Synthesis, conformational analysis, and evaluation of the multidrug resistance-reversing activity of the triamide and proline analogs of hapalosin

Article abstract of DOI:10.1021/jo9708396

Four analogs were synthesized which have trans-4-hydroxyl-L-proline replacing the N-Me-L-phenylalanine moiety in hapalosin. The triamide analog of hapalosin containing two secondary amide bonds in lieu of the two ester bonds in hapalosin was also synthesized. Conformations of hapalosin, the triamide analog, and two of the four proline analogs in chloroform were calculated utilizing distance constraints between NOESY-correlated protons. The lowest-energy, distance-constrained conformation of hapalosin is similar to that of the triamide analog and does not differ substantially from that of the two proline analogs. All conformations have an s-cis tertiary amide bond. The analogs ability to reverse P-glycoprotein-mediated multidrug resistance was evaluated in cytotoxicity and drug accumulation assays using MCF-7/ADR cells which overexpress P-glycoprotein. Two of the proline analogs are more potent than hapalosin (which has a similar activity as verapamil) whereas the other two proline analogs and the triamide analog are less active than hapalosin.

Full text of DOI:10.1021/jo9708396

J . Org. Chem. 1997, 62, 6773-6783
6773
Syn th esis, Con for m a tion a l An a lysis, a n d Eva lu a tion of th e
Mu ltid r u g Resista n ce-Rever sin g Activity of th e Tr ia m id e a n d
P r olin e An a logs of Ha p a losin
Tam Q. Dinh,1a Xiaohui Du,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
Received May 9, 1997X
Four analogs were synthesized which have trans-4-hydroxyl-L-proline replacing the N-Me-L-
phenylalanine moiety in hapalosin. The triamide analog of hapalosin containing two secondary
amide bonds in lieu of the two ester bonds in hapalosin was also synthesized. Conformations of
hapalosin, the triamide analog, and two of the four proline analogs in chloroform were calculated
utilizing distance constraints between NOESY-correlated protons. The lowest-energy, distance-
constrained conformation of hapalosin is similar to that of the triamide analog and does not differ
substantially from that of the two proline analogs. All conformations have an s-cis tertiary amide
bond. The analogs’ ability to reverse P -glycoprotein-mediated multidrug resistance was evaluated
in cytotoxicity and drug accumulation assays using MCF-7/ADR cells which overexpress P-
glycoprotein. Two of the proline analogs are more potent than hapalosin (which has a similar
activity as verapamil) whereas the other two proline analogs and the triamide analog are less active
than hapalosin.
In tr od u ction
non-N-Me analog was found to possess substantially
weaker MDR-reversing property than hapalosin.5f,6
trans-4-Hydroxy-L-proline (7) was selected as a com-
ponent in a series of analogs (3-6) for several reasons.
First, we wanted to see how the significant structural
change from the N-Me-phenylalanine moiety in hapalosin
to proline affects the conformations and anti-MDR activ-
ity. Computation suggested that utilization of L-proline
will also favor the s-cis rotamer. Second, cycloamidation
(forming 19) was expected to be higher-yielding with a
cyclic secondary amine than with an acyclic secondary
amine. Third, the hydroxyl group on the proline ring can
be functionalized.
The efficacy of cancer chemotherapy is often limited
by multidrug resistance (MDR). This phenomenon is
characterized by the resistance of tumor cells to a wide
range of seemingly unrelated drugs. One of the principal
mechanisms of MDR is the expulsion of structurally
diverse drugs by the transmembrane ATPase P-glyco-
protein (P-gp).2 Hapalosin (1) is a novel cyclic depsipep-
tide which reverses MDR putatively via inhibition of
P-gp.3 Extant anti-MDR agents have not exhibited
satisfactory clinical activity.4 Hapalosin5 represents a
new class of potential MDR reversal agents.
We have been interested in the structure-activity
relationship of hapalosin analogs. With the aid of 1H,1H-
NOESY data, computation revealed that the major s-cis
rotamer of hapalosin and the single s-trans rotamer of
the non-N-Me hapalosin analog (having a secondary
N-H amide bond instead of the tertiary N-Me amide
bond in hapalosin) have very different conformations. By
contrast, the minor s-trans rotamer of hapalosin and the
non-N-Me analog have very similar conformations.5d The
X Abstract published in Advance ACS Abstracts, September 15, 1997.
(1) (a) University of California, Los Angeles. (b) Fox Chase Cancer
Center.
(2) For reviews on multidrug resistance, including the involvement
of P-glycoprotein, see: (a) Simon, S. M.; Schindler, M. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 3497. (b) Gottesman, M. M.; Pastan, I. Annu.
Rev. Biochem. 1993, 62, 385. (c) Ford, J . M.; Hait, W. N. Pharmacol.
Rev. 1990, 42, 155. (d) Endicott, J . A.; Ling, V. Annu. Rev. Biochem.
1989, 58, 137.
(3) Stratmann, K.; Burgoyne, D. L.; Moore, R. E.; Patterson, G. M.
L.; Smith, C. D. J . Org. Chem. 1994, 59, 7219.
(4) (a) Ford, J . M. Eur. J . Cancer 1996, 32A, 991. (b) Sikic, B. I. J .
Clin. Oncol. 1993, 11, 1629. (c) Raderer, M.; Scheithauer, W. Cancer
1993, 72, 3553.
(5) 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) Dinh, T. Q.; Du, X.; Armstrong, R. W. J . Org. Chem. 1996,
61, 6606. (e) Wagner, B.; Beugelmans, R.; Zhu, J . Tetrahedron Lett.
1996, 37, 6557. (f) Okuno, T.; Ohmori, K.; Nishiyama, S.; Yamamura,
S.; Nakamura, K.; Houk, K. N.; Okamoto, K. Tetrahedron 1996, 52,
14723.
The triamide analog of hapalosin, 2, also seemed
intriguing from several standpoints. First, the two
additional amide bonds of the triamide analog are less
likely to be cleaved in vivo than the two ester bonds of
hapalosin. Second, the triamide analog should be more
water-soluble than hapalosin. Third, the conformation-
(s) of the triamide analog may differ from those of
hapalosin because of potential intramolecular hydrogen
bonding involving the two secondary amide bonds. In
this paper, we present the synthesis, conformational
analysis, and evaluation of the anti-MDR activity of the
triamide and proline analogs of hapalosin.7
(6) Dinh, T. Q.; Smith, C. D.; Armstrong, R. W. J . Org. Chem. 1997,
62, 790.
(7) The synthesis and MDR-reversing property of the proline analogs
were first described in a communication in this journal (ref 6).
S0022-3263(97)00839-6 CCC: $14.00 © 1997 American Chemical Society

6774 J . Org. Chem., Vol. 62, No. 20, 1997
Dinh et al.
Sch em e 1
Sch em e 2
Resu lts a n d Discu ssion
Syn th esis. The synthesis of the four proline analogs
of hapalosin, 3-6, is illustrated in Scheme 1. trans-4-
Hydroxy-L-proline (7) was tris-protected as ester 8. The
ester was converted to an aldehyde which underwent
Brown allylboration8 to produce homoallylic alcohol 9 in
>90% de. The alcohol was protected with p-methoxy-
benzyl 2,2,2-trichloroacetimidate (PMBTCAI),9 and the
olefin was transformed to acid 12. The acid was coupled
to alkenol 135d using 1-ethyl-3-[3-(dimethylamino)propyl]-
carbodiimide (EDC), and the resulting olefin was oxidized
to acid 16. After the acid was coupled to alcohol 17,5d
the Cbz carbamate and benzyl ester were selectively
deprotected in the presence of the PMB ether by hydro-
genation over W-2 Raney nickel.10 Cycloamidation of the
amino acid with bis(2-oxo-3-oxazolidinyl)phosphinic chlo-
i
ride (BOP-Cl) and Pr2NEt (DIPEA) in toluene at 85 °C11
provided 19 in a good yield of 58% over the two steps.
The four proline analogs 3-6 were easily generated
from macrolactam 19. Desilation of 19 produced analog
3 whose PMB ether was deprotected to form analog 5.
Reaction of analog 3 with benzyl isocyanate yielded
analog 4 whose benzyl group accounts for the benzyl
group in hapalosin. Deblocking of the PMB ether of
analog 4 resulted in analog 6. The benzyl ether and the
phenol ether (with the stereocenter inverted) of analog
3 could not be synthesized because the hydroxyl group
of analog 3 was unreactive with benzyl 2,2,2-trichloro-
acetimidate/TfOH12 and with DIAD/phenol/PPh3.13 The
synthesis of the analogs was designed to be linear so that
an alcohol different than 13 or 17 can be introduced at
the particular position.
octanal underwent Brown crotylboration14 with trans-2-
butene and (+)-B-methoxydiisopinocampheylborane to
form anti homoallylic alcohol 2015 in >95% de and ee.16
Mitsunobu reaction of anti alcohol 20 with diphenylphos-
phoryl azide (DPPA) as the azide source produced syn
azide 21.17 The azide was reduced to an amine with
PPh3/H2O,18 and the amine was protected with benzyl
chloroformate. Olefin 22 was then coverted to acid 24.
The rest of the synthesis of the triamide analog is
depicted in Scheme 3. Amino alcohol 255d was coupled
to N-Boc-L-valine with (benzotriazol-1-yloxy)tris(dimeth-
ylamino)phosphonium hexafluorophosphate [BOP re-
agent (rgt)].19 Carbamate 26 was deblocked and coupled
with acid 24 to provide alcohol 27.20 The alcohol was
silated,21 and the olefin was oxidized to acid 30. After
Synthesis of the triamide analog of hapalosin, 2, used
L-valine and â-amino acid 24 to form the two secondary
amide bonds. In the synthesis of acid 24 (Scheme 2),
(14) (a) Brown, H. C.; Bhat, K. S. J . Am. Chem. Soc. 1986, 108, 5919.
(b) Brown, H. C.; J adhav, P. K.; Bhat, K. S. J . Am. Chem. Soc. 1988,
110, 1535.
(8) J adhav, P. K.; Bhat, K. S.; Perumal, P. T.; Brown, H. C. J . Org.
Chem. 1986, 51, 432.
(9) Nakajima, N.; Horita, K.; Abe, R.; Yonemitsu, O. Tetrahedron
Lett. 1988, 29, 4139.
(10) Horita, K.; Yoshioka, T.; Tanaka, T.; Oikawa, Y.; Yonemitsu,
O. Tetrahedron 1986, 42, 3021.
(11) Evans, D. A.; Miller, S. J .; Ennis, M. D. J . Org. Chem. 1993,
58, 471.
(15) Mitsunobu reaction of syn alcohol 13 with benzoic acid, PPh3,
and DIAD in THF was very slow and did not consume much of 13.
(16) Although the specific rotation of alcohol 20 in CHCl3 is zero,
its ee is >95% since coupling of acid 24 and L-Val-OMe with BOP
reagent quantitatively resulted in only one isomer of the amide product.
(17) Lal, B.; Pramanik, B. N.; Manhas, M. S.; Bose, A. K. Tetrahe-
dron Lett. 1977, 1977-80.
(18) Nagarajan, S.; Ganem, B. J . Org. Chem. 1987, 52, 5044.
(19) Castro, B.; Dormoy, J .-R.; Dourtoglou, B.; Evin, G.; Selve, C.;
Ziegler, J .-C. Synthesis 1976, 751.
(12) Iversen, T.; Bundle, D. R. J . Chem. Soc., Chem. Commun. 1981,
1240.
(13) Hughes, D. L. Org. React. 1992, 42, 335.

Multidrug Resistance-Reversing Activity of Hapalosin
J . Org. Chem., Vol. 62, No. 20, 1997 6775
Sch em e 3
the carbamate was deprotected, the amino acid was
cyclized with BOP-Cl and DIPEA in toluene at 85 °C to
afford the TBS ether of the triamide analog, 31, in 66%
yield over the two steps. Desilation resulted in the
triamide analog of hapalosin, 2.
(v.4.5)22 using AMBER* force field and GB/SA chloroform
solvation.23 Conformational searches were conducted
employing Still’s internal coordinate Monte Carlo proto-
col.24 The search was done on blocks of 1000 Monte Carlo
steps until no additional conformation was found to be
of lower energy than the current global minimum. To
simplify the number of conformations found and save
computational time, the n-heptyl chain was replaced by
a methyl group in hapalosin and the three analogs.
1H,1H-NOESY at 500 MHz was conducted on the modu-
lators in CDCl3 at 25 °C over three different mixing
timess1.10, 1.80, and 2.50 s. The compounds engen-
dered positive NOE’s. In the computation, distance
constraints for protons of the major conformers which
exhibited NOESY crosspeaks of at least medium strength
were set between 1.5 and 5.0 Å.25
With no distance constraint applied, hapalosin and
analogs 2, 5, and 6 have s-cis and s-trans rotamers with
the lowest-energy conformation of all four compounds
bearing an s-cis tertiary amide bond.26 The s-cis popula-
tions within a 5 kcal/mol energy difference from the
lowest-energy conformation are 69% for hapalosin, 76%
for analogs 5 and 6, and 78% for the triamide analog 2.
In the s-trans rotamers of analogs 5 and 6, the steric
repulsion between the isopropyl group and the proline
ring is very obvious while in the s-trans amide conforma-
tions of hapalosin the steric repulsion between the
isopropyl group and the benzyl group is not as strong.
