Synthesis and conformational analysis of the multidrug resistance-reversing agent hapalosin and its non-N-methyl analog

Article abstract of DOI:10.1021/jo9608329

Hapalosin was initially synthesized by macrolactonization, and a second synthesis was achieved by cycloamidation. In both syntheses, three of the five stereocenters in hapalosin were established by two Brown allylboration reactions. The synthesis of the non-N-Me analog of hapalosin involved chelation-controlled reduction of a γ-amino-β-keto ester and cycloamidation. In CDCl₃ at 25°C, synthetic hapalosin exists as a 2.3:1 mixture of conformers, while its non-N-Me analog exists only as a single conformer. ¹H,¹H-NOESY and computation reveal that the configuration of the amide bond is responsible for the conformations of the two compounds. The major conformer of hapalosin is found to be an s-cis amide, the minor conformer an s-trans amide, and the non-N-Me analog an s-trans amide. Applying distance constraints to protons that exhibit NOESY correlations, computation shows that the major conformer of hapalosin and the non-N-Me analog have very different conformations. By contrast, the minor conformer of hapalosin and the non-N-Me analog have very similar conformations.

Full text of DOI:10.1021/jo9608329

6606
J . Org. Chem. 1996, 61, 6606-6616
Syn th esis a n d Con for m a tion a l An a lysis of th e Mu ltid r u g
Resista n ce-Rever sin g Agen t Ha p a losin a n d Its Non -N-m eth yl
An a log
Tam Q. Dinh, Xiaohui Du, and Robert W. Armstrong*
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095
Received May 6, 1996X
Hapalosin was initially synthesized by macrolactonization, and a second synthesis was achieved
by cycloamidation. In both syntheses, three of the five stereocenters in hapalosin were established
by two Brown allylboration reactions. The synthesis of the non-N-Me analog of hapalosin involved
chelation-controlled reduction of a γ-amino-â-keto ester and cycloamidation. In CDCl3 at 25 °C,
synthetic hapalosin exists as a 2.3:1 mixture of conformers, while its non-N-Me analog exists only
as a single conformer. 1H,1H-NOESY and computation reveal that the configuration of the amide
bond is responsible for the conformations of the two compounds. The major conformer of hapalosin
is found to be an s-cis amide, the minor conformer an s-trans amide, and the non-N-Me analog an
s-trans amide. Applying distance constraints to protons that exhibit NOESY correlations,
computation shows that the major conformer of hapalosin and the non-N-Me analog have very
different conformations. By contrast, the minor conformer of hapalosin and the non-N-Me analog
have very similar conformations.
In tr od u ction
be clinically tested for MDR reversal.3 Hapalosin poten-
tiates the cytotoxicity of drugs known to be transported
by P-gp such as taxol, vinblastine, and actinomycin D.
It achieves MDR reversal by elevating the intracellular
concentrations of drugs putatively via inhibition of P-gp.2
Many cytotoxic drugs are quite effective during the
initial stage of cancer chemotherapy. Over time, how-
ever, the tumor cells that survive the treatment often
become resistant not only to the particular drugs em-
ployed but also to a broad spectrum of seemingly un-
related drugs. This phenomenon is known as multidrug
resistance, or MDR.1 The most consistent and dominant
feature of MDR is reduced accumulation of cytotoxic
drugs inside tumor cells. Higher doses of drugs must be
used, with limited efficacy, while the side effects are more
severe.
Of the various potential mechanisms of MDR, the most
generally accepted one concerns the existence of a drug
efflux pump called P-glycoprotein (P-gp). Indeed, tumor
cells from chemotherapy patients often manifest in-
creased P-gp expression and activity. P-gp is a 170-200
kDa transmembrane protein that is homologous with
several bacterial transport ATPases. It utilizes ATP
energy to extrude structurally diverse drugs such as
taxol, Vinca alkaloids, and gramicidin D from the cyto-
plasm of tumor cells. An important strategy in chemo-
therapy is to use a chemosensitizer that blocks P-gp. The
chemosensitizer is not necessarily cytotoxic itself. Rather,
coadministration of the chemosensitizer with cytotoxic
drugs allows the the intracellular concentrations of the
drugs to remain high so that they can damage the
cytoskeleton and DNA of tumor cells.
Hapalosin was concurrently synthesized by our group4
and by Ghosh et al.5 Our synthetic hapalosin exists as
a 2.3:1 mixture of conformers in CDCl3 at 25 °C. Its
1H,1H-NOESY spectrum showed a very strong correlation
between H-9 and H-12 for the major conformer, indicative
of an s-cis amide. On the other hand, there was a strong
correlation between H-1′′ and H-12 for the minor con-
former, suggesting an s-trans amide, but there was none
for the major conformer. Since secondary amides have
a proclivity to exist in the s-trans conformation, we also
wanted to synthesize the non-N-Me analog of hapalosin
(2). It would be informative to relate the conformations
of hapalosin and its non-N-Me analog to their MDR-
reversing activity.
Hapalosin (1) is a novel cyclic depsipeptide that was
recently isolated from the cyanobacterium Hapalosiphon
welwitschii.2 It exhibits a greater MDR-reversing activ-
ity than verapamil, which was one of the first drugs to
Resu lts a n d Discu ssion
Syn th esis. Hapalosin was initially synthesized by
macrolactonization to form the O-5-C-6 ester bond
(Scheme 1).4 The synthesis converged at fragments 4 and
5. Brown allylboration of an aldehyde derivative of L-Phe
led to amino alcohol 4. A second Brown allylboration of
X Abstract published in Advance ACS Abstracts, August 1, 1996.
(1) 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) Ozols, R. F.; Cunnion, R. E.; Klecker, R. W.; Hamilton, T. C.;
Ostchega, Y.; Parrillo, J . E.; Young, R. C. J . Clin. Oncol. 1987, 5, 641.
(4) Dinh, T. Q.; Armstrong, R. W. J . Org. Chem. 1995, 60, 8118.
(5) Ghosh, A. K.; Liu, W.; Xu, Y.; Chen, Z. Angew. Chem., Int. Ed.
Engl. 1996, 35, 74.
(2) Stratmann, K.; Burgoyne, D. L.; Moore, R. E.; Patterson, G. M.
L. J . Org. Chem. 1994, 59, 7219.
S0022-3263(96)00832-8 CCC: $12.00 © 1996 American Chemical Society

Synthesis and Conformational Analysis of Hapalosin
J . Org. Chem., Vol. 61, No. 19, 1996 6607
Sch em e 1
Sch em e 3
Sch em e 4
Sch em e 2
octanal set the two contiguous stereocenters in acid 5. A
third key step was the macrolactonization of hydroxy acid
3.
yield, and the olefin was ozonized to the aldehyde (67%
yield). Oxidation of the aldehyde with sodium chlorite12
afforded acid 13 (100% yield). The acid was converted
to the acid chloride, which reacted with (S)-R-hydroxy-
isovaleric acid to produce ester acid 5 (52% yield).
Six steps after coupling of fragments 4 and 5 provided
hapalosin (Scheme 4). Amide 14 was formed by reacting
amino alcohol 4 with the acid chloride derivative of ester
acid 5 (70% yield). The hydroxyl group was protected13
with TBSOTf (89% yield), and the PMB ether was
deprotected with DDQ (85% yield). Alkenol 16 was
ozonized (85% yield), and the aldehyde was oxidized to
the acid with sodium chlorite. Macrolactonization of
crude hydroxy acid 3 under modified Mukaiyama condi-
tions14 and deprotection of the TBS (tert-butyldimethyl-
silyl) ether of the resulting macrolide with TBAF pro-
duced hapalosin (1) in 13% yield for the final three
steps.15 Synthetic hapalosin was identical in all respects
(1H NMR, 13C NMR, [R]D, IR, and HRMS) to the natural
Amino alcohol 4 was synthesized in four steps starting
with L-N-Boc-Phe-OMe 6 (Scheme 2). The amino ester
was N-methylated with NaH and MeI6 (97% yield) and
reduced to the aldehyde with DIBAH (72% yield).7
Aldehyde 8 underwent Brown allylboration8 to give the
desired diastereomer 9 in 64% yield and 90% de, the de
being determined at the stage of amino alcohol 4. All
N,N-dialkylamides and -carbamates in Schemes 2 and 4
exist as rotamers in CDCl3 at 25 °C. This characteristic
complicates the interpretation of 1H NMR spectra ac-
quired at 25 °C of compounds more advanced than 8.
Synthesis of the second fragment, ester acid 5, is
illustrated in Scheme 3. Brown allylboration9 of octanal
furnished homoallylic alcohol 10 in 90% yield and 90%
de.10 The hydroxyl group was protected with p-meth-
oxybenzyl 2,2,2-trichloroacetimidate (PMBTCAI)11 in 80%
(6) Cheung, S. T.; Benoiton, N. L. Can. J . Chem. 1977, 55, 906.
(7) To minimize racemization of aldehyde 8, solvents were removed
from 8 at e30 °C and flash chromatography with silica gel was done
in <30 min.
(8) J adhav, P. K.; Bhat, K. S.; Perumal, P. T.; Brown, H. C. J . Org.
Chem. 1986, 51, 432.
(9) (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.
(10) Subsequent formation of a single diastereomer of ester acid 5
proved that the ee of alcohol 10 was >95%.
(11) Nakajima, N.; Horita, K.; Abe, R.; Yonemitsu, O. Tetrahedron
Lett. 1988, 29, 4139.
(12) Hillis, L. R.; Ronald, R. C. J . Org. Chem. 1985, 50, 470.
(13) Several attempts to macrolactonize the diolacid corresponding
to 3 were unsuccessful.
(14) The original Mukaiyama conditions are in: Mukaiyama, T.;
Usui, M.; Saigo, K. Chem. Lett. 1976, 49. The modification of Mukai-
yama conditions is based on Evans’ modification of Keck conditions
for macrolactonization with DCC: Evans, D. A.; Kaldor, S. W.; J ones,
T. K.; Clardy, J .; Stout, T. J . J . Am. Chem. Soc. 1990, 112, 7001.
(15) The low yield for the three steps was due to macrolactonization.
The TBS ether of hapalosin could not be isolated in sufficient purity
for the yield of the macrolactonization step to be accurately known.

6608 J . Org. Chem., Vol. 61, No. 19, 1996
Dinh et al.
Sch em e 5
product. Other methods of macrolactonization did not
give better results: (1) 1-cyclohexyl-3-(2-morpholino-
ethyl)carbodiimide metho-p-toluenesulfonate, DMAP‚
HCl, DMAP,16 (2) 2,2′-dipyridyl disulfide, PPh3,17 (3)
2,4,6-trichlorobenzoyl chloride, N,N-diisopropylethyl-
amine (DIPEA), DMAP,18 (4) pivaloyl chloride, DIPEA,
DMAP,19 and (5) bis(2-oxo-3-oxazolidinyl)phosphinic chlo-
ride (BOP-Cl), DIPEA, and DMAP.20
The TBS ether of hapalosin was also synthesized by
cycloamidation. Bis-deprotection of TBS ether 1821 via
hydrogenation over Pearlman’s catalyst in MeOH and
cyclization of the resulting amino acid with BOP-Cl and
DIPEA in PhMe at 85 °C22 produced the TBS ether of
hapalosin in 16% yield for the two steps. The two
corresponding steps provided the TBS ether of the non-
N-Me analog of hapalosin in 60% yield (see Scheme 6).
