Total synthesis and conformational studies of hapalosin, N-desmethylhapalosin and 8-Deoxyhapalosin

Article abstract of DOI:10.1016/S0968-0896(98)00208-9

Hapalosin (2), a 12-membered cyclic depsipeptide possessing MDR-reversing activity, and analogues (3) and (4) have been synthesized using macrolactamization as an important ring-forming step. Three building blocks: (2S, 3R)-3-(tert-butyldimethylsilyloxy)-2-methyl-decanoic acid (13), benzyl (S)-2-hydroxy-3-methylbutanate (14), and (4S,3R)-4-(benzyloxycarbonyl-methylamino)-3-methoxymethoxy-5-phenyl-pentanoic acid (28) were prepared from Evans's chiral imide (9), l-valine, and l-N-Boc phenylalanine (17), respectively, and were assembled together by applying twice Yamaguchi's coupling methodology. A new and efficient selective N-methylation of γ-hydroxy-β-amino ester taking advantage of the vicinal amino alcohol function was uncovered in the course of this study. Thus, treatment of compound 19 with HCHO in the presence of catalytic amount of pTsOH followed by reduction (NaBH₃CN, TFA, CH₂Cl₂) of the so-formed oxazolidine 24 gave the N-methylated product 25. Furthermore, a dual role of oxazolidine as protecting group of vicinal amino alcohol and latent N-methyl function was established which allowed synthesizing both hapalosin (2) and N-desmethylhapalosin (3) from the same linear precursor 32 in a step-efficient and atom economic way. In contrast to hapalosin (2) and N-desmethyl analogue (3), the amide bond of 8-deoxy hapalosin (4) exists at room temperature (CDCl₃) exclusively in s-cis conformation as evidenced by NOE studies. This observation has been explained on the basis of computational studies. No significant MDR reversing activity of 8-deoxy hapalosin (4) was observed in K562 R and S/Adriblastine against human erythroleucemic cell lines indicating thus the important contribution of hydroxy group to the bioactivity of hapalosin. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00208-9

Bioorganic & Medicinal Chemistry 7 (1999) 737±747
Total Synthesis and Conformational Studies of Hapalosin,
N-Desmethylhapalosin and 8-Deoxyhapalosiny
Bjorn Wagner, Gabriel Islas Gonzalez, Marie Elise Tran Hun Dau and Jieping Zhu*
Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-sur-Yvette, France
Received 2 July 1998; accepted 15 September 1998
AbstractÐHapalosin (2), a 12-membered cyclic depsipeptide possessing MDR-reversing activity, and analogues (3) and (4) have
been synthesized using macrolactamization as an important ring-forming step. Three building blocks: (2S, 3R)-3-(tert-butyldi-
methylsilyloxy)-2-methyl-decanoic acid (13), benzyl (S)-2-hydroxy-3-methylbutanate (14), and (4S,3R)-4-(benzyloxycarbonyl-
methylamino)-3-methoxymethoxy-5-phenyl-pentanoic acid (28) were prepared from Evans's chiral imide (9), l-valine, and l-N-Boc
phenylalanine (17), respectively, and were assembled together by applying twice Yamaguchi's coupling methodology. A new and
ecient selective N-methylation of g-hydroxy-b-amino ester taking advantage of the vicinal amino alcohol function was uncovered
in the course of this study. Thus, treatment of compound 19 with HCHO in the presence of catalytic amount of pTsOH followed by
reduction (NaBH3CN, TFA, CH2Cl2) of the so-formed oxazolidine 24 gave the N-methylated product 25. Furthermore, a dual role
of oxazolidine as protecting group of vicinal amino alcohol and latent N-methyl function was established which allowed synthe-
sizing both hapalosin (2) and N-desmethylhapalosin (3) from the same linear precursor 32 in a step-ecient and atom economic
way. In contrast to hapalosin (2) and N-desmethyl analogue (3), the amide bond of 8-deoxy hapalosin (4) exists at room tempera-
ture (CDCl3) exclusively in s-cis conformation as evidenced by NOE studies. This observation has been explained on the basis of
computational studies. No signi®cant MDR reversing activity of 8-deoxy hapalosin (4) was observed in K562 R and S/Adriblastine
against human erythroleucemic cell lines indicating thus the important contribution of hydroxy group to the bioactivity of hapa-
losin. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
tine, vincristine), cochicine, taxoides (taxol, taxotere)
along with others such as mitomycin, topotecan, etopo-
sides, to name a few. The ability of P-gp to bind and
transport a range of structurally dissimilar compounds
is truly remarkable and has provided impetus for the
identi®cation of new targets for chemotherapy.1±3
One of the major problems in cancer therapy is the cel-
lular resistance to a wide range of structurally unrelated
cytotoxic drugs. This phenomenon, known as multidrug
resistance (MDR),1 is commonly developed following
upon administration of a single anticancer agent.
Reduced accumulation of the drug inside tumor cells
appears to be one principal cause of this undesired sce-
nario.2 A wide variety of biochemical mechanisms have
been identi®ed in multidrug resistant cell lines, the most
consistent of which is the increased expression of a drug
e‚ux pump called P-glycoprotein (P-gp).1,2 In fact, it
has been observed that the level of P-gp expression cor-
relates with the degree of drug resistance in a variety of
di€erent cell types. Since the ®rst observation made at
the beginning of the 1970s, the list of MDR drugs has
grown progressively including tetracycline antibiotics
(daunorubricin, adriamycin), vinca alkaloids (vinblas-
Among numerous prospects for circumventing MDR,
one possibility is the use of chemosensitizers to block
the action of P-gp,3 thus potentiating the cytotoxicity of
the anticancer agents. Verapamil (1, Fig. 1), a calcium
channel blocker, was one of the ®rst MDR reversing
agents tested in clinic4 and became the standard with
which all other MDR modulators are compared.
Hapalosin (2), a 12-membered cyclic depsipeptide was
recently identi®ed by Moore and co-workers5 from the
screening extracts of blue-green algae (cyanobacteria).
Hapalosin was mildly cytotoxic and was found to have
better MDR reversing activity than verapamil, espe-
cially in promoting taxol accumulation in SKVLB1
cells. Shortly after its isolation, synthesis of hapalosin
and analogues have been accomplished by Arm-
strong,6,10 Ghosh,7 Yamamura8 and our group9
employing both macrolactonization and macro-
lactamization methodology as key ring forming steps.
We report herein in full details our previous route to
Key words: Hapalosin; anti-MDR; oxazolidine; selective N-methyla-
tion; macrolactamization.
*Corresponding author. Fax:+33-1-69077247; e-mail: zhu@icsn.cnrs-gif.fr
y
Dedicated with a€ection and respect to the memory of Professor Sir
Derek H. R. Barton in recognition of his distinguished contributions
to science and in sincere gratitude for the immense degree of inspira-
tion and encouragement he provided to one of us (J. Zhu).
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00208-9

738
B. Wagner et al. / Bioorg. Med. Chem. 7 (1999) 737±747
hapalosin and 8-deoxy-hapalosin, together with addi-
tional studies in which the dual role of oxazolidine as
protecting group of vicinal amino alcohol and latent N-
methyl group was established and exploited for the
development of an improved synthesis of hapalosin and
N-desmethyl analogues. The general strategy employing
macrolactamization as a key ring forming step, similar
to that employed by Ghosh7 and Yamamura,8 was
depicted retrosynthetically in Scheme 1.
Results and Discussion
The b-hydroxy acid 13 was synthesized as shown in
Scheme 2. Reaction of boron enolate of chiral imide (9)
with n-octanaldehyde (10) under Evans' conditions11
furnished syn aldol 11 in excellent yield and diastereo-
selectivity. Installation of TBS protecting group fol-
lowed by removal of chiral auxiliary gave then the b-
hydroxy acid 13 uneventfully. Esteri®cation of 13 with
benzyl
(S)-(+)-2-hydroxy-3-methylbutanate
(14),
obtained in two straightforward steps from l-valine,12
was carried out under di€erent conditions and was best
realized using Yamaguchi's reagent13 to provide com-
pound 15 in 88% yield. Removal of silyl ether was
proved to be more dicult than expected due most
probably to the presence of sensitive b-hydroxy ester
entity. Among a range of reagents tested, only HF in
MeCN14 was found to be highly ecient in our hands to
furnish 16 in 90% yield.
Synthesis of suitably protected b-hydroxy-g-amino acid
28 was accomplished as shown in Scheme 3.15 Treat-
ment of N-Boc Phe (17) in THF with carbonyl diimida-
zole (CDI) gave the corresponding imidazolide which
was reacted directly with lithium enolate of ethyl acetate
to provide b-ketone ester 18 in 92% yield.16 Reduction
of 18 with NaBH4 in ethanol17 at 78 ꢀC gave amino
alcohol (de 80%) from which the diastereomerically
pure anti product 19 could be isolated in 74% yield by
simple recrystallization (ether/heptane). The diastereo-
selectivity of this reduction resulted from the chelation
controlled process and the stereochemistry was con-
®rmed by converting 19 and its syn diastereomer 20
(Fig. 2) into the corresponding oxazolidinone 21 and 22
Figure 1.