This may explain why the two proline analogs have a
more biased conformational ratio than hapalosin in
chloroform (about 6:1 vs 2.3:1).
Con for m a tion a l An a lysis. The conformational ra-
tios of the modulators in CDCl3 or MeOD at 25 °C are
interesting. The conformations of all the compounds are
due to the configurations of their tertiary amide bond.
In CDCl3, the three proline analogs with a â-PMB
ethers3, 4, and 19shave a conformational ratio from
1.1:1 to 1.5:1 while the ratio is about 6:1 for the two
analogs with a free â-hydroxyl groups5 and 6. On the
other hand, the conformational ratio increases from 2.3:1
for hapalosin to 3.2:1 for the PMB ether of hapalosin in
CDCl3. With respect to the triamide analog 2 and its TBS
ether 31, both exist only as one conformer in CDCl3. In
MeOD, the conformational ratios are 4.3:1 for hapalosin,
1.9:1 for analog 3 (vs 1.1:1 for 3 in CDCl3), and 2.5:1 for
analog 6. The triamide analog 2 also exists only as a
single conformer in MeOD.
Nuclear magnetic resonance experiments were con-
ducted to determine the effects of D2O and KOAc on the
conformations of analog 6 in MeOD at 25 °C. Analog 6
dissolved in a maximum of 30% D2O/MeOD at a concen-
tration of 12 mM. (The triamide analog 2 was not
1
significantly more water-soluble than analog 6.) The H
NMR spectrum of analog 6 in 30% D2O/MeOD differed
only a little from that of 6 in 100% MeOD as the chemical
shifts of a few protons changed slightly. The conforma-
tional ratio of analog 6 remained the same (2.5:1)
regardless of the amount of D2O added. One-dimensional
NOE difference spectroscopy demonstrated that the same
amide bond rotamer was favored at all concentrations of
D2O in MeOD. To test whether a biologically ubiquitous
cation could chelate to the Lewis basic heteroatoms of
analog 6 and affect its conformations, a solution of KOAc
in 25% D2O/MeOD was added to a solution of analog 6
in 25% D2O/MeOD. Varying amounts of KOAc up to 5
Application of distance constraints to pairs of NOESY-
correlated protons confirmed that the triamide analog 2
and the major conformers of hapalosin and analogs 5 and
6 all possess an s-cis tertiary amide bond in chloroform
(Figure 1). Only s-cis amide conformations were found
for hapalosin and analogs 2 and 6. For analog 5, 81% of
the distance-constrained conformations, including the
lowest-energy one, have the s-cis configuration.27 Figure
1
equiv were added, but the H NMR spectra of analog 6
(22) 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.
(23) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J .
Am. Chem. Soc. 1990, 112, 6127.
did not change.
For hapalosin (1) and analogs 2, 5, and 6, molecular
modeling studies were performed with Macromodel
(24) Chang, G.; Guida, W. C.; Still, W. C. J . Am. Chem. Soc. 1989,
111, 4379.
(25) Molecular modeling of the modulators in methanol was also
desired. For Macromodel programs, the GB/SA solvation model is the
best solvation model, but it is equipped with parameters only for
chloroform or water.
(20) After the ester generated by the coupling of acid 24 with L-Val-
OMe was hydrolyzed, the resulting acid could not be coupled with
amino alcohol 25 via various methods, including BOP reagent and
HATU, to make alcohol 27.
(21) Cycloamidation in the presence of a free â-hydroxyl group was
initially attempted with BOP-Cl at 85 °C, but the 1H NMR of the crude
product was inauspicious.
(26) With or without distance constraints, the two secondary amide
bonds of the triamide analog 2 are s-trans.

6776 J . Org. Chem., Vol. 62, No. 20, 1997
Dinh et al.
F igu r e 1. Stereoviews of the lowest-energy conformations
found for hapalosin 1 (a) and analogs 2 (b), 5 (c), and 6 (d) in
conformational searches with distance constraints. The com-
putation was conducted with a methyl group replacing the
n-heptyl group. All conformations have an s-cis tertiary amide
bond. Protons on carbon atoms are removed for clarity. Dark
circles, C; spotted circles, O; grey circles, N; white circles, H.
F igu r e 2. Stereoviews of the superimposed lowest-energy,
distance-constrained conformations of (a) hapalosin 1 and
triamide 2, (b) 1 and analog 5, (c) 1 and analog 6, and (d) 5
and 6. The n-heptyl group is replaced with a methyl group.
Protons on carbon atoms are removed for clarity. Dark circles,
C.; spotted circles, O.; grey circles, N.; white circles, H.
amide analog 2 (Figure 2a) differ in the orientation of
the phenyl, isopropyl methyl, and â-hydroxyl groups.
Nevertheless, they have very similar ring conformations
as indicated by an rms (root mean squared) of 0.138 Å
for the superimposition of the 12 ring atoms. The
hydrogen bond between the amide N-H1 proton and the
C6 carbonyl oxygen of analog 2 (Figure 1b) has little
impact on its conformation. Contrasting hapalosin and
analog 5 (Figure 2b), the â-hydroxyl, isopropyl methyl,
and C2 carbonyl groups point in different directions. The
overlay of the ring conformations (rms of 0.412 Å) is quite
good. With respect to hapalosin and analog 6 (Figure
2c), the structural change from phenylalanine to proline
again does not substantially perturb the ring conforma-
tion (the superimposition rms is 0.474 Å). Noticeable
features are the different orientations of the â-hydroxyl
group and the opposite directions of the C6 carbony group.
Comparing to the proline analogs 5 and 6 (Figure 2d),
the isopropyl methyl and C6 carbonyl groups are oriented
differently and the ring overlap is surprisingly not as
good (the superimposition rms is 0.601 Å) as the overlay
between hapalosin and analog 5 or 6. The principal
discrepancy between the two proline analogs, however,
is that analog 6 bears a benzyl carbamate group instead
of a hydroxyl group in analog 5.
1 depicts the lowest-energy, distance-constrained confor-
mations of the triamide analog 2 (b) and of the major
conformers of hapalosin (a) and analogs 5 (c) and 6 (d).
All four conformations surprisingly do not exhibit in-
tramolecular hydrogen bonding of the â-hydroxyl group.
The conformation of the triamide analog 2, however,
contains a transannular hydrogen bond between the
amide N-H1 proton and the C6 carbonyl oxygen.28 This
agrees with the results of a deuterium exchange experi-
ment in which D2O was added to a solution of 2 in CDCl3.
The amide N-H5 proton completely disappeared when less
than 20 equiv of D2O was added whereas the integration
of the N-H1 proton did not diminish at all regardless of
the amount of D2O added.
To compare the lowest-energy, distance-constrained
conformations of the four modulators, the conformations
are superimposed in Figure 2. Hapalosin and the tri-
(27) In ref 6, it was reported that computation employing distance
constraints resulted in seven conformations for analog 5, all containing
an s-cis amide bond. This is an error which stemmed from an incorrect
proton being inputted in the distance constraints.
(28) The distance between the amide N-H5 proton and the C2
carbonyl oxygen (3.28 Å) nearly equals that between the amide N-H1
proton and the C6 carbonyl oxygen (3.25 Å). The O-H5-N angle is
79° while the O-H1-N angle is 104°. Since the former angle is <90°,
it does not meet the requirement of a hydrogen bond.

Multidrug Resistance-Reversing Activity of Hapalosin
J . Org. Chem., Vol. 62, No. 20, 1997 6777
F igu r e 3. Reversal of MDR by hapalosin analogs. MCF-7/ADR cells were incubated with the indicated concentrations of
modulators in the presence of phosphate-buffered saline (as a control) (0), 25 nM actinomycin D (O), or 2 µM daunomycin (b).
Modulators 1 and 3-6 in (a) were tested at a different time than modulators 1, 2, and verapamil in (b). Cell survival after 48 h
was determined as indicated in the Experimental Section. Values represent the mean ( SD of triplicate samples.
Mu ltid r u g Resista n ce-Rever sin g Activity. The
anti-MDR activities of synthetic hapalosin and its ana-
logs (modulators) were determined by cytotoxicity and
drug accumulation assays using MCF-7/ADR cells which
overexpress P-gp.29 Verapamil was also tested as a posi-
tive control. In the cytotoxicity assay (Figure 3), cells
were exposed to one of eight doses of a modulator alone
(the PBS curves) or in the presence of 25 nM actinomycin
D, 2 µM daunomycin, or 2 µM cisplatin. Cisplatin was
included as a non-P-gp substrate to demonstrate selective
enhancement of killing by actinomycin D and daunomy-
cin, which are transported by P-gp. Moreover, the effects
of the modulators on the killing of drug-sensitive MCF-7
cells, which do not overexpress P-gp, were tested. In the
drug accumulation assay (Figure 4), MCF-7/ADR cells
were treated with one of eight doses of a modulator and
then incubated with [3H]vinblastine, which is also trans-
ported by P-gp. Reversal of MDR is demonstrated if
minimally cytotoxic doses of a modulator selectively
increase cell killing by actinomycin D and daunomycin
and increase the intracellular concentration of [3H]-
vinblastine.
The results of the cytotoxicity and drug accumulation
assays are depicted in Figure 3a,b and Figure 4a,b,
respectively. All the modulators in Figures 3a and 4a
were tested at one time, and the same was true for the
modulators in Figures 3b and 4b. The modulators in
Figures 3a and 4a, however, were tested 5 months prior
to the assays of the modulators in Figures 3b and 4b.
Therefore, the results for the two different times are
displayed separately. Nevertheless, synthetic hapalosin
was evaluated on both occasions and serves as a reference
by which the modulators in Figures 3a and 4a can be
compared to those in Figures 3b and 4b. Data for
cisplatin and MCF-7 cells are omitted since none of the
modulators altered the sensitivity of MCF-7/ADR cells
F igu r e 4. Effects of hapalosin analogs on [3H]vinblastine
accumulation by MDR cells. MCF-7/ADR cells were incubated
with the indicated concentrations of modulators 1 (4), 3 (b),
4 (2), 5 (0), or 6 (O) in (a) and 1 (4), 2 (O), or verapamil (2) in
(b). The modulators in (a) were tested at a different time than
those in (b). The intracellular accumulation of [3H]vinblastine
was then determined as indicated in the Experimental Section.
Values represent the mean ( SD of triplicate samples.
(29) These cytotoxicity and drug accumulation assays have been
employed to test the MDR-reversing activity of hapalosin (ref 3) and
dendroamide A and welwitindolinone analogs, respectively: (a) Ogino,
J .; Moore, R. E.; Patterson, G. M. L.; Smith, C. D. J . Nat. Prod. 1996,
59, 581. (b) Smith, C. D.; Zilfou, J . T.; Stratmann, K.; Patterson, G.
M. L.; Moore, R. E. Mol. Pharm. 1995, 47, 241.
to cisplatin or the sensitivity of MCF-7 cells to any of
the drugs, indicating that the modulators act on P-gp.

6778 J . Org. Chem., Vol. 62, No. 20, 1997
Dinh et al.
To test the effects of drugs on growth, cells were seeded in
96-well tissue culture dishes at approximately 15% confluency
and were allowed to attach and recover for at least 24 h.
Varying concentrations of drugs alone or combined with a
modulator were then added to each well, and the plates were
incubated for an additional 48 h. The number of surviving
cells was then determined by staining with sulforhodamine B
as previously described.33 The percentage of cells killed was
calculated as the percentage decrease in sulforhodamine B
binding compared with control cultures. Control cultures
included equivalent amounts of ethanol, which does not
modulate the growth or drug-sensitivity of these cells at does
utilized in these studies. Reversal of MDR is defined as the
ability of a compound to potentiate the cytotoxicity of P-
glycoprotein-transported drugs.
[3H]Dr u g Accu m u la tion Assa y. MCF-7/ADR cells were
plated into 24-well tissue culture dishes and allowed to grow
to 90% confluency. The cells were washed with phosphate-
buffered saline (PBS) and then incubated in 0.5 mL RPMI 1640
medium containing a modulator and 10-20 nM [3H]vinblastine
sulfate (Amersham Corporation) for 60 min at 37 °C. The
cultures were rapidly washed three times with ice-cold PBS.
Intracellular [3H]drug was solubilized with 0.3 mL of 1%
sodium dodecyl sulfate in water and quantified by liquid
scintillation counting.
Comparing the ability of the proline analogs to potenti-
ate the cytotoxicity of actinomycin D and daunomycin to
that of hapalosin (1) (Figure 3a), analogs 330 and 6 are
better while analogs 4 and 5 are worse. However, analog
4 is much less intrinsically cytotoxic (the PBS curve) than
the other four modulators. Hapalosin and verapamil are
equally effective in causing cell death whereas the
triamide analog 2 is worse, but verapamil is inherently
the least cytotoxic (Figure 3b). With regard to enhance-
ment of the intracellular concentration of [3H]vinblastine
(Figure 4a), analogs 3 and 6 are better than hapalosin,
analog 4 is slightly worse, and analog 5 is substantially
worse. Hapalosin and verapamil promote vinblastine
accumulation similarly well while the triamide analog 2
is slightly worse (Figure 4b). In summary of the modula-
tors’ MDR-reversing property, analogs 3 and 6 are the
most active, hapalosin and verapamil have similar activ-
ity, analogs 2 and 4 are less active than hapalosin, and
analog 5 is substantially weaker than hapalosin. Analog
4 and verapamil (and the non-N-Me analog of hapalosin6),
however are much less intrinsically cytotoxic than the
other modulators. The reasons for the difference in the
cytotoxicity of the modulators are not known.
