Total synthesis of amamistatin A, an antiproliferative linear peptide from an actinomycete

Article abstract of DOI:10.1016/S0040-4020(00)00170-8

Amamistatin A, a linear lipopeptide and a growth inhibitor of human tumor cell lines from an actinomycete, was efficiently synthesized by a convergent approach. The asymmetric synthesis of β-hydroxy acid fragment was achieved by using chiral oxazaborolidinone mediated aldol reaction. The oxazole ring was constructed from N-acylthreonine via side-chain oxidation and cyclodehydration. The synthesis of the linear peptide was carded out in a stepwise manner from the cyclic hydroxamic acid fragment, and the final deprotection provided amamistatin A. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00170-8

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 3027±3034
Total Synthesis of Amamistatin A, an Antiproliferative Linear
Peptide from an Actinomycete
*
Fumiaki Yokokawa, Kentaro Izumi, Junko Omata and Takayuki Shioiri
*
Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
Received 18 November 1999; revised 1 February 2000; accepted 17 February 2000
AbstractÐAmamistatin A, a linear lipopeptide and a growth inhibitor of human tumor cell lines from an actinomycete, was ef®ciently
synthesized by a convergent approach. The asymmetric synthesis of b-hydroxy acid fragment was achieved by using chiral oxazaboro-
lidinone mediated aldol reaction. The oxazole ring was constructed from N-acylthreonine via side-chain oxidation and cyclodehydration. The
synthesis of the linear peptide was carried out in a stepwise manner from the cyclic hydroxamic acid fragment, and the ®nal deprotection
provided amamistatin A. q 2000 Elsevier Science Ltd. All rights reserved.
Amamistatin A (1a) was isolated, together with Amamista-
tin B (1b), by Uemura and co-workers from an actinomycete
collected on Amami Island in Kagoshima prefecture, Japan,
and its absolute stereostructure was determined by spectro-
scopic and chemical analysis as shown in Fig. 1.1 The struc-
tures of Amamistatins are closely related to those of
formobactin,2 nocobactin,3 and mycobactins,4 bacterial
siderophores. These compounds are linear lipopeptides
and consist of hydroxamic acids, oxazoles or oxazolines,
and b-hydroxy acids. Amamistatin A does not show a
cell-killing effect but shows an antiproliferative effect
against several kinds of human tumor cell lines. The IC50
values of Amamistatin A are 0.48, 0.56, and 0.24 mM
against MCF-7 breast, A-549 lung, and MKN 45 stomach
cancer cell lines, respectively. Amamistatin A is a structu-
rally and biologically attractive natural product. As a part of
our program toward the synthesis and study of biologically
active peptides,5 we have recently investigated the total
synthesis of Amamistatin A. In this paper, we wish to
describe the details of our synthetic efforts toward
Amamistatin A.
Our retrosynthetic analysis of Amamistatin A is shown in
Fig. 2. Disconnection of the ester bond and two amide bonds
gives four fragments, cyclic hydroxamic acid 2, b-hydroxy
acid 3, acyclic hydroxamic acid 6, and oxazole fragment 7.
Cyclic hydroxamic acid 2 has been already synthesized
from l-lysine by Miller and co-workers.6 The b-hydroxy
Figure 1. Amamistatin A and its related compounds.
Keywords: Amamistatin; linear peptide; total synthesis; oxazole; aldol reaction.
* Corresponding authors. Tel.: 181-52-836-3440; fax: 181-52-834-4172; e-mail: yokokawa@phar.nagoya-cu.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00170-8

3028
F. Yokokawa et al. / Tetrahedron 56 (2000) 3027±3034
Figure 2. Retrosynthetic analysis.
Scheme 1.
acid 3 can be prepared by asymmetric aldol reaction of
octanal (4) with silyl ketene acetal (5). The acyclic hydrox-
amic acid 6 can be also synthesized from d-lysine using
Miller's methodology.6 The oxazole fragment 7 can be
synthesized from the salicylic acid derivative 8 and
threonine methyl ester (9).
have positive values (Dd.0) and D values on the left side of
the plane must have negative values (Dd,0). These results
established that the absolute stereochemistry of 11 was S.
Saponi®cation of the b-hydroxy ester 11 provided the
b-hydroxy acid 3 in 93% yield, which was one partner for
the upcoming fragment condensation.
The synthesis of the acyclic hydroxamic acid fragment
commenced with the nitrone 13, which was derived from
d-lysine by Miller's methodology (Scheme 2).6 After treat-
ment of the nitrone 13 with hydroxylamine hydrochloride,
the product hydroxylamine was reacted with an excess of
formic acid by the use of 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (EDCI´HCl) to give the
N- and O-formate moiety. Subsequent removal of the
O-formate group afforded the hydroxamate 14 in 71%
yield. Reaction of 14 with [2-(trimethylsilyl)ethoxy]methyl
chloride (SEMCl) in the presence of N,N-diisopropylethyl-
amine and 4-(dimethylamino)pyridine (DMAP) in dichloro-
methane provided the SEM protected ester 15 in 92% yield.
Results and Discussion
The stereoselective synthesis of the b-hydroxy acid
fragment was achieved by the enantioselective aldol reac-
tion developed by Kiyooka (Scheme 1).7 The aldol reaction
of octanal (4) with commercially available methyl
trimethylsilyl dimethylketene acetal (5) by the use of a
stoichiometric amount of the chiral oxazaborolidinone 10
derived from d-valine in dichloromethane proceeded to give
the (S)-b-hydroxy ester 11 with 91% ee in 62% yield. We
also found that the use of tetrahydrofuran (THF) as a solvent
allowed this aldol reaction to proceed without loss of both
enantioselectivity and chemical yield (98% ee, 54% yield),
and the use of toxic dichloromethane could be avoided.
The enantiomeric purity was determined by 1H NMR
analysis in the presence of chiral shift reagent Eu(hfc)3.
The absolute con®guration of 11 was ascertained by trans-
formation into the corresponding (S)- and (R)-MTPA esters
12, and comparison of 1H NMR spectra as shown in Fig. 3.8
Due to the diamagnetic effect of the benzene ring, Dd values
(dS2dR ppm) on the right side of the MTPA plane must
Figure 3. Determination of the absolute con®guration; Dd (dS±dR) values
(ppm) obtained from 1H NMR spectral data in CDCl3.

