Stereocontrolled synthesis of a novel pharmacophore of the tubulin- depolymerizing marine natural product spongistatin

Article abstract of DOI:10.1016/S0040-4039(00)00054-X

We report here the synthesis and X-ray structure of a novel spiroketal pyran, SPIKET-P1, as a pharmocophore for the tubulin-depolymerizing marine natural product spongistatin 1. Following its retro-synthetic analysis, SPIKET-P1 was prepared using a versatile 11-step synthetic scheme in a stereocontrolled fashion. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00054-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 1699–1702
Stereocontrolled synthesis of a novel pharmacophore of the
tubulin-depolymerizing marine natural product spongistatin
He Huang, Chen Mao, Shyi-Tai Jan and Fatih M. Uckun ∗
Drug Discovery Program, Departments of Chemistry and Structural Biology and Parker Hughes Cancer Center,
Parker Hughes Institute, St. Paul, MN 55113, USA
Received 6 December 1999; accepted 26 December 1999
Abstract
We report here the synthesis and X-ray structure of a novel spiroketal pyran, SPIKET-P1, as a pharmocophore for
the tubulin-depolymerizing marine natural product spongistatin 1. Following its retro-synthetic analysis, SPIKET-
P1 was prepared using a versatile 11-step synthetic scheme in a stereocontrolled fashion. © 2000 Elsevier Science
Ltd. All rights reserved.
Spongistatins are macrocyclic lactone compounds containing six pyran type rings and four of the six
pyran rings are incorporated into two spiro[5,5]ketal moieties.1 Spongistatin 1 (SP) (R=Cl, Fig. 1) is a
potent tubulin-depolymerizing natural product which has been isolated from an Eastern Indian Ocean
sponge in the genus Spongia.1,2 SP exhibited potent cytotoxicity with subnanomolar IC50 values against
the members of the NCI panel of 60 human cancer cell lines.1 We examined the electron crystallographic
structure of tubulin3 using graphics programs including Grasp and InsightII to identify a possible binding
site for SP which would have suitable dimensions to accommodate the large molecular volumn of SP and
contain a cluster of hydrophobic residues near the protein surface. The search resulted in the discovery
of a unique candidate binding pocket on the tubulin surface which is large enough to accommodate
SP and can also provide a highly hydrophobic environment for extensive molecular interactions.4 The
SP molecule was docked into this candidate binding site using the affinity module within the InsightII
program. The docking simulation results indicated that the putative SP binding pocket which is located
on the surface of tubulin is approximately 8 Å wide×18 Å long×11 Å deep. The pocket consists of an
unusual cluster of 10 aromatic residues situated in close proximity that includes Y108, W103, Y185,
W407, Y408, F399, F404, F395, F418 and H406 (Fig. 1B).
Advanced modeling studies of the interactions of SP with this putative SP binding pocket indicated
that the two spiroketal groups of SP are in close contact with protein residues lining the binding pocket
and may therefore serve as the critical binding components of SP. The identification of the spiroketal
subunits as the likely tubulin binding elements of SP prompted the hypothesis that simple synthetic
spiroketal pyrans (‘SPIKET-P compounds’) representing these subunits, such as compound SPIKET-P1
∗
Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00054-X
tetl 16334

1700
Fig. 1. Modeling design of SPIKET-P as a new pharmocophore of tubulin-depolymerizing agents. A: Structures of spongistatin
and SPIKET-P1. B: SPIKET-P1 as a spongistatin pharmacophore. The spiroketal ring is sandwiched between phenylalanine 404
and tryptophan 407 and close to histidine 406. The spiroketal ring of the SPIKET-P1 is in close contact with the surrounding
hydrophobic residues
(Fig. 1A), could serve as SP pharmacophores.4 Docking studies indicated that, when bound to tubulin,
the spiroketal ring of SPIKET-P1 would be sandwiched between aromatic residues F404 and W407 in the
binding pocket and would provide favorable hydrophobic interactions and van der Waals contacts with
these residues (Fig. 1B). The spiroketal group of SPIKET-P1 has a molecular surface of 218 Å,2 75% of
which would be covered by the aforementioned two aromatic rings. Therefore, SPIKET-P1 was selected
as our first synthetic target.
Retro-synthetic analysis (Scheme 1) was started by converting the spiroketal group in SPIKET-P1 to
the carbonyl and hydroxyl groups in 1, which was further converted to two segments 2 and 3. Both 2 and
3 could be derived from the commercially available benzyl (R)-(−)-glycidyl ether 5. The synthesis was
initiated by opening the commercially available epoxide 5 using vinylmagnesium bromide to obtain the
alcohol 6 which was protected as a tert-butyldimethylsilyl ether to form 7 (Scheme 2). Hydroboration
of the terminal olefin in 7 yielded the primary alcohol 8, which was then converted into the mesylate 4.
The mesylate group in 4 was substituted by bromide to form 3. Also the mesylate in 4 was substituted
by cyanide to form 9 which was further converted to aldehyde 2 by DIBAL reduction followed by acid-
Scheme 1. Retro-synthetic analysis