1
(Fig. 2), respectively. From H±1H decoupling experi-
ment, J values of 7.3 Hz and 4.6 Hz between protons Ha
and Hb were determined for compounds 21 and 22
Scheme 2. Reagents and conditions: (a) Bu2BOTf, Et3N, then n-
C7H15CHO (10), 92%; (b) TBSOTf, 2,6-lutidine, 95%; (c) LiOH,
H2O2, 98%; (d) 2,4,6-trichlorobenzoyl chloride, (S)-(+)-benzyl 2-
hydroxy-3-methylbutanate (14), Et3N, 85%; (e) HF-MeCN, 90%.
Scheme 1.

B. Wagner et al. / Bioorg. Med. Chem. 7 (1999) 737±747
739
respectively, in accord with the assigned stereo-
structure.18,19 Furthermore, the signi®cant NOE e€ect
observed between protons Ha and Hb in the NOEDIFF
spectra of oxazolidinone 21 reinforced the assignment of
their cis orientation and, thus, the anti stereochemistry
of amino alcohol 19. The N-methylated keto ester 23
was also synthesized (Fig. 2) whose reduction under
various conditions gave predominantely the undesired
syn amino alcohol20 (structure not shown ) according to
Felkin±Ahn model.21
Selective N-methylation of b-hydroxy-g-amino ester of
type 19 was reported to be troublesome20,22 probably
due to the competitive b-elimination, retro-Aldol and
pyrrolidinone ring forming process. After several
unsuccessful trials, we devised a new method taking
advantage of the proximity of the amino alcohol func-
tion (Scheme 3). Thus, reaction of 19 with aqueous for-
maldehyde in the presence of a catalytic amount of
pTsOH gave smoothly the corresponding oxazolidine
24. It is worth noting that the formation of tetra-
hydroisoquinoline derivative via Pictet±Spengler reac-
tion was not observed under these mild conditions.23
Reduction of oxazolidine 24 with NaBH3CN in a mix-
ture of solvents TFA±CH2Cl2 a€orded selectively the N-
methylated compound 25 in 81% yield. This two-step
procedure is reminiscent of that developed by Frei-
dinger et al. for the preparation of Fmoc protected N-
alkyl amino acid.24 It is worth noting that classic one-
step reductive amination conditions failed to give the
desired product even under recently modi®ed condi-
tions.25 Selective N-acylation of 25 with CbzOSu26
under Schotten±Baumann conditions a€orded carba-
mate 26 whose secondary hydroxyl group was protected
as MOM ether27 to give compound 27 uneventfully.
Finally, the ethyl ester function was hydrolyzed
(K2CO3, MeOH, re¯ux, 3 h) to provide the appro-
priately protected acid 28. A remarkable solvent e€ect
was observed for this simple transformation as no
hydrolysis occurred in EtOH under otherwise identical
conditions. The reaction course had to be carefully
monitored as prolonged reaction time led to the forma-
tion of a variable amount of inseparable b-elimination
product 29 which was used for the synthesis of 8-deoxy-
hapalosin (vide infra).
The synthesis of hapalosin 2 and 8-deoxyhapalosin 4
was accomplished as shown in Scheme 4. Coupling of
diester 16 with acid 28 using Yamaguchi's reagent gave
the triester 30 which had been transformed into the
hapalosin by Ghosh et al. in three steps.7 On the other
hand, the compound 4 was synthesized in 2 steps from
fully protected amino ester 31. Thus, simultaneous
removal of benzyl ester, Cbz functions and 1,4-reduc-
tion of enoate by hydrogenolysis (Pearlmans catalyst,
H2, 1 atm) followed by standard DPPA mediated mac-
rolactamization28 a€orded 8-deoxyhapalosin 4 in 45%
overall yield.
Scheme 3. Reagents and conditions: (a) CDI, then lithium salt of ethyl
acetate, 92%; (b) NaBH4, EtOH, 78 ꢀC, 74%; (c) HCHO, pTSOH,
toluene, Dean±Stark, 77%; (d) NaBH3CN, CH2Cl2, TFA, 81%; (e)
CbzOSu, NaHCO3, acetone, H2O; (f) MOMBr, Pr2NEt, 87%; (g)
K2CO3, MeOH, re¯ux, 90%.
i
At this stage, we wondered if we could take advantage
of the oxazolidine function as a protecting group of
vicinal amino alcohol to obtain the triester 32 (Fig. 3)
from which both hapalosin and N-desmethyl hapalosin
would be accessible. Moreover, if realizable, most of the
redundant protection±deprotection steps found in
Schemes 3 and 4 would then be avoided. The realization
of this idea was shown in Scheme 5.
Selective hydrolysis of ester 24 to acid 33 was thought to
be delicate due to the sensitivity of oxazolidine function.
After several trials, the most classic method turned out
to be the most ecient one. Thus, treatment of 24 with
1 M KOH in MeOH at room temperature for 10 min
gives acid 33 in quantitative yield which was puri®ed by
Figure 2.

740
B. Wagner et al. / Bioorg. Med. Chem. 7 (1999) 737±747
simple acid±base extraction. Coupling of acid 33 with
hydroxy ester 16 under Yamaguchi's conditions gave
the triester 32 in 76% yield. The synthesis was diverged
at this point allowing access to both hapalosine (2) and
N-desmethyl analogue (3). Thus, mild acidic hydrolysis
of oxazolidine (TFA±CH2Cl2) gave the compound 34.
Hydrogenolysis followed by DPPA mediated macro-
lactamization a€orded N-desmethyl analogue 3 in 48%
yield. On the other hand, reduction of oxazolidine
(NaBH3CN, CH2Cl2, TFA) a€orded the N-methylated
derivative 35 which was transformed into hapalosin
following the same two step sequence in 18% overall
yield. The physical data of compounds 2 and 3 are
identical with those described in the literatures.5,6,8
Conformational studies
The conformation of both hapalosin (2)5±8 and its N-
desmethyl analogue (3) has been carefully determined
by way of NMR and computational studies.5,6,8 While
the natural product exists at room temperature (CDCl3)
as a mixture of two rotamers in favor of s-cis amide
linkage, the N-desmethyl analogue exists only as a single
s-trans conformer. Interestingly, we found that the 8-
deoxyhapalosin (4) exists exclusively as a s-cis con-
former at room temperature in CDCl3 as evidenced by
the presence of a strong NOE correlation between pro-
tons H9 and H12 in NOESY spectra. Other character-
istic NOE cross peaks were listed as follows: H12±H15,
0
NMe±H17, NMe±H8, H12±Ha (Ha ), H9±Ha (Ha ), NMe±
0
0
Ha (Ha ), H17±Ha (Ha ).
0
Figure 3.
Scheme 4.
Scheme 5. Reagents and conditions: (a) 2,4,6-trichlorobenzoyl chloride, 16, Et3N, 76%; (b) TFA, CH2Cl2, 0 ꢀC; (c) TFA, CH2Cl2, NaBH3CN, 0 ꢀC;
(d) Pd/C, EtOH; (e) DPPA, iPr2NEt.

B. Wagner et al. / Bioorg. Med. Chem. 7 (1999) 737±747
741
To understand this conformational preference, a com-
putational study of compound 4 was performed. For
the purpose of reducing the number of available con-
formations as well as the computational time, the heptyl
side chain was replaced by a methyl group. Three thou-
sand conformations of 8-deoxy-hapalosin (4) were gen-
erated by random search Monte Carlo method29 and
optimized by TNCG Truncated Newton minimization
method30 using the Macromodel (Version 5.5) pro-
gram31 with the MM2* force ®eld.32 (All of the compu-
tations discussed below were also conducted using the
MM2* force ®eld and GB/SA water solvation which led
to similar results). Both s-cis amide 4a and s-trans amide
4b were used as the starting geometry and the search
was carried out on blocks of 1000 Monte Carlo steps
until no additional conformation was found to be of
lower energy than the current minimum. Duplicated
conformations as well as those that had chirality chan-
ges were discarded.
between H9 and H12 was found in the most stable s-
trans amide form of compound 4.
The two lowest energy conformations of s-cis and the
lowest s-trans amide conformers of compound 4 toge-
ther with that of natural hapalosin (2)34 were shown in
Figure 4. Careful analysis of these structures revealed
that the ring conformation of the most stable s-trans
amide form of 8-deoxy-hapalosin (4) was very close to
that of hapalosin (2). However, a slight conformational
di€erence of the lowest energy s-cis conformers between
compounds 4 and 2 became also evident from this
examination. Thus, a C8±C7±C6±O5 torsion angle of
19.3ꢀ was found for the most stable (E=115.9 kJ/mol)
conformation of s-cis amide form of the 8-deoxy-hapa-
losin (4) which was not the most stable one in the s-cis
amide form of the hapalosin. The most stable con-
formation of s-cis amide form of the hapalosin corre-
sponded in fact to the second lowest energy conformer
(E=121.7 kJ/mol) of 8-deoxy-hapalosin, in which the
C8±C7±C6±O5 torsion angle was 73.6ꢀ (Table 1). Mini-
mizing the electrostatic repulsion between the oxygen of
the hydroxyl group borne by the C8 carbon atom and
that of the C6 ester function in hapalosin may account
for the deviation of this conformational preference. As a
matter of fact, the pro R proton of C8 in the second
lowest s-cis amide conformation of compound 4 was
equidistant to the two oxygen atoms of C6 ester (3.2 A),
while in the lowest energy conformer, the distance
between this pro R proton and one of the ester oxygen
atom was reduced to 2.6 A disfavoring thus this con-
former in the case of hapalosin because of the electro-
static repulsion.