F. Yokokawa et al. / Tetrahedron 56 (2000) 3027±3034
3029
The oxazole fragment was synthesized by the use of Wipf's
developed variant of the Robinson±Gabriel oxazole
synthesis (Scheme 3).9 Commercially available 5-methoxy-
salicylic acid (16) was reacted with an excess of benzyl
bromide in the presence of potassium carbonate, followed
by hydrolysis of the benzyl ester to afford the benzoic acid
derivative 810 in 70% yield. Coupling of the acid 8 with
l-threonine methyl ester was carried out using diethyl
phosphorocyanidate (DEPC, (EtO)2P(O)CN)11 to give the
amide 17 in 98% yield. Oxidative cyclocondensation of
the threonine residue by side-chain oxidation with Dess±
Martin periodinane12 followed by exposure to triphenyl-
phosphine (Ph3P) and hexachloroethane (Cl3CCCl3)
provided the oxazole 18 in 85% yield. Hydrogenolytic
removal of the benzyl ether from 18 gave the phenol 19
in 84% yield. Subsequent saponi®cation of 19 with NaOH
in aqueous THF afforded the oxazole fragment 7 in 85%
yield.
The construction of whole carbon skeleton for Amamistatin
A was carried out by stepwise elongation from the cyclic
hydroxamate (Scheme 4). After removal of the Cbz group
from 26 hydrogenolytically, the corresponding amine was
condensed with the b-hydroxy acid 3 using the DEPC
methodology to give the dipeptide 20 in 61% yield. After
saponi®cation of 15, coupling of the resulting acid with the
Scheme 2.
Scheme 3.
Scheme 4.

3030
F. Yokokawa et al. / Tetrahedron 56 (2000) 3027±3034
sterically hindered secondary alcohol of the dipeptide 20
required considerable optimization of yields and was
accomplished in the presence of 1.5 equiv. of N,N-dicyclo-
hexylcarbodiimide (DCC) and 1.5 equiv. of DMAP in
toluene. The desired coupling product 21 was puri®ed in
43% yield by chromatography on silica gel, and unreacted
20 was recovered in 27% yield. Yamaguchi conditions,13
Mitsunobu conditions,14 and the use of O,O-di(2-pyridyl)-
thiocarbonate15 as a coupling reagent did not lead to the
effective condensation. Subsequent hydrogenolytic removal
of the Cbz group from the ester 21 gave the corresponding
amine, which was then coupled with the oxazole fragment 7
using EDCI´HCl to give the protected Amamistatin A 22 in
47% yield. Final deprotection of the SEM and TBDPS (tert-
butyldiphenylsilyl) groups using tri¯uoroacetic acid (TFA)
in dichloromethane provided Amamistatin A (1) in 61%
yield. Our synthetic Amamistatin A was identi®ed with
the natural product by 1H NMR, 13C NMR, high-resolution
FAB mass spectra, and TLC.
thickness plates. Elementary analysis (Anal) and high
resolution mass spectra (HRMS) were done at the Analyti-
cal Facility at Nagoya City University.
Solvents for extraction and chromatography were reagent
grade. Liquid chromatography was performed with forced
¯ow (¯ash chromatography of the indicated solvent mixture
on silica gel BW-820MH or BW-200 (Fuji Davison Co.)).
Tetrahydrofuran (THF) was distilled from sodium metal/
benzophenone ketyl. Dichloromethane (CH2Cl2) was
distilled from calcium hydride. Toluene and N,N-dimethyl-
Ê
formamide (DMF) were dried over 4-A molecular sieves.
Triethylamine and N,N-diisopropylethylamine were dried
over potassium hydroxide. All other commercially obtained
reagents were used as received.
Methyl (3S)-3-hydroxy-2,2-dimethyldecanoate (11). To a
solution of N-tosyl-d-Val-OH (694.8 mg, 2.56 mmol) in
THF (25.6 ml) at room temperature under Ar, BH3±THF
(1 M in THF, 2.56 ml, 2.56 mmol) was added. The solution
was stirred at room temperature for 0.5 h. To the resulting
solution was successively added octanal (4) (0.4 ml,
2.56 mmol) and 1-(trimethylsiloxy)-1-methoxy-2-methyl-
1-propene (5) (0.572 ml, 2.82 mmol) at 2788C. After
being stirred at 2788C for 1 h, the reaction mixture was
quenched by the addition of pH 7 buffer solution. The
mixture was extracted with ether (£1), and the organic
layer was washed with saturated NaHCO3 and brine. The
organic layer was dried over MgSO4, ®ltered, and concen-
trated. The residue was puri®ed by ¯ash chromatography
(silica gel BW-820MH, hexane±EtOAcˆ10:1) to afford
the desired product 11 as a colorless oil (320 mg, 54%,
98% ee). The enantiomeric excess of 11 (11.6 mg) was
In summary, we have developed a straightforward conver-
gent strategy for the preparation of the structurally and
biologically attractive liner lipopeptide Amamistatin A.
Our synthesis of Amamistatin A has not only proved the
proposed structure, but also promised availability of this
compound in quantities. We are currently evaluating the
spectra of biological activities and iron-chelating properties
of Amamistatin A.
Experimental
General information
1
determined by H NMR analysis in the presence of chiral
shift reagent Eu(hfc)3 (6.0 mg). 11: [a]2D5ˆ221.6 (c 1.0
Melting points were measured with YANACO melting
point apparatus (hot plate) or Yamato melting point
apparatus Model MP-21 and are uncorrected. Infrared
spectra were recorded on a SHIMADZU FT IR-8100 spec-
trometer. Optical rotations were measured on a DIP-1000
digital polarimeter with a sodium lamp (lˆ589 nm, D line)
and are reported as follows: [a]TD(c g/100 ml, solvent).
neat
max
1
CHCl3); IR n
cm21 3496, 2928, 1732, 1271; H NMR
(270 MHz, CDCl3) d 0.88 (3H, t, Jˆ6.8 Hz, CH3CH2), 1.16
(3H, s, CH3), 1.18 (3H, s, CH3), 1.28±1.62 (12H, m,
CH3(CH2)6CH), 2.35 (1H, d, Jˆ16.9 Hz, OH), 3.59 (1H,
m, CH), 3.70 (3H, s, CO2CH3); 13C NMR (67.8 MHz,
CDCl3) d 14.3, 20.7, 22.4, 22.9, 27.0, 29.5, 29.8, 32.0,
32.1, 47.5, 52.1, 76.9, 178.5. Anal. Calcd for C13H26O3: C,
67.79; H, 11.38. Found: C, 67.55; H, 11.50.