1701
Scheme 2. Stereocontrolled synthesis and structural characterization of the SP pharmacophore SPIKET-P1:6 (a) vinylMgBr,
CuBr, 2 h, 0°C; 81%. (b) TBDMSCl, imidazole, DMAP, 1 h at 0°C, 3 h at rt; 98%. (c) (1) BH3–THF, (2) H2O2, NaOH; 70%.
(d) MsCl, Et3N, 2 h, 0°C; 94%. (e) NaCN, DMSO, 15-crown-5, 40°C, overnight; 95%. (f) DIBAL-H, −78°C, 3 h, 10% tartaric
acid; 93%. (g) LiBr, acetone, reflux, 0.5 h; 80%. (h) Mg/THF; 78%. (i) oxalyl chloride, DMSO, Et3N, −78°C; 83%. (j) 5%
HF/CH3CN, 0.5 h, rt; 100%. (k) LDBB, 0°C; 50%
Fig. 2. X-Ray crystal structure of SPIKET-P1, molecule D (30% probability ellipsoids, T=22°C, solvent=chloroform/ether).
SPIKET-P1: Space group: P21, unit cell: a=11.4510(15) Å, b=8.9628(11) Å, c=22.942(3) Å, α=90°, β=93.396(3)°, γ=90°,
volume=2350.5(5) Å3, Z=8, θ range for data collection=1.78 to 28.32° (λ=0.71073 Å), total reflections collected=12806,
independent reflections=9044, data/restraints/parameters=9044/1/542, R1 [I>2σ(I)]=0.139, wR2=0.187, goodness-of-fit on
F2=1.009
catalyzed hydrolysis in a one pot reaction. Compound 3 was coupled with 2 by first converting 5 to a
Grignard reagent and then reacting it with aldehyde 2 to form 10 (Scheme 2). Swern oxidation converted
10 to 1. Deprotection of the two tert-butyldimethylsilyl protected hydroxyl groups in 1 followed by
acid-catalyzed acetal formation to give 11, the immediate benzyl-protected precursor of SPIKET-P1,
was carried out in a one pot reaction by treating 1 with 5% HF in acetonitrile at room temperature

1702
for 30 minutes. After the unsuccessful attempt to remove the two protecting benzylic groups in 11 by
platinum-catalyzed hydrogenation, SPIKET-P1 was obtained by treating 11 with lithium 4,40-di-tert-
butylbiphenylide (LDBB).5
The structure of SPIKET-P1 was confirmed by X-ray crystallography (Fig. 2). SPIKET-P1 caused
tubulin-depolymerization and exhibited potent cytotoxicity against taxol-resistant and vincristine-
resistant human cancer cells at nanomolar concentrations.7
References
1. Pettit, G. R.; Cichacz, Z. A.; Gao, F.; Herald, C. L.; Boyd, M. R. J. Org. Chem. 1993, 58, 1302.
2. Bai, R.; Cichacz, Z. A.; Herald, C. L.; Pettit, G. R.; Hamel, E. Mol. Pharmacol. 1993, 44, 757; Bai, R.; Taylor, G. F.; Cichacz,
Z. A.; Herald, C. L.; Kepler, J. A.; Pettit, G. R.; Hamel, E. Biochemistry 1995, 34, 9714.
3. Nogales, E.; Whittaker, M.; Milligan, R. A.; Downing, K. H. Cell 1999, 96, 79; Nogales, E.; Wolf, S. G.; Downing, K. H.
Nature 1998, 391, 199.
4. Mao, C.; Huang, H.; Jan, S.-T.; Navara, C.; Narla, R. K.; Uckun, F. M. Design and Synthesis of Novel Functional Analogs
of Spongistatin as Anti-Cancer Agents. Proceedings of the American Association for Cancer Research (AACR 90th Annual
Meeting), Vol. 40; Pharmacology and Experimental Therapeutics 36 — Novel Agents and Mechanisms of Action; Abstract
#4108; p. 623, Philadelphia, PA; April 10–14, 1999.
5. Cohen, T.; Jeong, I.-H.; Mudryk, B.; Bhupathy, M.; Awad, M. M. A. J. Org. Chem. 1990, 55, 1528.
6. Characterization data: [(2R,8R)-8-(hydroxymethyl)-1,7-dioxaspiro[5,5]undec-2-yl]methan-1-ol (SPIKET-P1): 1H NMR (300
MHz, CDCl3) δ 1.26–1.53 (m, 6H), 1.63 (m, 4H), 1.88 (m, 2H), 2.14 (bs, 2H), 3.52 (dd, J=11.0, 7.0 Hz, 2H), 3.62 (dd, J=11.0,
3.5 Hz, 2H), 3.75 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 96.0, 69.7, 66.2, 35.2, 26.3, 18.2; GC/MS m/z 216 (M+); IR (neat):
3373, 2939, 2871 cm−1; [α]D22 −59.4 (c 0.7, CHCl3).
7. Uckun, F. M.; Mao, C.; Jan, S.-T. Synthetic spiroketal pyrans (SPIKET-P) as a novel class of tubulin-depolymerizing anti-
cancer agents. AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics, Abstract #269;
Washington, DC; November 16–19, 1999.