Chemoenzymatic and ring E-molecular approach to the (-)-podophyllotoxin skeleton. Synthesis of 3',4',5'-tridemethoxy-(-)-podophyllotoxin

Full text of DOI:10.1021/ja961489s

9426
J. Am. Chem. Soc. 1996, 118, 9426-9427
Scheme 1
Chemoenzymatic and Ring E-Modular Approach to
the (-)-Podophyllotoxin Skeleton. Synthesis of
3′,4′,5′-Tridemethoxy-(-)-podophyllotoxin
David B. Berkowitz,*,† Jun-Ho Maeng,† Anne H. Dantzig,‡
Robert L. Shepard,‡ and Bryan H. Norman‡
Department of Chemistry
UniVersity of NebraskasLincoln
Lincoln, Nebraska 68588-0304
Cancer Research, Eli Lilly and Company
Indianapolis, Indiana 46285
ReceiVed May 5, 1996
the first synthesis and biological characterization of 3′,4′,5′-
tridemethoxy-(-)-podophyllotoxin (2), the ring E deoxygenated
analogue of (-)-podophyllotoxin.
(-)-Podophyllotoxin (1) acts as an antimitotic, inhibiting
tubulin assembly. Its semisynthetic derivatives, etoposide (3)
and teniposide (4), though not antimitotics, are important clinical
chemotherapeutic agents.1 Several in Vitro studies assign
functional roles to ring E in etoposide. For example, etoposide
promotes topoisomerase II-mediated DNA scission,2 and ring
E oxygenation may be required for this activity.3 On the other
hand, etoposide can be “activated” in Vitro by dealkylative
oxidation of ring E to produce derivatives (e.g. the semiquinone
or the o-quinone) capable of cleaving DNA4a or of covalently
binding to proteins4b,c and DNA.4d
Podophyllotoxin has captured the attention of organic chem-
ists for some time.5 Yet only recently have enantioselective
approaches to the natural product appeared.6,7 Philosophically,
our approach differs from these syntheses in two fundamental
ways: (1) absolute stereochemistry is introduced catalytically,
by means of an enzyme-catalyzed transformation upon an
unnatural substrate,8,9 and (2) ring E is introduced as late as
possible in the synthesis (Scheme 1).
Among several meso intermediates of the general structure
7, 10 proved to be the most useful as an enzyme substrate.
Diacetate 10 is readily constructed in seven steps (45% yield;
Scheme 2).10 The key step is an isobenzofuran Diels-Alder
reaction in which DMAD serves as both solvent and dieno-
phile.11 PPL selectively deacetylates the (R)-acetoxymethyl arm
of 10 to furnish (+)-11 (95% ee) in 83% yield.12
Aldehyde 12 is available from 11 via silylation, deacetylation,
and Swern oxidation (Scheme 3). Acetyl migration is not
(5) For a review of synthetic approaches to the Podophyllum lignans,
see: (a) Ward, R. S. Synthesis 1992, 719-730. For syntheses of (()-
podophyllotoxin, see: (b) Gensler, W. J.; Gastonis, C. G. J. Org. Chem.
1966, 31, 4004-4008. (c) Kende, A. S.; Liebeskind, L. S.; Mills, J. E.;
Rutledge, P. S.; Curran, D. P. J. Am. Chem. Soc. 1977, 99, 7082-7083.
(d) Murphy, W. S.; Wattanasin, S. J. Chem. Soc., Chem. Commun. 1980,
262-263. (e) Kende, A. S.; King, M. L.; Curran, D. P. J. Org. Chem. 1981,
46, 2826-2828. (f) Rajapaksa, D.; Rodrigo, R. J. Am. Chem. Soc. 1981,
103, 6208-6209. (g) Jung, M. E.; Lam, P. Y.; Mansuri, M. M.; Speltz, L.
