Analogs incorporating trans-4-hydroxy-L-proline that reverse multidrug resistance better than hapalosin

Full text of DOI:10.1021/jo962180u

790
J . Org. Chem. 1997, 62, 790-791
Raney nickel.8 Cycloamidation of the crude amino acid
An a logs In cor p or a tin g
with bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-
tr a n s-4-Hyd r oxy-L-p r olin e Th a t Rever se
Mu ltid r u g Resista n ce Better Th a n
Ha p a losin
i
Cl) and Pr2NEt (DIPEA) in PhMe at 85 °C9 provided 15
in a good yield of 58% for the two steps. The four proline
analogs 4-7 were easily generated subsequently. The
hydroxyl group of analog 4 was unreactive with benzyl
2,2,2-trichloroacetimidate/TfOH10 and with DIAD/phenol/
PPh3.11 The synthesis was designed to be linear so that
an alcohol different than 12 or 14 could be introduced at
the particular position.
Tam Q. Dinh,1a Charles D. Smith,1b and
Robert W. Armstrong*,1a
Department of Chemistry and Biochemistry, University of
California, Los Angeles, California 90095, and Fox Chase
Cancer Center, Philadelphia, Pennsylvania 19111
The conformational ratios of the five macrolactams in
Scheme 1 are interesting. The three compounds with a
â-PMB ethers15, 4, and 5shave a conformational ratio
from 1.1:1 to 1.5:1 in CDCl3 at 25 °C. By contrast, the
two analogs with a free â-hydroxyl groups6 and 7sexist
as a mixture of about 6:1 conformers. Applying distance
constraints (1.5-5.0 Å) to protons exhibiting NOESY
crosspeaks, computation with Macromodel (v. 4.5)12 using
the AMBER* force field and GB/SA chloroform solvation13
resulted in seven possible conformations for analog 6, all
containing an s-cis amide bond. Analog 7 exhibits
NOESY correlations similar to those for 6.
The anti-MDR activities of synthetic hapalosin and its
analogs (modulators) were determined by cytotoxicity and
drug accumulation assays using MCF-7/ADR cells, which
overexpress P-gp. In the cytotoxicity assay (Figure 1),
cells were exposed to a modulator alone or in the presence
of actinomycin D, daunomycin, or cisplatin. Cisplatin
was included as a non-P-gp substrate to demonstrate
selective enhancement of killing by actinomycin D and
daunomycin, which are transported by P-gp. In the drug
accumulation assay (Figure 2), MCF-7/ADR cells were
treated with a modulator and then incubated with [3H]-
vinblastine, which is also transported by P-gp. Reversal
of MDR is indicated if minimally cytotoxic doses of a
modulator selectively increase cell killing by actinomycin
D and daunomycin and increase the intracellular con-
centration of [3H]vinblastine.
Received November 22, 1996
Chemotherapy often fails over time due to multidrug
resistance (MDR), one of whose principal mechanisms is
expulsion of a wide spectrum of drugs from tumor cells
by P-glycoprotein (P-gp).2 The cyclic depsipeptide hapal-
osin, 1, reverses MDR presumably by inhibiting P-gp.3
Computation with the aid of 1H,1H-NOESY data revealed
that the major s-cis rotamer of hapalosin4 and the single
s-trans rotamer of the non-N-Me analog 2 have very
different conformations, whereas the minor s-trans rota-
mer of hapalosin and analog 2 have very similar confor-
mations.5 Analog 2 and the PMB ether of hapalosin (3)
were found to possess substantially lower anti-MDR
activity than hapalosin. Four proline analogs of hapal-
osin, 4-7, were also synthesized. Analogs 4 and 7
reverse MDR better than hapalosin, while analogs 5 and
6 were less effective than hapalosin.
Comparing the ability of the analogs to potentiate the
cytotoxicity of actinomycin D and daunomycin to that of
synthetic hapalosin (Figure 1), analogs 4 and 7 are better,
analogs 5 and 6 are significantly worse, and the non-N-
Me analog 2 is about two times worse. The PMB ether
of hapalosin (3) has only marginal activity. With regard
to the modulators’ enhancement of the intracellular
concentration of [3H]vinblastine (Figure 2), analogs 4 and
7 are better than hapalosin, while analogs 2, 3, 5, and 6
are significantly worse. None of the modulators altered
the sensitivity of MCF-7/ADR cells to cisplatin or the
sensitivity of MCF-7 cells, which do not express P-gp, to
any of the drugs.
The synthesis of the four proline analogs of hapalosin,
4-7, is illustrated in Scheme 1. trans-4-Hydroxy-L-Pro
(8) was tris-protected as ester 9. The ester was converted
to an aldehyde that underwent Brown allylboration6 to
produce homoallylic alcohol 10 in >90% de. The alcohol
was protected with p-methoxybenzyl 2,2,2-trichloroace-
timidate (PMBTCAI),7 and the olefin was transformed
to acid 11. The acid was coupled to alkenol 125 using
1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC),
and the resulting olefin was oxidized to acid 13. After
the acid was coupled to alcohol 14,5 the Cbz carbamate
and benzyl ester were selectively deprotected in the
presence of the PMB ether by hydrogenation over W-2
The bioassay results suggest that a free hydroxyl and
an aromatic group may be important for the anti-MDR
activity of hapalosin and its analogs.14 Except for the
non-N-Me analog 2, all the analogs less potent than
(1) (a) University of California, Los Angeles. (b) Fox Chase Cancer
Center.
(2) For reviews, see: (a) Simon, S. M.; Schindler, M. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 3497. (b) Gottesman, M. M.; Pastan, I.
Annual Rev. Biochem. 1993, 62, 385.
(3) Stratmann, K.; Burgoyne, D. L.; Moore, R. E.; Patterson, G. M.
L.; Smith, C. D. J . Org. Chem. 1994, 59, 7219.
(4) For the synthesis of hapalosin, see: (a) Dinh, T. Q.; Armstrong,
R. W. J . Org. Chem. 1995, 60, 8118. (b) Ghosh, A. K.; Liu, W.; Xu, Y.;
Chen, Z. Angew. Chem., Int. Ed. Engl. 1996, 35, 74. (c) Ohmori, K.;
Okuno, T.; Nishiyama, S.; Yamamura, S. Tetrahedron Lett. 1996, 37,
3467. (d) Wagner, B.; Beugelmans, R.; Zhu, J . Tetrahedron Lett. 1996,
37, 6557.
(5) Dinh, T. Q.; Du, X.; Armstrong, R. W. J . Org. Chem. 1996, 61,
6606.
(6) J adhav, P. K.; Bhat, K. S.; Perumal, P. T.; Brown, H. C. J . Org.
Chem. 1986, 51, 432.
(7) Nakajima, N.; Horita, K.; Abe, R.; Yonemitsu, O. Tetrahedron
Lett. 1988, 29, 4139.
(8) Horita, K.; Yoshioka, T.; Tanaka, T.; Oikawa, Y.; Yonemitsu, O.
Tetrahedron 1986, 42, 3021.
(9) Evans, D. A.; Miller, S. J .; Ennis, M. D. J . Org. Chem. 1993, 58,
471.
(10) Iversen, T.; Bundle, D. R. J . Chem. Soc., Chem. Commun. 1981,
1240.
(11) Hughes, D. L. Org. React. 1992, 42, 335.
(12) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J .
Comput. Chem. 1990, 11, 440.
(13) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J .
Am. Chem. Soc. 1990, 112, 6127.
(14) NMR experiments on the conformations of modulators in
solvent systems more biologically pertinent than CDCl3 are being
conducted.
S0022-3263(96)02180-9 CCC: $14.00 © 1997 American Chemical Society