From this search, nine conformations were found within
3 kcal/mol from the global minimum. Seven of them
have an s-cis amide bond and two have an s-trans amide
bond. The lowest energy conformation of the s-cis
amide form of 8-deoxy-hapalosin 4 was calculated to be
1.6 kcal/mol more stable than the s-trans amide form.
Assuming the Boltzman distribution33 and taking into
account the energy of the seven possible conformations
of s-cis amide conformers (E1,. . .,E7=115.9, 121.7,
123.3, 124.5, 125.1, 126.8, 127.8 kJ/mol) as well as the
two possible s-trans amide isomer (E1, E2=122.6,
122.7 kJ/mol), the calculated population of s-cis amide
conformers were greater than 90% which correlated
nicely with our experimental observations. The calcu-
lated steric energies (MM2) as well as the most relevant
geometrical parameters of these structures are summar-
ized in Table 1. The distance between the H9 and H12
hydrogens (the dotted line) of the two lowest s-cis amide
conformers was 2.2 A (Fig. 4) which was consistent with
a strong NOE correlation observed between these two
protons in NOESY spectrum. A distance of 4.5 A
Biological activity
The ability of 8-deoxy-hapalosin (4) to reverse P-gp-
mediated multidrug resistance was evaluated in drug
accumulation assays using K562 R and S/Adriblastine
against human erytholeucemic cell lines. At 10 mM con-
centration of compound 4, the IC50 value of adriblastine
Table 1
8-Deoxy-hapalosin
(cis-amide) 4a 1st conf
8-Deoxy-hapalosin
(cis-amide) 4a 2nd conf
8-Deoxy-hapalosin
(trans-amide) 4b
E (kJ/mol)
ÁE (kJ/mol)
ÁE (kcal/mol)
CH8±O5
CH8±O6
H12±H9
115.9
0
0
2.6
4.1
2.2
3.3
129.1
50.2
64.8
19.3
177.7
70.0
44.5
103.4
179.3
140.7
74.8
121.7
5.8
1.4
3.2
3.2
122.6
6.7
1.6
4.5
3.9
2.2
3.3
4.5
C
12±C11±N10±C9
C11±N10±C9±C8
10±C9±C8±C7
175.0
115.6
52.8
84.3
141.5
174.1
151.5
52.8
31.2
169.7
106.4
64.2
133.1
53.9
84.4
73.6
170.2
171.4
49.3
89.9
177.8
140.6
80.9
N
C9±C8±C7±C6
C8±C7±C6±O5
C7±C6±O5±C4
C6±O5±C4±C3
O5±C4±C3±C2
C4±C3±C2±O1
C3±C2±O1±C12
C2±O1±C12±C11
O1±C12±C11±N10
aThe distances are in A and the torsion angles are in degrees.

742
B. Wagner et al. / Bioorg. Med. Chem. 7 (1999) 737±747
Figure 4. Conformations of the lowest cis- and trans-amides of 8-deoxyhapalosin (top) and of hapalosin (bottom) optimized by MM2* force ®eld
calculations.
(5Â10 6 M) against resistance K562
R
cell lines
the internal standard (d ppm). Flash chromatography
was performed using Kieselgel 60 (230±400 mesh, E.
Merck) and usually employed a stepwise solvent polar-
ity gradient, correlated with TLC mobility. Solvents and
reagents were puri®ed according to standard laboratory
techniques. Optical rotations were determined on a
Perkin±Elmer automatic polarimeter at room tempera-
ture. Mass spectra were run on AEI MS-50 (EI), AEI
MS-9 (CI), and Kratos MS-80 (FAB), respectively. All
reactions requiring anhydrous conditions or inert
atmosphere were conducted under Argon.
remained almost unchanged indicating the lack of sig-
ni®cant anti MDR activity of 4. This result may infer
that hydroxy group, in addition to the conformation of
the amide bond (s-cis versus s-trans), played an impor-
tant role in the bioactivity of the natural product (2).
Conclusion
A concise synthesis of hapalosin and its 8-deoxy, N-
desmethyl analogues have been developed. The dual
role of oxazolidine as protecting group of vicinal amino
alcohol and latent N-methyl function was uncovered in
the course of this study. While the generality of this
methodology remains to be fully exploited, its applica-
tion to the synthesis of other complex natural pro-
ducts20,22 might be rewarding. The conformational
studies and the bioassay of synthetic analogue 4 indi-
cated that the s-cis amide conformation is necessary but
not sucient to account for the bioactivity of hapalosin.
The hydroxy group is indispensable to the anti MDR
activity of hapalosin-type compounds.
(4S)-3[(20S,30R)-20-Methyl-30-hydroxydecanoyl)-4-phenyl-
methyl-2-oxazolidinone (11). To a cooled solution of 9
(1.24 g, 5.32 mmol) in anhydrous dichloromethane (15 mL)
were added triethylamine (0.97 mL, 6.96 mmol) followed
by n-Bu2BOTf (6.1 mL, 1 M solution in dichloro-
methane). The reaction mixture was then cooled down
to 78 ꢀC and n-hexanal 10 (0.91 mL, 5.85 mmol) was
added dropwise over a period of 5 min. After being
stirred at 78 ꢀC for 20 min and at 0 ꢀC for 1 h, the
reaction mixture was quenched by addition of saturated
NH4Cl solution and methanol (20 mL). To this cloudy
solution was added H2O2 (30% solution, 10 mL) at such
a rate as to keep the internal temperature below 10 ꢀC.
After stirring for one additional hour at 0 ꢀC, the vola-
tile was removed under reduced pressure. The resulting
mixture was extracted with dichloromethane. The com-
bined organic extracts were washed with aqueous
NaHCO3 solution and then brine, dried over Na2SO4
and concentrated to a€ord a light yellow oil. Puri®cation
by ¯ash chromatography (SiO2, eluent: Et2O:heptane=
3:2) gave the syn aldol 11 (1.77 g, 92%): [a]d=+36.6 (c
Experimental
Melting points were determined with a Ko¯er apparatus
and were uncorrected. Infrared (IR) spectra were recorded
1
on a Nicolet-205 spectrometer. H NMR spectra were
measured on Brucker AC-200 (200 MHz), Bruker AC-
250 (250 MHz), Bruker (300 MHz), and Bruker WM-
400 (400 MHz) spectrometers with tetramethylsilane as

B. Wagner et al. / Bioorg. Med. Chem. 7 (1999) 737±747
743
1.6, CHCl3); IR (CHCl3) n 2931, 2853, 1771, 1679,
1384 cm
13 (637.0 mg, 2.01 mmol) in anhydrous THF (5 mL)
were added triethylamine (0.33 mL, 2.41 mmol) and
2,4,6-trichlorobenzoyl chloride (0.31 mL, 2.00 mmol),
successively. After being stirred at room temperature for
30 min, a solution of S-benzyl 2-hydroxy-3-methyl
butanate 14 (1.00 g, 4.81 mmol) and DMAP (490.00 mg,
4.02 mmol) in toluene (15 mL) were added. The reaction
mixture was stirred at room temperature for another
18 h and was then diluted with aqueous NH4Cl solution.
The volatile was removed under reduced pressure and
the remaining aqueous solution was extracted with
CH2Cl2. The combined organic extracts were washed
with a saturated NH4Cl solution, dried, and evaporated.
Puri®cation by ¯ash chromatography (SiO2, eluent:
heptane:ether=40:1) a€orded 15 (870 mg, 85%):
[a]d= 20 (c 0.9, CHCl3); IR (CHCl3) n 2925, 2843,
1737, 1450, 1250, 1106, 1050 cm 1; 1H NMR (300 MHz,
CDCl3) d 0.02 (s, 3H), 0.05 (s, 3H), 0.86 (br s, 12H),
0.96 (d, J=6.9 Hz, 3H), 0.98 (d, J=6.9 Hz, 3H), 1.18 (d,
J=7.0 Hz, 3H), 1.2±1.4 (m, 12H), 1.50 (m, 1H), 2.63 (d
of septet, J=4.5, 6.9 Hz, 1H), 3.99 (q, J=5.2 Hz, 1H),
4.84 (d, J=4.5 Hz, 1H), 5.14, 5.20 (AB system,
J=12.3 Hz, 2H), 7.34 (m, 5H); 13C NMR( CDCl3) d
4.3, 4.0, 12.7, 14.4, 17.6, 19.1, 23.7, 25.6, 26.0, 26.6,
30.3, 32.9, 34.7, 36.0, 45.8, 67.8, 74.4, 78.1, 129.3, 129.4,
129.5, 137.0, 170.8, 176.1; MS (CI) m/z 507 (M+H)+.