M. J. Org. Chem. 1985, 50, 1087-1105. (h) Jung, M. E.; Lowen, G. T.
Tetrahedron Lett. 1986, 27, 5319-5322. (i) Van der Eycken, J.; De Clerq,
P.; Vandewalle, M. Tetrahedron Lett. 1985, 26, 3871-3874. (j) Macdonald,
D. I.; Durst, T. J. Org. Chem. 1986, 51, 4749-4750. (k) Vyas, D. M.;
Skonezny, P. M.; Jenks, T. A.; Doyle, T. W. Tetrahedron Lett. 1986, 27,
3099-3102. (l) Kaneko, T.; Wong, H. Tetrahedron Lett. 1987, 28, 517-
520. (m) Peterson, J. R.; Hoang, D. D.; Rogers, R. D. Synthesis 1991, 275-
277. (n) Jones, D. W.; Thompson, A. M. J. Chem. Soc., Chem. Commun.
1987, 1797-1798. (o) Kraus, G. A.; Wu, Y. J. Org. Chem. 1992, 57, 2922-
2925.
(6) (a) Andrews, R. C.; Teague, S. J.; Meyers, A. I. J. Am. Chem. Soc.
1988, 110, 7854-7858. (b) Bush, E. J.; Jones, D. W. J. Chem. Soc., Perkin
Trans. 1 1996, 151-155.
(7) Two formal syntheses of 1 have also been reported. (a) (-)-
Epipodophyllotoxin: Van Speybroeck, R.; Guo, H.; Van der Eycken, J.;
Vandewalle, M. Tetrahedron 1991, 47, 4675-4682. (b) (-)-Neo-
podophyllotoxin: Charlton, J. L.; Koh, K. J. Org. Chem. 1992, 57, 1514-
1516.
However, it remains uncertain whether the degree of oxy-
genation or the oxidation state of ring E is related to the
oncolytic properties of podophyllotoxin or etoposide, in ViVo.
Herein we describe the a synthetic approach to the (-)-podo-
phyllotoxin skeleton that is modular in ring E, as a tool for the
study of its functional role. As proof of principle, we report
* To whom correspondence should be addressed.
† University of Nebraska-Lincoln.
‡ Eli Lilly and Company; inquiries specifically regarding the cytotoxicity
assays should be directed to these authors.
(1) Doyle, T. W. In Etoposide (VP-16). Current Status and New
DeVelopments; Academic Press: New York, 1984.
(2) (a) Robinson, M. J.; Osherhoff, N. Biochemistry 1991, 30, 1807-
1813. (b) Chen, G. L.; Yang, L.; Rowe, T. C.; Halligan, B. D.; Tewey, K.
M.; Liu, L. F. J. Biol. Chem. 1984, 259, 13560-13566 and references
therein.
(8) For recent reviews of chemoenzymatic natural product synthesis,
see: (a) Johnson, C. R. Tetrahedron 1996, 52, 3769-3826. (b) Mori, K.
Synlett 1995, 1097-1109.
(3) It has been suggested that a free 4′-OH is essential for DNA breakage
activity: (a) Long, B. H.; Musial, S. T.; Brattain, M. G. Biochemistry 1984,
23, 1183-1188. (b) Loike, J. D.; Horwitz, S. B. Biochemistry 1976, 15,
5443-5448. However, a related, “ring E”-deoxygenated lignan displays
potent topoisomerase II inhibition activity: (c) Kamal, A.; Atchinson, K.;
Daneshtalab, M.; Micetich, R. G. Anti-Cancer Drug Des. 1995, 10, 545-
554.
(4) (a) Sinha, B. K.; Eliot, H. M.; Kalayanaraman, B. FEBS Lett. 1988,
227, 240-244. (b) Haim, N.; Nemec, J.; Roman, J.; Sinha, B. K. Biochem.
Pharm. 1987, 36, 527-536. (c) Van Maanen, J. M. S.; de Ruiter, C.; de
Vries, J.; Kootstra, P. R.; Gobas, F.; Pinedo, H. M. Eur. J. Cancer Clin.
Oncol. 1985, 21, 1099-1106. (d) Van Maanen, J. M. S.; de Vries, J.; Pappie,
D.; van der Akker, E.; Vincent, M.; Lafleur, M.; Retel, J.; van der Greef,
J.; Pinedo, H. M. Cancer Res. 1987, 47, 4658-4662.