Gen er a l P r oced u r es. All water-sensitive reactions were
conducted in oven- or flame-dried glassware under a nitrogen
atmosphere. The starting materials were azeotroped two or
three times with benzene before the reactions. Solvents were
distilled immediately prior to use: CH2Cl2 from P2O5, PhMe
from CaH2, MeOH from magnesium metal, and THF from
sodium metal/benzophenone ketyl. Anhydrous DMF and
MeCN were purchased from the Aldrich Chemical Co. and
utilized without further purification. Most commercially
available reagents were distilled before use. Thin-layer chro-
matography (TLC) was performed on silica gel-coated plates
(Merck, Kieselgel 60 F254, 0.25 mm thickness for analytical and
0.5 mm for preparative TLC) and visualized by UV light and/
or p-anisaldehyde, ninhydrin, or bromocresol green (for car-
boxylic acids) staining. After all aqueous extractions of crude
reaction products, the combined organic layers were dried with
MgSO4 and concentrated in vacuo before further treatment.
(4R)-N-(Ben zyloxyca r bon yl)-4-[(ter t-bu tyld im eth ylsi-
lyl)oxy]-L-p r olin e Meth yl Ester (8). trans-4-Hydroxy-L-
proline (7) (8.033 g, 61.26 mmol, Aldrich) was dissolved in 10%
aqueous Na2CO3 (80 mL), and THF (50 mL) was added. Cbz-
Cl (8.33 mL, 58.3 mmol) was added slowly at 25 °C. Much
white precipitate soon formed in the slightly warming mixture.
The mixture was stirred fast at 25 °C for 15 h, diluted with
water (50 mL), cooled to 0 °C, carefully acidified to pH 1 with
concentrated HCl (about 7 mL), and extracted in EtOAc (2 ×
30 mL) with 0.1 N NaHSO4 (50 mL).
Con clu sion
The lowest-energy, distance-constrained conformations
of hapalosin (1) and analogs 2, 5, and 6 in chloroform all
possess an s-cis tertiary amide bond. The conformation
of hapalosin is similar to that of the triamide analog 2
and is not substantially different than that of analogs 5
and 6. In regard to reversal of P-gp-mediated MDR,
analogs 3 and 6 are more effective than hapalosin while
analogs 2, 4, and 5 are weaker. Considering that the
conformations of hapalosin and analogs 5 and 6 are not
that dissimilar, the weak activity of analog 5 suggests
that an aromatic group may be important for MDR
reversal.
It was originally conjectured that a simpler scaffold
which mimics the core structure of hapalosin and pre-
sents the appropriate peripheral functionalities in the
proper orientations may be bioactive.31 Although the
triamide analog 2 and hapalosin are structurally and
conformationally similar, the triamide analog has a
significantly weaker anti-MDR activity. This fact sug-
gests that important contacts with P-gp may be lost in
the ester-to-amide backbone permutation or that the
more rigid triamide backbone may be less effective at
presenting substituent groups in the appropriate fashion.
To a solution of the above crude hydroxy acid in MeCN (70
mL) was slowly added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU,
7.49 mL, 50.0 mmol) at 25 °C. After the slightly warmed-up
solution cooled to about 25 °C in approximately 7 min, MeI
(3.12 mL, 50.0 mmol) was added and the flask was sealed. The
solution was stirred at 25 °C for 15 h and extracted in EtOAc
(2 × 300 mL) with 0.1 N NaHSO4 (250 mL).
Exp er im en ta l Section
Cell Cu ltu r e a n d Cytotoxicity Assa y. MCF-7 breast
carcinoma cells and MCF-7/ADR cells, an MDR subline,32 were
obtained from the Division of Cancer Treatment of the
National Cancer Institute and were grown in RPMI 1640
containing 10% fetal bovine serum and 50 µg/mL gentamycin
sulfate.
To a solution of the above crude alcohol in DMF (35 mL)
were added imidazole (6.40 g, 94.0 mmol) followed by a solution
of tBuMe2SiCl (TBS-Cl, 11.33 g, 75.19 mmol) in DMF (20 mL).
(In order for TBS-Cl to dissolve in DMF, the mixture had to
be heated with a heatgun.) The solution was stirred at 25 °C
for 15 h and extracted in EtOAc (2 × 300 mL) with 0.1 N
NaHSO4 (300 mL). Flash chromatography with silica gel
(gradient to 60% EtOAc/hexanes) afforded TBS ether 8 (15.546
(30) The PMB ether of hapalosin has only marginal anti-MDR
activity (ref 6).
g, 68% yield for three steps) as a colorless oil: [R]22 -43° (c
D
(31) Hirschmann et al elegantly created nonpeptidal mimetics of the
cyclic hexapeptide L-363,301 which utilize â-D-glucose as the scaffold
and are agonists of the somatostatin receptor. See: Hirschmann, R.;
Nicolaou, K. C.; Pietranico, S.; Leahy, E. M.; Salvino, J .; Arison, B.;
Cichy, M. A.; Spoors, P. G.; Shakespeare, W. C.; Sprengeler, P. A.;
Hamley, P.; Smith, A. B., III; Reisine, T.; Raynor, K.; Maechler, L.;
Donaldson, C.; Vale, W.; Freidinger, R. M.; Cascieri, M. R.; Strader,
C. D. J . Am. Chem. Soc. 1993, 115, 12550.
0.039, CHCl3); IR (neat) 1117, 1416, 1713, 1752, 2953 cm-1
;
1H NMR (400 MHz, CDCl3), both rotamers unless stated
otherwise, δ 0.04-0.06 (4 singlets, 6 H), 0.85 (s, 9 H, 1
rotamer), 0.86 (s, 9 H, 1 rotamer), 2.04 (m, 1 H), 2.20 (m, 1
H), 3.42 (m, 1 H, 1 rotamer), 3.51 (m, 1 H, 1 rotamer), 3.54 (s,
(32) 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.
(33) Skehan, P.; Stoneng, R.; Scudiero, D.; Monks, A.; McMahon,
J .; Vistica, D.; Warren, J . T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J .
Natl. Cancer Inst. 1990, 82, 1107.

Multidrug Resistance-Reversing Activity of Hapalosin
J . Org. Chem., Vol. 62, No. 20, 1997 6779
3 H, 1 rotamer), 3.66 (m, 1 H), 3.76 (s, 3 H, 1 rotamer), 4.43
(m, 1 H), 4.48 (m, 1 H), 5.04 (d, J ) 12.4 Hz, 1 H, 1 rotamer),
5.10 (d, J ) 12.4 Hz, 1 H, 1 rotamer), 5.20 (d, J ) 12.4 Hz, 1
H), 7.26-7.37 (m, 5 H); 13C NMR (101 MHz, CDCl3), both
rotamers, δ -5.0, -4.93, -4.91, -4.88, 17.86, 17.92, 25.60,
25.64, 38.9, 39.8, 52.0, 52.3, 54.7, 55.2, 57.8, 58.0, 67.1, 69.7,
70.3, 127.7, 127.8, 127.88, 127.92, 128.3, 128.4, 136.4, 136.6,
154.3, 155.0, 173.1, 173.3; HRCI calcd for C20H32O5NSi [(M +
H)+] 394.2050, found 394.2048.
(2S,2(1R),4R)-N-(Ben zyloxyca r bon yl)-2-(1-h yd r oxybu t-
3-en yl)-4-[(ter t-bu tyld im eth ylsilyl)oxy]p yr r olid in e (9). A
solution of ester 8 (14.32 g, 36.38 mmol) in PhMe (100 mL)
was cooled to -78 °C, and DIBAH (72.8 mL in hexanes, 72.8
mol) was added over a 5 min period. After being stirred for
2.5 h at -78 °C, the solution was carefully quenched with -78
°C MeOH (35 mL) and stirring continued for an additional 5
min at -78 °C. The reaction solution was poured with swirling
into a separatory funnel containing ice-cold 1.2 M HCl (250
mL). Extraction with EtOAc (2 × 250 mL) and flash chroma-
tography with silica gel (gradient from 10% EtOAc/hexanes
to 100% EtOAc) provided the aldehyde (8.20 g, 62% yield) as
a colorless oil. The aldehyde underwent allylboration within
1 day.
72.7, 73.1, 77.5, 78.3, 113.6, 113.7, 116.99, 117.02, 127.6, 127.8,
127.9, 128.38, 128.41, 129.20, 129.22, 130.7, 131.0, 134.6,
134.8, 136.7, 137.1, 154.9, 155.0, 159.0, 159.1; HREI calcd for
C30H43O5NSi (M+) 525.2910, found 525.2915.
(2S,2(1R),4R)-N-(Ben zyloxyca r bon yl)-4-[(ter t-bu tyld i-
m eth ylsilyl)oxy]-2-[2-for m yl-1-[(p-m eth oxyben zyl)oxy]-
eth yl]p yr r olid in e (11). Ozone was bubbled into a solution
of olefin 10 (3.40 g, 6.47 mmol) in CH2Cl2 (150 mL) at -78 °C
with stirring for 10 min when TLC showed no more olefin.
Argon was bubbled into the colorless solution for 15 s, and
PPh3 (2.38 g, 9.06 mmol) was added. After the solution was
stirred at -78 °C for 15 min, stirring continued for 15 h at 25
°C. Flash chromatography with silica gel (gradient to 40%
EtOAc/hexanes) resulted in aldehyde 11 (2.513 g, 74% yield)
as a colorless oil: [R]22D -59° (c 0.041, CHCl3); IR (neat) 1109,
1250, 1414, 1701, 1725, 2930, 2953 cm-1; 1H NMR (400 MHz,
CDCl3, reference is CHCl3), both rotamers (2.0:1) unless stated
otherwise, δ 0.00 (s, 3 H), 0.01 (s, 3 H), 0.84 (s, 9 H), 1.80 (m,
1 H), 2.15 (m, 1 H), 2.33-2.58 (m, 2 H), 3.34-3.59 (m, 2 H),
3.77 (s, 3 H), 3.92-4.03 (m, 1 H), 4.28-4.53 (m, 3 H), 4.69 (m,
1 H), 5.06-5.21 (m, 2 H), 6.83 (d, J ) 8.5 Hz, 2 H), 7.10 (d, J
) 8.5 Hz, 2 H, 1 rotamer), 7.12 (d, J ) 8.5 Hz, 2 H, 1 rotamer),
7.27-7.39 (m, 5 H), 9.60 (broad m, 1 H, minor rotamer), 9.68
(broad m, 1 H, major rotamer); 13C NMR (101 MHz, CDCl3),
both rotamers, δ -5.0, -4.8, 17.9, 25.7, 34.3, 35.0, 46.9, 47.0,
55.2, 55.6, 60.1, 60.8, 66.7, 67.0, 70.0, 70.4, 72.7, 73.3, 73.37,
73.43, 113.7, 127.7, 127.9, 128.1, 128.4, 128.5, 129.4, 129.5,
130.0, 130.2, 136.6, 136.8, 155.0, 155.4, 159.2, 200.0, 200.4;
HRFAB calcd for C29H42O6NSi [(M + H)+] 528.2781, found
528.2790.
To a solution of (+)-B-methoxydiisopinocampheylborane
(9.28 g, 29.3 mmol) in THF (70 mL) at -78 °C was slowly
added allylmagnesium bromide (27.1 mL in Et2O, 27.1 mmol).
The mixture was stirred for 15 min at -78 °C and for 1 h
without the -78 °C bath. The resulting solution was recooled
to -78 °C, and a solution of the above aldehyde (8.20 g, 22.6
mmol) in THF (40 mL) was added slowly. The solution was
stirred for 3 h at -78 °C and for 2 h without the -78 °C bath.