In Ghosh’s synthesis of hapalosin, bis-deprotection of
MOM ether 19 by hydrogenation over Pearlman’s cata-
lyst in EtOAc and cycloamidation with 1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide (EDC), HOBt, and
triethylamine in DMF at 60 °C afforded the MOM ether
of hapalosin in 30-37% yield for the two steps.5 Cyclo-
amidation to form the TBS ether of hapalosin might have
been sterically hindered by the TBS group, which is
bulkier than MOM.23 For example, bis-deprotection of
TBS ether 18 was not complete after 15 h, whereas it
was finished in 5 h for MOM ether 19.5 Furthermore,
N-methylation of TBS ether 20 with NaH and MeI in
DMF at 25 °C was poor while Ghosh could N-methylate
MOM ether 21 with NaH and MeI in THF/DMF (10:1)
at 25 °C in a good yield.5
to a pyrrole overnight at 25 °C by losing the hydroxyl
and ring TBS ether groups. Two possible ways to obviate
pyrrolidinol formation were to protect the amide nitrogen
or to convert the olefin to a carboxylic acid in one step.
However, the simpler compound 20 was unreactive
toward protection of the carbamate nitrogen with Cbz-
Cl or Boc2O and it could not be transformed into a
carboxylic acid by NaIO4/KMnO4,24 NaIO4/RuCl3,25 or O3/
H2O2.26
The non-N-Me analog of hapalosin was synthesized by
cycloamidation after bis-deprotection of TBS ether 22
(Scheme 5). Compound 22 derived from chiral γ-amino-
â-keto ester 23. The necessary (R)-â-hydroxyl group was
created by a chelation-controlled reduction of the amino
ketone. Coupling of the enolate of acetate 24 to an
activated L-N-Cbz-Phe-formed γ-amino-â-keto ester 23.
The chemistry concerning 23 was based on the work of
J oullie et al. in the synthesis of the didemnins.27
An efficient synthesis of the non-N-Me analog of
hapalosin is illustrated in Scheme 6. Addition of the
enolate of acetate 2428 to the imidazolide of L-N-Cbz-Phe
furnished γ-amino-â-keto ester 23 (72% yield).27 Chela-
tion-controlled KBH4 reduction of the keto group resulted
in two inseparable diastereomers.27 The alcohols were
separated as TBS ethers in a 4.4:1 diastereomeric ratio
(92% yield for two steps).
It was originally thought that synthesis of the non-N-
Me analog of hapalosin could be achieved by macro-
lactonization as for hapalosin. The major problem,
however, was that during ozonolysis, the amide nitrogen
quickly cyclized onto the aldehyde group to form a
pyrrolidinol (eq 1). The pyrrolidinol partially rearranged
The prediction that KBH4 reduction of γ-amino-â-keto
ester 23 would favor the chelation-controlled product was
verified by experiments with the very similar compound
(21) Characterization data of 18 are in the Experimental Section
and its 1H and 13C NMR spectra are in the supporting information.
(22) Evans; D. A.; Miller, S. J .; Ennis, M. D. J . Org. Chem. 1993,
58, 471.
(23) In a work published after this paper was submitted, cycloami-
dation of the amino acid with a free â-hydroxyl group using diphenyl
phosphorazidate and DIPEA in DMF generated hapalosin in 44%
yield: Ohmori, K.; Okuno, T.; Nishiyama, S.; Yamamura, S. Tetrahe-
dron Lett. 1996, 37, 3467.
(24) Aristoff, P. A.; J ohnson, P. D.; Harrison, A. W. J . Am. Chem.
Soc. 1985, 107, 7967.
(25) Carlsen; P. H. J .; Katsuki, T.; Martin, V. S.; Sharpless, K. B.
J . Org. Chem. 1981, 46, 3936.
(26) Bailey, P. S. Ind. Eng. Chem. 1958, 50, 993.
(27) (a) Li, W.-R.; Ewing, W. R.; Harris, B. D.; J oullie, M. M. J . Am.
Chem. Soc. 1990, 112, 7659. (b) Harris, B. D.; J oullie, M. M.
Tetrahedron 1988, 44, 3489.
(28) Acetate 24 was quantitatively obtained by acetylation of alcohol
10 with AcCl, pyridine, and catalytic DMAP in CH2Cl2 at 0-25 °C.
(16) The Evans paper in ref 14.
(17) Corey, E. J .; Nicolaou, K. C. J . Am. Chem. Soc. 1974, 96, 5614.
(18) Inanaga, J .; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M.
Bull. Chem. Soc. J pn. 1979, 52, 1989.
(19) Roush, W. R.; Blizzard, T. A. J . Org. Chem. 1984, 49, 1772.
(20) Corey, E. J .; Hua, D. H.; Pan, B.-C.; Seitz, S. P. J . Am. Chem.
Soc. 1982, 104, 6818.

Synthesis and Conformational Analysis of Hapalosin
J . Org. Chem., Vol. 61, No. 19, 1996 6609
Sch em e 6
Sch em e 7
of 22 by hydrogenation over Pearlman’s catalyst and
cyclization of the resulting amino acid with BOP-Cl and
DIPEA in PhMe at 85 °C22 generated the TBS ether of
the non-N-Me analog of hapalosin, 29, in 60% yield for
the two steps.31 As stated before, the two corresponding
reactions for 18 produced the TBS ether of hapalosin in
only 16% yield. Cyclization of the non-N-Me amino acid
with BOP-Cl was superior to that with 2-chloro-N-
methylpyridinium iodide/DIPEA (55% yield),14 1-cyclo-
hexyl-3-(2-morpholinoethyl)carbodiimide metho-p-tolu-
enesulfonate/DMAP/DMAP‚HCl (33% yield),16 or 2-mesi-
tylenesulfonyl chloride/DIPEA (40% yield).32 Deblocking
of TBS ether 29 with 2.0 equiv of TBAF in THF at 0 °C
for 0.5 h resulted in about 50% epimerization of the non-
N-Me analog of hapalosin, 2 (the site of epimerization is
unknown). This was a surprising observation consider-
ing that TBAF did not cause any epimerization in the
formation of hapalosin. Removal of the TBS group for
the non-N-Me analog was effected by HF/pyridine with-
out any epimerization (71% yield).
Con for m a tion a l An a lysis. In CDCl3 at 25 °C, syn-
thetic hapalosin exists as a 2.3:1 mixture of conformers
whereas its non-N-Me analog exists only as a single
conformer. Their conformations in CDCl3 at 25 °C were
analyzed by 1H,1H-NOESY at 500 MHz over three
different mixing timess1.15, 2.30, and 3.45 s (0.80 s was
too short for hapalosin). The two compounds tumbled
rapidly in the positive NOE regime. Crosspeaks of at
least medium strength that were used in the conforma-
tional computation of the major conformer of hapalosin
and the non-N-Me analog are listed in Table 1. Due to
peak overlap of H-1′′′b/H-7b and H-1′′/H-7a for the major
conformer of hapalosin, some potential correlations in-
volving these protons were not considered. Attempts to
crystallize the two compounds were unsuccessful.
Since rotational isomerism about the amide bond is
common for tertiary N,N-dialkylamides and secondary
amides tend to favor the s-trans configuration, these
characteristics were conjectured to be responsible for the
conformations of hapalosin and its non-N-Me analog.
30 (Scheme 7). KBH4 reduction of γ-amino-â-keto ester
30 produced two separable diastereomers in a 4.4:1 ratio.
L-Selectride (Aldrich) reduction of 3016 resulted in a
substantially greater diastereomeric excess, but the
major diastereomer in this reaction was the minor
diastereomer in the KBH4 reaction. The major dia-
stereomer in the L-Selectride reaction, 31, was converted
to oxazolidinone 32. 1H,1H-NOESY of oxazolidinone 32
showed that the two ring protons, Hb and Hc, were trans.
For instance, there were crosspeaks correlating Hb to both
Hd protons and to the o-phenyl protons, and there was
no crosspeak correlating either of the Ha protons to either
of the Hd protons. The trans relationship in 32 proved
that L-Selectride reduction of 30 favored the Felkin-Anh
product 31. These results agreed with J oullie’s findings
that KBH4 reductions of similar chiral N-Boc- and N-Cbz-
γ-amino-â-keto esters favored chelation-controlled prod-
ucts.27,29
The desired TBS ether, 25, was ozonized to the
aldehyde (85% yield), which was then oxidized to the acid
with sodium chlorite (100% yield). Acid 27 was coupled
to hydroxybenzyl ester 2830 with EDC and DMAP to
furnish tris-protected 22 (85% yield). Bis-deprotection
(30) Hydroxybenzyl ester 28 was made in 87% yield by reaction of
(S)-R-hydroxyisovaleric acid, BnBr, and DBU in MeCN at 25 °C. The
protocol was culled from: Rao, C. G. Org. Prep. Proc. Int. 1980, 12,
225.
(31) Since it was best not to purify the zwitterionic amino acid
resulting from bis-deprotection of 22, the yield of this step is not
specifically known but is probably good.
(32) Baker, R.; Castro, J . L. J . Chem. Soc. Perkin Trans. 1 1990,
47.
(29) KBH4 reduction of the N-Me variant of 23 for the synthesis of
hapalosin would probably demonstrate insignificant stereoselectivity
since this was so when MOM-protected trans-4-hydroxy-L-proline was
used in lieu of the L-phenylalanine moiety in 23.

6610 J . Org. Chem., Vol. 61, No. 19, 1996
Dinh et al.
Ta ble 1. NOESY Cor r ela tion s betw een P r oton sa
attached to C-11, N-10, the hydroxyl oxygen, C-2, and
C-6 for the non-N-Me analog. Superimposition of the
s-trans conformation of hapalosin with the non-N-Me
analog was done by Biosym’s INSIGHT II (v. 2.3.5). The
carbonyl atoms C-2, C-6, and C-11 were superimposed.
hapalosin (1)b
non-N-Me analog (2)
9-12
2′-3′′′/7′′′
1′-1′′
7b-1′′′a
8-1′′
2′-4′′′/6′′′
2′-5′′′
2′-1′′
7b-1′′′b
1′′-1′′′b
3-1′′′′′
1′′′′a-1′′′′′
12-3′′′/7′′′
3′-1′′
1′′′′a-3
From the conformational search of hapalosin, 32
conformations were found within 5 kcal/mol from the
global minimum. Twenty-one of them have an s-cis
amide bond, and 11 have an s-trans amide bond. The
lowest-energy conformation found has an s-cis amide
bond. The distance between H-9 and H-12 for all s-cis
conformations ranges from 2.0 to 3.0 Å except for one of
3.3 Å, while that for all s-trans conformations is above
4.4 Å. This is in accordance with the very strong NOESY
correlation observed between H-9 and H-12 for the major
conformer of hapalosin. These results strongly suggest
that the major conformer has an s-cis amide bond and
the minor conformer has an s-trans amide bond. With
regard to the non-N-Me analog of hapalosin, 12 confor-
mations were found within 5 kcal/mol from the global
minimum. All have an s-trans amide bond. This agrees
with the NOESY data and the fact that only one
1′′-3′′′/7′′′
9-3′′′/7′′′
12-1′′
1′′′a-1′′
1′′′b-1′′
8-1′′
1′′′′a-1′′′′′
1′′′′b-1′′′′′
3′-3′′′/7′′′
1′-3′′′/7′′′
1′′-3′′′/7′′′
9-3′′′/7′′′
8-3′′′/7′′′
OH-9
7a-9
8-1′′′a
8-1′′′b
1H,1H-NOESY of the two compounds in CDCl3 was conducted
at 25 °C at 500 MHz over three different mixing times-1.15, 2.30,
and 3.45 s. All the correlations are of at least medium strength.
a
b
The major conformer.