1H NMR spectra were recorded on a JEOL EX-270
(270 MHz), ALPHA 500 (500 MHz), or LAMBDA
(500 MHz) spectrometer. Chemical shifts are reported in
ppm from tetramethylsilane as the internal standard. Data
are reported as follows: chemical shift, integration, multi-
plicity (sˆsinglet, dˆdoublet, tˆtriplet, qˆquartet,
brˆbroad, mˆmultiplet), coupling constants (Hz), and
assignment. Amamistatin A numbering is used for assign-
ments on all intermediates. 13C NMR spectra were recorded
on a JEOL EX-270 (67.8 MHz), ALPHA 500 (125.7 MHz),
or LAMBDA 500 (125.4 MHz) spectrometer with complete
proton decoupling. Chemical shifts are reported in ppm
from tetramethylsilane with the solvent as the internal
standard (deuterochloroform: d 77.0 ppm).
(3S)-3-Hydroxy-2,2-dimethyldecanoic acid (3). To a solu-
tion of the ester 11 (53 mg, 0.23 mmol) in methanol (2.3 ml)
at 08C, 4N aqueous NaOH (0.173 ml, 0.69 mmol) was
added at 08C. The reaction mixture was stirred at room
temperature for 1 h, and then an additional 4N aqueous
NaOH (0.345 ml, 1.38 mmol) was added. After the mixture
was stirred at room temperature for 1.5 h, an additional 4N
aqueous NaOH (0.173 ml, 0.69 mmol) was added. After
being stirred at room temperature for 1 h, the mixture was
quenched by the addition of 1N HCl and the mixture was
extracted with ether. The organic extracts were washed with
brine, dried over MgSO4, ®ltered, and concentrated. The
residue was puri®ed by ¯ash chromatography (silica gel
BW-820MH hexane±EtOAcˆ5:1) to afford the desired
product 3 as a pale yellow oil (46 mg, 93%): [a]2D6ˆ223.0
(c 1.0, CHCl3); IR nmneaaxt cm21 3500±2500, 3450, 1700, 1279;
1H NMR (270 MHz, CDCl3) d 0.88 (1H, t, Jˆ6.93 Hz,
CH3CH2), 1.19 (3H, s, CH3), 1.23 (3H, s, CH3), 1.28±1.56
(14H, m, CH3(CH2)6), 3.61 (1H, m, CHOH), 6.75 (1H, br,
Analytical thin layer chromatography was performed on
Merck Art. 5715, Kieselgel 60F254/0.25 mm thickness
plates. Visualization was accomplished with UV light,
phosphomolybdic acid, or ninhydrin solution followed by
heating. Preparative thin layer chromatography was
performed on Merck Art. 5744, Kiselgel 60F254/0.5 mm

F. Yokokawa et al. / Tetrahedron 56 (2000) 3027±3034
3031
CO2H); 13C NMR (67.8 MHz, CDCl3) d 14.0, 20.1, 22.4,
22.6, 26.6, 29.2, 29.5, 31.5, 31.8, 47.0, 76.5, 183.2. Anal.
Calcd for C12H24O3: C, 66.63; H, 11.18. Found: C, 66.65; H,
11.41.
To a solution of HCO2H (0.095 ml, 2.50 mmol) in CH2Cl2
(2 ml) was added EDCI´HCl (475 mg, 2.48 mmol) at 08C.
After the solution was stirred at 08C for 15 min, to the
resulting solution was added a solution of the hydroxyl-
amine in CH2Cl2 (4 ml). After the mixture was stirred at
08C for 50 min and then at room temperature for 1 h, an
additional HCO2H (0.095 ml, 2.50 mmol) and EDCI´HCl
(475 mg, 2.48 mmol) were added to the reaction mixture
at 08C. After stirring at 08C for 40 min, the bulk of solvent
was removed. The residue was diluted with EtOAc, then
washed with H2O (£2) and brine. The organic layer was
dried over Na2SO4, ®ltered, and concentrated.
(R)-MTPA ester ((R)-12). (R)-MTPA (216 mg, 0.92 mmol)
and oxalyl chloride (2 ml) were re¯uxed together at 808C for
6 h. After the excess oxalyl chloride was removed by
vacuum evaporation, the residue was distilled to give the
(R)-MTPA anhydride, bp 130±1558C (1 mmHg). To a solu-
tion of (R)-MTPA anhydride (55 mg, 0.122 mmol) in
CH2Cl2 (0.2 ml) at 08C was added the aldol 11 (7 mg,
0.030 mmol), DMAP (1.9 mg, 0.016 mmol) and triethyl-
amine (0.017 ml, 0.122 mmol). The mixture was stirred at
room temperature for 1 h and puri®ed by ¯ash chromato-
graphy (silica gel BW-200 hexane±EtOAcˆ20:1) to afford
The residue was treated with i-Pr2NEt (0.24 ml) in MeOH
(4 ml) at room temperature for 62 h. The resulting mixture
was concentrated and puri®ed by ¯ash chromatography
(BW-820MH, hexane±EtOAcˆ1:2±0:1) to afford the
the desired product (R)-12 as a colorless oil (17 mg, quant.);
neat
max
[a]2D5ˆ17.77 (c 1.1, CHCl3); IR n
cm21 2930, 2857,
desired product 14 as a colorless oil (149 mg, 71% in 3
steps): [a]2D6ˆ28.2 (c 1.1, CHCl3); IR n
cm21 3325,
neat
max
1744, 1497, 1468, 1393, 1256, 1188, 1169, 1123, 1082,
1019, 766; H NMR (270 MHz, CDCl3) d 0.87 (3H, t,
1
1738, 1717, 1669, 1532, 1456, 1439, 1383, 1352, 1217;
1H NMR (270 MHz, CDCl3) d 1.30±1.38 (2H, m, CH2),
1.63±1.88 (4H, m, CH2£2), 3.37 (2H, t, Jˆ6.3 Hz,
NCH2), 3.75 (3H, s, CH3 ester), 4.31±4.41 (1H, m, CH),
5.10 (2H, s, CH2Ph), 5.71 (1H, d, Jˆ7.9 Hz, NH), 7.35
(5H, s, ArH), 7.76 (1H, s, NCHO), 9.11 (1H, br, NOH);
13C NMR (67.8 MHz, CDCl3) d 21.4, 25.9, 31.6, 49.5,
52.3, 53.5, 66.9, 127.8, 128.1, 128.4, 136.1, 156.0, 157.0,
172.7. HRMS (EI) m/z Calcd for C16H22N2O6: 338.1477.
Found: 338.1489.