Anal. calcd for C29H50O5Si: C, 68.73; H, 9.94. Found:
C, 69.03; H, 10.06.
1
;
1H NMR (200 MHz, CDCl3) d 0.90 (t,
J=7.0 Hz, 3H), 1.25 (m, 15H), 2.78 (dd, J=9.4,
13.4 Hz, 1H), 2.91 (d, J=2.9 Hz, 1H), 3.24 (dd, J=3.3,
13.4, 1H), 3.76 (qd, J=2.7 Hz, 7.0 Hz, 1H), 3.94 (m, 1H),
4.20 (m, 2H), 4.70 (m, 1H), 7.1±7.4 (m, 5H); 13C NMR
(CDCl3) d 10.7, 14.4, 22.9, 26.3, 29.5, 29.8, 32.1, 34.2,
38.1, 42.4, 55.4, 66.4, 71.8, 127.6, 129.5, 129.8, 135.3,
153.3, 177.8; MS (CI) m/z 362 (M+H), 234. Anal. calcd
for C21H31NO4: C, 69.77; H, 8.64; N, 3.87. Found: C,
69.61; H, 8.48; N, 3.95.
(4S)-3[(20S,30R)-20-Methyl-30-(tert-butyldimethylsilyloxy)-
decanoyl)-4-phenylmethyl-2-oxazolidinone (12). To
a
solution of compound 11 (880.00 mg, 2.43 mmol) and
2,6-lutidine (0.55 mL, 4.70 mmol) in anhydrous dichlor-
omethane was added dropwise TBDMSOTf (0.82 mL,
3.60 mmol) at 0 ꢀC. After being stirred at 0 ꢀC for 1 h
and then at room temperature for 2.5 h, the reaction
mixture was diluted with CH2Cl2 and the organic phase
was washed with 5% aqueous citric acid followed by a
saturated NH4Cl solution and dried over Na2SO4. After
evaporation of the combined organic phases, the crude
product was puri®ed by ¯ash chromatograhy (SiO2,
heptane:EtOAc=9:1) to a€ord compound 12 (1.06 g,
95%): mp 42±46 ꢀC; [a]d=+59 (c 0.8, CHCl3); IR
(CHCl3) d 2938, 2868, 1771, 1693, 1468, 1391, 1110,
1
821 cm 1; H NMR (200 MHz, CDCl3) d 0.01 (s, 3H),
0.02 (s, 3H), 0.90 (br s, 12H), 1.2±1.7 (m, 15H), 2.55 (dd,
J=9.8, 13.2 Hz, 1H), 3.30 (dd, J=3.0, 13.2 Hz, 1H),
3.85 (m, 1H), 4.02 (q, J=5.2 Hz, 1H), 4.20 (m, 2H), 4.61
(m, 1H), 7.1±7.3 (m, 5H); 13C (CDCl3) d 4.8, 4.1,
11.5, 14.1, 18.1, 22.6, 25.0, 25.8, 29.2, 29.8, 31.8, 35.6,
37.6, 42.8, 55.8, 66.0, 72.9, 127.3, 128.9, 129.5, 135.4,
153.1, 175.3; MS (CI) m/z 476 (M+H). Anal. calcd for
C27H45NO4Si: C, 68.17; H, 9.53; N, 2.94. Found: C,
68.19; H, 9.33; N, 3.12.
(S)-1-Carboxy-2-methylpropyl(2S,3R)-3-(tert-butyldimethyl-
silyloxy)-2-methyl-decanoic acid (16). A solution of
compound 15 (363.0 mg, 0.72 mmol) in a mixture of
solvent acetonitrile:aqueous HF (95:5, 10 mL) was stir-
red at room temperature for 1 h. The reaction mixture
was diluted with water and the volatile was removed
under reduced pressure. The remaining aqueous solu-
tion was extracted with CH2Cl2 and the combined
organic extracts were washed with a saturated NH4Cl
solution, dried and evaporated. The crude product was
puri®ed by ¯ash chromatography (SiO2, eluent: hep-
tane:ether=1:1) to a€ord compound 16 (253 mg, 90%):
[a]d= 11.0 (c 0.2, CHCl3); IR (CHCl3) n 3500, 2925,
2862, 1725, 1443, 1181, 1131 cm 1; 1H NMR (200 MHz,
CDCl3) d 0.90 (t, J=6.9 Hz, 3H), 0.94 (d, J=6.9 Hz,
3H), 1.00 (d, J=6.9 Hz, 3H), 1.17 (d, J=7.1 Hz, 3H),
1.3±1.7 (m, 12H), 2.28 (m, 1H), 2.69 (dq, J=3.1, 7.1 Hz,
1H), 2.90 (d, J=4.8 Hz, 1H), 4.08 (m, 1H), 5.00 (d,
J=4.0 Hz, 1H), 5.14, 5.25 (AB system, J=12.2 Hz, 2H),
7.34 (m, 5H); 13C NMR (CDCl3) d 9.6, 14.2, 18.9, 22.8,
26.3, 29.4, 29.7, 30.1, 31.9, 33.6, 44.6, 67.4, 72.0, 76.5,
128.6, 128.5, 128.6, 137.2, 170.4, 175.3; MS (CI) m/z 393
(M+H)+, 333, 285, 209, 203. Anal. calcd for C23H36O5:
C, 70.38; H, 9.24. Found: C, 70.18; H, 9.16.
(2S,3R)-3-(tert-Butyldimethylsilyloxy)-2-methyl-decanoic
acid (13). To a cooled (0 ꢀC) solution of 12 (1.24 g,
2.61 mmol) in THF/H2O (4/1, 50 mL) was added H2O2
(30% in water, 5.46 mL, 53.4 mmol) and then LiOH
hydrate (1.12 g, 26.7 mmol) in one portion. The tem-
perature was allowed to come to room temperature and
stirring was continued overnight. After evaporation of
the volatile, the residue was suspended in water, and the
mixture was acidi®ed with citric acid and extracted sev-
eral times with dichloromethane. The combined organic
extracts were washed with brine, dried over Na2SO4,
and concentrated to dryness. Puri®cation by ¯ash chro-
matography (SiO2, eluent: heptane:ether=9:1) a€orded
pure compound 13 (823.0 mg, 98%): [a]d=+25 (c 1,
1
CHCl3); H NMR (200 MHz, CDCl3) d 0.10 (s, 3H),
0.11 (s, 3H), 0.90 (s, 9H, tBu), 0.91 (t, J=7.1 Hz, 3H),
1.13 (d, J=7.0 Hz, 3H), 1.2±1.3 (m, 12H), 2.60 (m, 1H),
3.95 (m 1H); 13C NMR (CDCl3) 4.2, 4.6, 10.9, 14.2,
18.1, 22.8, 25.5, 25.9, 29.3, 29.8, 31.9, 34.5, 44.5, 73.6,
179.3; MS (CI) m/z 317 (M+H). Anal. calcd for
C17H36O3Si: C, 64.50; H, 11.46. Found: C, 64.74; H,
11.65.
(4S)-4-tert-Butoxycarbonyl amino-3-oxo-5-phenyl-pentanoic
acid ethyl ester (18). To a solution of l-N-Boc-phenyla-
lanine 17 (7.35 g, 27.73 mmol) in dry THF (150 mL) was
added carbonyldiimidazole (4.95 g, 30.51 mmol) in one
portion at 0 ꢀC. The solution was stirred at 0 ꢀC for
30 min and at room temperature for 1.5 h. The forma-
tion of the amide can be monitored by TLC (EtOA-
c:heptane=1:2). To another ¯ask, butyllithium (1.6 M
solution in hexane, 78.00 mL, 124.8 mmol) was added
(S)-1-Carboxy-2-methylpropyl(2S,3R)-3-(tert-butyldimethyl-
silyloxy)-2-methyl-decanoate (15). To a solution of acid

744
B. Wagner et al. / Bioorg. Med. Chem. 7 (1999) 737±747
dropwise to diisopropylamine (18.37 mL, 131.04 mmol)
in THF (50 mL) at 0 ꢀC. The light yellow suspension
was stirred for 15 min and was then cooled down to
78 ꢀC. Ethyl acetate (12.2 mL, 124.8 mmol) was added
and stirring was continued for 30 min. The so formed
light-yellow solution of the lithium enolate of ethyl
acetate was then added during 5 min to a precooled
solution of the imidazolide. After stirring at 78 ꢀC for
1.5 h, the cooling bath was removed and the reaction
temperature was raised to ambient. The suspension was
then quenched by addition of saturated NH4Cl solution.
After evaporation of the volatile, the aqueous solution
was extracted with dichloromethane. The combined
organic phases were washed with 1 N HCl and satu-
rated NH4Cl solution, dried over Na2SO4, and evapo-
rated to dryness. Puri®cation by ¯ash chromatography
(SiO2, EtOAc:heptane=1:4) give compound 18 (8.55 g,
92%): mp 62±64 ꢀC; [a]d= 2.5 (c 1.1, CHCl3); IR
129.4, 138.2, 155.5, 173.0; MS (CI) m/z 338 (M+H),
238, 282. Anal. calcd for C18H27NO5: C, 64.07; H, 8.06;
N, 4.15. Found: C, 63.79; H, 8.11; N, 4.09.