After being cooled to 0 °C, the solution was carefully quenched
with 3 M aqueous NaOH (24.5 mL) and 30% aqueous H2O2
(10.5 mL). The ice bath was allowed to warm up to 25 °C,
and stirring transpired for 12 h. Much THF was removed in
vacuo from the supernatant of the reaction mixture, and
extraction was performed in EtOAc (2 × 300 mL) with brine
(250 mL) followed by water (200 mL). Most isopinocampheyl
alcohol was removed under high vacuum at 90 °C using a
Kugelrohr apparatus. Flash chromatography with silica gel
(gradient to 70% EtOAc/hexanes) furnished alcohol 9 (5.50 g,
(2S,2(1R),4R)-N-(Ben zyloxyca r bon yl)-4-[(ter t-bu tyld i-
m eth ylsilyl)oxy]-2-[2-ca r boxy-1-[(p-m eth oxyben zyl)oxy]-
eth yl]p yr r olid in e (12). To a solution of aldehyde 11 (2.483
g, 4.705 mmol) and 2-methyl-2-butene (4.70 mL in THF, 9.41
mmol) in tBuOH (24 mL) was slowly added a solution of
sodium chlorite (80% purity, 691 mg, 6.12 mmol) and NaH2-
PO4 (677 mg, 5.65 mmol) in water (6 mL). The flask was
sealed and the mixture was vigorously stirred at 25 °C for 15
h. Extraction in CH2Cl2 (2 × 150 mL) with 0.1 N NaHSO4
(100 mL) followed by water (75 mL) gave clean acid 12 (2.558
g, 100% yield) as a colorless oil: [R]22 -59° (c 0.039, CHCl3);
D
60% yield) as a colorless oil in >90% de: [R]22 -39° (c 0.039,
IR (neat) 1109, 1250, 1418, 1705, 1734, 2930, 2955, 3166
D
CHCl3); IR (neat) 835, 1111, 1420, 1686, 2930, 2953, 3441 cm-1
;
(shoulder) cm-1 1H NMR (400 MHz, CDCl3, reference is
;
1H NMR (400 MHz, CDCl3, 2.7:1 rotamers), the major rotamer,
δ 0.03 (s, 3 H), 0.05 (s, 3 H), 0.85 (s, 9 H), 1.88 (m, 1 H), 1.94
(m, 1 H), 2.12 (m, 2 H), 3.21 (broad s, 1 H), 3.38 (dd, J ) 11.4,
4.1 Hz, 1 H), 3.56 (d, J ) 11.4 Hz, 1 H), 4.07 (broad m, 1 H),
4.15 (m, 1 H), 4.36 (broad m, 1 H), 5.06-5.20 (m, 4 H), 5.88
(m, 1 H), 7.27-7.38 (m, 5 H); 13C NMR (101 MHz, CDCl3), the
major rotamer, δ -4.9, -4.8, 17.9, 25.7, 35.5, 37.3, 56.2, 61.8,
67.0, 70.2, 70.9, 117.1, 127.7, 127.9, 128.5, 135.2, 136.6, 156.5;
HREI calcd for C22H36O4NSi [(M + H)+] 406.2413, found
406.2415.
(2S,2(1R),4R)-N-(Ben zyloxyca r bon yl)-4-[(ter t-bu tyld i-
m eth ylsilyl)oxy]-2-[1H[(p-m eth oxyben zyl)oxy]bu t-3-en yl]-
p yr r olid in e (10). To a solution of alcohol 9 (5.00 g, 12.3
mmol) in THF (60 mL) was added PMBTCAI (5.11 mL, 24.6
mmol). A solution of TfOH (6.5 µL, 0.074 mmol) in THF (2
mL) was then added slowly. The solution was stirred at 25
°C for 2 h, and TfOH was quenched with triethylamine (20
µL). Flash chromatography with silica gel (gradient to 15%
EtOAc/hexanes) produced the PMB ether 10 (5.22 g, 81% yield)
as a colorless oil: [R]22D -51° (c 0.041, CHCl3); IR (neat) 1107,
1250, 1414, 1514, 1705, 2930, 2953 cm-1; 1H NMR (400 MHz,
CDCl3, reference is CHCl3), both rotamers (1.7:1) unless stated
otherwise, δ 0.01 (s, 3 H), 0.02 (s, 3 H), 0.84 (s, 9 H), 1.78 (m,
1 H), 2.03 (m, 1 H, 1 rotamer), 2.13 (m, 1 H, 1 rotamer), 2.18
(m, 1 H), 2.29 (m, 1 H), 3.35-3.54 (m, 2 H), 3.77 (s, 3 H, major
rotamer), 3.79 (s, 3 H, minor rotamer), 3.89 (m, 1 H, 1
rotamer), 4.03 (m, 1 H), 4.17 (m, 1 H, 1 rotamer), 4.27-4.52
(m, 3 H), 4.97-5.18 (m, 4 H), 5.68 (m, 1 H, minor rotamer),
5.85 (m, 1 H, major rotamer), 6.83 (d, J ) 8.6 Hz, 2 H, major
rotamer), 6.84 (d, J ) 8.6 Hz, 2 H, minor rotamer), 7.14 (d, J
) 8.6 Hz, 2 H, 1 rotamer), 7.16 (d, J ) 8.6 Hz, 2 H, 1 rotamer),
7.27-7.38 (m, 5 H); 13C NMR (101 MHz, CDCl3), both rotam-
ers, δ -4.93, -4.90, -4.86, -4.8, 17.9, 25.7, 33.9, 34.6, 36.9,
37.2, 55.1, 55.19, 55.23, 55.5, 59.4, 60.2, 66.5, 66.8, 70.2, 70.6,
CHCl3), both rotamers unless stated otherwise, δ 0.00 (s, 3
H), 0.02 (s, 3 H), 0.83 (s, 9 H), 1.81 (m, 1 H), 2.15 (m, 1 H),
2.34-2.51 (m, 2 H), 3.33 (dd, J ) 11.1, 4.7 Hz, 1 H, 1 rotamer),
3.41 (m, 1 H), 3.54 (broad m, 1 H, 1 rotamer), 3.75 (s, 3 H),
3.97-4.07 (m, 1 H), 4.29-4.44 (m, 3 H), 4.53-4.62 (m, 1 H),
5.10-5.21 (m, 2 H), 6.81 (d, J ) 8.5 Hz, 2 H), 7.11 (d, J ) 8.5
Hz, 2 H, minor rotamer), 7.15 (d, J ) 8.5 Hz, 2 H, major
rotamer), 7.26-7.39 (m, 5 H); 13C NMR (101 MHz, CDCl3), both
rotamers, δ -5.0, -4.9, -4.8, 17.9, 25.7, 34.4, 34.9, 37.9, 55.1,
55.2, 55.5, 59.9, 60.4, 66.8, 67.0, 70.0, 70.4, 73.5, 73.6, 74.6,
75.2, 113.6, 113.7, 127.8, 127.9, 128.1, 128.4, 129.4, 129.5,
130.2, 130.5, 136.6, 136.8, 155.1, 155.4, 159.1, 159.2, 176.0,
176.6; HRFAB calcd for C29H42O7NSi [(M + H)+] 544.2731,
found 544.2729.
(1R,2R)-1-Hep tyl-2-m eth ylbu t-3-en yl (3R,3(2S,4R))-3-
[N-(Ben zyloxyca r bon yl)-4-[(ter t-bu tyld im eth ylsilyl)oxy]-
2-pyr r olidin yl]-3-[(p-m eth oxyben zyl)oxy]pr opan oate (14).
To a solution of acid 12 (2.211 g, 4.067 mmol) and alcohol 13
(825 mg, 4.47 mmol) in CH2Cl2 (30 mL) was added DMAP (497
mg, 4.07 mmol) followed by EDC (1.09 g, 5.69 mmol). The
solution was stirred at 25 °C for 15 h and extracted in CH2Cl2
(2 × 100 mL) with 0.1 N NaHSO4 (100 mL). Flash chroma-
tography with silica gel (gradient to 20% EtOAc/hexanes)
provided ester 14 (2.168 g, 75% yield) as a colorless oil: [R]22
D
-37° (c 0.040, CHCl3); IR (neat) 1107, 1250, 1414, 1705, 1734,
2928 cm-1 1H NMR (400 MHz, CDCl3, reference is CHCl3),
;
both rotamers unless stated otherwise, δ -0.01 (s, 3 H), 0.00
(s, 3 H), 0.82 (s, 9 H), 0.87 (t, J ) 6.8 Hz, 3 H), 0.96 (d, J ) 6.8
Hz, 3 H, minor rotamer), 1.00 (d, J ) 6.8 Hz, 3 H major
rotamer), 1.15-1.35 (broad m, 10 H), 1.51 (broad m, 2 H), 1.78
(m, 1 H), 2.15 (m, 1 H), 2.30-2.51 (m, 3 H), 3.31 (dd, J ) 11.1,
4.7 Hz, 1 H, major rotamer), 3.37 (dd, J ) 11.3, 4.6 Hz, 1 H,
minor rotamer), 3.42 (broad d, 1 H, 1 rotamer), 3.55 (broad d,
1 H, 1 rotamer), 3.76 (s, 3 H, major rotamer), 3.78 (s, 3 H,

6780 J . Org. Chem., Vol. 62, No. 20, 1997
Dinh et al.
minor rotamer), 4.01 (m, 1 H), 4.27-4.48 (m, 3 H), 4.60 (d, J
) 10.9 Hz, 1 H, 1 rotamer), 4.64 (broad m, 1 H, 1 rotamer),
4.86 (broad m, 1 H), 4.99-5.20 (m, 4 H), 5.73 (m, 1 H), 6.80
(d, J ) 8.6 Hz, 2 H), 7.11 (d, J ) 8.6 Hz, 2 H, 1 rotamer), 7.14
(d, J ) 8.6 Hz, 2 H, 1 rotamer), 7.27-7.40 (m, 5 H); 13C NMR
(101 MHz, CDCl3), both rotamers, δ -4.93, -4.89, -4.85, 14.1,
15.1, 15.2, 17.9, 22.6, 25.5, 25.7, 29.1, 29.5, 31.3, 31.8, 34.3,
35.0, 38.2, 38.3, 41.1, 41.2, 55.2, 55.6, 59.8, 60.6, 66.6, 66.8,
70.0, 70.5, 73.4, 73.8, 74.8, 75.6, 113.5, 113.6, 115.1, 127.7,
127.8, 128.0, 128.4, 129.2, 130.5, 131.0, 136.8, 137.0, 139.9,
140.0, 155.08, 155.13, 158.97, 159.05, 170.9; HRFAB calcd for
C41H64O7NSi [(M + H)+] 710.4452, found 710.4452.
mL). Flash chromatography with silica gel (gradient to 20%
EtOAc/hexanes) generated triester 18 (997 mg, 86% yield) as
a colorless oil: [R]22 -43° (c 0.034, CHCl3); IR (neat) 1109,
D
1250, 1705, 1742, 2930 cm-1
;
1H NMR (400 MHz, CDCl3,
reference is CHCl3), both rotamers unless stated otherwise, δ
-0.01 (s, 3 H), 0.00 (s, 3 H), 0.82 (s, 9 H), 0.87 (t, J ) 6.8 Hz,
3 H), 0.95 (d, J ) 6.8 Hz, 3 H), 0.98 (d, J ) 6.9 Hz, 3 H), 1.17
(d, J ) 7.2 Hz, 3 H, 1 rotamer), 1.20 (d, J ) 7.2 Hz, 3 H, 1
rotamer), 1.12-1.37 (broad m, 10 H), 1.58 (broad m, 2 H), 1.78
(broad m, 1 H), 2.13 (m, 1 H), 2.24 (m, 1 H), 2.32-2.50 (m, 2
H), 2.75 (m, 1 H), 3.31 (dd, J ) 11.1, 4.8 Hz, 1 H, 1 rotamer),
3.37 (dd, J ) 11.1, 4.6 Hz, 1 H, 1 rotamer), 3.41 (broad dd, 1
H, 1 rotamer), 3.54 (broad dd, 1 H, 1 rotamer), 3.76 (s, 3 H,
major rotamer), 3.77 (s, 3 H, minor rotamer), 3.99 (broad m, 1
H), 4.28 (d, J ) 10.8 Hz, 1 H, 1 rotamer), 4.35 (d, J ) 10.8 Hz,
1 H, 1 rotamer), 4.38 (broad m, 1 H), 4.46 (d, J ) 10.8 Hz, 1
H, 1 rotamer), 4.48 (broad m, 1 H, 1 rotamer), 4.60 (d, J )
10.8 Hz, 1 H, 1 rotamer), 4.64 (broad m, 1 H, 1 rotamer), 4.85
(d, J ) 4.3 Hz, 1 H), 5.08-5.19 (m, 4 H), 5.24 (broad m, 1 H),
6.80 (d, J ) 8.6 Hz, 2 H), 7.11 (d, J ) 8.6 Hz, 2 H, 1 rotamer),
(2S,3R)-2-F or m yl-3-d ecyl (3R,3(2S,4R))-3-[N-(Ben zyl-
oxyca r b on yl)-4-[(ter t-b u t yld im et h ylsilyl)oxy]-2-p yr r o-
lidin yl]-3-[(p-m eth oxyben zyl)oxy]pr opan oate (15). Ozone
was bubbled into a solution of olefin 14 (2.146 g, 3.022 mmol)
in CH2Cl2 (70 mL) at -78 °C with stirring until the solution
turned moderately blue (the color quickly faded). Argon was
bubbled into the solution for 15 s, and PPh3 (1.03 g, 3.93 mmol)
was added. After the solution was stirred at -78 °C for 15
min, stirring continued for 15 h at 25 °C. Flash chromatog-
raphy with silica gel (gradient to 25% EtOAc/hexanes) fur-
7.14 (d, J ) 8.6 Hz, 2 H, 1 rotamer), 7.24-7.41 (m, 10 H); 13
C
NMR (101 MHz, CDCl3), both rotamers, δ -4.94, -4.89, -4.8,
12.2, 12.3, 14.1, 17.1, 17.9, 18.8, 22.6, 25.4, 25.7, 29.1, 29.4,
30.0, 31.8, 31.9, 34.3, 35.1, 38.0, 38.1, 42.8, 42.9, 55.2, 55.6,
59.9, 60.5, 66.6, 66.8, 70.0, 70.5, 73.6, 73.9, 74.3, 74.8, 75.5,
76.8, 113.5, 113.6, 127.7, 127.8, 127.9, 128.0, 128.2, 128.26,
128.32, 128.4, 128.5, 129.2, 129.3, 130.6, 131.0, 135.3, 135.4,
136.8, 137.0, 155.1, 158.97, 159.04, 169.1, 170.7, 173.3, 173.4;
HRFAB calcd for C52H76O11NSi [(M + H)+] 918.5188, found
918.5179.