Jˆ6.8 Hz, CH3CH2), 1.14 (3H, s, (CH3)2C), 1.15 (3H, s,
(CH3)2C), 1.57 (12H, m, CH3(CH2)6), 3.54 (3H, d,
Jˆ1.3 Hz, OCH3), 3.63 (3H, s, CO2CH3), 5.46 (1H, dd,
Jˆ9.4 Hz, CH-OMTPA), 7.38±7.59 (5H, m, ArH); 13C
NMR (CDCl3) d 175.8, 165.9, 132.0, 129.5, 128.3, 127.5,
121.3, 80.3, 55.3, 52.0, 46.7, 31.7, 30.7, 29.3, 29.0, 26.2,
22.6, 21.5, 20.3, 14.0. Anal. Calcd for C23H33F3O5: C, 61.87;
H, 7.45. Found: C, 61.97; H, 7.56.
(S)-MTPA ester ((S)-12). (S)-MTPA (201 mg, 0.86 mmol)
and oxalyl chloride (2 ml) were re¯uxed together at 808C for
2 days. After the excess oxalyl chloride was removed by
vacuum evaporation, the residue was distilled to give the
(S)-MTPA chloride. To a solution of (S)-MTPA chloride
(0.030 ml, 0.160 mmol) in CH2Cl2 (2.5 ml) at 08C was
added aldol 11 (9.5 mg, 0.041 mmol), DMAP (2.0 mg,
0.016 mmol) and triethylamine (0.023 ml, 0.165 mmol).
The mixture was stirred at room temperature for 2 days
and puri®ed by ¯ash chromatography (silica gel BW-200
hexane±EtOAcˆ20:1) to afford the desired product (S)-12
Na-Cbz-Ne -Formyl-Ne -[[2±(trimethylsilyl)ethoxy]-
methoxy]-d-lysine methyl ester (15). To a solution of the
formate 14 (119 mg, 0.352 mmol) in CH2Cl2 (2 ml) were
added i-Pr2NEt (0.31 ml, 1.78 mmol), DMAP (2 mg,
0.016 mmol) and SEMCl (0.16 ml, 0.904 mmol) at 08C.
After being stirred at 08C for 30 min and then at room
temperature for 17 h, the mixture was diluted with ether.
The mixture was washed with 1 M aqueous KHSO4,
water, saturated aqueous NaHCO3, water, and brine. The
organic layer was dried over MgSO4, ®ltered, and concen-
trated. The residue was puri®ed by ¯ash chromatography
(silica gel BW-820MH, hexane±EtOAcˆ5:1±2:1±1:1) to
as a colorless oil (15 mg, 81%); [a]2D5ˆ232.1 (c 0.8,
cm21 2930, 2857, 1744, 1499, 1468,
neat
CHCl3); IR n
max
1
1393, 1258, 1186, 1169, 1123, 1082, 1017, 766; H NMR
(270 MHz, CDCl3) d 0.87 (3H, t, Jˆ6.8 Hz, CH3CH2), 1.16
(3H, s, (CH3)2C), 1.18 (3H, s, (CH3)2C), 1.48 (12H, m, CH3
(CH2)6), 3.54 (3H, d, Jˆ1.3 Hz, OCH3), 3.64 (3H, s,
CO2CH3), 5.44 (1H, dd, Jˆ9.4 Hz, CH-OMTPA), 7.37±
7.59 (5H, m, ArH); 13C NMR (CDCl3) d 175.9, 166.0,
131.9, 129.6, 128.3, 127.6, 121.2, 80.3, 55.3, 52.0, 46.6,
31.7, 30.3, 29.2, 29.0, 25.9, 22.6, 21.3, 20.9, 14.0. Anal.
Calcd for C23H33F3O5: C, 61.87; H, 7.45. Found: C, 62.09;
H, 7.68.
afford the desired product 15 as a colorless oil (151 mg,
92%): [a]2D5ˆ27.3 (c 1.0, CHCl3); IR n
cm21 3333,
neat
max
1725, 1678, 1530, 1456, 1437, 1354, 1250; 1H NMR
(270 MHz, CDCl3) d 0.03 (9H, s, (CH3)3Si), 0.96 (2H, dd,
Jˆ8.3, 6.9 Hz, TMSCH2CH2), 1.34±1.36 (2H, m, CH2),
1.67±1.85 (4H, m, CH2£2), 3.58 (2H, m, NCH2), 3.73
(5H, s and br CH3 ester and TMSCH2CH2), 4.37 (1H, m,
CH), 3.73 (2H, s, OCH2O), 5.10 (2H, s, CH2Ph), 5.33 (1H, d,
Jˆ7.9 Hz, NH), 7.30±7.36 (5H, m, ArH), 8.30 (1H, s,
NCHO); 13C NMR (67.8 MHz, CDCl3) d 20.38, 19.1,
23.3, 27.3, 33.0, 45.8, 53.4, 54.8, 68.0, 68.2, 99.3, 129.1,
129.2, 129.6, 137.4, 157.0, 164.6, 173.9. Anal. Calcd for
C22H36N2O7Si: C, 56.39; H, 7.74; N, 5.98. Found: C,
56.14; H, 7.77; N, 5.83.