(9) Kutney has described an ambitious biotechnological approach to these
lignans. Thus, the combination of H2O2 and crude plant cell extracts (e.g.,
from P. peltatum or N. sylVestris) has been reported to effect a ring
C-forming cyclization reaction on appropriately substituted dibenzylbu-
tanolides. However, in these cyclizations the unnatural stereochemistry at
C1 (S) apparently predominates (i.e., for C2-H: dd, J ) 11, 14 Hz) providing
epiisopodophyllotoxins: (a) Kutney, J. P.; Du, X.; Naidu, R.; Stoynov, N.
M.; Takemoto, M. Heterocycles 1996, 42, 479-484. (b) Kutney, J. P.; Chen,
Y. P.; Gao, S.; Hewitt, G. M.; Kuri-Brena, F.; Milanova, R. K.; Stoynov,
N. M. Ibid. 1993, 36, 13-20.
(10) Bromination of piperonal proceeds in 84% yield: (a) Conrad, P.
C.; Kwiatkowski, P. L.; Fuchs, P. L. J. Org. Chem. 1987, 52, 586-591.
Acetalization provides 8 in 96% yield: (b) Keay, B. A.; Plaumann, H. P.;
Rajapaksa, D., Rodrigo, R. Can. J. Chem. 1983, 61, 1987-1995.
S0002-7863(96)01489-8 CCC: $12.00 © 1996 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 118, No. 39, 1996 9427
Scheme 2a
Scheme 3a
a (a) n-BuLi, THF, (CH2O)n, 82%; (b) HOAc, DMAD, 80 °C, 92%;
(c) H2, Pd/C, 99%; (d) LiAlH4, Et2O, reflux, 88%; (e) Ac2O, Pyr,
DMAP, -5 °C, 100%; (f) PPL, 10% DMSO 50 mM KPO4 buffer, pH
8, 66% (83% corr.), 95% ee.
observed under the silylation conditions nor is silyl migration
observed under the deacetylation conditions as established by
Mosher esterification.13 Efficient retro-Michael ring opening14
of 12 unveils the (methylenedioxy)cinnamyl system 5 envisioned
as a vehicle for the introduction of ring E. Following protection
of the C4-OH and aldehyde oxidation, the acyl oxazolidinone
functionality is installed.
Michael acceptor 14 was designed to bias the system toward
conjugate addition from the re face with the following con-
siderations: (a) the TIPS ether might sterically block approach
from the si face, (b) the SEM ether could conceivably coordinate
to copper to direct re face attack,15 and (c) the relatively
sterically demanding substituents at C3 and C4 might be disposed
pseudoequatorially in the transition state, thereby enforcing re
face addition of the cuprate (pseudoaxial trajectory of approach).
Pleasingly, CuI-mediated conjugate addition of PhMgBr to 14
occurs exclusively from the desired re face and is quite efficient
(78%, Scheme 3).
a (a) TIPSCI, imidazole, DMF, rt, 100%; (b) K2CO3, MeOH, rt, 97%;
(c) (COCl)2, DMSO, CH2Cl2, NEt3, -78 °C, 100%; (d) NaOMe, MeOH,
rt, 90%; (e) SEMCl, i-Pr2NEt, CH2Cl2, rt 93%; (f) NaClO2, NaH2PO4,
t-BuOH, 2-methyl-2-butene, 100%; (g) CDI, THF, 100%; then n-BuLi,
oxazolidinone, -78 °C, 60%.
Scheme 4a
a (a) TBAF, THF, 50 °C, 80%; (b) LDA, -78 °C; then pyr-HCl
quench, 94%; 2:1 ratio (trans-cis-lactone); (c) MgBr2, EtSH, Et2O-
PhH (4:1) 0 °C f rt, 17 (32%) and 2 (47%).
Only three steps separate conjugate addition product 15 from
the targeted analogue of (-)-podophyllotoxin 2 (Scheme 4).