nished aldehyde 15 (1.629 g, 76% yield) as a colorless oil: [R]22
D
-38° (c 0.012, CHCl3); IR (neat) 1107, 1250, 1414, 1705, 1734,
2930 cm-1 1H NMR (400 MHz, CDCl3, reference is CHCl3),
;
both rotamers (1.4:1) unless stated otherwise, δ -0.01-0.00
(4 singlets, 6 H), 0.82 (s, 9 H), 0.87 (t, J ) 6.8 Hz, 3 H), 1.06
(d, J ) 7.1 Hz, 3 H, minor rotamer), 1.10 (d, J ) 7.0 Hz, 3 H,
major rotamer), 1.17-1.38 (broad m, 10 H), 1.55 (broad m, 1
H), 1.63 (broad m, 1 H), 1.79 (broad m, 1 H), 2.12 (m, 1 H),
2.33 (m, 1 H), 2.43 (m, 1 H), 2.54 (m, 1 H), 3.28-3.56 (m, 2
H), 3.76 (s, 3 H, major rotamer), 3.78 (s, 3 H, minor rotamer),
4.00 (m, 1 H), 4.27-4.44 (m, 3 H), 4.54-4.62 (m, 1 H), 5.08-
5.18 (m, 2 H), 5.30 (m, 1 H), 6.79-6.82 (2 overlapping doublets,
2 H), 7.09-7.17 (2 overlapping doublets, 2 H), 7.27-7.40 (m,
5 H), 9.65 (s, 1 H, minor rotamer), 9.70 (s, 1 H, major rotamer);
13C NMR (101 MHz, CDCl3 both rotamers, δ -4.93, 4.91,
-4.88, -4.8, 7.9, 8.1, 14.1, 17.9, 22.6, 25.6, 25.7, 29.1, 29.3,
31.7, 31.8, 34.3, 35.0, 38.0, 38.2, 49.7, 49.8, 55.2, 55.6, 59.9,
60.5, 66.6, 66.9, 70.0, 70.5, 72.9, 73.1, 73.6, 73.8, 74.7, 74.8,
75.5, 113.6, 113.7, 127.76, 127.84, 127.9, 128.0, 128.4, 128.5,
129.2, 129.6, 130.5, 130.8, 136.8, 137.0, 155.1, 155.2, 159.0,
159.2, 170.7, 202.2, 202.5; HRFAB calcd for C40H62O8NSi [(M
+ H)+] 712.4245, found 712.4219.
Ma cr ola cta m 19. A 50 mL round-bottom flask containing
a solution of triester 18 (335 mg, 0.365 mmol) in EtOH (14
mL) was equipped with a 3.2-cm-long egg-shape stir bar. A
bottle of Raney nickel (equivalent to W-2, 50% slurry water,
Aldrich) was shaken well, and 55 drops of the Raney Ni from
a Pasteur pipet were added. The flask was purged with N2,
and H2 was bubbled into the mixture for 6 min. After the
solution was stirred for 18 h at 25 °C under a balloon full of
H2, H2 was again bubbled into the mixture for 6 min and
stirring continued for 20 h more. After the flask was purged
with N2, the mixture was filtered through a small column of
Celite and the Raney Ni clinging to the stir bar and the column
were washed well with EtOH. 1H NMR of the crude product
(232 mg colorless film, 92% crude yield) showed that the Cbz
and benzyl ester protecting groups were completely removed
and the PMB ether was intact.
(2S,3R)-2-Ca r boxy-3-d ecyl [3R,3(2S,4R)]-3-[N-(Ben zyl-
oxyca r b on yl)-4-[(ter t-b u t yld im et h ylsilyl)oxy]-2-p yr r o-
lid in yl]-3-[(p-m eth oxyben zyl)oxy]p r op a n oa te (16). The
procedure for sodium chlorite oxidation of aldehyde 15 to acid
16 was based on that for making acid 12. The crude product
contained clean acid 16 (1.640 g, 100% yield) as a colorless
To a solution of the above crude amino acid (232 mg, 0.334
mmol) in PhMe (250 mL) were successively added DIPEA (582
µL, 3.34 mmol) and BOP-Cl (595 mg, 2.34 mmol). The mixture
was stirred for 16 h at 85 °C and extracted with 0.1 N NaHSO4
(60 mL). The aqueous layer was back-extracted with EtOAc
(60 mL). The toluene layer from the previous extraction was
again extracted with 0.1 N NaHSO4 (60 mL), and the aqueous
layer was back-extracted with the EtOAc layer from the
previous back-extraction. Flash chromatography with silica
gel (gradient to 20% EtOAc/hexanes) furnished macrolactam
19 (144 mg, 58% for two steps) as a white solid: [R]22D -33° (c
0.030, CHCl3); IR (neat) 1171, 1250, 1651, 1732, 1748, 2930
oil: [R]22 -39° (c 0.033, CHCl3); IR (neat) 1109, 1250, 1707,
D
1740, 2930, 3178 (shoulder) cm-1; 1H NMR (400 MHz, CDCl3,
reference is CHCl3), both rotamers unless stated otherwise, δ
-0.01-0.01 (4 singlets, 6 H), 0.82 (s, 9 H), 0.87 (t, J ) 6.9 Hz,
3 H), 1.14 (d, J ) 7.1 Hz, 3 H, minor rotamer), 1.17 (d, J ) 7.1
Hz, 3 H, major rotamer), 1.55 (broad m, 1 H), 1.62 (broad m,
1 H), 1.80 (broad m, 1 H), 2.13 (m, 1 H), 2.31-2.52 (m, 2 H),
2.68 (m, 1 H), 3.31-3.55 (m, 2 H), 3.76 (s, 3 H, major rotamer),
3.77 (2, 3 H, minor rotamer), 4.01 (m, 1 H), 4.27 (d, J ) 10.8
Hz, 1 H, minor rotamer), 4.33 (d, J ) 10.9 Hz, 1 H, major
rotamer), 4.41 (broad m, 2 H), 4.44 (d, J ) 10.8 Hz, 1 H, minor
rotamer), 4.58 (d, J ) 10.9 Hz, 1 H, major rotamer), 5.07-
5.17 (m, 2 H), 5.25 (m, 1 H), 6.80 (d, J ) 8.6 Hz, 2 H), 7.11 (d,
J ) 8.6 Hz, 2 H, minor rotamer), 7.14 (d, J ) 8.6 Hz, 2 H,
major rotamer), 7.26-7.41 (m, 5 H); 13C NMR (101 MHz,
CDCl3), both rotamers, δ -5.0, -4.9, -4.8, 11.4, 11.7, 14.1,
17.9, 22.6, 25.5, 25.7, 29.1, 29.3, 31.5, 31.7, 31.8, 34.2, 35.0,
38.0, 38.2, 42.5, 55.1, 55.2, 55.6, 59.8, 60.5, 66.8, 66.9, 70.1,
70.5, 73.4, 73.5, 74.3, 74.5, 75.1, 75.6, 113.58, 113.64, 127.8,
127.9, 128.0, 128.4, 129.2, 129.3, 130.5, 130.8, 136.76, 136.81,
155.2, 159.0, 159.1, 170.6, 170.7, 177.8, 178.4; HRFAB calcd
for C40H62O9NSi [(M + H)+] 728.4194, found 728.4200.
cm-1 1H NMR (400 MHz, CDCl3, reference is CHCl3), both
;
conformers (about 1.5:1) unless stated otherwise, δ -0.03 (s,
3 H, 1 conformer), -0.02 (s, 3 H, 1 conformer), -0.002 (s, 3 H,
1 conformer), 0.001 (s, 3 H, 1 conformer), 0.79-0.85 (a masked
pair of doublets, 3 H), 0.82 (s, 9 H, 1 conformer), 0.84 (s, 9 H,
1 conformer), 0.88 (t, J ) 7.0 Hz, 3 H), 0.95 (d, J ) 6.5 Hz, 3
H, 1 conformer), 0.99 (d, J ) 6.7 Hz, 3 H, 1 conformer), 1.15
(d, J ) 7.2 Hz, 3 H, 1 conformer), 1.21 (d, J ) 7.2 Hz, 3 H, 1
conformer), 1.22-1.32 (broad m, 10 H), 1.54 (m, 1 H), 1.63 (m,
1 H), 1.78 (m, 1 H), 1.88 (m, 1 H), 2.10 (m, 1 H, major
conformer), 2.31 (m, 1 H, minor conformer), 2.42 (m, 1 H), 2.64
(m, 1 H), 3.08 (m, 1 H, minor conformer), 3.24 (m, 1 H, major
conformer), 3.41 (dd, J ) 11.9, 5.5 Hz, 1 H, 1 conformer), 3.48
(dd, J ) 11.9, 3.0 Hz, 1 H, 1 conformer), 3.60 (dd, J ) 12.4,
6.7 Hz, 1 H, 1 conformer), 3.68 (dd, J ) 12.4, 4.9 Hz, 1 H, 1
conformer), 3.75 (m, 1 H, major conformer), 3.87 (m, 1 H, minor
conformer), 4.27 (d, J ) 10.9 Hz, 1 H, 1 conformer), 4.31 (d, J
) 11.6 Hz, 1 H, 1 conformer), 4.34 (m, 1 H, 1 conformer), 4.53
(m, 1 H, 1 conformer), 4.58 (d, J ) 11.6 Hz, 1 H, 1 conformer),
Tr iester 18. To a solution of acid 16 (919 mg, 1.26 mmol)
and alcohol 17 (289 mg, 1.39 mmol) in CH2Cl2 (14 mL) were
added successively DMAP (154 mg, 1.26 mmol) and EDC (338
mg, 1.76 mmol). The solution was stirred at 25 °C for 15 h
and extracted in CH2Cl2 (2 × 70 mL) with 0.1 N NaHSO4 (70

Multidrug Resistance-Reversing Activity of Hapalosin
J . Org. Chem., Vol. 62, No. 20, 1997 6781
4.57 (a masked doublet, 1 H, 1 conformer), 4.65 (m, 1 H, major
conformer), 4.74 (m, 1 H), 4.95 (m, 1 H, minor conformer), 5.15
(d, J ) 10.9 Hz, 1 H, major conformer), 5.46 (d, J ) 4.8 Hz, 1
H, minor conformer), 6.85 (d, J ) 8.7 Hz, 2 H, minor
conformer), 6.89 (d, J ) 8.7 Hz, 2 H, major conformer), 7.17
(d, J ) 8.7 Hz, 2 H, minor conformer), 7.27 (d, J ) 8.7 Hz, 2
H, major conformer); 13C NMR (101 MHz, CDCl3), both
conformers, δ -5.02, -5.00, -4.9, -4.8, 12.3, 12.9, 14.1, 17.7,
17.9, 18.0, 18.4, 19.0, 19.7, 22.6, 25.7, 25.8, 27.4, 28.6, 29.1,
29.2, 29.8, 31.72, 31.74, 32.3, 35.1, 35.6, 37.4, 39.1, 41.0, 41.8,
54.6, 55.20, 55.24, 56.7, 58.0, 58.8, 68.7, 70.6, 71.4, 71.6, 75.6,
76.5, 78.9, 79.92, 79.94, 82.3, 113.8, 113.9, 129.3, 129.71,
129.74, 159.3, 159.4, 167.4, 169.8, 170.0, 171.4, 172.6, 173.2;
HRFAB calcd for C37H62O8NSi [(M + H)+] 676.4245, found
676.4250.