Na-Cbz-Ne-Hydroxy-Ne-formyl-d-lysine methyl ester
(14). To
a
solution of the nitrone 136 (217 mg,
0.619 mmol) in MeOH (3 ml) at 408C was added hydroxyl-
amine hydrochloride (60 mg, 0.930 mmol). After the solu-
tion was stirred at 408C for 12 min, the solvent was
removed. The residue was dissolved in saturated aqueous
NaHCO3 (20 ml) and then extracted with CHCl3. The
combined organic layers were dried over Na2SO4, ®ltered,
and concentrated to afford the corresponding hydroxyl-
amine.
2-Benzyloxy-5-methoxybenzoic acid (8). 5-Methoxy-
salicylic acid (16) (2 g, 11.89 mmol) was dissolved in
DMF (40 ml). K2CO3 (8.22 g, 59.45 mmol) and BnBr
(4.24 ml, 35.67 mmol) were added at 08C. The solution
was stirred at 08C for 30 min and then at room temperature
for 21 h. After dilution with EtOAc, the mixture was washed

3032
F. Yokokawa et al. / Tetrahedron 56 (2000) 3027±3034
with 1 M aqueous KHSO4, water, saturated aqueous
NaHCO3, and brine. The organic layer was dried over
MgSO4, ®ltered, and concentrated. The residue was
dissolved in MeOH (38 ml) and 40% aqueous NaOH
(7 ml). The resulting mixture was stirred at room tempera-
ture for 1 h. After dilution with water, the mixture was
washed with ether. The aqueous layer was acidi®ed by the
addition of 1N aqueous HCl and salted out. The mixture
was extracted with ether (£3). The combined organic
extracts were dried over MgSO4, ®ltered, and concentrated
to afford the desired product 8 as yellow crystals (2.15 g,
product 18 as white crystals (146 mg, 0.41 mmol, 85%):
KBr
mp 98±1008C (hexane±EtOAc); IR n
cm21 2361,
max
2341, 1713, 1616, 1541, 1491, 1468, 1448; 1H NMR
(270 MHz, CDCl3) d 2.68 (3H, s, C29-CH3), 3.83 (3H, s,
OCH3), 3.95 (3H, s, CO2Me), 5.14 (2H, s, PhCH2), 6.99
(2H, m, C33 and C34-H), 7.35 (6H, m, C34-H and ArH);
13C NMR (67.8 MHz, CDCl3) d 11.9, 51.7, 55.7, 71.5,
114.2, 115.8, 116.9, 118.4, 126.8, 127.6, 128.0, 136.9,
150.9, 153.7, 156.1, 158.3, 162.8. Anal. Calcd for
C20H19NO5: C, 67.98; H, 5.42; N, 3.96. Found: C, 67.94;
H, 5.45; N, 3.89.
70%): mp 85±878C (hexane±EtOAc) (Ref. 8 mp 93±
94.58C); IR n
cm21 3034, 1728, 1620, 1589, 1502,
Methyl 2-[(2-hydroxy-5-methoxy)phenyl]-5-methyloxa-
zole-4-carboxylate (19). To a solution of 18 (70 mg,
0.20 mmol) in MeOH (1 ml) was added Pd/C (5%,
40 mg). The mixture was stirred under H2 (1 atm) at room
temperature for 2.5 h. The mixture was ®ltered and concen-
trated to afford the desired product 19 as yellow crystals
KBr
max
1
1406, 1290, 1197, 1151; H NMR (270 MHz, CDCl3) d
3.82 (3H, s, OCH3), 4.70 (1H, s, CO2H), 5.25 (2H, s,
PhCH2), 7.08 (2H, dd, Jˆ1.64, 2.97 Hz C33 and C34-H),
7.42 (5H, m, ArH), 7.70 (1H, s, C36-H); 13C NMR
(67.8 MHz, CDCl3) d 56.0, 72.7, 114.9, 116.2, 118.5,
121.6, 127.7, 128.9, 134.5, 151.4, 154.3, 165.3. Anal. Calcd
for C15H14O4: C, 69.76; H, 5.46. Found: C, 69.68; H, 5.64.
(44 mg, 0.17 mmol, 84%): mp 139±1428C (hexane±
KBr
max
EtOAc); IR n
cm21 3856, 3676, 2361, 1719, 1620,
1560, 1500, 1443, 1352, 1285, 1100, 1051; 1H NMR
(270 MHz, CDCl3) d 2.73 (3H, s, C29-CH3), 3.83 (3H, s,
OCH3), 3.93 (3H, s, CO2Me), 7.00 (3H, m, C33 and C34
and C36-H), 10.38 (1H, s, OH); 13C NMR (67.8 MHz,
CDCl3) d 11.9, 51.9, 55.9, 109.0, 109.8, 118.3, 120.2,
127.0, 151.6, 152.4, 155.0, 159.2, 162.0. HRMS (EI) m/z
Calcd for C13H13NO5: 263.0794. Found: 263.0794.
N-(2-Benzyloxy-5-methoxy)benzoyl-l-threonine methyl
ester (17). To a solution of 8 (6.47 g, 25.1 mmol) and
l-Thr-OMe´HCl (4.25 g, 25.1 mmol) in DMF (84 ml) was
successively added dropwise DEPC (3.8 ml, 25.1 mmol)
and triethylamine (10.4 ml, 75.3 mmol). After being stirred
at 08C and then at room temperature for 12 h, the reaction
mixture was diluted with EtOAc and washed with 1 M
aqueous KHSO4, water, saturated aqueous NaHCO3, and
brine. The organic layer was dried over MgSO4, ®ltered,
and concentrated. The residue was puri®ed by ¯ash chro-
matography (silica gel BW-820MH, hexane±EtOAcˆ1:1)
to afford the desired product 17 as yellow crystals (9.18 g,
98%): mp 75±778C (hexane±EtOAc); [a]2D4ˆ13.86 (c 1.17,
CHCl3); IR nKmBaxr cm21 3383, 3076, 2949, 2843, 1740, 1610,
2-[(2-Hydroxy-5-methoxy)phenyl]-5-methyloxazole-4-
carboxylic acid (7). To a solution of the methyl ester 19
(51 mg, 0.184 mmol) in THF (1.8 ml) was added 4N
aqueous NaOH (0.2 ml, 0.8 mmol) at 08C. After the mixture
was stirred at room temperature for 40 min, the additional
4N aqueous NaOH (0.2 ml, 0.8 mmol) and THF (1 ml)
were added to the reaction mixture. After being stirred at
room temperature for 30 min, the reaction mixture was
quenched by the addition of 1N HCl. The mixture was
extracted with EtOAc (£1). The organic layer was washed
with brine, dried over Na2SO4, ®ltered and concentrated.