(4S,5R)-(4-Benzyl-2-oxo-oxazolidin-5-yl) acetic acid ethyl
ester (21). To a solution of 19 (56.0 mg, 0.16 mmol) in
dichloromethane (0.5 mL) was added TFA (0.5 mL) at
0 ꢀC. After being stirred at room temperature for
10 min, the reaction mixture was diluted with 20 mL
ether and concentrated in vacuo. The remaining TFA
was removed azeotropically with ether, toluene, and
dried under high vacuum. To the solution of crude
amino alcohol in dry dichloromethane (1.5 mL) were
added at 0 ꢀC, diisopropylethylamine (72.0 mL, 0.41 mmol)
and carbonyldiimidazole (67.00 mg, 0.41 mmol), succes-
sively. After being stirred at room temperature for 24 h,
the reaction mixture was diluted with dichloromethane
and washed with aqueous citric acid solution (10%) and
saturated NH4Cl solution. The collected organic phases
were dried over Na2SO4 and evaporated to dryness. The
residue was puri®ed by preparative TLC (SiO2, heptane:
ethyl acetate=1:1) to a€ord compound 21 (28.1 mg,
67%). [a]d= 38 (c 1.8, CHCl3); IR (CHCl3) n 3437,
2975, 1768, 1506, 1406, 1181 cm 1; 1H NMR (200 MHz,
CDCl3) d 1.31 (t, J=7.1 Hz, 3H), 2.6±2.90 (m, 3H), 2.92
(dd, J=6.9, 16.7 Hz, 1H), 4.12 (m, 1H), 4.20 (q,
J=7.1 Hz, 2H), 4.85 (br s, 1H), 5.12 (q, J=7.3 Hz, 1H),
7.1±7.4 (m, 5H); 13C (CDCl3) d 14.5, 34.9, 36.7, 56.7,
61.7, 75.8, 127.8, 129.3, 129.5, 136.5, 155.5, 169.9; MS
(CI) m/z 264 (M+H). Anal. calcd for C14H17NO4: C,
63.87; H, 6.51; N, 5.32. Found: C, 63.45; H, 6.61; N,
5.66; In NOEDIFF spectrum, irradiation at O2=
4135.422 Hz (250 MHz), corresponding to the OCH
proton (d=5.12 ppm) leading to the 8% enhancement
of the signal of NCH proton at d=4.12 ppm. The
1H-1H decoupling experiment allowed the calculation of
coupling constant between Ha and Hb (J=7.3) which is
also indicative of cis relationship between these two
protons.
1
(CHCl3) n 3012, 1728, 1506, 1362, 1325, 1162 cm 1; H
NMR (200 MHz, CDCl3) d 1.25 (t, J=7.1 Hz, 3H), 1.42
(s, 9H), 2.94 (dd, J=7.6, 14.0 Hz, 1H), 3.16 (dd, J=5.8,
14.0 Hz, 1H), 3.42, 3.52 (AB system, J=16.0 Hz, 2H),
4.16 (q, J=7.1 Hz, 2H), 4.55 (br q, J=7.8, 1H), 5.22 (d,
J=7.8 Hz, 1H), 7.1±7.4 (m, 5H); 13C (CDCl3) d 14.0,
28.2, 36.7, 46.7, 60.4, 61.3, 80.0, 126.8, 128.6, 129.2,
136.3, 155.2, 166.8, 201.9; MS (CI) m/z 336 (M+H),
280. Anal. calcd for C18H25NO5: C, 64.46; H, 7.51; N,
4.17. Found: C, 64.38; H, 7.22; N, 4.21.
(3R,4S)-4-tert-Butoxycarbonyl amino-3-hydroxy-5-phenyl-
pentanoic acid ethyl ester (19). To a solution of 18
(2.02 g, 6.03 mmol) in ethanol (50 mL) was added
NaBH4 (573.0 mg, 15.07 mmol). After being stirred at
78 ꢀC for 2 h, the reaction mixture was quenched by
addition of water (10 mL) and 1 N HCl (10 mL). After
evaporation of the volatile, the suspension was extrac-
ted with dichloromethane. The combined organic pha-
ses were washed with a saturated NH4Cl solution, dried
over Na2SO4 and evaporated to dryness. Recrystalliza-
tion from EtOAC/hexane gave 19 (1.27 g). The ®ltrate
was evaporated and puri®ed by ¯ash chromatography
(SiO2, eluent: EtOAC:hexane=1:4) to a€ord another
portion of compound 19 (237.0 mg, total yield of 19:
74%) and its (3S,4S) diastereomer 20 (158.0 mg, 7.8%).
Compound 19: mp 143±144 ꢀC; [a]d +3.8ꢀ (c 0.9,
(4S,5S)-(4-Benzyl-2-oxo-oxazolidin-5-yl)acetic acid ethyl
ester (22). [a]d= 89 (c 1.5, CHCl3); d 1.31 (t,
J=7.1 Hz, 3H), 2.60 (dd, J=6.9, 16.3 Hz, 1H), 2.65 (dd,
J=6.2, 16.3 Hz, 1H), 2.83 (dd, J=8.1, 13.4 Hz, 1H),
2.96 (dd, J=5.4, 13.4 Hz, 1H), 3.80 (m, 1H), 4.15 (q,
J=7.1 Hz, 2H), 4.70 (dt, J=4.8, 6.5 Hz, 1H), 5.20 (br s,
1H), 7.1±7.4 (m, 5H); 13C (CDCl3) d 14.7, 39.7, 42.3,
59.5, 61.9, 78.0, 127.9, 129.5, 129.7, 136.4, 158.7, 169.8;
MS (CI) m/z 264 (M+H); The 1H±1H decoupling
experiment allows the calculation of coupling constant
between Ha and Hb (J=4.6), indicative of trans rela-
tionship between these two protons.
CHCl3); IR (CHCl3) n 3347, 1778, 1714, 1510, 1194,
1
864 cm
;
1H NMR (200 MHz, CDCl3) d 1.25 (t,
J=7.1 Hz, 3H), 1.40 (s, 9H), 2.50 (m, 2H), 2.82 (dd,
J=7.5, 13.8 Hz, 1H), 2.96 (dd, J=4.5, 13.8 Hz, 1H),
3.60 (br s, 1H), 3.7±3.9 (m, 2H), 4.15 (q, J=7.1 Hz, 2H),
4.62 (br d, J=9.2 Hz, 1H), 7.1±7.4 (m, 5H); 13C (CDCl3)
a 13.7, 27.8, 35.4, 37.8, 54.8, 60.4, 69.7, 79.8, 125.9,
127.9, 129.0, 137.2, 155.3, 172.5; MS (CI) m/z 338
(M+H), 282. Anal. calcd for C18H27NO5: C, 64.07; H,
8.06; N, 4.15. Found: C, 63.96; H, 7.95; N, 4.25. The syn
diastereomer 20: mp 87 ꢀC; [a]d=20.6 (c 0.2, CHCl3);
(4S,5R)-4-Benzyl-3-(tert-butoxycarbonyl)-oxazolidin-5-yl)-
acetic acid ethyl ester (24). To a solution of 19 (1.22 g,
3.62 mmol) in toluene (40 mL) were added aqueous
HCHO solution (30% in water, 3.62 mL, 36.2 mmol)
and p-toluenesulfonic acid (34.4 mg, 0.18 mmol). The
mixture was heated to re¯ux using a Dean±Stark appa-
ratus for removing water. After being re¯uxed for 20 h,
the reaction mixture was diluted with aqueous NH4Cl
solution, the volatile was evaporated in vacuo and the
aqueous residue was extracted with dichloromethane.