An a log 3. A stock solution in a polyethylene bottle was
prepared of 70% HF/pyridine (1.0 mL, purchased from Ald-
rich), pyridine (2.0 mL), and THF (8.0 mL). Most of this stock
solution (9.5 mL) was added to a solution of TBS ether 19 (124
mg, 0.183 mmol) in THF (5.5 mL) in another polyethylene
bottle. After being stirred for 15 h at 25 °C, the solution was
extracted in CH2Cl2 (2 × 60 mL) with 0.1 N NaHSO4 (3 × 40
mL). Preparative TLC (55% EtOAc/hexanes) produced analog
3 (Rf ) 0.53, 88 mg, 85% yield) as a colorless film: [R]22D -37°
(c 0.059, CHCl3); IR (neat) 1171, 1250, 1514, 1626, 1730, 1748,
2928, 3428 cm-1; 1H NMR (500 MHz, CDCl3), both conformers
(about 1.1:1) unless stated otherwise, δ 0.81 (d, J ) 6.6 Hz, 3
H, 1 conformer), 0.84 (d, J ) 6.4 Hz, 3 H, 1 conformer), 0.88
(t, J ) 6.9 Hz, 3 H), 0.95 (d, J ) 6.1 Hz, 3 H, 1 conformer),
1.00 (d, J ) 6.4 Hz, 3 H, 1 conformer), 1.15 (d, J ) 6.9 Hz, 3
H, 1 conformer), 1.22 (d, J ) 7.0 Hz, 3 H, 1 conformer), 1.23-
1.41 (broad m, 10 H), 1.55 (broad m, 1 H, 1 conformer), 1.65
(broad m, 1 H), 1.84 (broad m, 1 H), 1.96 (broad m, 1 H), 2.14
(broad m, 1 H, 1 conformer), 2.32-2.51 (broad m, 2 H), 2.63
(m, 1 H), 3.06 (broad m, 1 H, 1 conformer), 3.25 (m, 1 H, 1
conformer), 3.43 (m, 1 H, 1 conformer), 3.59 (broad m, 1 H),
3.80 (s, 3 H), 3.82 (masked m, 1 H, 1 conformer), 3.90 (broad
m, 1 H), 4.32 (m, 1 H), 4.45 (broad m, 1 H, 1 conformer), 4.54
(broad m, 1 H, 1 conformer), 4.57 (d, J ) 11.3 Hz, 1 H), 4.67
(broad m, 1 H, 1 conformer), 4.76 (broad m, 1 H), 4.98 (broad
m, 1 H, 1 conformer), 5.17 (d, J ) 10.9 Hz, 1 H, 1 conformer),
5.46 (broad d, 1 H, 1 conformer), 6.87 (broad pair of doublets,
2 H), 7.17 (d, J ) 8.0 Hz, 2 H, 1 conformer), 7.26 (d, J ) 8.0
Hz, 2 H, 1 conformer); 13C NMR (126 MHz, CDCl3), both
conformers, δ 12.2, 12.8, 14.0, 17.5, 18.1, 18.9, 19.7, 22.5, 25.7,
27.4, 28.5, 29.1, 29.8, 31.7, 32.3, 34.8, 35.1, 37.2, 38.9, 40.9,
41.8, 54.1, 55.2, 56.7, 57.8, 58.6, 68.4, 70.67, 70.71, 71.3, 75.6,
76.5, 79.3, 79.5, 79.8, 82.2, 113.7, 113.9, 129.1, 129.4, 129.5,
129.7, 159.2, 159.4, 167.7, 169.9, 170.2, 171.4, 172.6, 173.3;
HRFAB calcd for C31H48O8N [(M + H)+] 562.3380, found
562.3380.
conformer), 6.88 (d, J ) 8.5 Hz, 2 H, major conformer), 7.18
(d, J ) 8.5 Hz, 2 H, minor conformer), 7.23-7.34 (m, 5 H;
masked doublet, 2 H, major conformer); 13C NMR (126 MHz,
CDCl3), both conformers, δ 12.4, 12.9, 14.0, 17.4, 18.0, 18.9,
19.8, 22.5, 25.7, 27.2, 28.5, 29.0, 29.12, 29.13, 30.2, 31.66, 31.69,
32.2, 32.6, 35.2, 35.9, 37.0, 40.9, 41.8, 44.9, 45.0, 52.5, 53.9,
55.1, 55.2, 57.3, 58.4, 70.8, 71.0, 72.0, 74.7, 75.6, 79.2, 79.3,
79.8, 81.8, 113.8, 114.0, 127.37, 127.42, 127.5, 127.6, 128.57,
128.58, 128.7, 129.5, 129.6, 138.1, 138.3, 155.6, 155.9, 159.3,
159.4, 167.2, 169.8, 169.9, 171.2, 172.6, 173.4; HRFAB calcd
for C39H55O9N2 [(M + H)+] 695.3908, found 695.3911.
An a log 5. To a mixture of analog 3 (29 mg, 0.052 mmol)
in CH2Cl2 (1.0 mL) and water (0.06 mL) was added DDQ (29
mg, 0.13 mmol). The flask was sealed, and the mixture was
stirred vigorously for 2.5 h at 25 °C. After extraction in EtOAc
(2 × 15 mL) with saturated NaHCO3 (2 × 15 mL), preparative
TLC (90% EtOAc/hexanes) produced analog 5 (Rf ) 0.51, 19
mg, 83% yield) as a white solid: [R]22 -41° (c 0.019, CHCl3);
D
IR (neat) 1167, 1626, 1732, 1748, 2928, 3401 cm-1; H NMR
1
(400 MHz, CDCl3, about 6.5:1 conformers), the major con-
former, δ 0.88 (t, J ) 6.9 Hz, 3 H), 0.92-1.05 (2 coincidental
pairs of doublets, 6 H), 1.18 (d, J ) 7.0 Hz, 3 H), 1.22-1.41
(broad m, 10 H), 1.54 (broad m, 1 H), 1.85 (broad m, 1 H),
2.09 (broad m, 1 H), 2.18 (broad m, 1 H), 2.27 (broad m, 1 H),
2.48 (broad d, 1 H, the hydroxyl group on the proline ring),
2.56 (m, 2 H), 3.13 (m, 1 H), 3.49 (broad m, 2 H, including the
â-hydroxyl group), 3.67 (broad m, 1 H), 4.07 (broad m, 1 H),
4.48 (broad m, 1 H), 4.55 (broad m, 1 H), 4.83 (broad m, 1 H),
5.27 (d, J ) 10.4 Hz, 1 H); 13C NMR (101 MHz, CDCl3), the
major conformer, δ 11.2, 14.1, 17.9, 19.8, 22.6, 25.4, 28.6, 29.1,
29.3, 29.5, 31.7, 38.8, 39.5, 41.0, 54.7, 62.1, 70.5, 72.9, 76.2,
82.0, 170.9, 171.3, 172.6; HRFAB calcd for C23H40O7N [(M +
H)+] 442.2805, found 442.2808.
An a log 6. The procedure for the synthesis of analog 5 was
used. Preparative TLC (60% EtOAc/hexanes) afforded analog
6 (Rf ) 0.55, 17.5 mg, 71% yield) as a colorless film: [R]22
D
-27° (c 0.017, CHCl3); IR (neat) 1167, 1258, 1636, 1727, 2928,
3341 cm-1; 1H NMR (400 MHz, CDCl3, about 6.0:1 conformers),
the major conformer, δ 0.86 (d, J ) 5.2 Hz, 3 H), 0.88 (t, J )
6.8 Hz, 3 H), 0.93 (d, J ) 6.3 Hz, 3 H), 1.17 (d, J ) 7.1 Hz, 3
H), 1.22-1.39 (broad m, 10 H), 1.52 (m, 1 H), 1.84 (m, 1 H),
2.00 (m, 1 H), 2.30 (m, 2 H), 2.48 (dd, J ) 16.7, 10.2 Hz, 1 H),
2.68 (dd, J ) 16.7, 4.3 Hz, 1 H), 3.12 (m, 2 H, including the
hydroxyl group), 3.52 (dd, J ) 13.0, 3.7 Hz, 1 H), 3.96 (d, J )
13.0 Hz, 1 H), 4.10 (m, 1 H), 4.29 (dd, J ) 14.9, 5.8 Hz, 1 H),
4.38 (dd, J ) 14.9, 6.3 Hz, 1 H), 4.43 (m, 1 H), 4.81 (m, 1 H),
5.12 (t, J ) 6.0 Hz, 1 H (the N-H)), 5.20 (broad m, 1 H), 5.26
(d, J ) 10.8 Hz, 1 H), 7.23-7.35 (m, 5 H); 13C NMR (101 MHz,
CDCl3), the major conformer, δ 11.1, 14.1, 17.8, 19.9, 22.6, 25.4,
28.7, 29.1, 29.3, 29.7, 31.7, 35.6, 39.6, 41.0, 45.0, 53.1, 62.5,
72.5, 74.4, 76.3, 81.6, 127.6, 127.7, 128.7, 138.1, 155.6, 170.9,
171.1, 172.5; HRFAB calcd for C31H47O8N2 [(M + H)+] 575.3332,
found 575.3330.
An a log 4. To a solution of analog 3 (59 mg, 0.10 mmol) in
CH2Cl2 (1.1 mL) were successively added DMAP (19 mg, 0.16
mmol) and benzyl isocyanate (45 µL, 0.37 mmol). The flask
was sealed, and the solution was stirred for 15 h at 25 °C.
After extraction in CH2Cl2 (2 × 20 mL) with 0.1 N NaHSO4
(20 mL), preparative TLC (33% EtOAc/hexanes) provided
(3R,4S)-3-Meth ylu n d ec-1-en -4-ol (20). To a mixture of
t
sublimed BuOK (2.733 g, 24.35 mmol) in THF (30 mL) at -78
°C were added trans-2-butene (about 6 mL) and then nBuLi
(13.4 mL, 22.14 mmol, 1.65 M in pentane). The bright yellow
mixture was stirred at -78 °C for 30 min, and a solution of
(+)-B-methoxydiisopinocampheylborane (8.40 g, 26.57 mmol)
in THF (25 mL) was added, resulting in the disappearance of
much of the yellow color. The solution was stirred at -78 °C
for 30 min, and then BF3‚OEt2 (3.65 mL, 28.78 mmol) was
added, giving a white mixture. A solution of octanal (3.46 mL,
22.14 mmol) in THF (25 mL) was immediately added, and the
thickened mixture was stirred for 2.5 h at -78 °C and 1.5 h
without the -78 °C bath. The mixture was cooled to 0 °C,
and 30% aqueous H2O2 (8 mL) and 3 M NaOH (16 mL) were
added slowly. After the mixture was stirred overnight at 25
°C, most THF was removed in vacuo and extraction in EtOAc
(2 × 150 mL) was done successively with brine (200 mL) and
water (150 mL). Flash chromatography with silica gel (gradi-
ent to 8% EtOAc/hexanes) and concentration in vacuo at 30
°C provided alcohol 20 (3.50 g, 86% yield) as a colorless liquid
in >95% de and >95% ee. (Though the specific rotation is zero,
the ee of alcohol 20 is >95% since coupling of acid 24 and L-Val-
OMe with BOP reagent quantitatively resulted in only one
analog 4 (Rf ) 0.45, 60 mg, 82% yield) as a colorless film: [R]22
D
-17° (c 0.058, CHCl3); IR (neat) 1248, 1514, 1725, 2928, 3335
cm-1; 1H NMR (500 mHz, CDCl3), both conformers (about 1.4:
1) unless stated otherwise, δ 0.80 (d, J ) 6.6 Hz, 3 H, major
conformer), 0.86-0.91 (1 triplet, 3 H; 1 pair of doublets, 3 H;
1 doublet, 3 H, minor conformer), 1.14 (d, J ) 7.1 Hz, 3 H,
major conformer), 1.20 (d, J ) 7.2 Hz, 3 H, minor conformer),
1.22-1.40 (broad m, 10 H), 1.55 (m, 1 H, 1 conformer), 1.65
(m, 1 H), 1.81 (m, 1 H, 1 conformer), 1.90 (m, 1 H, 1 conformer),
2.02 (m, 1 H; m, 1 H, 1 conformer), 2.38 (m, 1 H), 2.49-2.64
(m, 2 H), 3.06 (m, 1 H, minor conformer), 3.27 (m, 1 H, major
conformer), 3.42 (dd, J ) 13.7, 4.5 Hz, 1 H, major conformer),
3.66 (m, 1 H, 1 conformer), 3.78 (s, 3 H, minor conformer),
3.80 (s, 3 H, major conformer), 3.83 (m, 1 H, 1 conformer), 3.87
(m, 1 H), 4.05 (dd, J ) 13.3, 3.1 Hz, 1 H, minor conformer),
4.25-4.42 (m, 3 H), 4.56-4.59 (2 doublets, 1 H), 4.69 (m, 1 H,
1 conformer), 4.74 (m, 1 H), 4.97 (m, 1 H, 1 conformer), 507
(m, 1 H, 1 conformer), 5.13 (d, J ) 11.0 Hz, 1 H), 5.16 (m, 1 H,
1 conformer), 5.29 (m, 1 H, minor conformer), 5.43 (d, J ) 4.5
Hz, 1 H, minor conformer), 6.86 (d, J ) 8.5 Hz, 2 H, minor
isomer of the amide product.) 20: [R]22 0° (c 0.029, CHCl3);
D

6782 J . Org. Chem., Vol. 62, No. 20, 1997
IR (neat) 912, 999, 1460, 3368 cm-1 1H NMR (500 MHz,
CDCl3) δ 0.88 (t, J ) 6.9 Hz, 3 H), 1.03 (d, J ) 6.9 Hz, 3 H),
1.22-1.40 (broad m, 10 H), 1.48 (broad m, 2 H), 2.21 (m, 1 H),
3.39 (m, 1 H), 5.08-5.12 (m, 2 H), 5.76 (m, 1 H); 13C NMR
(126 MHz, CDCl3) δ 14.1, 16.3, 22.6, 25.7, 29.3, 29.7, 31.8, 34.2,
44.1, 74.7, 116.1, 140.4; HREI calcd for C12H24O (M+) 184.1827,
found 184.1822.
Dinh et al.
;
26.3, 29.1, 29.3, 31.5, 31.7, 43.8, 53.2, 66.8, 128.0, 128.1, 128.5,
136.5, 156.2, 179.8; HRFAB calcd for C19H30O4N [(M + H)+]
336.2175, found 336.2180.
