Chan, L. K.; Goess, B. C.; Joseph, R.; Shair, M. D.
J. Am. Chem. Soc. 2000, 122, 422-423.
(10) Lippincott-Schwartz, J.; Roberts, T. H.; Hirschberg, K. Annu. ReV.
Cell DeV. Biol. 2000, 16 557-589.
(18) All combinations of 11 were synthesized in parallel for quality control.
Following a pool step, R3 and R4 building blocks were incorporated in parallel
to generate 13. See Supporting Information.
10.1021/ja016093h CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/16/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 27, 2001 6741
Scheme 1a
i
a Reagents: (a) 6, CH(OCH3)3-CH2Cl2, wash then NaBH3CN, AcOH, MeOH-THF, 23 °C. (b) allylchloroformate, Pr2EtN, CH2Cl2, 23 °C. (c)
piperidine, THF, 23 °C. (d) PhI(OAc)2, (CF3)2CHOH-CH2Cl2, 23 °C. (e) Pd(PPh3)4, morpholine-THF, 23 °C. (f) R1OH, PPh3, DIAD, THF, 0 °C
n
(2×). (g) R2SH, 2,6-lutidine, BuLi, THF 0 f 40 °C. (h) R3CHO, AcOH, MeOH-THF, then NaBH3CN in MeOH, 23 °C or R3COCl, 2,6-lutidine,
CH2Cl2, 23 °C or R3NCO, CH2Cl2, 23 °C, (i) R4NH2, AcOH, MeOH-CH2Cl2, 23 °C. (j) HF-pyridine, THF, 23 °C then TMSOMe.
Figure 2. Building blocks used in the library synthesis.
Natural products, such as brefeldin A,20 are powerful reagents
for studying the secretory pathway, a process in cells that shuttles
proteins from the endoplasmic reticulum (ER) to the plasma
membrane via the Golgi apparatus (GA). In an effort to expand
the limited pool of molecules that perturb this process,21 our
library was screened using a cell-based, phenotypic assay. The
fluorescent fusion protein VSVG-GFP was used to monitor the
ability of individual library members to block protein trafficking
(Figure 3).22 Screening at ∼750 nM identified 15 as a potent
inhibitor of VSVG-GFP movement from the GA to the plasma
membrane. In analogy to the yeast sec mutants23 and as a result
of its secondary amine and the phenotype it induces, we have
named this compound secramine. Cell-based experiments with
secramine confirmed its ability to block protein trafficking from
the GA to the plasma membrane at 2 µM.24 Notably, galanthamine
(2) had no observable effect on the secretory pathway at up to
100 µM.
Figure 3. Discovery of sercramine, a galanthamine-based molecule that
perturbs protein trafficking.
In conclusion, a library of 2527 molecules based upon
galanthamine was synthesized using solid-phase biomimetic
reactions. Even though the library was based on a natural product
that had no effect on the secretory pathway, diversity-oriented
synthesis combined with phenotypic screening was successful in
identifying an active molecule that may emerge as an important
probe reagent for exploring protein trafficking. The success of
this synthesis and screen underscores the power of this approach
to discover small molecules with utility in cell biology.25
Acknowledgment. The Harvard ICCB is supported by grants from
the National Cancer Institute, Merck & Co., and Merck KGaA. Additional
support for this project is acknowledged from HFSP (RG-293/98), and
awards to M.D.S from Bristol-Myers Squibb, Astra-Zeneca, the Sloan
Foundation, and Eli-Lilly and to T.K. from the NIH (GM62566). H.E.P
was supported by an NDSEG fellowship. We thank Michel Calderini,
Jason Chen, and Michael Dunn for their assistance.
(19) Bead fragmentation during the synthesis may account for the unidenti-
fied 14% of the potential compounds.
(20) Doms, R. W.; Russ, G.; Yewdell, J. W. J. Cell Biol. 1989, 109, 61-
72. Lippincott-Schwartz, J.; Yuan, L. C.; Bonifacino, J. S.; Klausner, R. D.
Cell 1989, 56, 801-813.
(21) Dinter, A.; Berger, E. G. Histochem. Cell Biol. 1998, 109, 571-590.
(22) Presley, J. F.; Cole, N. B.; Schroer, T. A.; Hirschberg, K.; Zaal, K. J.
M.; Lippincott-Schwartz, J. Nature 1997, 389, 81-85. This screen was
developed by Yan Feng and Tomas Kirchhausen, and its details will be
reported elsewhere.
(23) Novick, P.; Field, C.; Schekman, R. Cell 1980, 21, 205-215.
(24) Cell-staining experiments have confirmed that secramine does not
disrupt microtubule or actin structure. Additional cell-based experiments have
demonstrated that secramine does not perturb clathrin-dependent endocytosis
up to 100 µM.
(25) (a) Gura, T. Nature 2000, 407, 282-284. (b) http://sbweb.med.har-
vard.edu/∼iccb/
Supporting Information Available: Syntheses of solid-phase precur-
sors, library synthesis protocols, MS analysis for the structural assignment
of all 2527 library members, LC analysis for purity assessment, and
representative compound synthesis (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
JA016093H