IR (CHCl3) n 3550, 3443, 2981, 1720, 1605, 1506,
1
1368 cm
;
1H NMR (200 MHz, CDCl3) d 1.20 (t,
J=7.2 Hz, 3H), 1.49 (s, 9H), 2.48 (dd, J=2.9, 16.8 Hz,
1H), 2.60 (m, 1H), 2.8±3.0 (m, 2H), 3.46 (br s, 1H), 3.71
(m, 1H), 3.90 (m, 1H), 4.10 (q, J=7.2 Hz, 2H), 4.98 (br
d, J=9.5 Hz, 1H), 7.1±7.4 (m, 5H); 13C (CDCl3) d 14.1,
28.3, 38.6, 55.4, 60.8, 67.1, 70.1, 80.2, 126.4, 128.5,

B. Wagner et al. / Bioorg. Med. Chem. 7 (1999) 737±747
745
The combined organic phases were washed with brine,
dried, and evaporated to dryness. The crude product
was puri®ed by ¯ash chromatography (silica, EtOAc:
heptane=1:4) to a€ord 24 (973 mg, 77%): mp 68 ꢀC;
[a]d= 32 (c 0.9, CHCl3); IR (CHCl3) n 2980, 1743,
(30 mL) was added MeOCH2Br (1.59 mL, 19.52 mmol)
and Pr2NEt (3.73 mL, 21.47 mmol) at 0 ꢀC. After being
i
stirred at room temperature for 2 days, the reaction
mixture was diluted with aqueous NH4Cl solution and
extracted with CH2Cl2. The combined organic extracts
were washed with brine, dried, and evaporated. Pur-
i®cation by ¯ash chromatography (SiO2, eluent: EtOAc:
heptane=1:4) a€orded compound 27 (1.45 g, 87%):
[a]d= 39 (c 0.4, CHCl3); 1H NMR (200 MHz, CDCl3):
two rotamers d 1.25 (t, J=7.1 Hz, 3H), 2.60, 2.70 (s,
3H), 2.5±3.0 (m, 3H), 3.20 (m, 1H), 3.41, 3.42 (s, 3H),
4.10±4.35 (m, 4H), 4.75 (m, 2H), 4.90 (m, 1H), 5.10 (s,
1H), 7.0±7.4 (m, 10H); 13C NMR (CDCl3) d 14.4, 34.2
(32.4), 38.3 (38.5), 56.1 (56.2), 60.5 (60.7), 61.9, 66.7,
67.4, 77.1, 97.4, 126.2 (126.4), 127.4 (127.8), 128.1
(128.2), 128.5 (128.6), 129.0 (129.1), 136.5 (136.9), 138.3
(138.5), 156.3 (156.4), 171.0 (171.3); MS (CI) m/z 430
(M+H), 398. Anal. calcd for C24H31NO6: C, 67.11; H,
7.27; N, 3.26. Found: C, 67.26; H, 7.22; N, 3.15.
1
1693, 1393 cm 1; H NMR (200 MHz, CDCl3): due to
1
the presence of two rotamers, signals in H NMR spec-
trum were broadened d 1.1±1.3 (m, 12H), 2.5±2.7 (m,
4H), 4.00 (m, 2H), 4.40 (m, 2H), 4.90 (m, 2H), 7.1±7.4
(m, 5H); 13C NMR (CDCl3) d 14.1, 28.2, 34.8, 35.2, 59.1,
61.0, 77.1, 77.9, 80.3, 126.3, 128.4, 129.5, 138.0, 170.3; MS
(CI) m/z 350 (M+H), 294. Anal. calcd for C19H27NO5:H,
7.79; N, 4.01. Found: C, 65.69; H, 7.62; N, 3.91.
(4S,3R)-4-(Benzyloxycarbonyl-methylamino)-3-hydroxy-
5-phenyl-pentanoic acid ethyl ester (26). To a solution
of CH2Cl2 (10 mL) and TFA (10 mL), cooled at 0 ꢀC,
was added NaBH3CN (2.07 g, 32.81 mmol) in a small
portion. A solution of compound 21 (2.29 g, 6.56 mmol)
in CH2Cl2 (10 mL) was then added in such a rate as the
internal temperature did not exceed 0 ꢀC. After stirring
at the same temperature for 1 h, the volatile was
removed and the residue was redissolved in CH2Cl2 and
aqueous NaHCO3 solution. The aqueous phase was
extracted with CH2Cl2, the combined organic extracts
were washed with H2O, brine, dried and evaporated
to give crude compound 25 which was used directly for
the next step. A small amount of pure 25 was obtained
by preparative TLC (SiO2, CH2Cl2:MeOH=9:1):
(4S,3R)-4-(Benzyloxycarbonyl-methylamino)-3-methoxy-
methoxy-5-phenyl-pentanoic acid (28). To a solution of
ester 27 (1.28 g, 2.98 mmol) in MeOH (40 mL) and water
(10 mL) was added K2CO3 (824.8 mg, 5.98 mmol). After
being re¯uxed for 3 h, the reaction mixture was diluted
with aqueous NH4Cl solution, the volatile was removed
and the remaining aqueous phase was extracted with
hexane to remove any neutral materials. The aqueous
phase was then acidi®ed with 2 N HCl and extracted
with CH2Cl2. The combined organic extracts were
washed with brine, dried, and evaporated to give acid 28
which was used without further puri®cation (1.08 g,
90%): [a]d= 30 (c 1.3, CHCl3); IR (CHCl3) n 3068,
[a]d= 3.22 (c 1.2, CHCl3); IR (CHCl3) n 3612, 3012,
2968, 1750, 1681, 1443, 1412, 1125, 1043 cm
1
;
1H
NMR (200 MHz, CDCl3) d 1.25 (t, J=7.1 Hz, 3H), 2.50
(m, 2H), 2.70 (s, 3H), 3.10 (d, J=7.3 Hz, 2H), 3.60 (m,
1H), 4.15 (q, J=7.1 Hz, 2H), 4.45 (m, 1H), 7.1±7.4 (m,
5H), 7.6 (br s, 2H); 13C NMR (CDCl3) d 13.9, 32.1,
32.8, 36.9, 61.7, 65.0, 65.6, 128.0, 128.9, 129.5, 134.6,
172.5. To the solution of crude amino alcohol 25 in H2O
(30 mL) and acetone (30 mL) was added CbzOSu
(2.45 g, 9.84 mmol) and NaHCO3 (827 mg, 9.84 mmol).
After being stirred at room temperature for 16 h, the
reaction mixture was diluted with H2O (30 mL), the
volatile was removed under reduced pressure and the
remaining aqueous phase was extracted with CH2Cl2.
The combined organic extracts were washed with brine,
dried, and evaporated. Puri®cation by ¯ash chromato-
graphy (SiO2, eluent: EtOAc:heptane=1:4 then 1:3)
a€orded the compound 26 (1.74 g, 69% in two steps):
[a]d= 33 (c 1.5, CHCl3); IR (CHCl3) n 3400, 2980,
1740, 1681, 1468, 1325, 1137 cm 1; 1H NMR (200 MHz,
CDCl3) d 1.28 (t, J=7.2 Hz, 3H), 2.42 (dd, J=8.6,
17.0 Hz, 1H), 2.60 (m, 1H), 2.61 (s, 3H), 3.00 (dd,
J=11.6, 14.4, 1H), 3.21 (dd, J=4.0, 14.4 Hz, 1H), 3.45
(d, J=4.4 Hz, 1H), 4.12 (m, 1H), 4.18 (q, J=7.2 Hz,
2H), 4.28 (m, 1H), 5.10 (s, 2H), 7.0±7.4 (m, 10H); 13C
NMR (CDCl3) d 14.2, 33.2, 38.2 (38.8), 60.8, 64.5, 66.9
(70.5), 69.2, 70.1, 126.5, 127.5, 127.9, 128.4, 128.9,
139.0, 159.5, 172.5; MS (CI) m/z 386 (M+H). Anal.
calcd for C22H27NO5: C, 68.55; H, 7.06; N, 3.63. Found:
C, 68.03; H, 7.04; N, 3.78.
1
2981, 1706, 1425 cm 1; H NMR (200 MHz, CDCl3) d
two rotamers 2.5±3.0 (m, 6H), 3.20 (m, 1H), 3.38, 3.40
(s, 3H), 4.2 (m, 1H), 4.75 (m, 1H), 4.9±5.2 (m, 4H) 7.0±
7.4 (m, 10H); 13C NMR (CDCl3) d 33.9 (34.1), 36.9,
55.8 (55.9), 57.3 (57.5), 66.7 (66.9), 67.3, 76.4 (76.5),
96.8, 126.0±129.1 (complex), 135.9 (136.3), 137.9
(138.0), 156.3 (156.4), 175.0 (175.5); MS (CI) m/z 402
(M+H), 370, 340.
Compound 30 and 31. To a solution of 28 (141.00 mg,
0.35 mmol) in anhydrous toluene (5 mL) was added
Et3N (59.00 mL, 0.42 mmol) and 2,4,6-trichloro-
benzoylchloride (55.00 mL, 0.35 mmol) at room tem-
perature. After stirring at room temperature for 1.5 h, a
solution of 16 (264.00 mg, 0.67 mmol) and 4-dimethyl-
aminopyridine (428.00 mg, 3.51 mmol) was added at
room temperature and stirring was continued for 1.5 h.