Indeed, the NOESY spectra showed prima facie that the
major conformer of hapalosin is an s-cis amide, the minor
conformer is an s-trans amide, and the non-N-Me analog
is an s-trans amide. There was a very strong correlation
between H-9 and H-12 for the major conformer of
hapalosin, suggesting an s-cis amide. By contrast, there
was a strong correlation between H-1′′ and H-12 for the
minor conformer, implying an s-trans amide, but no such
correlation existed for the major conformer. The non-N-
Me analog of hapalosin appeared to be an s-trans amide
due to the presence of crosspeaks correlating all four
types of isobutyl protons (H-12, -1′, -2′, and -3′) to H-1′′.
Computation corroborated the NOESY interpretations.
Molecular modeling studies were performed with Mac-
romodel (v. 4.5)33 using the AMBER* force field and GB/
SA chloroform solvation.34 (All of the computations
discussed below were also conducted using the MM3*
force field, and the results are similar to those with the
AMBER* force field.) Conformational searches were
performed using Still’s internal coordinate Monte Carlo
protocol.35 The search was done on blocks of 1000 Monte
Carlo steps until no lower-energy conformation was found
compared to the current global minimum. For hapalosin,
different cis and trans conformations were used as the
starting geometry, and no lower energy minimum was
found. To simplify the number of conformations found
in the computation, the carbon atoms of the heptyl side
chain were not compared; i.e., if two conformations had
exactly the same coordinates on all atoms except for the
heptyl side chain, they were considered to be the same
conformation. Duplicate conformations as well as those
that had chirality change were discarded. Internuclear
distance constraints for protons that have NOESY cor-
relations were set between 1.5 and 5.0 Å. Table 1
contains the pairs of protons used in the distance
constraints for the major conformer of hapalosin and the
non-N-Me analog. Clustering of conformations of the
same structure was done employing the Xcluster program
implemented with Macromodel. The clustering was
based on atomic root mean square displacement between
pairs of structures following rigid-body superimposition.
Atoms C-11, the oxygen attached to C-11, N-10, C-1′′, the
hydroxyl oxygen, C-2, and C-6 were compared during
clustering for hapalosin and atoms C-11, the oxygen
conformer exists in CDCl3
at 25 °C.
Though hapalosin is not a big molecule, it is quite
flexible. Twelve clusters could be found among all
conformations.36 They have different ring shapes and
different carbonyl orientations (Figure 1a). Ring shapes
1-7 belong to the s-cis amide configuration and ring
shapes 8-12 to the s-trans amide configuration. There
are four very different combinations of the relative
positions of the carbonyl groups: uuu, uud, pdu, and udu
in the order of C-11, C-6, and C-2 (with respect to the
ring, “u” denotes up, “d” down, and “p” parallel). Most
of the conformations do not have any hydrogen bonding
between the hydroxyl group and a carbonyl oxygen. By
contrast, the non-N-Me analog of hapalosin is much less
variable due to the intramolecular hydrogen bonds
formed. In every conformer, the amide H-1′′ hydrogen
bonds with O-1 and the oxygen attached to C-6 and the
hydroxyl group hydrogen bonds with at least one carbonyl
oxygen. The three clusters of the non-N-Me analog37 vary
in their ring shape but have similar carbonyl orientations
(Figure 1b).
Conformational searches with distance constraints for
protons that have correlations in the NOESY of the major
conformer of hapalosin and the non-N-Me analog (Table
1) were also conducted. Nine conformations were found
for the major conformer of hapalosin. They all have an
s-cis amide bond, indicating again that the major con-
former of hapalosin has an s-cis amide bond. The nine
conformations can be separated into four clusters, and
their ring shapes can be represented by ring shapes 1,
2, 4, and 7 of hapalosin in Figure 1a.38 The superimposi-
tion of the nine conformations is shown in Figure 2a. The
distance constraints helped to locate the isopropyl group
and the benzyl group very well, and their positions
remain relatively unchanged through all nine conforma-
tions. Four conformations were found for the non-N-Me
analog, separated into two clusters represented by ring
(36) The number of conformations contained in clusters 1-12 of
hapalosin is six, four, five, two, two, one, one, five, two, two, one, and
one, respectively.
(37) The number of conformations contained in clusters 1-3 of the
non-N-Me analog is three, seven, and two, respectively.
(38) The number of conformations belonging to ring shapes 1, 2, 4,
and 7 for the major conformer of hapalosin is four, two, one, and two,
respectively.
(33) 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.
(34) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J .
Am. Chem. Soc. 1990, 112, 6127.
(35) Chang, G.; Guida, W. C.; Still, W. C. J . Am. Chem. Soc. 1989,
111, 4379.

Synthesis and Conformational Analysis of Hapalosin
J . Org. Chem., Vol. 61, No. 19, 1996 6611
F igu r e 1. Ring conformations of the clusters for hapalosin (a) and for the non-N-Me analog (b) within 5 kcal/mol of the found
global minimum. All the side chains and protons on the ring carbon atoms are removed for clarity. Key: dark, C; spotted, O;
gray, N; white, H.
F igu r e 2. Stereoviews: superimposition of the nine conformations of the major conformer of hapalosin (a) and superimposition
of the four conformations of the non-N-Me analog (b) that satisfy the distance constraints, with the four atoms of the amide bond
superimposed. The heptyl group is labeled as R. The protons on carbon atoms are removed for clarity. Key: dark, C; spotted, O;
gray, N; white, H.
shapes 1 and 2 in Figure 1b.39 The four conformations
are superimposed in Figure 2b. The lowest-energy
conformation from the major cluster of s-cis hapalosin
and that for the non-N-Me analog are displayed in Figure
3a,b, respectively. Comparison of Figure 3a to b shows
that the major conformer of hapalosin and the non-N-
Me analog have different orientations of side chains and
very different ring shapes as a result of the difference in
amide bond geometry. Considering such dissimilar
conformations, the major conformer of hapalosin and the
non-N-Me analog may have very different biological
actvities. The non-N-Me analog is currently undergoing
biological evaluation for its ability to reverse MDR.
In contrast, there are many similarities between the
lowest energy conformation from the major cluster of the
s-trans amide conformations of hapalosin and the lowest
energy, distance-constrained conformation from the ma-
jor cluster of the non-N-Me analog (Figure 4). The two
rings superimpose quite well on the left half from C-12
to C-6 as exhibited in Figure 4. Both amide bonds are
s-trans, and all three carbonyl groups are in similar
orientations. The isopropyl and benzyl groups are also
in similar orientations. The difference on the right side
of the ring is a result of the orientation of the hydroxyl
group. In the s-trans conformer of hapalosin, the hy-
droxyl group is sticking out while in the non-N-Me
analog, the hydroxyl group rotates inward to form a
hydrogen bond with the carbonyl oxygen attached to C-2.
The computation suggests that the non-N-Me analog and
minor conformer of hapalosin, having similar conforma-
tions, may share similar biological activities.
Con clu sion
NOESY and computation demonstrate that the major
conformer of hapalosin has an s-cis amide bond, the
minor conformer has an s-trans amide bond, and the non-
N-Me analog has an s-trans amide bond. With the aid
of the NOESY data, computation shows that the major
(39) The number of conformations belonging to ring shapes 1 and 2
for the non-N-Me analog is three and one, respectively.

6612 J . Org. Chem., Vol. 61, No. 19, 1996
Dinh et al.
F igu r e 3. Stereoviews: the lowest-energy conformation from the major cluster of hapalosin with an s-cis amide (a) and that for
the non-N-Me analog (b) that satisfy the distance constraints. The heptyl group is labeled as R. The protons on carbon atoms are
removed for clarity. Key: dark, C; spotted, O; gray, N; white, H.
F igu r e 4. Stereoview: superimposition of the lowest-energy conformation from the major cluster of the non-N-Me analog that
satisfies the distance constraints and the lowest-energy conformation from the major cluster of hapalosin with an s-trans amide.
The carbonyl atoms C-2, C-6, and C-11 are superimposed. The heptyl group is labeled as R. The protons on carbon atoms are
removed for clarity. Key: dark, C; spotted, O; gray, N; white, H.
Boc-Phe-OMe (6) can be bought from Aldrich. Most com-
mercially available reagents were distilled before use. Thin-
layer chromatography (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 carboxylic acids) staining. After all aqueous extrac-
tions of crude reaction products, the combined organic layers
were dried over MgSO4, filtered, and concentrated in vacuo
before further treatment.
conformer of hapalosin and the non-N-Me analog have
very different conformations. On the other hand, the
minor conformer of hapalosin and the non-N-Me analog
exhibit very similar conformations. Knowledge of the
MDR-reversing activity of the non-N-Me analog may
reveal whether the major or minor conformer of hapalosin
accounts for its MDR-reversing property.
Exp er im en ta l Section
L-N-(tBu tyloxycar bon yl)-N-m eth ylph en ylalan in e Meth -
yl Ester (7). A solution of L-N-Boc-Phe-OMe (6) (10.00 g, 35.8
mmol) and MeI (4.5 mL, 72 mmol) in DMF (130 mL) was cooled
to 0 °C. NaH (1.12 g, 46.5 mmol) was added in 200 mg
portions, and more was added when bubbling subsided. After
all the NaH was added, the ice bath was removed, and the
mixture was stirred at 25 °C for 15 h. The mixture was
quenched with saturated aqueous NH4Cl (5 mL), and much
DMF was removed in vacuo. Extraction in EtOAc (3 × 200
mL) with H2O (250 mL) gave pure 7 (10.18 g, 97% yield) as
an oil: [R]22D -54° (c 0.043, CHCl3); IR (neat) 1144, 1393, 1699,
Gen er a l P r oced u r es. 1H and 13C NMR spectra were
obtained at 25 °C with a Bruker ARX-400 or -500 spectrometer.
1H NMR chemical shifts are referenced to TMS (0.00 ppm) and
those for 13C NMR to CDCl3 (77.0 ppm). IR spectra were
recorded with a Nicolet 510P FT-IR spectrometer. Optical
rotations were measured with a Perkin-Elmer 241MC pola-
rimeter, and concentrations (c) are reported in g/mL. High-
resolution mass spectroscopy (HRMS) was performed with a
VG Autospec instrument for the EI and CI methods and a VG
ZAB-SE instrument for the FAB method.