The residue was recrystallized from EtOAc±hexane to
1
1579, 1433, 1385, 1371, 1207, 1167; H NMR (270 MHz,
CDCl3) d 1.10 (3H, d, Jˆ6.3 Hz, Thr-CH3), 3.71 (3H, s,
CO2Me), 3.84 (3H, s, CH3O), 4.26 (1H, m, N-CH-
CO2Me), 4.76 (1H, d, Jˆ5.6 Hz, CHOH), 5.18 (2H, m,
PhCH2), 7.02 (2H, s, C33 and C34-H), 7.35±7.51 (5H, m,
ArH), 7.77 (1H, s, C36-H), 8.65 (1H, d, Jˆ8.6 Hz, NH);
13C NMR (67.8 MHz, CDCl3) d 19.7, 52.1, 55.6, 57.8,
67.7, 71.7, 114.3, 115.5, 119.5, 121.7, 128.2, 128.4, 128.6,
135.6, 151.1, 153.8, 165.3, 171.2. Anal. Calcd for
C20H23NO6: C, 64.33; H, 6.21; N, 3.75. Found: C, 64.22;
H, 6.23; N, 3.74.
afford the desired product 7 as white crystals (39 mg,
KBr
max
85%): mp 190±1918C; IR n
cm21 3475, 3500±2000,
1701, 1613, 1561, 1493, 1331, 1287, 1233, 1202; 1H
NMR (270 MHz, DMSO-d6) d 2.67 (3H, s, C29-CH3), 3.76
(3H, s, CH3O), 6.97±7.06 (2H, m, C33 and C34-CH), 7.22
(1H, d, Jˆ2.6 Hz, C36-CH), 10.03 (1H, s, OH), 12.8±13.4
(1H, br, CO2H); 13C NMR (67.8 MHz, DMSO-d6) d 12.1,
55.9, 109.6, 110.4, 118.4, 120.4, 127.5, 150.8, 152.5, 155.5,
158.4, 162.6. HRMS (EI) m/z Calcd for C12H11NO5:
249.0637. Found: 249.0612.
Methyl 2-[(2-benzyloxy-5-methoxy)phenyl]-5-methyloxa-
zole-4-carboxylate (18). To a solution of 17 (208 mg,
0.56 mmol) in CH2Cl2 (2 ml) was added Dess±Martin
periodinane (354 mg, 0.84 mmol) in one portion. The
mixture was stirred at room temperature for 1 h, and
concentrated. The residue was puri®ed by ¯ash chromato-
graphy (silica gel BW-820MH, hexane±EtOAcˆ3:1) to
afford the corresponding ketone (180 mg), which was used
for the next step without further puri®cation. To a solution
of the ketone (180 mg, 0.49 mmol) in CH2Cl2 (1.6 ml) was
added Ph3P (191 mg, 0.73 mmol) and triethylamine
(0.30 ml, 2.18 mmol) under Ar at 2108C. After addition
of hexachloroethane (172 mg, 0.73 mmol), the mixture
was stirred at 2108C for 30 min. The mixture was con-
centrated, and puri®ed by ¯ash chromatography (silica gel
BW-820MH, hexane±EtOAcˆ3:1) to afford the desired
Lactam Derivative (20). To a solution of the lactam 26
(47 mg, 0.091 mmol) in MeOH (0.9 ml) was added
Pd(OH)2 on carbon (20 mg). After being stirred at room
temperature for 1.5 h under H2 (1 atm), the reaction mixture
was ®ltered and concentrated to afford the corresponding
amine. To the mixture of amine and carboxylic acid 3
(20 mg, 0.092 mmol) in DMF (0.3 ml) were successively
added DEPC (0.015 ml, 0.099 mmol) and i-Pr2NEt
(0.018 ml, 0.103 mmol) at 08C. After being stirred at 08C
for 1 h and then at room temperature for 3.5 h, the reaction
mixture was diluted with EtOAc and washed with 1 M
aqueous KHSO4, water, saturated aqueous NaHCO3, water

F. Yokokawa et al. / Tetrahedron 56 (2000) 3027±3034
3033
and brine. The organic layer was dried over Na2SO4,
®ltered, and concentrated. The residue was puri®ed by
preparative thin layer chromatography (0.5 mm thickness,
hexane±EtOAcˆ2:1) to afford the desired product 20 as a
colorless oil (32 mg, 61% in 2 steps): [a]2D4ˆ114.2 (c 1.0,
CHCl3); IR nnmeaaxt cm21 3407, 1674, 1640, 1510, 1462, 1429,
1115; 1H NMR (270 MHz, CDCl3) d 0.88 (3H, t, Jˆ7.3 Hz,
CH3(CH2)6), 1.11 (3H, s, CH3), 1.15 (9H, s, SiC(CH3)3),
1.19 (3H, s, CH3), 1.21±1.28 (12H, m, CH3(CH2)6), 1.55±
1.59 (4H, m, C4 and C5-CH2), 1.77±1.81 (2H, m, C3-CH2),
3.38±3.48 (3H, m, C6-CH2, C9-CH), 4.13±4.19 (1H, m, C2-
CH), 7.09 (1H, d, Jˆ5.9 Hz, NH), 7.33±7.48 (6H, m, ArH),
7.71±7.77 (4H, m, ArH); 13C NMR (67.8 MHz, CDCl3) d
14.1, 19.5, 20.7, 22.6, 23.6, 25.4, 26.7, 26.9, 27.3, 29.3,
29.6, 30.6, 31.7, 31.8, 45.7, 51.5, 54.2, 127.5, 127.6,
130.2, 131.6, 132.1, 136.1, 136.2, 170.1, 177.5. HRMS
(EI) m/z Calcd for C30H43N2O4Si (M12t-Bu): 523.2992.