(2S ,3R )-2-[N -(N -(t er t -Bu t yloxyca r b on yl)-L-va lyl)-N -
m eth yla m in o]-1-p h en ylh ex-5-en -3-ol (26). To a solution
of amino alcohol 25 (225 mg, 1.10 mmol) and N-Boc-L-valine
(262 mg, 1.21 mmol) in MeCN (9 mL) were successively added
DIPEA (344 µL, 1.97 mmol) and BOP reagent (628 mg, 1.42
mmol). After the solution was stirred at 25 °C for 15 h, brine
(2 mL) and AcOH (44 µL, 0.77 mmol, to quench excess DIPEA)
were added to the brown solution and stirring was continued
for 10 min. Much MeCN was removed in vacuo until bumping
became a problem. Extraction in EtOAc (3 × 40 mL) with
brine (40 mL) followed by 0.1 M NaHSO4 (40 mL) and flash
chromatography with silica gel (gradient to 25% acetone/
hexanes) resulted in amide 26 (372 mg, 84% yield) as a
colorless oil: [R]22D -36° (c 0.026, CHCl3); IR (neat) 1173, 1368,
1497, 1630, 1705, 3416 cm-1; 1H NMR (500 MHz, CDCl3, about
4.5:1 rotamers), the major rotamer, δ 0.89 (d, J ) 6.8 Hz, 3
H), 0.91 (d, J ) 6.7 Hz, 3 H), 1.43 (s, 9 H), 1.80 (m, 1 H), 2.26-
2.36 (m, 3 H), 2.76 (s, 3 H), 3.08 (broad m, 2 H), 3.93 (broad
m, 1 H), 4.26 (dd, J ) 9.4, 5.2 Hz, 1 H), 5.11 (d, J ) 8.8 Hz,
carbamate N-H), 5.15-5.17 (m, 2 H), 5.84 (m, 1 H), 7.14-7.29
(m, 5 H); 13C NMR (126 MHz, CDCl3), both rotamers, δ 16.7,
16.9, 19.0, 19.9, 22.6, 25.2, 28.3, 29.0, 30.6, 30.7, 31.5, 32.2,
34.2, 34.6, 38.7, 39.5, 54.5, 55.6, 72.3, 72.8, 79.4, 118.5, 119.0,
126.4, 126.7, 128.4, 128.7, 128.9, 129.2, 134.3, 134.5, 138.4,
138.8, 155.8, 173.2, 173.5; HRFAB calcd for C23H37O4N2 [(M
+ H)+] 405.2753, found 405.2748.
(3R,4R)-4-Azid o-3-m eth yl-1-u n d ecen e (21). To a solu-
tion of alcohol 20 (512 mg, 2.78 mmol) and triphenylphosphine
(1.24 g, 4.72 mmol) in THF (17 mL) at 0 °C were successively
added diisopropyl azodicarboxylate (DIAD, 929 µL, 4.72 mmol)
dropwise and diphenylphosphoryl azide (DPPA, 1.02 mL, 4.72
mmol). The milky beige mixture was stirred at 0 °C for 5 min
and at 25 °C for 15 h. Addition of CHCl3 (3 mL) dissolved the
mixture and concentration in vacuo resulted in a thick oil.
Flash chromatography with silica gel (eluent was 100%
hexanes) and concentration in vacuo at 30 °C furnished azide
21 (550 mg, 95% yield) as a colorless liquid: [R]22 +32° (c
D
0.032, CHCl3); IR (neat) 918, 1252, 1274, 1456, 2099 cm-1; 1H
NMR (500 MHz, CDCl3) δ 0.89 (t, J ) 6.9 Hz, 3 H), 1.07 (d, J
) 6.7 Hz, 3 H), 1.22-1.38 (broad m, 10 H), 1.47 (m, 1 H), 1.55
(m, 1 H), 2.33 (m, 1 H), 3.15 (m, 1 H), 5.04-5.10 (m, 2 H),
5.75 (m, 1 H); 13C NMR (126 MHz, CDCl3) δ 14.1, 15.9, 22.6,
26.4, 29.2, 29.4, 31.8, 31.9, 42.4, 67.6, 115.4, 140.4; HREI calcd
for C12H24N [(M - N2 + H)+] 182.1909, found 182.1903.
(3R,4R)-4-[N-(Ben zyloxyca r bon yl)a m in o]-3-m eth yl-1-
u n d ecen e (22). Water (142 µL, 7.89 mmol) was added to a
solution of azide 21 (550 mg, 2.63 mmol) and triphenylphos-
phine (896 mg, 3.42 mmol) in THF (10 mL). After the solution
was stirred at 25 °C for 30 h, DIPEA (916 µL, 5.26 mmol) and
benzyl chloroformate (488 µL, 3.42 mmol) were added. Stirring
at 25 °C for 6 h, concentration in vacuo, and flash chroma-
tography with silica gel (gradient to 10% EtOAc/hexanes)
furnished carbamate 22 (527 mg, 63% yield for two steps) as
Alcoh ol 27. Trifluoroacetic acid (585 µL, 7.59 mmol) was
added to a solution of Boc carbamate 26 (307 mg, 0.759 mmol)
in CH2Cl2 (5 mL). After the solution was stirred at 25 °C for
2 h, TFA and CH2Cl2 were removed in vacuo. Extraction in
EtOAc (2 × 40 mL and 2 × 30 mL) with 1 M NaOH (40 mL)
and then water (30 mL) gave the desired amino alcohol (227,
mg, 98% crude yield) as a colorless oil.
flat, colorless crystals: [R]22 +25° (c 0.028, CHCl3); IR (neat)
D
696, 916, 1243, 1542, 1687, 3319 cm-1
;
1H NMR (500 MHz,
CDCl3) δ 0.87 (t, J ) 7.0 Hz, 3 H), 1.01 (d, J ) 6.9 Hz, 3 H),
1.13-1.33 (broad m, 10 H), 1.35 (broad m, 1 H), 1.54 (broad
m, 1 H), 2.30 (m, 1 H), 3.58 (m, 1 H), 4.55 (d, J ) 9.7 Hz, 1 H),
5.00-5.14 (m, 4 H), 5.71 (m, 1 H), 7.29-7.36 (m, 5 H); 13C NMR
(126 MHz, CDCl3) δ 14.1, 16.0, 22.6, 26.1, 29.2, 29.5, 31.76,
31.79, 42.6, 55.1, 66.5, 115.3, 127.97, 128.00, 128.5, 136.8,
140.5, 156.2; HREI calcd for C20H32O2N [(M + H)+] 318.2433,
found 318.2430.
To a solution of the above crude amino alcohol (205 mg,
0.673 mmol) and acid 24 (249 mg, 0.741 mmol) in MeCN (8
mL) were added DIPEA (211 µL, 1.21 mmol) and BOP reagent
(387 mg, 0.875 mmol). After the solution was stirred at 25 °C
for 15 h, brine (1.3 mL) and AcOH (27 µL) were added to the
brown solution and stirring was continued for 10 min. After
removing much MeCN in vacuo, extraction in EtOAc (3 × 40
mL) with brine (30 mL) followed by 0.1 M NaHSO4 (30 mL)
and flash chromatography with silica gel (gradient to 50%
EtOAc/hexanes) provided alcohol 27 (350 mg, 84% yield for
(2S,3R)-3-[N-(Ben zyloxyca r b on yl)a m in o]-2-m et h yl-1-
d eca n a l (23). Ozone was bubbled into a solution of olefin 22
(468 mg, 1.47 mmol) in CH2Cl2 (20 mL) at -78 °C for 4 min.
Triphenylphosphine (503 mg, 1.92 mmol) was added, the -78
°C bath was removed, and the solution was stirred at 25 °C
for 12 h. Concentration in vacuo and flash chromatography
with silica gel (gradient to 20% EtOAc/hexanes) produced
two steps) as a colorless oil: [R]22 -1.1° (c 0.038, CHCl3); IR
D
(neat) 699, 1255, 1455, 1534, 1624, 1636, 1696, 3304 cm-1; 1H
NMR (500 MHz, CDCl3, 4:1 rotamers), the major rotamer, δ
0.84-0.89 (overlapping d and t, 6 H), 0.90 (d, J ) 6.9 Hz, 3
H), 1.14 (d, J ) 7.0 Hz, 3 H), 1.16-1.32 (broad m, 10 H), 1.38
(broad m, 1 H), 1.48 (broad m, 1 H), 1.83 (broad m, 1 H), 2.27
(broad m, 3 H), 2.52 (broad m, 1 H), 2.76 (s, 3 H), 3.07 (broad
m, 2 H), 3.70 (broad m, 1 H), 3.91 (broad m, 1 H), 4.57 (dd, J
) 8.6, 4.6 Hz, 1 H), 5.09-5.18 (m, 4 H), 5.65 (d, J ) 9.1 Hz, 1
H), 5.83 (m, 1 H), 6.07 (d, J ) 8.1 Hz, 1 H), 7.13-7.39 (m, 10
H); 13C NMR (126 MHz, CDCl3), both rotamers, δ 14.1, 14.2,
16.3, 16.8, 19.5, 20.1, 22.59, 22.61, 26.36, 26.44, 29.1, 29.2,
29.30, 29.32, 29.6, 29.7, 29.9, 30.6, 31.7, 31.8, 32.2, 34.0, 38.7,
39.5, 45.2, 54.2, 54.4, 66.4, 72.7, 118.6, 118.8, 126.4, 126.7,
127.80, 127.84, 127.88, 127.90, 128.38, 128.40, 128.7, 128.9,
129.0, 129.1, 134.36, 134.43, 136.7, 136.8, 138.4, 138.7, 156.2,
156.3, 173.2, 173.3, 174.0, 174.1; HRFAB calcd for C37H56O5N3
[(M + H)+] 622.4220, found 622.4212.
aldehyde 23 (421 mg, 90% yield) as colorless crystals: [R]22
D
+47° (c 0.022, CHCl3); IR (neat) 1254, 1536, 1692, 1720, 3316
1
cm-1; H NMR (500 MHz, CDCl3) δ 0.88 (t, J ) 7.0 Hz, 3 H),
1.08 (d, J ) 7.2 Hz, 3 H), 1.20-1.35 (broad m, 10 H), 1.47
(broad m, 2 H), 2.55 (m, 1 H), 4.11 (m, 1 H), 4.76 (d, J ) 9.3
Hz, carbamate N-H), 5.05 (d, J ) 12.2 Hz, 1 H), 5.10 (d, J )
12.2 Hz, 1 H), 7.29-7.39 (m, 5 H), 9.72 (s, 1 H); 13C NMR (126
MHz, CDCl3) δ 8.5, 14.0, 22.6, 26.3, 29.1, 29.2, 31.7, 32.6, 50.6,
51.2, 66.8, 128.0, 128.2, 128.5, 136.3, 156.0, 203.5; HRFAB
calcd for C19H30O3N [(M + H)+] 320.2226, found 320.2233.
(2S,3R)-3-[N-(Ben zyloxyca r b on yl)a m in o]-2-m et h yl-1-
d eca n oic Acid (24). To a solution of aldehyde 23 (350 mg,
t
1.10 mmol) in BuOH (3.0 mL) were added 2-methyl-2-butene
(1.10 mL, 2.19 mmol, 2.0 M in THF) and then a solution of
80% sodium chlorite (149 mg, 1.31 mmol) and NaH2PO4 (131
mg, 1.10 mmol) in water (1.0 mL). The round-bottom flask
was sealed, and the mixture was stirred vigorously at 25 °C
for 15 h. Extraction in EtOAc (3 × 40 mL) with 0.1 M NaHSO4
(30 mL) followed by water (30 mL) gave clean acid 24 (367
mg, 100% yield) as a white solid: [R]22D +29° (c 0.018, CHCl3);
TBS Eth er 28. To a solution of alcohol 27 (213 mg, 0.342
mmol) in CH2Cl2 (5 mL) at 0 °C were successively added 2,6-
lutidine (80 µL, 0.685 mmol) and TBSOTf (118 µL, 0.514
mmol). After the solution was stirred at 0 °C for 1 h, extraction
in EtOAc (3 × 30 mL) with aqueous NaHCO3 (20 mL) followed
by 0.1 M NaHSO4 (30 mL) and flash chromatography with
silica gel (gradient to 30% EtOAc/hexanes) afforded TBS ether
IR (neat) 1258, 1541, 1692, 3318 cm-1
;
1H NMR (500 MHz,
28 (219 mg, 87% yield) as a colorless oil: [R]22 -11° (c 0.042,
D
CDCl3) δ 0.87 (t, J ) 6.9 Hz, 3 H), 1.18 (d, J ) 7.1 Hz, 3 H),
1.20-1.33 (broad m, 10 H), 1.36 (broad m, 1 H), 1.52 (broad
m, 1 H), 2.70 (m, 1 H), 3.87 (m, 1 H), 5.03 (d, J ) 9.9 Hz, 1 H),
5.07 (d, J ) 12.2 Hz, 1 H), 5.12 (d, J ) 12.2 Hz, 1 H), 7.29-
7.36 (m, 5 H); 13C NMR (126 MHz, CDCl3) δ 13.2, 14.1, 22.6,
CHCl3); IR (neat) 696, 776, 836, 1063, 1257, 1535, 1641, 1694,
3296 cm-1; 1H NMR (500 MHz, CDCl3, 4:1 rotamers), the major
rotamer, δ 0.11 (s, 6 H), 0.82-0.88 (overlapping d and t, 6 H),
0.89 (d, J ) 6.9 Hz, 3 H), 0.94 (s, 9 H), 1.14 (d, J ) 7.1 Hz, 3
H), 1.16-1.32 (broad m, 10 H), 1.37 (broad m, 1 H), 1.46 (broad

Multidrug Resistance-Reversing Activity of Hapalosin
J . Org. Chem., Vol. 62, No. 20, 1997 6783
m, 1 H), 1.82 (broad m, 1 H), 2.18-2.35 (m, 3 H), 2.47 (broad
m, 1 H), 2.75 (broad m, 1 H), 2.86 (dd, J ) 14.3, 10.7 Hz, 1 H),
2.99 (s, 3 H), 3.16 (dd, J ) 14.3, 3.5 Hz, 1 H), 3.68 (broad m,
1 H), 4.51 (broad m, 1 H), 5.03-5.18 (m, 4 H), 5.91 (m, 1 H),
5.97 (d, J ) 8.8 Hz, an N-H), 6.04 (d, J ) 9.1 Hz, an N-H),
7.05-7.41 (m, 10 H); 13C NMR (126 MHz, CDCl3), both
rotamers, δ -4.7, -4.6, -3.8, -3.6, 14.1, 14.2, 14.4, 15.6, 16.7,
18.02, 18.04, 19.9, 20.2, 22.59, 22.62, 25.89, 25.93, 26.5, 29.1,
29.2, 29.26, 29.34, 30.0, 30.2, 30.5, 31.8, 32.2, 40.4, 45.2, 45.3,
54.2, 54.4, 62.3, 66.3, 66.4, 75.1, 117.8, 118.5, 126.2, 126.6,
127.8, 127.9, 128.2, 128.29, 128.34, 128.4, 128.6, 128.9, 129.1,
133.8, 134.0, 136.9, 137.0, 138.8, 156.2, 172.0, 172.8, 173.87,
173.91; HREI calcd for C43H69O5N3Si (M+) 735.5006, found
735.5000.