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 im-
mediately prior to use: CH2Cl2 from P2O5, PhMe from CaH2,
MeOH from magnesium metal, and THF from sodium metal/
benzophenone ketyl. Anhydrous DMF and MeCN and (S)-R-
hydroxyisovaleric acid were purchased from the Aldrich
Chemical Co. and utilized without further purification. L-N-
1
1746, 2977 cm-1; H NMR (400 MHz, CDCl3, 1.3:1 rotamers)
major rotamer δ 1.33 (s, 9 H), 2.72 (s, 3 H), 3.01 (dd, J ) 14.2,
10.9 Hz, 1 H), 3.27 (m, 1 H), 3.75 (s, 3 H), 4.54 (dd, J ) 10.7,
4.4 Hz, 1 H), 7.12-7.24 (m, 3 H), 7.28 (m, 2 H), minor rotamer
δ 1.37 (s, 9 H), 2.70 (s, 3 H), 3.01 (dd, J ) 14.2, 10.9 Hz, 1 H),
3.31 (m, 1 H), 3.73 (s, 3 H), 4.94 (dd, J ) 10.5, 5.3 Hz, 1 H),
7.12-7.24 (m, 3 H), 7.28 (m, 2 H); 13C NMR (101 MHz, CDCl3)
both rotamers δ 28.1, 28.2, 31.8, 32.5, 35.0, 35.5, 52.1, 59.4,

Synthesis and Conformational Analysis of Hapalosin
J . Org. Chem., Vol. 61, No. 19, 1996 6613
61.6, 79.9, 80.2, 126.4, 126.6, 128.3, 128.5, 128.9, 129.0, 137.3,
137.6, 154.9, 155.7, 171.5, 171.9; HRFAB calcd for C16H24O4N
[(M + H)+] 294.1705, found 294.1710.
disappearance of much of the yellow color. The solution was
stirred at -78 °C for 30 min, and then BF3 OEt2 (14.5 mL,
.
118 mmol) was added, giving a white mixture. The mixture
thickened upon cannulation of a solution of octanal (15.4 mL,
98.5 mmol) in THF (110 mL) and was stirred for 2 h at -78
°C and 1.5 h without the dry ice/acetone bath. The mixture
was cooled to 0 °C, and aqueous 30% H2O2 (33.6 mL) and
aqueous 3 M NaOH (67 mL) were added slowly. After
oxidation occurred overnight at 25 °C, most THF was removed
in vacuo and extraction in EtOAc (3 × 400 mL) was conducted
successively with brine (600 mL) and H2O (600 mL). Flash
chromatography with silica gel (gradient to 10% EtOAc/
hexanes) and concentration in vacuo at 30 °C afforded 10
(15.84 g, 90% yield) as a colorless liquid in 90% de: [R]22D +28°
(c 0.022, CHCl3); IR (neat) 912, 1456, 2926, 3362 cm-1; 1H NMR
(400 MHz, CDCl3) δ 0.87 (t, J ) 6.9 Hz, 3 H), 1.01 (d, J ) 6.9
Hz, 3 H), 1.20-1.38 (broad m, 10 H), 1.48 (broad m, 2 H), 2.26
L-N-(tBu tyloxyca r bon yl)-N-m eth ylp h en yla la n in a l (8).
A solution of methyl ester 7 (9.64 g, 32.9 mmol) in PhMe (100
mL) was cooled to -78 °C. DIBAH (49.6 mL in hexanes, 49.6
mmol) was added over 15 min, and the solution was stirred
for 2.5 h at -78 °C and then quenched with -78 °C MeOH
(20 mL). After successive extractions in CH2Cl2 (3 × 200 mL)
with cold aqueous 1.2 M HCl (300 mL) and brine (300 mL)
and concentration of the combined organic layers in vacuo at
30 °C, flash chromatography with silica gel (gradient to 50%
EtOAc/hexanes) was performed in <30 min to minimize
racemization. Aldehyde 8 (6.26 g, 72% yield) was obtained as
a colorless oil: [R]22 -83° (c 0.023, CHCl3); IR (neat) 1152,
D
1368, 1686, 1740, 2977 cm-1; 1H NMR (400 MHz, CDCl3, 1.2:1
rotamers), major rotamer δ 1.43 (s, 9 H), 2.68 (s, 3 H), 2.89
(dd, J ) 14.1, 10.4 Hz, 1 H), 3.30 (dd, J ) 14.2, 4.5 Hz, 1 H),
4.01 (dd, J ) 10.4, 4.1 Hz, 1 H), 7.16 (d, J ) 7.3 Hz, 2 H), 7.23
(m, 1 H) 7.30 (m, 2 H), 9.66 (s, 1 H), minor rotamer δ 1.37 (s,
9 H), 2.62 (s, 3 H), 3.00 (dd, J ) 14.2, 10.3 Hz, 1 H), 3.30 (dd,
J ) 14.2, 4.5 Hz, 1 H), 4.19 (dd, J ) 10.3, 4.9 Hz, 1 H), 7.19
(d, J ) 7.6 Hz, 2 H), 7.23 (m, 1 H), 7.30 (m, 2 H), 9.66 (s, 1 H);
13C NMR (101 MHz, CDCl3), both rotamers δ 28.1, 28.2, 32.7,
33.5, 34.7, 34.9, 68.3, 69.5, 80.5, 81.2, 126.5, 126.7, 128.5, 128.7,
129.1, 137.6, 154.7, 155.8, 198.9, 199.3; HRFAB calcd for
C15H22NO3 [(M + H)+] 264.1600, found 264.1603.
(m, 1 H), 3.48 (m, 1 H), 5.05-5.12 (m, 2 H), 5.79 (m, 1 H); 13
NMR (101 MHz, CDCl3) δ 13.9, 14.1, 22.6, 26.1, 29.3, 29.6,
C
31.8, 34.0, 43.4, 74.7, 115.1, 141.1; HREI calcd for C12H24
O
(M+) 184.1827, found 184.1828.
(3R,4R)-4-p -[(Met h oxyb en zyl)oxy]-3-m et h ylu n d ec-1-
en e (11). To a solution of alcohol 10 (1.46 g, 7.92 mmol) in
THF (30 mL) were added PMBTCAI (4.5 mL, 22 mmol) and
then TfOH (2.1 µL, 0.024 mmol) as 350 µL of a stock solution
of 6 µL of TfOH in 1 mL of THF. The solution was stirred for
5 h at 25 °C. TfOH was quenched with triethylamine (about
25 µL), and the solution was concentrated in vacuo. Flash
chromatography with silica gel (gradient to 5% EtOAc/hex-
anes) led to PMB ether 11 (1.93 g, 80% yield) as a colorless
(2S,3R)-2-[N-(t Bu t yloxyca r b on yl)-N-m et h yla m in o]-1-
p h en ylh ex-5-en -3-ol (9). To a solution of (+)-B-methoxydi-
isopinocampheylborane (7.93 g, 25.1 mmol) in THF (65 mL)
at -78 °C was slowly added allylmagnesium bromide (23.6 mL
in Et2O, 23.6 mmol), and the mixture was stirred at -78 °C
for 15 min and then at 25 °C for 1 h. The reaction solution
was cooled to -78 °C, and a solution of amino aldehyde 8 (5.50
g, 20.9 mmol, azeotroped with PhMe under high vacuum at
<25 °C) in THF (40 mL) was added slowly. The solution was
stirred at -78 °C for 2 h and then for 1.5 h without the dry
ice/acetone bath. Oxidation of the boronate with aqueous 3
M NaOH (21 mL) and aqueous 30% H2O2 (9 mL) went
overnight at 25 °C. A great deal THF was removed in vacuo
before extraction in EtOAc (3 × 200 mL) with brine (300 mL).
Much isopinocampheyl alcohol was removed under high
vacuum at 90 °C. Flash chromatography with silica gel
(gradient to 50% EtOAc/hexanes) afforded 9 (4.07 g, 64% yield)
as a colorless oil: [R]22D -26° (c 0.067, CHCl3); IR (neat) 1169,
1366, 1667, 2977, 3428 cm-1; 1H NMR (400 MHz, CDCl3) both
rotamers δ 1.25 (broad s, 9 H), 1.39 (s, 9 H), 2.10-2.42 (m, 2
H), 2.47 (s, 3 H), 2.72 (broad s, 3 H), 2.96-3.24 (m, 2 H), 3.60-
3.97 (broad m, 2 H), 5.08-5.21 (m, 2 H), 5.87 (m, 1 H), 7.10-
7.30 (m, 5 H); 13C NMR (101 MHz, CDCl3) both rotamers δ
27.9, 28.1, 32.4, 34.1, 34.7, 38.8, 39.3, 71.4, 72.9, 79.2, 79.5,
117.7, 118.1, 125.8, 128.0, 128.8, 134.2, 134.7, 139.0, 155.4,
156.3; HRFAB calcd for C18H28NO3 [(M + H)+] 306.2069, found
306.2071.
oil: [R]22 +22° (c 0.018, CHCl3); IR (neat) 1248, 1514, 2930
D
1
cm-1; H NMR (400 MHz, CDCl3) δ 0.88 (t, J ) 6.9 Hz, 3 H),
1.03 (d, J ) 6.9 Hz, 3 H), 1.17-1.35 (broad m, 10 H), 1.45
(broad m, 2 H), 2.47 (m, 1 H), 3.23 (m, 1 H), 3.80 (s, 3 H), 4.43
(d, J ) 11.0 Hz, 1 H), 4.49 (d, J ) 11.0 Hz, 1 H), 5.02 (m, 2 H),
5.85 (m, 1 H), 6.87 (d, J ) 8.7 Hz, 2 H), 7.27 (d, J ) 8.7 Hz, 2
H); 13C NMR (101 MHz, CDCl3) δ 14.1, 15.6, 22.7, 25.6, 29.3,
29.8, 31.1, 31.9, 40.8, 55.3, 71.4, 82.6, 113.7, 114.1, 129.3, 131.2,
141.3, 159.0; HREI calcd for C20H32O2 (M+) 304.2402, found
304.2396.
(2S,3R)-3-p-[(Meth oxyben zyl)oxy]-2-m eth yldecan al (12).
A solution of olefin 11 (1.78 g, 5.85 mmol) in CH2Cl2 (75 mL)
was cooled to -78 °C, and ozone was bubbled in for 8 min with
stirring. PPh3 (2.7 g, 10 mmol) was added to the moderate
gray solution, and the solution was stirred overnight at 25 °C.
Concentration in vacuo and flash chromatography with silica
gel (gradient to 10% EtOAc/hexanes) provided aldehyde 12
(1.20 g, 67% yield) as a colorless oil: [R]22 +26° (c 0.019,
D
CHCl3); IR (neat) 1248, 1514, 1725, 2928 cm-1; 1H NMR (400
MHz, CDCl3) δ 0.89 (t, J ) 6.9 Hz, 3 H), 1.11 (d, J ) 7.0 Hz,
3 H), 1.17-1.34 (broad m, 10 H), 1.39 (m, 1 H), 1.50 (m, 1 H),
2.55 (m, 1 H), 3.79 (m, 1 H), 3.80 (s, 3 H), 4.43 (d, J ) 11.1 Hz,
1 H), 4.47 (d, J ) 11.1 Hz, 1 H), 6.87 (d, J ) 8.7 Hz, 2 H), 7.22
(d, J ) 8.7 Hz, 2 H), 9.74 (d, J ) 1.0 Hz, 1 H); 13C NMR (101
MHz, CDCl3) δ 8.1, 14.1, 22.6, 25.8, 29.2, 29.5, 31.6, 31.8, 49.6,
55.2, 71.3, 78.1, 113.7, 129.3, 130.3, 159.2, 204.9; HREI calcd
for C19H30O3 (M+) 306.2195, found 306.2192.