The reaction mixture was diluted with NH4Cl solution
and the organic phase was separated. The aqueous
phase was extracted with dichloromethane. The com-
bined organic phases were washed with brine, dried, and
evaporated. Puri®cation by ¯ash chromatography
(SiO2, heptane:hexane=6:1) a€orded 30 (186.2 mg,
68%) and a variable amount of b-elimination product
31 (the yield was variable depending on the purity of
acid 28 as well as the reaction time of this coupling
step). Prolonged reaction time increased the yield of 31
at the expense of that of compound 30. Compound 30:
(4S,3R)-4-(Benzyloxycarbonyl-methylamino)-3-methoxy-
methoxy-5-phenyl-pentanoic acid ethyl ester (27). To a
solution of compound 26 (1.50 g, 3.9 mmol) in CH2Cl2
[a]d= 25.7 (c 0.7, CHCl3); IR (CHCl3) n 2937, 2856,
1743, 1693, 1456, 1143 cm
1
;
1H NMR (250 MHz,

746
B. Wagner et al. / Bioorg. Med. Chem. 7 (1999) 737±747
CDCl3) d two rotamers 0.90±1.60 (m, 24H), 2.25 (m,
1H), 2.40±3.00 (m, 8H), 3.00 (m, 1H), 3.39, 3.40 (m,
3H), 4.30 (m, 1H), 4.60±5.30 (m, 7H), 7.00±7.40 (m,
15H); 13C (CDCl3): d two rotamers 12.3, 12.6, 14.0,
14.2, 17.1, 18.7, 22.6, 25.3, 29.1, 29.3, 29.7, 30.1, 31.7,
31.9, 34.3, 37.4, 37.7, 37.9, 42.8, 43.0, 56.2, 57.4, 60.6,
66.6, 66.8, 67.2, 74.4, 74.5, 76.6, 76.8, 97.2, 97.4, 122.4,
126.2, 126.3, 126.7, 127.4, 127.7, 127.9, 128.3, 128.4,
128.5, 128.8, 137.2, 138.5, 145.4, 166.5, 170.6, 177.5; MS
(CI) m/z 776 (M+H)+. Compound 31: [a]d= 10.0 (c
to give acid 33 which was used directly for the next step
without further puri®cation (166 mg, 100%): IR
(CHCl3) n 3450, 3075, 2981, 2943, 1730, 1693, 1406,
1
1200, 1187 cm 1; H NMR (300 MHz, CDCl3) d two
rotamers 1.47 (s, 9H), 2.21 (dd, J=2.5, 14.8 Hz, 1H),
2.42 (dd, J=8.5, 14.8, 1H), 2.80 (m, 2H), 3.20 (m, 1H),
3.94 (m, 1H), 4.60, 4.90 (m, 2H), 7.0±7.4 (m, 5H); MS
(CI) m/z 322 (M+H)+.
Compound 32. To a solution of acid 33 (112.00 mg,
0.35 mmol) in THF (4 mL) were added Et3N (48.00 mL,
0.35 mmol) and Yamaguchi's reagent (54.00 mL,
0.35 mmol). The resulting reaction mixture was stirred
at room temperature for 20 min and then a solution of
compound 16 (109.00 mg, 0.28 mmol) in THF was
introduced. After being stirred at room temperature for
3 h, the reaction mixture was diluted with aqueous
NH4Cl solution and extracted with EtOAc. The com-
bined organic extracts were washed with brine, dried,
and evaporated. Puri®cation by ¯ash chromatography
(SiO2, eluent: heptane:EtOAc=10:1 then 4:1) a€orded
compound 32 (149.00 mg, 76%): [a]d= 16.0 (c 0.3,
CHCl3); IR (CHCl3) n 2945, 2860, 1735, 1454, 1293,
1
0.8, CHCl3); H NMR (250 MHz, CDCl3): d two rota-
mers 0.80±1.70 (m, 24H), 2.25 (m, 1H), 2.60±3.00 (m,
6H), 3.9±4.1 (m, 1H), 4.8±5.3 (m, 6H), 5.87 (dd, J=1.8,
15.8 Hz, 1H), 6.92 (dd, J=4.6, 15.8 Hz, 1H), 7.10±7.40
(m, 15H); 13C NMR (CDCl3) d 12.6, 14.0, 17.1, 18.7,
22.6, 25.4, 29.1, 29.3, 29.4, 29.7, 30.1, 31.7, 31.9, 37.4,
42.9, 43.2, 57.3, 66.8, 67.1, 74.4, 76.3, 122.3, 126.3,
126.7, 127.5, 127.7, 127.9, 128.2, 128.3, 128.4, 128.5,
128.8, 129.2, 135.4, 136.9, 145.8, 165.5, 169.1, 173.3; MS
(FAB) m/z 720 (M+Li)+. Anal. calcd for C43H55NO8:
C, 72.34; H, 7.77; N, 1.96 Found: C, 72.41; H, 7.72; N,
1.98.
1
8-Deoxyhapalosin (4). A solution of compound 30
(40 mg, 56.0 mmmol) in EtOAc (2 mL) and MeOH
(1 mL) was hydrogenated at 1 atm in the presence of
Pearman's catalyst. After being stirred at room tem-
perature for 1 h, the reaction mixture was ®ltered
through a short pad of Celite. The ®ltrate was evapo-
rated to give crude seco acid (28 mg): MS (CI) m/z 492
(M+1)+; IR (CHCl3) n 3600, 3468, 2937, 1731, 160,
1193 cm 1. To the solution of this crude seco acid in
DMF (56 mL) were added at 0 ꢀC, DPPA (24 mL,
114 mmol) and Hunig's base (20 mL, 114 mmol), succes-
sively. After being stirred at 0 ꢀC for 24 h, the reaction
mixture was diluted with EtOAc (200 mL) and washed
with H2O, brine successively. The organic phase was
then dried and evaporated. Puri®cation by preparative
TLC (SiO2, EtOAc:heptane=1:3) a€orded compound 4
1208, 1131, 983 cm 1; H NMR (300 MHz, CDCl3) two
rotamers d 0.80 (d, J=6.9 Hz, 3H), 0.85 (t, J=7.0 Hz,
3H), 0.90 (d, J=6.9 Hz, 3H), 1.20 (d, J=7.0 Hz, 3H),
1.25±1.60 (m, 21H), 2.20±2.90 (m, 6H), 4.05 (m, 1H),
4.40 (m, 1H), 4.95 (d, J=4.0 Hz, 1H), 5.10±5.30 (m,
4H), 7.1±7.4 (m, 10H); 13C NMR (CDCl3) d 9.6, 14.2,
17.0, 17.2, 18.9, 22.7, 25.3, 26.3, 28.4, 29.6, 30.2, 31.8,
32.0, 33.6, 38.4, 44.6, 66.9, 67.4, 72.1, 76.5, 77.0, 78.1,
80.6, 126.4, 126.8, 128.4, 128.5, 128.7, 129.7, 137.5,
137.6, 170.4, 172.5, 175.3; MS (CI) m/z 696 (M+H)+.
N-Desmethyl hapalosin (3). A solution of compound 32
(32.00 mg, 46.00 mmol) in CH2Cl2 (2 mL) and TFA
(0.50 mL) was stirred at 0 ꢀC for 2 h. The volatile was
removed and the residue, dissolved in EtOH (2.0 mL),
was hydrogenated at 1 atm in the presence of Pd/C.
After being stirred at room temperature for 2 h, the
reaction mixture was ®ltered through a short pad of
Celite. The ®ltrate was evaporated under reduced pres-
sure and the residue was redissolved in DMF (40 mL,
10 3 M). To this solution cooled at 0 ꢀC were added
DPPA (30.00 mL, 0.14 mmol) and diisopropylethylamine
(48.00 mL, 0.28 mmol) dropwise. After being stirred at
0 ꢀC for 5 h and at room temperature for 18 h, the reac-
tion mixture was diluted with EtOAc (200 mL) and
washed with water, brine successively. The organic
phase was then dried and evaporated. Puri®cation by
¯ash chromatography gave the N-desmethyl hapalosin 3
(10.5 mg, 48%): [a]d= 54 (c 0.2, CHCl3);{Lit [6],
[a]d= 66 (c 0.0061, CHCl3); Lit. 7, [a]D= 32 (c 0.5,
CHCl3)}; IR (CHCl3) n 3425, 2932, 2856, 1742, 1675,
1650, 1519, 1460, 1272, 1179, 1085, 991 cm 1; 1H NMR
(300 MHz, CDCl3) 0.66 (d, J=6.8 Hz, 3H), 0.85 (d,
J=6.6 Hz, 3H), 0.88 (t, J=6.7 Hz, 3H), 1.19 (d,
J=7.0 Hz, 3H), 1.20±1.24 (m, 10H), 1.50±1.90 (m, 3H),
2.30 (m, 1H), 2.55 (dd, J=5.7, 13.9 Hz, 1H), 2.62 (dd,
J=3.7, 13.9 Hz, 1H), 2.88±3.02 (m, 3H), 4.10 (m, 1H),
4.55 (d, J=8.0 Hz, 1H), 4.65 (m, 1H), 5.45 (d,
J=10.5 Hz, 1H), 5.52 (dt, J=3.6, 7.0 Hz, 1H), 7.1±7.4
(m, 5H); 13C NMR (CDCl3) d 9.4, 14.2, 17.8, 18.6, 22.7,
1
(12 mg, 45%): [a]d=+12.7 (c 1.4, CHCl3); H NMR
(300 MHz, CDCl3) d 0.46 (d, J=6.7 Hz, 3H), 0.65 (d,
J=6.9 Hz, 3H), 0.89 (t, J=6.5 Hz, 3H), 1.13 (d, J=7.1,
3H), 1.20±1.40 (m, 10H), 1.55±1.72 (m, 3H), 1.95 (m,
1H), 2.17 (m, 1H), 2.27 (dd, J=2.2, 12.5 Hz, 1H), 2.48
(ddd, J=2.4, 5.7, 18.5 Hz, 1H), 2.74 (d, J=7.2 Hz, 2H),
2.84 (s, 3H), 3.24 (quintet, J=7.0 Hz, 1H), 3.95 (m, 1H),
4.57 (d, J=8.3 Hz, 1H), 4.87 (ddd, J=3.0, 6.3, 16.3,
1H), 7.15±7.35 (m, 5H); 13C NMR (CDCl3) d 12.8, 14.2,
17.8, 18.7, 22.8, 25.9, 27.4, 29.4, 29.3, 29.7, 31.9, 40.4,
40.8, 55.5, 74.3, 76.1, 127.1, 128.5, 128.9, 129.2, 129.5,
169.5, 170.4, 175.3; MS (CI) m/z 474 (M+H)+; HRMS
(CI) m/z 474.3224 (C28H43NO5 +H requires 474.3220).