Found: 523.2993.
under H2 (1 atm), the reaction mixture was ®ltered and
concentrated to afford the corresponding amine. To a
mixture of the amine and carboxylic acid 7 (7.6 mg,
0.030 mmol) in CH2Cl2 (0.3 ml) was added EDCI´HCl
(7 mg, 0.037 mmol) at 08C. After being stirred at room
temperature for 2 h, the reaction mixture was puri®ed by
preparative thin layer chromatography (0.5 mm thickness,
hexane±EtOAcˆ3:2) to afford the desired product 22 as a
colorless oil (16 mg, 47% in 2 steps): [a]2D5ˆ13.7 (c 0.70,
CHCl3); IR nnmeaaxt cm21 3368, 1744, 1663, 1501, 1456, 1429,
1
1364, 1248; H NMR (500 MHz, CDCl3) d 0.01 (9H, s,
(CH3)3Si), 0.82 (3H, t, Jˆ6.7 Hz, C16-CH3), 0.95 (2H, t,
Jˆ8.5 Hz, TMSCH2CH2), 1.10 (9H, s, SiC(CH3)3), 1.11
(3H, s, CH3), 1.12 (3H, s, CH3), 1.16±1.29 (12H, m, C4
and C22-CH2, CH3CH2(CH2)4), 1.42±1.58 (4H, m, and C5
and C15-CH2), 1.60±1.81 (6H, m, C3 and C10 and C23-CH2),
1.97±1.98 (2H, m, C21-CH2), 2.76 (3H, s, C29-CH3), 3.48
(2H, m, TMSCH2CH2), 3.56 (1H, m, C24-CH2), 3.64 (1H, m,
C24-CH2), 3.73 (2H, m, C6-CH2), 3.83 (3H, s, C37-OCH3),
4.17 (1H, dd, Jˆ10.0, 6.1 Hz, C20-CH), 4.75 (1H, br,
C2-CH), 4.85 (2H, s, OCH2O), 5.07 (1H, dd, Jˆ9.8,
3.1 Hz, C9-CH), 6.97 (1H, s, C33-CH), 6.98 (1H, s,
C34-CH), 7.28±7.35 (5H, m, ArH, C36-CH), 7.38±7.43
(2H, m, ArH), 7.67 (2H, d, Jˆ6.7 Hz, ArH), 7.74 (2H, dd,
Jˆ7.9, 1.2 Hz, ArH), 7.84 (2H, br, NH£2), 8.33 (1H, s,
NCHO), 9.93 (1H, s, OH); 13C NMR (125.7 MHz, CDCl3)
d 21.5, 11.6, 14.0, 18.0, 19.5, 22.0, 22.5, 22.6, 22.8,
25.1, 25.9, 26.3, 26.8, 27.3, 29.0, 29.3, 30.0, 30.5, 31.0,
31.7, 44.7, 46.1, 51.9, 52.3, 54.3, 56.0, 67.1, 79.2, 98.1,
109.3, 118.3, 120.2, 127.5, 128.8, 130.1, 130.2, 131.7,
132.0, 136.0, 136.2, 151.3, 152.5, 152.6, 158.2, 161.2,
163.7, 169.9, 174.3. Anal. Calcd for C59H87N5O12Si2:
C, 63.58; H, 7.87; N, 6.28. Found: C, 63.62; H, 8.25; N,
5.96.
Ester (21). To a solution of the methyl ester 15 (44 mg,
0.939 mmol) in THF (0.44 ml) was added 0.5N aqueous
LiOH (0.28 ml, 0.14 mmol) at 08C. After being stirred at
08C for 30 min and then at room temperature for 1 h, the
reaction mixture was quenched by the addition of 1 M
aqueous KHSO4. The mixture was extracted with EtOAc,
dried over Na2SO4, ®ltered, and concentrated to afford the
corresponding carboxylic acid, which was used for the next
step without further puri®cation.
To a solution of the carboxylic acid, lactam derivative 20
(45 mg, 0.077 mmol), and DMAP (19 mg, 0.156 mmol) in
toluene (0.5 ml) was added DCC (31 mg, 0.150 mmol).
After being stirred at room temperature for 39 h, the mixture
was concentrated. The residue was puri®ed by preparative
thin layer chromatography (0.5 mm thickness, hexane±
EtOAcˆ2:1) to afford the desired product 21 as a colorless
Amamistatin A (1a). The protected Amamistatin A 22
(17 mg, 0.015 mmol) was stirred in TFA/CH2Cl2 (0.3 ml,
1:1) at room temperature for 1 h. The mixture was concen-
trated and then puri®ed by ¯ash chromatography (silica gel
BW-820MH, hexane±acetoneˆ1:1) to afford the desired
oil (34 mg, 43%) followed by the recovered 20 (12 mg,
neat
max
27%). 21: [a]2D8ˆ114.6 (c 0.81, CHCl3); IR n
cm21
3405, 3345, 1727, 1680, 1653, 1514, 1456, 1429, 1364,
1250; 1H NMR (270 MHz, CDCl3) d 0.03 (9H, s,
(CH3)3Si), 0.85 (3H, t, Jˆ6.9 Hz, C16-CH3), 0.96 (2H, t,
Jˆ8.6 Hz, TMSCH2CH2), 1.02 (3H, s, CH3), 1.09 (3H, s,
CH3), 1.12 (9H, s, SiC(CH3)3), 1.21±1.25 (14H, m, C4 and
C22-CH2, CH3(CH2)5CH2), 1.37±1.40 (4H, m, C5 and
C23-CH2), 1.51±1.60 (2H, m, C10-CH2), 1.67±1.73 (2H,
m, C3-CH2), 1.79±1.84 (2H, m, C21-CH2), 3.38±3.44 (4H,
m, C6 and C24-CH2), 3.56±3.73 (2H, m, TMSCH2CH2),
4.03±4.09 (1H, m, C20-CH), 4.32±4.34 (1H, m, C2-CH),
4.85 (2H, s, OCH2O), 4.94±4.97 (1H, m, C9-CH), 5.11
(2H, d, Jˆ6.3 Hz, CH2Ph), 6.31 (1H, d, Jˆ9.2 Hz,
CbzNH), 7.29±7.41 (12H, m, ArH, NH), 7.67 (2H, d,
Jˆ6.6 Hz, ArH), 7.73 (2H, d, Jˆ6.6 Hz, ArH), 8.31 (1H, s,
NCHO); 13C NMR (67.8 MHz, CDCl3) d 21.5, 14.0, 18.0,
19.6, 22.3, 22.6, 22.8, 23.7, 25.3, 25.6, 26.1, 27.2, 29.0, 29.2,
30.2, 30.6, 31.5, 31.7, 44.7, 45.8, 51.6, 54.1, 54.6, 66.5, 67.1,
79.4, 98.1, 127.4, 127.5, 127.7, 127.9, 128.4, 130.1, 130.2,
131.7, 131.9, 135.9, 136.0, 156.3, 163.5, 169.5, 171.5, 173.8.