(2S,3R)-2-(Meth yla m in o)-1-p h en ylh ex-5-en -3-ol (4). To
a solution of 9 (615 mg, 2.01 mmol) in CH2Cl2 (14 mL) was
added TFA (3.9 mL, 50 mmol), and the stirring transpired for
1.5 h at 25 °C. TFA was removed in vacuo, and extraction in
EtOAc (3 × 75 mL) with saturated aqueous Na2CO3 (100 mL)
(2S,3R)-3-p -[(m et h oxyb en zyl)oxy]-2-m et h yld eca n oic
Acid (13). To a solution of aldehyde 12 (1.15 g, 3.75 mmol)
provided pure 4 (413 mg, 100% yield) as a white solid: [R]22
D
t
+6° (c 0.009, CHCl3); IR (neat) 698, 914, 2946, 3069, 3291 cm-1
;
and 2-methyl-2-butene (0.7 mL, 6.4 mmol) in BuOH (25 mL)
1H NMR (500 MHz, CDCl3) δ 2.29 (m, 1 H), 2.29 (s, 3 H), 2.40
(m, 1 H), 2.58 (dd, J ) 13.8, 10.2 Hz, 1 H), 2.70 (m, 1 H), 2.87
(dd, J ) 13.8, 4.0 Hz, 1 H), 3.83 (m, 1 H), 5.14 (dd, J ) 10.2,
1.6 Hz, 1 H), 5.19 (dd, J ) 17.1, 1.6 Hz, 1 H), 5.92 (m, 1 H),
7.19 (d, J ) 7.4 Hz, 2 H), 7.23 (t, J ) 7.4 Hz, 1 H), 7.31 (dd, J
) 7.4, 7.4 Hz, 2 H); 13C NMR (126 MHz, CDCl3) δ 34.1 34.4,
36.9, 64.1, 68.4, 117.2, 126.5, 128.7, 129.1, 135.2, 138.8; HRCI
calcd for C13H18NO [(M - H)+] 204.1388, found 204.1382.
(3R,4R)-3-Meth ylu n d ec-1-en -4-ol (10). To a mixture of
was added slowly a solution of 80% NaClO2 (508 mg, 4.5 mmol)
and NaH2PO4 (540 mg, 4.5 mmol) in H2O (6 mL). Once the
reaction solution was not too warm, the round-bottom flask
was glass-stoppered and parafilmed, and stirring was contin-
ued overnight at 25 °C. After most of tBuOH was removed in
vacuo and extractions in CH2Cl2 (2 × 150 mL) with aqueous
HCl (pH 1, 100 mL) and then H2O (100 mL) were done, pure
acid 13 (1.21 g, 100% yield) was obtained as a colorless oil:
[R]22 +20° (c 0.011, CHCl3); IR (neat) 1248, 1514, 1707, 2930
D
1
cm-1; H NMR (500 MHz, CDCl3) δ 0.88 (t, J ) 6.9 Hz, 3 H),
t
sublimed BuOK (11.05 g, 98.5 mmol) in THF (50 mL) at -78
1.19 (d, J ) 7.0 Hz, 3 H), 1.21-1.32 (broad m, 10 H), 1.53 (m,
2 H), 2.73 (m, 1 H), 3.68 (m, 1 H), 3.80 (s, 3 H), 4.49 (d, J )
11.0 Hz, 1 H), 4.54 (d, J ) 11.0 Hz, 1 H), 6.87 (d, J ) 8.7 Hz,
2 H), 7.25 (d, J ) 8.7 Hz, 2 H); 13C NMR (126 MHz, CDCl3) δ
11.7, 14.1, 22.6, 25.6, 29.2, 29.5, 31.4, 31.8, 42.4, 55.3, 71.9,
°C were added cis-2-butene (about 25 mL) via cannula and
then nBuLi (47.9 mL in pentane, 95.8 mmol). The bright
yellow mixture was stirred at -78 °C for 30 min, and a solution
of (-)-B-methoxydiisopinocampheylborane (35.8 g, 113 mmol)
in THF (110 mL) was cannulated into it, resulting in the

6614 J . Org. Chem., Vol. 61, No. 19, 1996
Dinh et al.
79.8, 113.8, 129.6, 129.8, 159.4, 178.5; HRFAB calcd for
C
169.3, 174.8; HRFAB calcd for C43H70O6NSi [(M + H)+]
724.4972, found 724.4964.
19H30O4 (M+) 322.2144, found 322.2144.
(S)-1-Ca r boxy-2-m eth ylp r op yl (2S,3R)-3-p-[(m eth oxy-
[(tBu tyld im eth ylsilyl)oxy]a lk en ol 16. DDQ (231 mg,
1.02 mmol) was added to a mixture of PMB ether 15 (368 mg,
0.51 mmol) in 18:1 (v/v) CH2Cl2/H2O (11.6 mL), and the
mixture was stirred vigorously for 2 h at 25 °C. After
extraction in CH2Cl2 (2 × 70 mL) with saturated aqueous
NaHCO3 (100 mL) and flash chromatography with silica gel
(gradient to 10% EtOAc/hexanes), 16 (262 mg, 85% yield) was
ben zyl)oxy]-2-m eth yld eca n oa te (5). To a solution of acid
13 (1.18 g, 3.66 mmol) in CH2Cl2 (40 mL) were added DIPEA
(637 µL, 3.66 mmol) and SOCl2 (400 µL, 5.49 mmol). The
solution was stirred for 2 h at 0 °C, and then excess SOCl2
was removed in vacuo carefully to obviate bumping. The crude
acid chloride was dissolved in CH2Cl2 (40 mL) and cooled to 0
°C. DIPEA (1.40 mL, 8.05 mmol) was added slowly followed
by (S)-R-hydroxyisovaleric acid (519 mg, 4.39 mmol). The
solution was stirred overnight at 25 °C and extracted in CH2Cl2
(2 × 100 mL) with aqueous HCl (pH 1, 100 mL). Flash
chromatography with silica gel (gradient to 60% EtOAc/
hexanes) furnished 5 (811 mg, 52% yield) as a brown oil and
obtained as a colorless oil: [R]22 -43° (c 0.0095, CHCl3); IR
D
(neat) 1645, 1738, 2930, 3432 cm-1; 1H NMR (400 MHz, CDCl3)
both rotamers δ 0.05-0.16 (m, 6 H), 0.84-1.00 (m, 15 H),
1.00-1.12 (m, 6 H), 1.23-1.37 (broad m, 10 H), 1.37-1.56
(broad m, 2 H), 1.56-1.91 (broad m, 1 H), 2.17-2.94 (m, 6 H),
3.00-3.19 (m, 2 H), 3.67-4.20 (broad m, 3 H), 4.88 (d, J ) 2.8
Hz, 1 H), 5.00-5.25 (m, 2 H), 5.91 (m, 1 H), 7.08-7.29 (m, 5
H); 13C NMR (101 MHz, CDCl3) both rotamers δ -4.7, -3.8,
9.0, 14.1, 15.5, 16.3, 18.0, 19.5, 20.0, 22.63, 22.65, 25.9, 26.4,
28.5, 28.8, 29.3, 29.7, 30.2, 31.8, 32.9, 39.3, 40.4, 44.4, 61.6,
72.16, 72.21, 74.7, 74.9, 75.0, 117.8, 126.2, 126.7, 128.3, 128.9,
129.0, 133.9, 134.3, 138.7, 170.2, 170.5, 174.9, 175.1; HRFAB
calcd for C35H62NO5Si [(M + H)+] 604.4397, found 604.4404.
no other diastereomer of 5: [R]22 +19° (c 0.012, CHCl3); IR
D
(neat) 1248, 1514, 1736, 2932 cm-1; 1H NMR (400 MHz, CDCl3)
δ 0.88 (t, J ) 6.9 Hz, 3 H), 1.00 (d, J ) 7.0 Hz, 3 H), 1.02 (d,
J ) 7.0 Hz, 3 H), 1.22 (d, J ) 7.1 Hz, 3 H), 1.14-1.34 (broad
m, 10 H), 1.52 (m, 2 H), 2.30 (m, 1 H), 2.82 (m, 1 H), 3.72 (m,
1 H), 3.79 (s, 3 H), 4.47 (d, J ) 10.9 Hz, 1 H), 4.53 (d, J ) 10.9
Hz, 1 H), 4.96 (d, J ) 4.0 Hz, 1 H), 6.86 (d, J ) 8.7 Hz, 2 H),
7.25 (d, J ) 8.7 Hz, 2 H); 13C NMR (101 MHz, CDCl3) δ 12.0,
14.1, 17.0, 18.8, 22.6. 25.5, 29.2, 29.5, 30.1, 31.8, 42.8, 55.2,
71.8, 76.1, 80.0, 113.7, 129.4, 130.2, 159.2, 174.2, 174.3;
HRFAB calcd for C24H38O6 (M+) 422.2668, found 422.2663.
Hyd r oxya ld eh yd e 17. Ozone was bubbled into a solution
of olefin 16 (252 mg, 0.42 mmol) in CH2Cl2 (22 mL) at -78 °C
until it turned blue. PPh3 (164 mg, 0.63 mmol) was added,
and the solution was stirred overnight at 25 °C. Concentration
in vacuo and flash chromatography with silica gel (gradient
to 20% EtOAc/hexanes) furnished aldehyde 17 (214 mg, 85%
yield) as a colorless oil: [R]22D -60° (c 0.010, CHCl3); IR (neat)
1644, 1732, 2930, 3430 cm-1; 1H NMR (400 MHz, CDCl3), both
rotamers δ 0.05-0.18 (m, 6 H), 0.80-0.98 (m, 15 H), 1.05 (d,
J ) 7.0 Hz, 3 H), 1.07 (d, J ) 7.0 Hz, 3 H), 1.20-1.36 (broad
m, 10 H), 1.36-1.58 (broad m, 2 H), 1.83 (broad m, 1 H), 2.50-
2.90 (m, 6 H), 3.04-3.30 (m, 2 H), 3.74-4.41 (m, 3 H), 4.82 (d,
J ) 2.6 Hz, 1 H), 7.09-7.31 (m, 5 H), 9.79 (m, 1 H); 13C NMR
(101 MHz, CDCl3), both rotamers δ -4.8, -4.7, -4.2, -4.1,
8.6, 8.7, 14.1, 15.6, 16.1, 17.9, 18.0, 19.7, 20.0, 22.6, 25.7, 25.8,
26.3, 26.4, 28.4, 29.2, 29.3, 29.55, 29.64, 31.8, 32.9, 33.0, 34.0,
34.5, 44.3, 44.5, 48.2, 64.0, 68.6, 69.1, 72.1, 72.2, 74.9, 75.1,
126.4, 126.9, 128.4, 128.9, 129.1, 138.1, 170.7, 171.1, 175.1,
[(p-Meth oxyben zyl)oxy]a lk en ol 14. To a solution of ester
acid 5 (699 mg, 1.65 mmol) in CH2Cl2 (20 mL) were added
DIPEA (288 µL, 1.65 mmol) and SOCl2 (190 µL, 2.60 mmol).
The solution was stirred for 2 h at 0 °C, and then excess SOCl2
was removed in vacuo carefully to obviate bumping. The crude
acid chloride was dissolved in CH2Cl2 (13 mL) and cooled to 0
°C. DIPEA (570 µL, 3.30 mmol) was added slowly followed
by a solution of amino alcohol 4 (373 mg, 1.82 mmol) in CH2Cl2
(7 mL). The ice bath was removed, and the solution was
stirred for 1.5 h. After extraction in CH2Cl2 (2 × 100 mL) with
saturated aqueous NH4Cl (70 mL), flash chromatography with
silica gel (gradient to 35% EtOAc/hexanes) provided 14 (707
mg, 70% yield) as a brown oil: [R]22 -36° (c 0.010, CHCl3);
D
IR (neat) 1248, 1514, 1640, 1732, 2930, 3424 cm-1; H NMR
1
175.3, 200.5; HREI calcd for
548.3407, found 548.3412.