(4S,5R)-4-Benzyl-3-(tert-butoxycarbonyl)-oxazolidin-5-yl)-
acetic acid (33). A solution of ester 24 (182 mg,
0.52 mmol) in EtOH (4 mL) and 1 N NaOH (4 mL) was
stirred at room temperature for 10 min. The reaction
was diluted with H2O (10 mL) and the volatile was
removed under reduced pressure. The aqueous phase
was extracted with Et2O to remove any neutral species.
The aqueous phase was then acidi®ed with citric acid
and extracted with EtOAc. The combined organic
extracts were washed with brine, dried, and evaporated

B. Wagner et al. / Bioorg. Med. Chem. 7 (1999) 737±747
747
25.7, 29.2, 29.4, 29.9, 31.1, 31.9, 37.7, 39.2, 41.4, 54.2,
70.8, 76.0, 81.8, 126.9, 128.6, 129.3, 137.2, 169.9, 173.9,
174.2; MS (CI) m/z 476 (M+H)+; HRMS (CI) m/z
476.3018 (C27H42NO6 requires 476.3012).
6. (a) Dinh, T. Q.; Armstrong, R. W. J. Org. Chem. 1995, 60,
8118. (b) Dinh, T. Q.; Du, X. H.; Armstrong, R. W. J. Org.
Chem. 1996, 61, 6606.
7. Ghosh, A. K.; Liu, W.; Xu, Y.; Chen, Z. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 74.
8. (a) Ohmori, K.; Okuno, T.; Nishiyama, S.; Yamamura, S.
Tetrahedron Lett. 1996, 37, 3467. (b) Okuno, T.; Ohmori, K.;
Nishiyama, S.; Yamamura, S.; Nakamura, K.; Houk, K. N.;
Okamoto, K. Tetrahedron 1996, 52, 14723.
9. Wagner, B.; Beugelmans, R.; Zhu, J. Tetrahedron Lett.
1996, 37, 6557.
10. (a) Dinh, T. Q.; Smith, C. D.; Armstrong, R. W. J. Org.
Chem. 1997, 62, 790. (b) Dinh, T. Q.; Du, X. H.; Smith, C. D.;
Armstrong, R. W. J. Org. Chem. 1997, 62, 6773.
11. Evans, D. A.; Bartroli, J.; Shih, T. L. J. Am. Chem. Soc.
1981, 103, 2127.
Hapalosin 2. To a mixture of solvent TFA (0.5 mL) and
CH2Cl2 (0.5 mL) cooled at 0 ꢀC were added NaBH3CN
(12.60 mg, 0.20 mmol) followed by a solution of com-
pound 29 (28.00 mg, 40.00 mmol) in CH2Cl2 (1 mL).
After stirring at 0 ꢀC for 2 h, the volatile was removed
and the residue, dissolved in EtOH (2 mL), was hydro-
genated at 1 atm in the presence of Pd/C for 2 h. The
reaction mixture was ®ltered through a short pad of
Celite. The ®ltrate was evaporated under reduced pres-
sure to give the seco acid: MS (CI) m/z 508 (M+H)+.
To this crude seco acid, redissolved in DMF (40 mL,
10 3 M) and cooled at 0 ꢀC, DPPA (26.00 mL,
0.12 mmol) and diisopropylethylamine (42.00 mL,
0.24 mmol) were added dropwise. After being stirred at
0 ꢀC for 5 h and then at room temperature for 72 h, the
reaction mixture was diluted with EtOAc (200 mL) and
washed with water, brine successively. The organic
phase was then dried and evaporated. Puri®cation by
¯ash chromatography gave the hapalosin 2 (3.5 mg,
18%): [a]d= 45 (c 0.1, CH2Cl2); {Lit. 5, [a]d= 49.2
12. Koch, P.; Nakatani, Y.; Luu, B.; Ourisson, G. Bull. Soc.
Chim. Fr. 1983, 11, 189.
13. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yama-
guchi, M. Bull. Chem. Soc. Jpn. 1979, 52, 1989.
14. Newton, R. F.; Reynolds, D. P.; Webb, C. F.; Roberts, S.
M. J. Chem. Soc., Perkin Trans. 1 1981, 2055.
15. Yokomatsu, T.; Yuasa, Y.; Shibuya, S. Heterocycles 1992,
33, 1051.
16. Li, W. R.; Ewing, W. R.; Harris, B. D.; Joullie, M. M. J.
Am. Chem. Soc. 1990, 112, 7659.
17. Harris, B. D.; Joullie, M. M. Tetrahedron 1988, 44, 3489.
18. Futagawa, S.; Inui, T.; Shiba, T. Bull. Chem. Soc. Jpn.
1973, 46, 3308.
19. Kiyooka, S.-I.; Nakano, M.; Shiota, F.; Fujiyama, R. J.
Org. Chem. 1989, 54, 5409.
20. Maugras, I.; Poncet, J.; Jouin, P. Tetrahedron 1990, 46,
2807.
21. (a) Cherest, M.; Felkin, H.; Prudent, N. Tetrahedron Lett.
1968, 2199. (b) Cherest, M.; Felkin, H. Tetrahedron Lett. 1968,
2204.
22. Roux, F.; Maugras, I.; Poncet, J.; Niel, G.; Jouin, P. Tet-
rahedron 1994, 50, 5345.
(c 0.35, CH2Cl2)}; IR (CHCl3) n 3435, 2950, 1735,
1
1638 cm
;
1H NMR (300 MHz, CDCl3) 0.22 (d,
J=6.9 Hz, 3H), 0.57 (d, J=6.9 Hz, 3H), 0.90 (d,
J=7.0 Hz, 3H), 1.2±1.4 (m, 13H), 1.60±2.0 (m, 3H),
2.61 (m, 1H), 2.65 (m, 1H), 2.85 (s, 3H), 2.91 (dd,
J=17.8, 5.1 Hz, 1H), 3.22 (m, 1H), 3.41 (dd, J=13.9,
2.5 Hz, 1H), 3.70 (dt, J=10.1, 2.5 Hz, 1H), 3.85 (m,
1H), 4.30 (d, J=8.4 Hz, 1H), 5.12 (m, 1H), 7.2±7.4 (m,
5H); MS (CI) m/z 490 (M+H)+; HRMS (CI) m/z
490.3179 (C28H44NO6 requires 490.3169).
23. Houpis, I. N.; Molina, A.; Reamer, R. A.; Lynch, J. E.;
Volante, R. P.; Reider, P. J. Tetrahedron Lett. 1993, 34, 2593.
24. Freidinger, R. M.; Hinkle, J. S.; Perlow, D. S.; Arison,
B. H. J. Org. Chem. 1983, 48, 77.
25. Luke, R. W. A.; Boyce, P. G. T.; Dorling, E. K. Tetra-
hedron Lett. 1996, 37, 263.
Acknowledgements
The authors thank Professor S. Yamamura (Keio Uni-
1
versity, Japan) for kindly sending us the copies of H
NMR spectra of compounds 2 and 3 and Ms. Chris-
tiane Gaspard of this institute for performing the
bioassays. A post-doctoral fellowship from `Ministere
des a€aires Etrangeres' to B. Wagner, and a SFERE-
CONACYT doctoral fellowship funded jointly by the
Mexican and French governments to G. Islas Gonzalez
are gratefully acknowledged.
26. Paquet, A. Can. J. Chem. 1982, 60, 976.
27. Stork, G.; Takahashi, T. J. Am. Chem. Soc. 1977, 99,
1275.
28. (a) Wipf, P. Chem. Rev. 1995, 95, 2115. (b) Meng, Q.;
Hesse, M. In Topics in Current Chemistry; Springer Verlag:
Berlin, 1991; 107±176.
29. Chang, G.; Guida, W. C.; Still, W. C. J. Am. Chem. Soc.
1989, 111, 4379.
30. Ponder, J. W.; Richards, F. M. J. Comput. Chem. 1987, 8,
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References and Notes
31. Mohamadi, F.; Richards, N. J. G.; Guida, W. C.; Lis-
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32. Allinger, N. L. J. Am. Chem. Soc. 1977, 99, 8127.
33. Eliel, E. L.; Allinger, N. L.; Angyal, S. J.; Morrison, G. A.
Conformational Analysis; John Wiley & Sons: New York,
1965.
34. The most stable conformations of s-cis and s-trans amide
forms of hapalosin were obtained using the same calculation
method as metioned for 8-deoxy hapalosin.
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