Synthesis of stipiamide and a new multidrug resistance reversal agent, 6,7-dehydrostipiamide

Full text of DOI:10.1021/ja9641923

J. Am. Chem. Soc. 1997, 119, 2327-2328
2327
Synthesis of Stipiamide and a New Multidrug
Resistance Reversal Agent, 6,7-Dehydrostipiamide
Merritt B. Andrus*,† and Salvatore D. Lepore
Purdue UniVersity, Department of Chemistry
West Lafayette, Indiana 47907
ReceiVed December 5, 1996
Stipiamide,1 a new member of a growing class of insecticidal
polyene antibiotics,2 has recently been patented (under the name
phenalamid A1) for its anti-HIV and antifungal activity.3 Most
remarkably, this compound has also been shown to reverse
P-glycoprotein-mediated multidrug resistance (MDR), a condi-
tion common to many cancer cell lines.4 The ability to
potentiate the cytotoxicity of antitumor drugs toward drug
resistant cells has been demonstrated by verapamil, cyclosporin-
A,5 GF120918,6 and more recently hapalosin.7 However, the
clinical performance of MDR reversal agents has not been
adequate, making the identification of new agents of great
importance.8 To this end a flexible route to stipiamide was
pursued to make possible the synthesis of new MDR reversal
agents. In spite of their wide range of activity, a synthetic route
to the polyene antibiotics has not been reported.9 Herein, we
report the first total synthesis of stipiamide and the design and
synthesis of 6,7-dehydrostipiamide (a new non-natural com-
pound), now shown to potently reverse MDR in human MCF-7
adriamycin resistant breast cancer cells (MCF-7adrR).10 Also,
6,7-dehydrostipiamide is remarkably less toxic relative to
stipiamide.
The vinyl iodide was derived from an alcohol containing both
a mono and trisubstituted olefin. A critical problem to be
overcome then was the selective oxidative cleavage of the
terminal olefin. The vinyltin fragment was planned to be
assembled using a higher order tributylstannyl cuprate to
acetylene to alkynyl ester tandem addition reaction, the first
synthetic application of this kind.
The synthesis of the left side of stipiamide began with acyl
oxazolidinone 1 methylated according to the procedure of
Evans11 with 25:1 selectivity. The mixture of diastereomers
was reacted with LAH to remove the auxiliary, followed by
oxidation to the aldehyde, and treatment with (carbethoxyethy-
lidine) triphenylphosphorane to give 2 as an 8:1 diastereomeric
mixture which was not separated. The unsaturated aldehyde
was generated using DIBAL followed by tetrapropylammonium
perruthenate (TPAP), 4-methylmorpholine N-oxide (NMO)
oxidation.12 Reaction with diisopinocampheyl E-crotylborane
derived from (-)-(R)-pinene according to Brown13 then gave
the anti-homoallylic alcohol 3 in good overall yield. Attempted
modification of the Evans aldol reaction using added aluminum
chloride to give the anti-product14 was explored without success.
Use of the efficient crotylborane addition then required the
development of a selective olefin oxidation. Differential olefin
dihydroxylation proceeded very slowly and with little selectivity
using catalytic OsO4 and NMO.15 The problem was overcome
by simply reacting 4 with Sharpless’ AD-mix-R reagent in tert-
butyl alcohol and water.16 The bulky cinchona alkaloid ligand‚
OsO4 complex cleanly favored reactivity at the terminal olefin
The challenging (E,E,Z,E,E)-olefin array common to all
members of the family was envisioned to arise from a Stille
coupling as the final step as shown.
† email: mandrus@chem.purdue.edu.
(1) Trowitzsch-Kienast, W.; Forche, E.; Wray, V.; Riechenbach, H.;
Hunsmann, G.; Ho¨fle, G. Liebigs Ann. Chem. 1992, 659.
(2) Jansen, R.; Reinfenstrahl, G.; Gerth, K.; Reichenbach, H.; Ho¨fle, G.
Liebigs Ann. Chem. 1983, 1801.
(3) Ho¨fle, G.; Bedorf, N.; Jansen, R.; Steinmetz, H.; Trowitzsch-Kienast,
W.; Reichenbach, H.; Forche, E.; Gerth,K.; Irschik, H.; Kunze, B.; Sasse,
F.; Hunsmann, G.; Jurkiewicz, E. Ger. Patent, DE 4041686A1, 1990.
Hunsmann, G.; Jurkiewicz, E.; Reichenbach, H.; Forche, E.; Gerth, K.;
Irschik, H.; Kunze, B.; Sasse, F.; Ho¨fle, G.; Bedorf, N.; Jansen, R.;
Steinmetz, H.; Trowitzsch-Kienast, W. Ger. Patent, DE 4041688A1, 1990.