C
30H50O6NSi [(M - tBu)+]
(400 MHz, CDCl3) both rotamers δ 0.88 (m, 3 H), 0.91 (d, J )
6.9 Hz, 3 H), 1.03 (d, J ) 6.7 Hz, 3 H), 1.23 (d, J ) 7.1 Hz, 3
H), 1.19-1.34 (broad m, 10H), 1.45 (m, 2 H), 1.99 (m, 1 H),
2.20-2.35 (m, 2 H), 2.67 (m, 1 H), 2.73 (s, 3 H), 3.01 (dd, J )
14.3, 3.8 Hz, 1 H), 3.17 (m, 1 H), 3.59 (broad m, 1 H), 3.76 (m,
1 H), 3.79 (s, 3 H), 3.93 (m, 1 H), 4.45 (m, 2 H), 4.84 (d, J )
5.3 Hz, 1 H), 5.10-5.22 (m, 2 H), 5.80 (m, 1 H), 6.85 (d, J )
8.6 Hz, 2 H), 7.14-7.29 (m, 7 H); 13C NMR (101 MHz, CDCl3)
major conformer δ 11.7, 14.1, 17.2, 19.3, 22.6, 25.5, 29.2, 29.3,
29.6, 31.75, 31.83, 32.7, 39.6, 43.4, 55.2, 60.3, 71.9, 72.8, 75.3,
79.7, 113.6, 118.1, 126.3, 128.4, 129.0, 129.2, 131.0, 134.8,
139.1, 159.0, 170.8, 174.9; HRFAB calcd for C37H56NO6 [(M +
H)+] 610.4108, found 610.4099.
Hyd r oxy Acid 3. To a solution of aldehyde 17 (200 mg,
0.33 mmol) and 2-methyl-2-butene (70 µL, 0.66 mmol) in
tBuOH (4.5 mL) was added slowly a solution of 80% NaClO2
(49 mg, 0.43 mmol) and NaH2PO4 (52 mg, 0.43 mmol) in H2O
(1.1 mL). The round-bottom flask was glass-stoppered and
parafilmed and the solution was stirred fast overnight at 25
°C. After removal of most of the tBuOH in vacuo and
successive extractions in CH2Cl2 (2 × 50 mL) with aqueous
HCl (pH 2, 40 mL) and H2O (40 mL), a colorless oil was
obtained that was used without further purification: [R]22
D
-51° (c 0.014, CHCl3); IR (neat) 1186, 1636, 1734, 2930, 3422
1
cm-1; H NMR (400 MHz, CDCl3) both rotamers δ 0.07-0.17
[(t Bu t yld im e t h ylsilyl)oxy][(p -m e t h oxyb e n zyl)oxy]-
a lk en e 15. To a solution of alkenol 14 (360 mg, 0.59 mmol)
in CH2Cl2 (6 mL) at 0 °C were added 2,6-lutidine (90 µL, 0.77
mmol) and TBSOTf (176 µL, 0.77 mmol). The solution was
stirred at 0 °C for 1.5 h and concentrated in vacuo. Flash
chromatography with silica gel (gradient to 16% EtOAc/
(m, 6 H), 0.77-0.98 (m, 15 H), 1.05 (d, J ) 7.2 Hz, 3 H), 1.08
(d, J ) 7.0 Hz, 3 H), 1.17-1.37 (broad m, 10 H), 1.37-1.57
(broad m, 2 H), 1.82 (broad m, 1 H), 2.43-2.95 (broad m, 6
H), 3.05-3.28 (broad m, 2 H), 3.92-4.34 (broad m, 3 H), 4.85
(d, J ) 2.4 Hz, 1 H), 7.09-7.30 (m, 5 H); 13C NMR (101 MHz,
CDCl3) both rotamers δ -4.99, -4.96, -4.3, -4.2, 8.6, 9.0, 14.0,
15.5, 16.1, 17.8, 17.9, 19.4, 20.0, 22.5, 22.6, 25.7, 25.8, 26.2,
26.3, 28.3, 28.5, 29.1, 29.2, 29.45, 29.54, 31.7, 32.7, 32.8, 33.3,
34.4, 38.9, 44.2, 62.6, 69.5, 70.4, 72.1, 72.3, 74.7, 75.1, 126.3,
126.8, 128.3, 128.9, 129.1, 138.1, 170.5, 171.2, 175.0, 175.4,
hexanes) afforded olefin 15 (379 mg, 89% yield) as an oil: [R]22
D
-47° (c 0.010, CHCl3); IR (neat) 835, 1061, 1250, 1514, 1663,
1732, 2930 cm-1; 1H NMR (400 MHz, CDCl3) both rotamers δ
0.04-0.14 (m, 6 H), 0.86-0.95 (m, 15 H), 1.03 (d, J ) 6.5 Hz,
3 H), 1.23 (d, J ) 7.1 Hz, 3 H), 1.16-1.34 (broad m, 10 H),
1.44 (broad m, 2 H), 1.94 (broad m, 1 H), 2.15-2.40 (m, 2 H),
2.64 (m, 4 H), 2.86-3.14 (m, 2 H), 3.71-3.83 (m, 5 H), 4.12
(m, 1 H), 4.41-4.49 (m, 2 H), 4.84 (d, J ) 2.2 Hz, 1 H), 5.03-
5.20 (m, 2 H), 5.92 (m, 1 H), 6.84 (d, J ) 8.6 Hz, 2 H), 7.10-
7.27 (m, 7 H); 13C NMR (101 MHz, CDCl3) both rotamers δ
-4.72, -4.68, -4.0, -3.6, 11.4, 11.6, 14.1, 15.8, 16.9, 17.98,
18.01, 19.5, 22.6, 25.5, 25.6, 25.7 25.9, 28.7, 29.2, 29.3, 29.55,
29.61, 29.8, 31.8, 32.1, 32.8, 33.1, 40.4, 43.6, 43.7, 55.2, 61.7,
71.9, 72.0, 74.4, 74.9, 75.0, 79.56, 79.62, 113.5, 113.6, 117.7,
118.3, 126.0, 126.5, 128.2, 128.3, 128.6, 129.0, 129.1, 129.28,
129.34, 131.0, 131.1, 134.2, 134.5, 138.9, 158.9, 159.0, 169.1,
175.7, 176.1; HRFAB calcd for
622.4139, found 622.4133.
C
34H60NO7Si [(M + H)+]
Ha p a losin (1). Crude hydroxy acid 3 (30 mg, 0.048 mmol)
was azeotroped with benzene once alone and once with DMAP
(29 mg, 0.24 mmol). A solution of 3 and DMAP in MeCN (20
mL) was added to a refluxing solution of 2-chloro-N-methyl-
pyridinium iodide (246 mg, 0.96 mmol) and DMAP (89 mg,
0.72 mmol) in MeCN (100 mL) by a syringe pump over 16 h.
After 18 h of refluxing, most MeCN was removed in vacuo and
extraction in CH2Cl2 (2 × 30 mL) with aqueous HCl (pH 2, 50
mL) was performed. Crude TBS ether of hapalosin was

Synthesis and Conformational Analysis of Hapalosin
J . Org. Chem., Vol. 61, No. 19, 1996 6615
collected in a broad area (Rf about 0.35) in preparative TLC
(10% EtOAc/hexanes).
1.43-1.58 (broad m, 2 H), 2.39 (m, 1 H), 3.01 (dd, J ) 14.1,
7.0 Hz, 1 H), 3.17 (dd, J ) 14.1, 6.2 Hz, 1 H), 3.44 (d, J ) 16.0
Hz, 1 H), 3.50 (d, J ) 16.0 Hz, 1 H), 4.67 (m, 1 H), 4.85 (m, 1
H), 5.00-5.10 (m, 4 H), 5.36 (d, J ) 7.8 Hz, 1 H), 5.71 (m, 1
H), 7.14 (d, J ) 6.5 Hz, 2 H), 7.20-7.37 (m, 8 H); 13C NMR
(101 MHz, CDCl3) δ 14.0, 15.1, 22.6, 25.3, 29.1, 29.3, 31.1, 31.7,
37.0, 41.1, 46.9, 60.9, 67.0, 78.5, 115.3, 127.1, 128.0, 128.2,
128.5, 128.7, 129.2, 135.7, 136.1, 139.6, 155.7, 166.5, 201.3;
HREI calcd for C31H41NO5 (M+) 507.2985, found 507.2996.
(1R,2R)-1-n Hep tyl-2-m eth yl-3-bu ten yl (3R,4S)-4-[(Ben -
zyloxyca r bon yl)a m in o]-3-[(ter t-bu tyld im eth ylsilyl)oxy]-
5-p h en ylp en ta n oa te (25). To a solution of γ-amino-â-keto
ester 23 (298 mg, 0.587 mmol) in EtOH (6.0 mL) at 0 °C was
added KBH4 (127 mg, 2.35 mmol). The mixture was stirred
at 0 °C for 6.5 h, carefully quenched with saturated aqueous
NH4Cl at 0 °C, and extracted in CH2Cl2 (2 × 40 mL)
successively with saturated aqueous NH4Cl (40 mL) and H2O
(40 mL). The crude product was a mixture of inseparable
diastereomers.
The crude alcohol (284 mg, 0.557 mmol) was dissolved in
CH2Cl2 (5.3 mL) and cooled to 0 °C. 2,6-Lutidine (130 µL, 1.11
mmol) and TBSOTf (154 µL, 0.668 mmol) were added, and the
solution was stirred at 0 °C for 30 min. After successive
extractions in CH2Cl2 (2 × 40 mL) with saturated aqueous
NaHCO3 (30 mL) and aqueous 0.1 N NaHSO4 (30 mL), flash
chromatography with silica gel (gradient to 10% EtOAc/
hexanes) afforded the undesired TBS ether diastereomer (62
mg, 17% yield for two steps) and the desired diastereomer 25
(275 mg, 75% yield for two steps) as a colorless oil: [R]22D -5.0°
(c 0.020, CHCl3); IR (neat) 837, 1051, 1252, 1732, 2928, 3349
cm-1; 1H NMR (400 MHz, CDCl3) δ 0.05 (s, 3 H), 0.08 (s, 3 H),
0.87 (t, J ) 7.0 Hz, 3 H), 0.90 (s, 9 H), 0.98 (d, J ) 6.8 Hz, 3
H), 1.17-1.35 (broad m, 10H), 1.51 (broad m, 2 H), 2.40 (m, 1
H), 2.56 (m, 2 H), 2.69 (m, 1 H), 2.98 (m, 1 H), 3.99 (broad m,
1 H), 4.33 (broad m, 1 H), 4.77 (d, J ) 7.8 Hz, 1 H), 4.84 (m,
1 H), 4.97-5.05 (m, 2 H), 5.73 (m, 1 H), 7.15-7.32 (m, 10 H);
13C NMR (101 MHz, CDCl3) δ -5.0, -4.5, 14.1, 15.2, 18.0, 22.6,
25.5, 25.9, 29.1, 29.5, 31.1, 31.8, 35.5, 39.7, 41.1, 56.6, 66.3,
70.5, 77.4, 115.1, 126.3, 27.76, 127.84, 128.3, 128.4, 129.1,
136.6, 138.0, 139.8, 155.7, 170.7; HRFAB calcd for C37H58NO5-
Si [(M + H)+] 624.4084, found 624.4091.