Communications
J . Org. Chem., Vol. 62, No. 4, 1997 791
F igu r e 1. Reversal of MDR by hapalosin analogs. MCF-7/ADR cells were incubated with the indicated concentrations of analogs
1-7 (left to right) in the presence of phosphate-buffered saline (as a control) (0), 25 nM actinomycin D (9), or 2 µM daunomycin
(1). Cell survival after 48 h was determined as indicated in the Supporting Information. Values represent the mean ( SD of
triplicate samples.
Sch em e 1
hapalosin (3, 5, and 6) have either two free hydroxyl or
two aromatic groups. By contrast, hapalosin and the two
analogs more potent than it (4 and 7) have a free
hydroxyl and an aromatic group. An objective is to
synthesize analogs that are more potent and water
soluble than 4 and 7 but are less intrinsically cytotoxic.
Ack n ow led gm en t. T.D. and R.A. thank Xiaohui Du
for conducting the computational modeling and the NIH
(Grant No. GM51095) and UCLA for financial support.
Studies on the biological activities of the compounds
were supported by NIH Grant No. CA64983 (C.S.).
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
F igu r e 2. Effects of hapalosin analogs on [3H]vinblastine
cedures for all the steps in Scheme 1; 1H and 13C NMR spectra
accumulation by MDR cells. MCF-7/ADR cells were incubated
and characterization data for compounds 3-7, 9-11, 13, and
with the indicated concentrations of analogs 1 (9), 2 (2), 3 (1),
15; and protocol for the cytotoxicity and drug accumulation
4 ([), 5 (b), 6 (0), or 7 (4). The intracellular accumulation of
[3H]vinblastine was then determined as indicated in the
Supporting Information. Values represent the mean ( SD of
triplicate samples.
assays (32 pages).
J O962180U