Chemoselective TIPS deprotection is effected by careful heating
of 15 with TBAF. Following lactonization, epimerization at
C2 using the conditions of Kende et al.5e,16 proceeds smoothly
to provide largely the trans-lactone. Modified Kim conditions
(EtSH, MgBr2)17 convert the previously inseparable cis-lactone
16 into the readily separable thioether 17 and effect SEM
deprotection of the trans-lactone to provide the title compound
2. The facile and stereocontrolled conversion of 14 to 2 in just
four steps attests to the potential of this synthetic route for the
generation of other ring E-modified analogues of (-)-podo-
phyllotoxin.
cytotoxic oncolytic etoposide (IC50 of 1.1 µM). An MDR18 cell
line CEM/VLB100, selected with vinblastine, is 12-fold resis-
tant to etoposide (IC50 of 12.9 µM). This resistant cell line
is, however, nearly as sensitive to 1 and 2 (IC50s of 11 and
57 nM, respectively) as is the parent cell line.19 Thus 2
maintains the favorable MDR profile of the natural product.
Therefore, the degree of oxygenation in ring E is apparently
not a strong determinant of either cytotoxicity or MDR profile
in the (-)-podophyllotoxin series. Its effects in the etoposide
series remain to be investigated.
Acknowledgment. Financial support from the American Cancer
Society (DHP-151) is gratefully acknowledged. This research was
facilitated by a seed grant from the Nebraska State Department of Health
(Cancer and Smoking Disease Program). Thanks are due to V. W.
Day and T. A. Eberspacher for X-ray crystallography,12 to R. K.
Shoemaker for technical assistance, and to the NIH (SIG 1-S10-
RR06301) and NSF (CHE-93000831) for NMR and GC/MS instru-
mentation, respectively. J.-H.M. thanks the UNL Center for Biotech-
nology for a fellowship.
Cytotoxicity Data. 1 and 2 both display potent cytotoxicity
against a drug-sensitive human leukemia CCRF-CEM cell line
(IC50s of 8 and 34 nM, respectively), in contrast to the less
(11) For a review of the use of isobenzofurans in natural product
synthesis, see: Rodrigo, R. Tetrahedron 1988, 44, 2093-2135.
(12) Porcine pancreatic lipase (PPL) was purchased from Sigma (10¢/
g) and displays largely one band at MW ≈ 50-52 kDa on SDS-PAGE
(see Supporting Information). The absolute stereochemistry of 11 was
established from the X-ray structure of its Mosher ester derived from (R)-
R-methoxy-R-(trifluoromethyl)phenylacetyl chloride.13 For details, see:
Berkowitz, D. B.; Maeng, J.-H. Tetrahedron: Asymmetry 1996, 7, 1577-
1580.
Supporting Information Available: Complete characterization data
and copies of the 1H NMR spectra for all synthetic intermediates;
comparison 1H NMR spectra of 2 and (-)-podophyllotoxin; 1H NMR
spectra of the Mosher esters used to determine the enantiomeric purity
of 11, and a photograph of an SDS-PAGE gel of PPL (29 pages). See
any current masthead page for ordering and Internet access instructions.
(13) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34,
2543-2549.
(14) Keay, B. A.; Rajapaksa, D.; Rodrigo, R. Can. J. Chem. 1984, 62,
1093-1098.
(15) (a) Hanessian, S.; Sumi, K. Synthesis 1991, 1083-1089. (b)
Hanessian, S.; Thavonekham, B.; DeHoff, B. J. Org. Chem. 1989, 54, 5831-
5833. (c) Dorigo, A. E.; Morokuma, K. J. Am. Chem. Soc. 1989, 111, 6524-
-6536.
JA961489S
(18) For a recent review, see: Bellamy, W. T. Annu. ReV. Pharmacol.
Toxicol. 1996, 36, 161-183.
(19) (a) Beck, W. T.; Mueller, M. J.; Tanzer, L. R. Cancer Res. 1979,
39, 2070-2076. (b) Denizot, F.; Lang, R. J. Immunol. Methods 1989, 89,
271-277.
(16) For an early articulation of this concept, see: Zimmerman, H. E. J.
Org. Chem. 1955, 20, 549-557.
(17) Kim, S.; Kee, I. S.; Park, Y. H.; Park, J. H. Synlett 1991, 183-184.