To a solution of the crude macrolide in THF (0.6 mL) was
added TBAF (15 µL in THF, 0.015 mmol). The solution was
stirred for 45 min at 0 °C and then extracted in CH2Cl2 (2 ×
13 mL) with aqueous NH4Cl (10 mL). Preparative TLC (50%
EtOAc/hexanes) furnished hapalosin (3.0 mg, Rf 0.45) in 13%
yield for the last three steps: [R]22 -48° (c 0.0029, CH2Cl2)
D
[natural [R]D -49.2° (c 0.35, CH2Cl2)]; IR (neat) 1632, 1734,
2926, 3420 cm-1; 1H NMR (500 MHz, CDCl3, 2.5:1 conformers),
major conformer δ 0.23 (d, J ) 7.0 Hz, 3 H), 0.55 (d, J ) 7.0
Hz, 3 H), 0.89 (3 H), 1.18 (d, J ) 7.0 Hz, 3 H), 1.20-1.40 (broad
m, 10 H), 1.68 (m, 1 H), 1.92 (m, 1 H), 2.01 (m, 1 H), 2.62 (m,
1 H), 2.65 (m, 1 H), 2.85 (s, 3 H), 2.92 (dd, J ) -17.8, 5.1 Hz,
1 H), 3.22 (m, 1 H), 3.41 (dd, J ) -14.0, 2.5 Hz, 1 H), 3.70 (dt,
J ) 10.1, 2.5 Hz, 1 H), 3.85 (m, 1 H), 4.31 (d, J ) 8.4 Hz, 1 H),
5.12 (m, 1 H), 7.18 (d, J ) 7.6 Hz, 2 H), 7.25 (t, J ) 7.6 Hz, 1
H), 7.33 (dd, J ) 7.6, 7.6 Hz, 2 H); 13C NMR (126 MHz, CDCl3)
major conformer δ 12.1, 14.1, 17.6, 18.3, 22.6, 26.0, 28.1, 28.8,
29.08, 29.15, 29.2, 31.7, 36.4, 37.1, 40.7, 61.4, 70.2, 73.8, 76.5
127.1, 128.9, 129.7, 137.4, 168.5, 168.7, 172.7; HREI calcd for
C28H43NO6 (M+) 489.3090, found 489.3094.
tBu tyldim eth ylsilyl Eth er 18: [R]22D -31° (c 0.036, CHCl3);
IR (neat) 1192, 1701, 1742, 2930 cm-1 1H NMR (500 MHz,
;
CDCl3) both rotamers δ 0.06-0.15 (four singlets, 6 H), 0.85-
0.94 (m, 6 H), 0.92 (s, 9 H), 0.96 (d, J ) 6.8 Hz, 3 H), 1.15 (d,
J ) 7.1 Hz, 3 H), 1.20-1.30 (broad m, 10 H), 1.53-1.65 (broad
m, 2 H), 2.23 (m, 1 H), 2.44-2.54 (m, 1 H), 2.57 (s, 3 H), 2.61-
2.77 (m, 3 H), 2.86 (m, 1 H), 3.13-3.21 (m, 1 H), 4.08-4.51
(broad multiplets, 2 H), 4.84 (d, J ) 4.2 Hz, 1 H), 4.97-5.19
(m, 4 H), 7.06 (d, J ) 7.1 Hz, 1 H), 7.10-7.36 (m, 14 H); 13C
NMR (126 MHz, CDCl3) both conformers δ -4.84, -4.77,
-4.12, -4.09, 12.1, 12.6, 13.0, 14.1, 17.1, 17.97, 18.00, 18.8,
22.6, 25.4, 25.86, 25.89, 25.93, 29.1, 29.2, 29.4, 30.0, 31.7, 32.0,
34.4, 40.0, 42.8, 43.1, 66.5, 66.77, 66.80, 67.2, 69.8, 70.1, 74.4,
74.5, 126.1, 126.2, 127.3, 127.7, 128.2, 128.27, 128.28, 128.33,
128.4, 128.5, 128.8, 128.9, 135.3, 135.4, 136.4, 136.9, 138.9,
156.3, 169.11, 169.14, 170.8, 173.27, 173.33; HRFAB calcd for
C49H72O9NSi [(M + H)+] 846.4976, found 846.4971.
(1R,2R)-1-n Hep tyl-2-m eth yl-3-bu ten yl Aceta te (24). To
a solution of alcohol 10 (1.750 g, 9.49 mmol) in CH2Cl2 (17
mL) at 0 °C were added successively pyridine (920 µL, 11.4
mmol), DMAP (116 mg, 0.95 mmol), and acetyl chloride (740
µL, 10.4 mmol) slowly. Stirring transpired for 10 min at 0 °C
and for 1 h without the ice bath. The mixture was extracted
in CH2Cl2 (2 × 150 mL) successively with 0.1 N NaHSO4 (100
mL) and H2O (100 mL). Concentration at water aspirator
pressure at 35 °C provided clean acetate 24 (2.149 g, 100%
(2S,3R)-2-F or m yl-3-d ecyl (3R,4S)-4-[(Ben zyloxyca r -
bon yl)a m in o]-3-[(ter t-bu tyld im eth ylsilyl)oxy]-5-p h en yl-
p en ta n oa te (26). Into a solution of olefin 25 (275 mg, 0.441
mmol) in CH2Cl2 (14 mL) and MeOH (1 mL) at -78 °C was
bubbled ozone for 3 min, at which time TLC showed no more
olefin. PPh3 (180 mg, 0.685 mmol) was added to the clear,
colorless solution, the -78 °C bath was removed, and stirring
transpired at 25 °C for 15 h. Flash chromatography with silica
gel (gradient to 20% EtOAc/hexanes) furnished aldehyde 26
yield) as a colorless liquid: [R]22 +24° (c 0.014, CHCl3); IR
D
(neat) 1240, 1742, 2928, 2957 cm-1; 1H NMR (400 MHz, CDCl3)
δ 0.88 (t, J ) 6.9 Hz, 3 H), 1.00 (d, J ) 6.9 Hz, 3 H), 1.18-
1.34 (broad m, 10 H), 1.43-1.57 (broad m, 2 H), 2.05 (s, 3 H),
2.39 (m, 1 H), 4.82 (m, 1 H), 5.01-5.06 (m, 2 H), 5.73 (m, 1
H); 13C NMR (101 MHz, CDCl3) δ 14.0, 15.2, 21.0, 22.6, 25.4,
29.1, 29.4, 31.3, 31.7, 41.4, 76.8, 115.0, 140.0, 170.8; HREI
calcd for C14H27O2 [(M + H)+] 227.2011, found 227.2008.
(1R,2R)-1-n Hep tyl-2-m eth yl-3-bu ten yl (4S)-4-[(Ben zyl-
(234 mg, 85% yield) as a colorless oil: [R]22 +7.1° (c 0.024,
D
CHCl3); IR (neat) 837, 1252, 1728, 2928, 3357 cm-1; H NMR
1
(500 MHz, CDCl3) δ 0.04 (s, 3 H), 0.07 (s, 3 H), 0.88 (t, J ) 7.0
Hz, 3 H), 0.89 (s, 9 H), 1.08 (d, J ) 7.1 Hz, 3 H), 1.20-1.39
(broad m, 10 H), 1.56 (broad m, 1 H), 1.65 (broad m, 1 H),
2.49-2.60 (m, 3 H), 2.67 (m, 1 H), 2.97 (m, 1 H), 3.96 (broad
m, 1 H), 4.30 (broad m, 1 H), 4.72 (d, J ) 7.8 Hz, 1 H), 4.97 (s,
2 H), 5.27 (m, 1 H), 7.16 (d, J ) 7.3 Hz, 2 H), 7.18-7.32 (m, 8
H); 13C NMR (126 MHz, CDCl3) δ -4.9, -4.6, 8.1, 14.0, 18.0,
22.6, 25.7, 25.8, 29.0, 29.2, 31.6, 31.7, 35.6, 39.6, 49.7, 56.7,
66.4, 70.6, 73.2, 126.4, 127.8, 127.9, 128.36, 128.44, 129.1,
136.6, 137.9, 155.7, 170.5, 202.2; HRFAB calcd for C36H56NO6-
Si [(M + H)+] 626.3877, found 626.3877.
oxyca r bon yl)a m in o]-3-oxo-5-p h en ylp en ta n oa te (23).
A
solution of L-N-Cbz-Phe (425 mg, 1.42 mmol) and 1,1′-car-
bonyldiimidazole (CDI, 253 mg, 1.56 mmol) in THF (13.5 mL)
was stirred at 25 °C for 2 h and then cooled to -78 °C. In the
meantime, a solution of acetate 24 (1.286 g, 5.68 mmol) and
LDA (4.73 mL, 5.96 mmol, 1.26 M in cyclohexane) in THF (22
mL) was stirred at -78 °C for 1 h. The enolate solution at
-78 °C was added via syringe with a big-bore needle to the
imidazolide solution at -78 °C at a moderate rate with fast
stirring. The resulting mixture was stirred at -78 °C for 15
min, the -78 °C bath was removed, and the mixture was
quenched with saturated aqueous NH4Cl and extracted in
CH2Cl2 (2 × 150 mL) with saturated aqueous NH4Cl (100 mL).
Flash chromatography with silica gel (gradient to 20% EtOAc/
hexanes) furnished acetate 24 (0.939 g) and γ-amino-â-keto
(2S,3R)-2-Ca r boxy-3-d ecyl (3R,4S)-4-[(Ben zyloxyca r -
bon yl)a m in o]-3-[(ter t-bu tyld im eth ylsilyl)oxy]-5-p h en yl-
p en ta n oa te (27). To a solution of aldehyde 26 (234 mg, 0.374
mmol) and 2-methyl-2-butene (374 µL, 0.75 mmol, 2.0 M in
t
THF) in BuOH (3.2 mL) was added dropwise a solution of 80%
sodium chlorite (55 mg, 0.49 mmol) and NaH2PO4 (58 mg, 0.49
mmol) in H2O (0.8 mL). The round-bottom flask was glass-
stoppered and parafilmed, and the solution was vigorously
stirred at 25 °C for 16 h. Successive extractions in CH2Cl2 (2
× 40 mL) with 0.1 N NaHSO4 (40 mL) and H2O (40 mL)
produced pure acid 27 (240 mg, 100% yield) as a colorless oil:
ester 23 (519 mg, 72% yield) as a colorless oil: [R]22 +22° (c
D
0.019, CHCl3); IR (neat) 698, 1046, 1250, 1499, 1712, 2928,
1
3333 cm-1; H NMR (400 MHz, CDCl3) δ 0.87 (t, J ) 6.8 Hz,
[R]22 -8.8° (c 0.016, CHCl3); IR (neat) 837, 1252, 1717, 1736,
D
1
3H), 0.98 (d, J ) 6.9 Hz, 3 H), 1.15-1.34 (broad m, 10 H),
2928, 3265, 3324 cm-1; H NMR (500 MHz, CDCl3) δ 0.07 (s,

6616 J . Org. Chem., Vol. 61, No. 19, 1996
Dinh et al.