Anal. Calcd for C55H84N4O10Si2: C, 64.93; H, 8.32; N, 5.51.
Found: C, 64.89; H, 8.48; N, 5.30.
product (1) as a purple oil (7 mg, 61%): [a]2D6ˆ26.8 (c
neat
max
0.19, MeOH); IR n
cm21 3240, 1740, 1667, 1661,
1651, 1646, 1559, 1501, 1456, 1385, 1285, 1260, 1235,
1184; 1H NMR (500 MHz, CDCl3) d 0.89 (3H, t,
Jˆ7.0 Hz, C16-CH3), 1.22 (6H, s, C17 and C18-CH3), 1.25
(4H, m, C12 and C14-CH2), 1.29 (3H, m, C11 and C15-CH2),
1.33 (3H, m, C11 and C13-CH2), 1.45 (2H, m, C10 and
C22-CH2), 1.55±1.60 (4H, m, C3, C5, C10 and C22-CH2),
1.81 (3H, m, C5 and C23-CH2), 1.91±1.97 (2H, m, C3 and
C4-CH2), 1.99±2.05 (2H, m, C21-CH2), 2.76 (3H, s,
C29-CH3), 3.57 (2H, m, C24-CH2), 3.68±3.71 (1H, m,
C6-CH2), 3.86 (3H, s, C37-CH3), 3.96 (1H, dd, Jˆ15.1,
12.4 Hz, C6-CH2), 4.56 (1H, m, C2-CH), 4.70 (1H, m,
C20-CH), 5.21 (1H, d, Jˆ10.4 Hz, C9-CH), 7.01 (1H, d,
Jˆ8.9 Hz, C33-CH), 7.07 (1H, d, Jˆ8.9 Hz, C34-CH), 7.39
(1H, s, C36-CH), 8.31 (1H, s, C25-CHO); 13C NMR
(125.4 MHz, CD3OD) d 11.8, 14.4, 21.4, 22.8, 23.1, 24.0,
24.2, 27.1, 27.7, 28.7, 30.2, 30.3, 31.0, 31.5, 31.6, 31.7,
31.8, 32.9, 47.1, 47.5, 51.0, 52.9, 53.9, 54.0, 54.1, 56.4,
80.2, 80.3, 110.7, 111.5, 119.3, 121.2, 129.9, 152.1, 154.3,
154.4, 159.7, 163.5, 164.0, 170.6, 172.6, 172.8, 176.8. The
1H NMR and 13C NMR spectra were identical with the
spectra of the natural Amamistatin A provided by Professor
Protected Amamistatin A (22). To a solution of the ester
21 (31 mg, 0.031 mmol) in MeOH (0.4 ml) was added Pd/C
(5%, 31 mg). After being stirred at room temperature for 2 h

3034
F. Yokokawa et al. / Tetrahedron 56 (2000) 3027±3034
4. (a) Snow, G. A. Biochem. J. 1965, 97, 166±175. (b) Snow, G.
A. Bacteriol. Rev. 1970, 34, 99±125.
Daisuke Uemura. HRMS (FAB) m/z Calcd for C37H56N5O11
(M1H)1: 746.3981. Found: 746.4000.
5. For our recent work in this area, see (a) Yokokawa, F.; Shioiri,
T. J. Org. Chem. 1998, 63, 8638±8639. (b) Yokokawa, F.;
Fujiwara, H.; Shioiri, T. Tetrahedron Lett. 1999, 40, 1915±1916.
6. (a) Hu, J.; Miller, M. J. J. Am. Chem. Soc. 1997, 119, 3462±
3468. (b) Hu, J.; Miller, M. J. J. Org. Chem. 1994, 59, 4858±4861.
7. Kiyooka, S.-I.; Kaneko, Y.; Komura, M.; Matsuo, H.; Nakano,
M. J. Org. Chem. 1991, 56, 2276±2278.
Acknowledgements
We thank Prof. Daisuke Uemura (Nagoya University) for
providing us the sample of natural Amamistatin A and its
NMR spectra. The high resolution FAB mass spectrum of
our synthetic Amamistatin A was determined by Drs.
Daisuke Nohara and Akito Nagatsu (Nagoya City
University), to whom the authors' thanks are due. This
work was ®nancially supported in part by Grants-in-Aid
from the Ministry of Education, Science, Sports and
Culture, Japan.
8. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc. 1991, 113, 4092±4096.
9. Wipf, P.; Miller, C. P. J. Org. Chem. 1993, 58, 3604±3606.
10. Six patents. Registry no. 53985-53-8.
11. Takuma, S.; Hamada, Y.; Shioiri, T. Chem. Pharm. Bull. 1982,
30, 3147±3153 and references cited therein.
12. (a) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277±7287. (b) Ireland, R. E.; Liu, L. J. Org. Chem. 1993, 58,
2899.
References
13. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M.
Bull. Chem. Soc. Jpn 1979, 52, 1989±1993.
1. Suenaga, K.; Kokubo, S.; Shinohara, C.; Tsuji, T.; Uemura, D.
Tetrahedron Lett. 1999, 40, 1945±1948.
14. (a) Mitsunobu, O. Synthesis 1981, 1±28. (b) Hughes, D. L.
Org. React. 1992, 42, 335±656.
2. Murakami, Y.; Kato, S.; Nakajima, M.; Matsuoka, M.; Kawai,
H.; Shin-Ya, K.; Seto, H. J. Antibiot. 1996, 49, 839±845.
3. Ratledge, C.; Snow, G. A. Bio. Chem. J. 1974, 139, 407±413.
15. Saito, K.; Shiina, I.; Mukaiyama, T. Chem. Lett. 1998,
679±680.