plug of Celite and thorough washing with MeOH and EtOAc
resulted in the desired amino acid (112 mg, 99% crude yield)
as white crystals.
To a solution of the above crude amino acid (110 mg, 0.177
mmol) in toluene (180 mL) were added DIPEA (308 µL, 1.77
mmol) and BOP-Cl (315 mg, 1.24 mmol). The mixture was
stirred at 85 °C for 15 h and then the solution was extracted
with 0.1 M NaHSO4 (40 mL). The aqueous layer was back-
extracted with EtOAc (3 × 30 mL), and the four organic layers
were separately extracted with 0.1 M NaHSO4 (30 mL). Flash
chromatography with silica gel (gradient to 3.0% MeOH/CH2-
Cl2) afforded the TBS ether of the triamide analog of hapalosin,
31 (71 mg, 66% yield for two steps), as a colorless film: [R]22
D
-15° (c 0.017, CHCl3); IR (neat) 830, 1063, 1530, 1654, 3281,
1
Ald eh yd e 29. Ozone was bubbled into a solution of olefin
28 (199 mg, 0.270 mmol) in CH2Cl2 (8 mL) at -78 °C for 3
min. Triphenylphosphine (99 mg, 0.38 mmol) was added, the
-78 °C bath was removed, and stirring transpired at 25 °C
for 12 h. Flash chromatography with silica gel (gradient to
40% EtOAc/hexanes) gave aldehyde 29 (183 mg, 92% yield)
3380 cm-1; H NMR (500 MHz, CDCl3, only one conformer) δ
0.06 (d, J ) 6.6 Hz, 3 H), 0.21 (s, 3 H), 0.22 (s, 3 H), 0.58 (d,
J ) 6.7 Hz, 3 H), 0.89 (t, J ) 6.9 Hz, 3 H), 1.00 (s, 9 H), 1.11
(d, J ) 7.1 Hz, 3 H), 1.18-1.35 (broad m, 10 H), 1.46 (broad
m, 1 H), 1.66 (m, 1 H), 1.82 (m, 1 H), 2.39 (dd, J ) 17.5, 1.8
Hz, 1 H), 2.51 (dd, J ) 13.4, 11.0 Hz, 1 H), 2.66 (dd, J ) 17.5,
5.0 Hz, 1 H), 2.85 (s, 3 H), 3.28-3.33 (m, 2 H), 3.67 (dd, J )
10.4, 8.5 Hz, 1 H), 3.75 (m, 1 H), 4.01 (m, 1 H), 4.08 (dt, J )
10.2, 2.6 Hz, 1 H), 6.12 (d, J ) 10.5 Hz, an N-H), 6.94 (d, J )
6.1 Hz, an N-H), 7.22-7.34 (m, 5 H); 13C NMR (126 MHz,
CDCl3) δ -4.9, -3.5, 13.1, 14.1, 17.6, 18.1, 19.0, 22.6, 26.0,
27.8, 28.3, 28.4, 29.26, 29.32, 30.0, 31.9, 36.5, 38.5, 41.2, 52.9,
53.4, 59.6, 71.8, 127.1, 128.9, 129.9, 137.2, 170.4, 171.7, 173.7;
HRFAB calcd for C34H60O4N3Si [(M + H)+] 602.4353, found
602.4359.
as a colorless oil: [R]22 -17° (c 0.036, CHCl3); IR (neat) 697,
D
777, 837, 1085, 1255, 1456, 1534, 1640, 1694, 1725, 3295 cm-1
;
1H NMR (400 MHz, CDCl3, 4:1 rotamers), the major rotamer,
δ 0.09 (s, 3 H), 0.14 (s, 3 H), 0.80 (d, J ) 6.4 Hz, 3 H), 0.85-
0.89 (overlapping d and t, 6 H), 0.92 (s, 9 H), 1.10 (d, J ) 6.0
Hz, 3 H), 1.15-1.33 (broad m, 10 H), 1.38 (broad m, 1 H), 1.46
(broad m, 1 H), 1.77 (broad m, 1 H), 2.50 (m, 1 H), 2.54 (m, 1
H), 2.63 (dd, J ) 5.6, 2.3 Hz, 1 H), 2.67 (dd, J ) 5.7, 2.4 Hz,
1 H), 2.73 (broad m, 1 H), 2.77 (m, 1 H), 2.85 (s, 3 H), 3.22
(broad m, 1 H), 3.67 (broad m, 1 H), 4.45 (broad m, 1 H), 5.13
(m, 2 H), 5.89 (d, J ) 8.1 Hz, 1 H), 7.09-7.41 (m, 10 H), 9.78
(t, J ) 2.1 Hz, 1 H); 13C NMR (101 MHz, CDCl3), both
rotamers, δ -4.8, -4.7, -4.3, -4.2, 14.1, 14.2, 15.6, 16.5, 17.9,
18.0, 20.0, 20.3, 22.6, 25.7, 25.9, 26.4, 26.5, 29.1, 29.2, 29.26,
29.30, 29.34, 29.6, 29.8, 30.2, 31.7, 31.8, 34.0, 34.4, 45.1, 48.8,
49.1, 54.2, 54.4, 65.0, 66.3, 66.4, 68.6, 68.7, 126.4, 126.8, 127.8,
127.9, 128.3, 128.35, 128.40, 128.5, 128.6, 128.8, 129.1, 136.8,
136.9, 138.4, 156.2, 156.3, 172.6, 173.3, 174.1, 174.2, 200.1,
200.9; HRFAB calcd for C42H68O6N3Si [(M + H)+] 738.4877,
found 738.4882.
Tr ia m id e An a log of Ha p a losin (2). TBAF (70 µL, 0.070
mmol, 1.0 M in THF) was added to a solution of the TBS ether
of the triamide analog, 31 (21 mg, 0.035 mmol), in THF (1.0
mL) and the solution was stirred at 0 °C for 1 h. Concentration
in vacuo without warming the water bath and preparative TLC
(45% acetone/CHCl3) resulted in a band (Rf ) 0.50, brown in
ninhydrin staining) containing the triamide analog of hapal-
osin, 2 (12 mg, 71% yield), as a colorless, glassy film: [R]22
D
-24° (c 0.0080, CH2Cl2); IR (neat) 700, 1051, 1225, 1406, 1455,
1545, 1635, 1651, 1748, 3262 cm-1; 1H NMR (500 MHz, CDCl3,
only one conformer) δ 0.17 (d, J ) 6.7 Hz, 3 H), 0.62 (d, J )
6.8 Hz, 3 H), 0.89 (t, J ) 6.9 Hz, 3 H), 1.11 (d, J ) 7.1 Hz, 3
H), 1.22-1.43 (broad m, 10 H), 1.72 (m, 1 H), 1.77 (m, 1 H),
1.85 (m, 1 H), 2.26 (dd, J ) 16.6, 1.2 Hz, 1 H), 2.64 (dd, J )
16.6, 5.6 Hz, 1 H), 2.70 (dd, J ) 13.7, 9.9 Hz, 1 H), 2.88 (s, 3
H), 3.31-3.36 (m, 2 H), 3.69 (m, 1 H), 3.78 (t, J ) 9.4 Hz, 1
H), 4.03 (dt, J ) 8.1, 3.3 Hz, 1 H), 4.05 (broad m, 1 H), 6.60 (d,
J ) 10.3 Hz, an N-H), 7.10 (d, J ) 5.8 Hz, an N-H), 7.24 (t, J
) 7.0 Hz, 1 H), 7.30 (d, J ) 7.0 Hz, 2 H), 7.33 (t, J ) 7.0 Hz,
2 H); 13C NMR (126 MHz, CDCl3) δ 13.0, 14.1, 17.8, 19.2, 22.6,
26.9, 27.8, 28.8, 29.2, 29.3, 30.2, 31.8, 35.7, 39.8, 41.1, 52.7,
53.9, 59.3, 70.9, 127.0, 128.8, 129.9, 137.3, 170.5, 171.6, 174.0;
HREI calcd for C28H45O4N3 (M+) 487.3410, found 487.3410.
Acid 30. To a solution of aldehyde 29 (162 mg, 0.219 mmol)
t
in BuOH (2.0 mL) were added successively 2-methyl-2-butene
(274 µL, 0.548 mmol, 2.0 M in THF) and a solution of 80%
sodium chlorite (32 mg, 0.28 mmol) and NaH2PO4 (26 mg, 0.22
mmol) in water (0.6 mL). The flask was sealed and the
mixture was stirred vigorously at 25 °C for 15 h. Extraction
in EtOAc (3 × 25 mL) with 0.1 M NaHSO4 (15 mL) and then
water (10 mL) furnished acid 30 (165 mg, 100% crude yield)
as a colorless oil: [R]22 -16° (c 0.021, CHCl3); IR (neat) 698,
D
836, 1082, 1253, 1454, 1535, 1618, 1709, 3300 cm-1; 1H NMR
(400 MHz, CDCl3), both rotamers, δ 0.13-0.17 (singlets, 6 H),
0.80-0.90 (m, 9 H), 0.93 (s, 9 H), 1.13 (overlapping doublets,
3 H), 1.16-1.50 (broad m, 12 H), 1.65 (broad m, 1 H), 2.29-
2.95 (broad m, 7 H), 3.04-3.27 (broad m, 2 H), 3.51-3.81
(broad m, 1 H), 4.25-4.56 (broad m, 1 H), 4.71-4.97 (broad
m, 1 H), 5.10 (m, 2 H), 5.28 (d, J ) 11.4 Hz, 1 H, major
rotamer), 5.96 (d, J ) 9.7 Hz, 1 H, minor rotamer), 6.22-6.58
(broad d, 1 H), 7.07-7.39 (m, 10 H); 13C NMR (101 MHz,
CDCl3), both rotamers, δ -5.0, -4.8, -4.3, -4.1, 14.1, 15.7,
17.8, 17.89, 17.94, 19.9, 21.0, 22.6, 25.8, 26.5, 26.8, 29.18, 29.24,
29.3, 29.4, 30.0, 30.7, 31.8, 34.2, 45.1, 46.5, 53.8, 54.0, 55.3,
66.5, 67.4, 71.3, 71.8, 126.5, 126.8, 127.7, 127.9, 128.37, 128.43,
128.5, 128.9, 129.0, 129.1, 136.1, 136.7, 138.4, 156.4, 158.2,
172.6, 175.1; HRFAB calcd for C42H68O7N3Si [(M + H)+]
754.4827, found 754.4822.
Ack n ow led gm en t. T. D., X. D., and R. A. thank the
NIH (Grant GM51095) and UCLA for financial support.
Computational resources from Professors Y. Rubin and
K. Houk’s groups and the Office of Academic Computing
at UCLA are greatly appreciated. Studies on the
biological activities of the compounds were supported
by NIH Grant CA64983 (C.S.).
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR
spectra of compounds in the three schemes (52 pages). This
material is contained in libraries on microfiche, immediately
follows this article in the microfilm version of the journal, and
can be ordered from the ACS; see any current masthead page
for ordering information.
TBS Eth er of th e Tr ia m id e An a log of Ha p a losin (31).
To a solution of acid 30 (138 mg, 0.183 mmol) in MeOH (4
mL) was added 10% Pd on activated carbon (35 mg). The
round-bottom flask was purged with N2, H2 was bubbled into
the mixture for 3 min, and stirring occurred at 25 °C for 3 h
with an H2-filled balloon attached. Filtration through a short
J O9708396