6 H), 0.87 (t, J ) 7.0 Hz, 3 H), 0.90 (s, 9 H), 1.18 (d, J ) 6.8
Hz, 3 H), 1.20-1.35 (broad m, 10 H), 1.54 (broad m, 1 H), 1.64
(broad m, 1 H), 2.50 (m, 1 H), 2.59 (dd, J ) 15.8, 7.1 Hz, 1 H),
2.64-2.76 (m, 2 H), 3.00 (m, 1 H), 4.00 (broad m, 1 H), 4.30
(broad m, 1 H), 4.80 (d, J ) 8.9 Hz, 1 H), 4.96 (s, 2 H), 5.25
(broad m, 1 H), 7.15-7.32 (m, 10H); 13C NMR (126 MHz,
CDCl3) δ -4.9, -4.6, 11.2, 14.1, 18.0, 22.6, 25.6, 25.8, 29.1,
29.3, 31.4, 31.7, 34.9, 40.0, 42.2, 56.0, 66.6, 71.2, 74.6, 126.4,
127.87, 127.95, 128.4, 129.3, 136.4, 137.9, 155.9, 170.5, 177.1;
HRFAB calcd for C36H56NO7Si [(M + H)+] 642.3826, found
642.3827.
followed by BOP-Cl (143 mg, 0.56 mmol). The mixture was
stirred at 85 °C for 15 h and then extracted with 0.1 N NaHSO4
(30 mL), and the aqueous layer was back-extracted with EtOAc
(30 mL). Preparative TLC with two silica gel-coated plates of
0.5 mm thickness (the eluent was 10% EtOAc/hexanes)
resulted in a green band (p-anisaldehyde stain, Rf ) 0.28)
containing the silylated analog of hapalosin, 29 (20 mg
colorless oil, 60% yield for two steps): [R]22 -45° (c 0.034,
D
CH2Cl2); IR (neat) 839, 1088, 1175, 1198, 1541, 1663, 1732,
1
1744, 2928, 3318 cm-1; H NMR (500 MHz, CDCl3) δ 0.13 (s,
3 H), 0.14 (s, 3 H), 0.56 (d, J ) 6.7 Hz, 3 H), 0.76 (d, J ) 6.6
Hz, 3 H), 0.88 (t, J ) 7.0 Hz, 3 H), 0.91 (s, 9 H), 1.17 (d, J )
7.1 Hz, 3 H), 1.20-1.37 (broad m, 10H), 1.55 (m, 1 H), 1.69
(m, 2 H), 2.48 (d, J ) 5.7 Hz, 2 H), 2.66 (dd, J ) 14.1, 9.8 Hz,
1 H), 3.07 (m, 1 H), 3.2 (dd, J ) 14.1, 4.5 Hz, 1 H), 4.22 (m, 1
H), 4.31 (m, 1 H), 4.80 (d, J ) 8.3 Hz, 1 H), 5.16 (m, 1 H), 5.53
(d, J ) 10.5 Hz, 1 H), 7.17 (t, J ) 7.2 Hz, 1 H), 7.19 (d, J ) 7.2
Hz, 2 H), 7.25 (t, J ) 7.2 Hz, 2 H); 13C NMR (126 MHz, CDCl3)
δ -4.7, -4.5, 10.7, 14.1, 17.8, 17.9, 18.2, 22.6, 25.68, 25.71,
29.1, 29.3, 29.5, 30.4, 31.7, 38.8, 40.5, 40.8, 55.6, 71.7, 75.7,
80.0, 126.4, 128.3, 129.2, 137.6, 168.9, 171.6, 173.0; HRFAB
calcd for C33H56NO6Si [(M + H)+] 590.3877, found 590.3878.
Non -N-Me An a log of Ha p a losin (2). A stock solution of
HF/pyridine was prepared from 0.25 mL of 70% HF/pyridine
(Aldrich), 0.5 mL of pyridine, and 2.0 mL of THF in a
polyethylene bottle. This stock solution of HF/pyridine (1.5
mL) was added to a solution of TBS ether 29 (13 mg, 0.022
mmol) in THF (0.8 mL) in a glass shell vial, and the solution
was stirred at 25 °C for 18 h. After extraction in CH2Cl2 (2 ×
20 mL) with 0.1 N NaHSO4 (2 × 15 mL), preparative TLC
with two silica gel-coated plates of 0.5 mm thickness (the
eluent was 40% EtOAc/hexanes) was performed. A broad
green band (p-anisaldehyde stain, Rf ) 0.33) contained the
non-N-Me analog of hapalosin, 2 (7.5 mg colorless solid, 71%
Ben zyl (2S)-2-Hyd r oxy-3-m eth ylbu ta n oa te (28). To a
solution of (S)-R-hydroxyisovaleric acid (272 mg, 2.30 mmol)
in MeCN (3.5 mL) were added successively 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU, 344 µL, 2.30 mmol), which warmed
up the solution, and BnBr (274 µL, 2.30 mmol). After being
stirred for 17 h at 25 °C, the solution was extracted in CH2Cl2
(2 × 40 mL) with 0.1 N NaHSO4 (40 mL). Flash chromatog-
raphy with silica gel (gradient to 20% EtOAc/hexanes) pro-
vided hydroxy ester 28 (418 mg, 87% yield) as a colorless oil:
[R]22 -11° (c 0.025, CHCl3); IR (neat) 1138, 1734, 2963, 3505
D
1
cm-1; H NMR (400 MHz, CDCl3) δ 0.83 (d, J ) 6.9 Hz, 3 H),
1.00 (d, J ) 6.9 Hz, 3 H), 2.09 (m, 1 H), 2.79 (d, J ) 6.2 Hz, 1
H), 4.08 (dd, J ) 6.2, 3.5 Hz, 1 H), 5.19 (d, J ) 12.1 Hz, 1 H),
5.24 (d, J ) 12.1 Hz, 1 H), 7.31-7.39 (m, 5 H); 13C NMR (101
MHz, CDCl3) δ 15.8, 18.7, 32.1, 67.2, 75.0, 128.4, 128.5, 128.6,
135.1, 174.8; HREI calcd for C12H16O3 (M+) 208.1099, found
208.1105.
Tr iester 22. Acid 27 (80 mg, 0.12 mmol) and alcohol 28
(26 mg, 0.12 mmol) were dissolved in CH2Cl2 (2.0 mL), and
DMAP (15 mg, 0.12 mmol) followed by 1-ethyl-3-[3-(dimethyl-
amino)propyl]carbodiimide (EDC, 31 mg, 0.16 mmol) were
added. The round-bottom flask was glass-stoppered and
parafilmed, and stirring transpired at 25 °C for 17 h. After
extraction in CH2Cl2 (2 × 25 mL) with 0.1 N NaHSO4 (25 mL),
flash chromatography with silica gel (gradient to 20% EtOAc/
hexanes) afforded triester 22 (88 mg, 85% yield) as a colorless
yield): [R]22 -66° (c 0.0061, CH2Cl2); IR (neat) 1194, 1545,
D
1661, 1728, 1742, 2921, 3303, 3385 (shoulder) cm-1; 1H NMR
(500 MHz, CDCl3) δ 0.66 (d, J ) 6.8 Hz, 3 H), 0.86 (d, J ) 6.6
Hz, 3 H), 0.89 (t, J ) 6.8 Hz, 3 H), 1.21 (d, J ) 7.0 Hz, 3 H),
1.23-1.39 (broad m, 10 H), 1.58 (broad m, 1 H), 1.71 (broad
m, 1 H), 1.77 (m, 1 H), 2.34 (d, J ) 11.2 Hz, 1 H), 2.57 (dd, J
) 13.9, 5.8 Hz, 1 H), 2.65 (dd, J ) 13.9, 3.6 Hz, 1 H), 2.92 (m,
1 H), 2.96 (m, 1 H), 3.01 (dd, J ) 14.2, 6.1 Hz, 1 H), 4.07 (broad
m, 1 H), 4.54 (d, J ) 8.0 Hz, 1 H), 4.64 (m, 1 H), 5.46 (d, J )
10.4 Hz, 1 H), 5.52 (m, 1 H), 7.20 (t, J ) 7.2 Hz, 1 H), 7.23 (d,
J ) 7.2 Hz, 2 H), 7.27 (t, J ) 7.2 Hz, 2 H); 13C NMR (126
MHz, CDCl3) δ 9.2, 14.1, 17.7, 18.5, 22.6, 25.5, 29.1, 29.3, 29.9,
31.0, 31.7, 37.5, 39.0, 41.3, 54.0, 70.6, 75.9, 81.8, 126.7, 128.6,
129.0, 137.0, 169.7, 173.7, 174.1; HREI calcd for C27H41NO6
(M+) 475.2934, found 475.2936.
oil: [R]22 -21° (c 0.022, CHCl3); IR (neat) 1252, 1740, 2930,
D
3386 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.05 (s, 3 H), 0.07 (s,
3 H), 0.87 (t, J ) 7.0 Hz, 3 H), 0.89 (s, 9 H), 0.92 (d, J ) 6.8
Hz, 3 H), 0.96 (d, J ) 6.9 Hz, 3 H), 1.18 (d, J ) 7.1 Hz, 3 H),
1.20-1.38 (broad m, 10 H), 1.57 (broad m, 1 H), 1.65 (broad
m, 1 H), 2.22 (m, 1 H), 2.55 (dd, J ) 16.3, 6.2 Hz, 1 H), 2.64
(dd, J ) 16.3, 5.6 Hz, 1 H), 2.71 (m, 1 H), 2.79 (m, 1 H), 2.97
(m, 1 H), 3.98 (broad m, 1 H), 4.35 (broad m, 1 H), 4.84 (1 H),
4.85 (d, J ) 4.4 Hz, 1 H), 4.97 (s, 2 H), 5.09 (d, J ) 12.3 Hz,
1 H), 5.15 (d, J ) 12.3 Hz, 1 H), 5.20 (m, 1 H), 7.15-7.36 (m,
10 H); 13C NMR (101 MHz, CDCl3) δ -5.0, -4.6, 12.2, 14.0,
17.1, 18.0, 18.7, 22.6, 25.5, 25.8, 29.1, 29.3, 30.0, 31.5, 31.7,
35.4, 39.5, 42.6, 56.7, 66.3, 66.8, 70.3, 74.5, 76.8, 126.3, 127.7,
127.8, 128.2, 128.32, 128.34, 128.5, 129.1, 135.3, 136.6, 138.2,
155.6, 169.2, 170.5, 173.3; HRFAB calcd for C48H70NO9Si [(M
+ H)+] 832.4820, found 832.4832.
Ack n ow led gm en t. We thank the following compu-
tational resources at UCLA: Prof. Ken Houk’s group,
Prof. Yves Rubin’s group, and the Office of Academic
Computing. We also thank Prof. Houk and Dr. Kensuke
Nakamura for helpful discussions about computation.
Financial support from UCLA and NIH (Grant No.
GM51095) is greatly appreciated.
TBS Eth er of th e Non -N-Me An a log of Ha p a losin (29).
The round-bottom flask containing a mixture of triester 22 (83
mg, 0.10 mmol) and moist Pd(OH)2/C (Pearlman’s catalyst, 56
mg) in MeOH (4.0 mL) was purged with N2, and H2 was then
bubbled into the mixture for 5 min. After the mixture was
stirred for about 15 h at 25 °C under a balloon full of H2, H2
was again bubbled into the mixture for 5 min. After the
mixture was stirred for an additional 15 h under the same
conditions, the flask was purged with N2 and the mixture was
filtered through a short column of Celite and washed well with
MeOH and EtOAc. 1H NMR of the crude product showed that
bis-deprotection was complete.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra of all new compounds in the synthesis of hapalosin
and its non-N-Me analog (50 pages). This material is con-
tained 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.
To a solution of the crude amino acid (34 mg, 0.056 mmol)
in PhMe (60 mL) were added DIPEA (146 µL, 0.84 mmol)
J O9608329