(4) Kim, Y. J.; Furihata, K.; Yamanaka, S.; Fudo, R.; Seto, H. J. Antibiot.
1991, 44, 553.
(5) Ford, J. M.; Hait, W. N. Pharacol. ReV. 1990, 42, 155.
(6) Hyafil, F.; Vergely, C.; Du, V. P.; Grand, P. T. Cancer Res. 1993,
53, 4595.
(7) (a)Dihn, T. Q.; Armstrong, R. W. J. Org. Chem. 1995, 60, 8118. (b)
Stratmann, K.; Burgoyne, D. L.; Moore, R. E.; Patterson, G. M. L. J. Org.
Chem. 1994, 59, 7219.
(8) (a) Ford, J. M. Eur. J. Cancer 1996, 32A, 991. (b) Raderer, M.;
Scheithauer, W. Cancer 1993, 72, 3553.
(11) Evans, D. A.; Ennis, M. D.; Mathre, D. J. J. Am. Chem. Soc. 1982,
104, 1737.
(12) Griffith, W. P.; Ley, S. V. Aldrichim. Acta 1990, 23, 13.
(13) Brown, H. C.; Bhat, K. S. J. Am. Chem. Soc. 1986, 108, 293.
(14) Walker, M. A.; Heathcock C. H. J. Org. Chem.1991, 56, 5747.
(15) Provencal, D. P.; Gardelli, C.; Lafontaine, J. A.; Leahy, J. W.
Tetrahedron Lett. 1995, 36, 6033.
(9) A partial synthesis of myxalamide D, a member of this polyene class,
has been reported. Cox, C. M.; Whiting, D. A. J. Chem. Soc., Perkin Trans.
I 1991, 1901, 1907.
(10) Fairchild, C. R.; Ivy, S. P.; Kao-Shan, C. S.; Whang-Peng, J.; Rosen,
N.; Israel, M. A.; Melera, P. W.; Cowan, K. H.; Goldsmith, M. E. Cancer
Res. 1987, 47, 5141.
(16) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem. ReV.
1994, 94, 2483.
S0002-7863(96)04192-3 CCC: $14.00 © 1997 American Chemical Society

2328 J. Am. Chem. Soc., Vol. 119, No. 9, 1997
Communications to the Editor
giving diol in 87% yield (∼1:1 de) with no product resulting
from oxidation of the internal trisubstituted olefin. The aldehyde
was then generated using sodium periodate, followed by the
Wittig reaction to provide unsaturated ester 5 in good yield and
selectivity. The resulting alcohol product of DIBAL reduction
of 5 was carefully purified by radial chromatography to remove
the minor diastereomers resulting from the two Wittig and the
crotyl boration reactions. This pure alcohol was then subjected
to TPAP/NMO to access the intermediate enal. Takai reaction16
using iodoform and flame dried chromous chloride was per-
formed in THF to produce vinyl iodide 6 in 70% yield with
high 20:1 E:Z selectivity.
A similar ratio of isomers was also observed in the authentic
material suggesting that stipiamide readily isomerizes once
formed.22 As a first step toward improved MDR reversal, 6,7-
dehydrostipiamide was designed and obtained from vinyl iodide
10 using Castro-Stephens coupling with acetylene 1123 in 50%
yield.24
MCF-7adrR in Vitro assays were then performed using
synthetic stipiamide and 6,7-dehydrostipiamide. Minimal re-
versal of MDR in the presence of chemotherapeutic drugs except
in the case of colchicine with stipiamide was found. This
synergistic effect was earlier noted with natural stipiamide using
colchicine resistant KB cells.4 Synthetic stipiamide in the
absence of the drug also proved to be highly toxic (ED50
)
0.03 nM, MCF-7adrR cells) possibly arising from ATP-synthesis
inhibition as noted previously with other members of the polyene
class.2 Conversely, we were gratified to find that 6,7-dehy-
drostipiamide showed excellent inhibition of MDR with near
complete removal of toxicity (ED50 ) 1 µM)! With 6,7-
dehydrostipiamide present (10 µM), the MCF-7adrR cell line
ceased to exhibit MDR (ED50 ) 0.2 nM for adriamycin).
Remarkably, this simple structural modification, from Z-olefin
to alkyne, greatly lowers toxicity, presumably through abroga-
tion of mitochondrial arrest, while promoting the ability to
interact with P-glycoprotein reversing MDR. For comparison,
in the same assay, a higher concentration of verapamil (22 µM),
the clinical standard, was clearly less effective in MDR reversal
(ED50 ) 10 nM).
Construction of the vinyltin portion of stipiamide began with
tin-cuprate syn addition to acetylene following the precedence
of Marino,17 Fleming, and Pulido.18 While conjugate additions
of the intermediate derived from reaction of a higher order
tributylstannylcuprate with acetylene to enones have been
reported,17,18 additions to alkynyl esters have not. Our route
now highlights the utility of ethyl propiolate as a substrate for
this reaction. The higher order tributylstannylcuprate reagent
was generated from hexabutylditin, methyllithium, and
copper(I) cyanide at -78 °C. Excess acetylene was then added
directly to the cold solution, followed by addition of ethyl
propiolate. After quenching with methanol,20 ester 7 was
obtained in 65% yield and 4:1 (E,Z:Z,Z) selectivity. DIBAL
reduction gave dienyl alcohol from which the pure (2E, 4Z)-
isomer was readily obtained using radial chromatography.
Oxidation with TPAP/NMO followed by a Horner-Emmons
reaction then gave triene ester 8 in good overall yield.
Hydrolysis of 8 was achieved using lithium hydroxide in water
added slowly to a tert-butyl alcohol solution of 8.19 The
resultant acid was coupled with (S)-alaninol using bromotris-
(pyrrolidino)phosphonium hexafluorophosphate (PyBrop)20 to
give 9.
To conclude, the synthesis of stipiamide was accomplished
in 16 steps in 3.7% overall yield using the powerful combination
of a Stille coupling and higher order tributylstannylcuprate
chemistry. The synthesis of new structural variants based on
this motif is currently underway.
Acknowledgment. We are grateful to the donors of the Petroleum
Research Fund administered by the ACS, Purdue Research Foundation,
and Proctor & Gamble for funding. We are also grateful to Professor
G. Ho¨fle for a sample of natural stipiamide, A. Rothwell for mass
spectroscopy, Dr. D. Lee for elemental analysis, and V. Croy for
cytotoxicity assays. Finally, we thank Dr. Ankush Argade for many
helpful discussions.
Supporting Information Available: Complete experimental pro-
cedures and cytotoxicity data for stipiamide and 6,7-dehydrostipiamide
(14 pages). See any current masthead page for ordering and Internet
access instructions.
Completion of the synthesis of stipiamide involved depro-
tection of E-vinyliodide 6 with TBAF to access alcohol 10. Stille
coupling of 10 and 9 employed catalytic palladium(II) chloride
bis-acetonitrile in N-methylpyrrolidinone21 to give in 80% yield
a 4:2:1 mixture of (-)-stipiamide, its all trans, and its 4Z isomer
respectively as indicated by NMR, HRMS, and optical rotation.
JA9641923
(22) Coste, J.; Frerot, E.; Jouin, P.; Castro, B. Tetrahedron Lett. 1991,
32, 1967.
(23) Evans, D. A.; Gage, J. R.; Leighton, J. L. J. Am. Chem. Soc. 1992,
114, 9434.
(24) This process, similar in nature to â-carotene isomerization, is known
to occur with silica gel or light. Doering, W. von E.; Sotiroiu-Leventis, C.;
Roth, W. R. J. Am. Chem. Soc. 1995, 117, 2747.
(25) Amide 11 was synthesized from known (2E,4E)-ethyl 5-(tributyl-
stannyl)-2-methyl-2,4-pentadienoate (see ref 21) by conversion of the
vinyltin to vinylbromide followed by Castro-Stephens coupling with TMS-
acetylene, removal of TMS, hydrolysis, and then amidation.
(26) Nicolaou, K. C.; Veale, C. A.; Webber, S. E.; Katerinopoulos, H.
J. Am. Chem. Soc. 1985, 107, 7515.
(17) Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986, 108,
7408.
(18) (a) Marino, J. P.; Long, J. K. J. Am. Chem.Soc. 1988, 110, 7916.
(b) Piers, E.; Chong, J. M.; Morton, H. E. Tetrahedron Lett. 1981, 22, 4905.
(19) Barbero, A.; Cuadrado, P.; Fleming, I.; Gonzalez, A. M.; Pulido,
F.; Rubio, R. J. Chem. Soc., Perkin Trans. I 1993, 1657.
(20) Corey, E. J.; Katzenellenbogen, J. A. J. Am. Chem. Soc. 1969, 91,
1853.
(21) Evans, D. A.; Black, W. C. J. Am. Chem. Soc. 1993, 115, 4497.