A practical diastereoselective synthesis of (?)-bestatin

Article abstract of DOI:10.1002/psc.3067

Diastereoselective addition of nitromethane to Boc-D-Phe-H in the presence of sodium hydride in diethyl ether/hexane containing 15-crown-5 and subsequent N,O-protection with 2,2-dimethoxypropane gave trans-oxazolidine in a diastereomeric ratio of >16:1. T

Full text of DOI:10.1002/psc.3067

Received: 22 September 2017
DOI: 10.1002/psc.3067
Revised: 29 November 2017
Accepted: 9 January 2018
R E S E A R C H A R T I C L E
A practical diastereoselective synthesis of (−)‐bestatin
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|
Suisheng Shang Andreas V. Willems Satendra S. Chauhan
Peptides International Inc., 11621 Electron
Diastereoselective addition of nitromethane to Boc‐D‐Phe‐H in the presence of sodium
Drive, Louisville, KY 40299, USA
hydride in diethyl ether/hexane containing 15‐crown‐5 and subsequent N,O‐protection with
2,2‐dimethoxypropane gave trans‐oxazolidine in a diastereomeric ratio of >16:1. The oxazolidine
was easily separated by column chromatography, which after Nef reaction was coupled to
H‐Leu‐OtBu. The 8‐step synthesis afforded (−)‐bestatin in an overall yield of 24.7% after
deprotection and ion exchange.
Correspondence
Satendra S. Chauhan, Peptides International
Inc., 11621 Electron Drive, Louisville, KY
40299, USA.
Email: schauhan@pepnet.com
KEYWORDS
Bestatin, Nitroaldol, Nef reaction, (2S, 3R)‐3‐amino‐2‐hydroxy‐4‐phenylbutanoic acid,
diastereoselective
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1
INTRODUCTION
In our approach, we utilized nitromethane as a masked functional-
ity for the creation of a carboxylic acid via Nef reaction to generate the
protected AHPBA moiety, the core peptidomimetic component of 1.16
In this publication, we would like to report that by choosing an
appropriate base and solvents mixture, desired stereochemical
outcome of nucleophilic addition of nitromethane to Boc‐D‐Phe‐H
can be obtained even in the absence of a chelating agent. These efforts
led to an efficient diastereoselective synthesis of bestatin in overall
good yield.
Bestatin (1; Figure 1), a competitive inhibitor of aminopeptidase B, is
currently used as a drug, either alone or in combination with other anti-
biotics or anticancer drugs, for the treatment of cancer and bacterial
infections.1,2 Bestatin has also shown potential for the treatment of
HIV as well as an anti‐inflammatory drug.3,4 Due to the therapeutic
importance of bestatin, several strategies for its synthesis have been
developed.5 These syntheses have utilized chiral auxiliaries, sugars,
chiral synthons, or asymmetric catalysis to influence the chirality. Our
goal was to develop a synthesis, which was robust, scalable, and
utilized no expensive chiral catalysts or auxiliaries.
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2
MATERIALS AND METHODS
Retrosynthetic analysis of bestatin showed that it contains a (2S,
3R)‐3‐amino‐2‐hydroxy‐4‐phenylbutanoic acid (2; AHPBA) moiety
and L‐leucine. AHPBA can be conveniently synthesized from
D‐phenylalanine aldehyde (3; D‐Phe‐H), which already contains a chiral
carbon with required configuration (Figure 1).
Except amino acid derivatives, all other reagents and solvents were
procured from Sigma‐Aldrich (Milwaukee, WI) and were used without
further purification unless otherwise stated. Analytical HPLC was
performed on a C18, reversed‐phase column (YMC, 150 × 4.6 mM,
5 μ) using Shimadzu LC10 equipped with dual pumps and a diode array
detector. Buffer A consisted of 0.05% TFA in water, and buffer B was
0.05% TFA in acetonitrile. Flow rate was 1.0 mL/min. Three methods
were used: Method A: a linear gradient from 30% buffer B to 80%
buffer B in 25 minutes, method B: a linear gradient from 60% buffer
B to 100% buffer B in 20 minutes, and method C: a linear gradient
from 15% buffer B to 75% buffer B in 30 minutes. The mass spectra
were obtained using in‐house MALDI‐MS (Voyager DE™ Pro
Biospectrometry™ Workstation, Applied Biosystems, CA) and Waters
Acquity H‐Class Xevo G2 TOF UPLC‐MS System (Waters, MA)
equipped with ESI probe. The 1H and 13C NMR spectra were obtained
using a 600‐MHz Varian Unity600 NMR spectrometer in CDCl3 or
DMSO‐d6 as solvent (Emory University, Atlanta, GA). Elemental
Thus, we envisioned that a suitably protected D‐Phe‐H can be
utilized as the starting material to synthesize bestatin. A literature
survey revealed that the N‐protected D‐Phe‐H has been exploited
for the synthesis of bestatin.6-12 Furthermore, the diastereomeric
ratios of the nucleophilic addition products varied from 1:1 to 9.5:1
depending on the nucleophile and other contributing factors as shown
in Figure 2. Generally, the formation of syn isomer was favored due to
stabilization of the chelation‐controlled Cram cyclic transition state.
However, when diprotected phenylalaninal, N,N‐(Bzl)2‐Phe‐H was
used for nucleophilic addition reaction or when the reaction was
influenced by a chiral catalyst (9), the anti‐isomer predominated,
suggesting that the reaction occurred via a non‐chelated intermediate
in conformance with Felkin‐Anh model.13-15
J Pep Sci. 2018;e3067.
wileyonlinelibrary.com/journal/psc
Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.
1 of 6
https://doi.org/10.1002/psc.3067

2 of 6
SHANG ET AL.
NH2
O
NH2 O
NH2
O
OH
H
N
H
OH
OH
O
OH
1; Bestatin (Ubenimex)
2; AHPBA
3; D-Phe-H
FIGURE 1 Retrosynthesis of bestatin
H
Z
TMSCN
CH2Cl2
N
H
Bz
CN
OTMS
N
CH2=CHMgBr
OR
5; (threo:erythro = 2.5:1; Ref. 6)
(threo:erythro = 3.5:1; SnCl4)
(threo:erythro = 1:1; BF3.OEt2)
12; (syn:anti = 1.1:1; Ref. 12)
H
Pf
N
H
Z
R
R'
O
1) NaHSO3
2) NaCN
N
N
MgBr
CN
H
OH
OH
6; (syn:anti = 1:1; Ref. 7)
4
11; (syn:anti = 9.5:1; Ref. 11)
interaction contribution)
(
H
Boc
CH3NO2
8 kbar
Bn
Bn
1) Wittig
N
N
2) AD-mix alpha
OH
NO2
OH
OH
10; (syn:anti = 0.2:1; Ref. 10)
7; (syn:anti = 1.1:1; Ref. 8)
Ph
O
O
Ph
P
H
Boc
N
TMSCN
CH2Cl2
O
O
CN
OTMS
8; (syn:anti = 4:96; Ref. 9)
9
Al
FIGURE 2 Diastereomeric ratios of
nucleophilic addition reaction to N‐protected
D‐Phe‐H
Cl
24
analysis was performed by Atlantic Microlab Inc. (Norcorss, GA).
Diastereomeric excess was measured using HPLC and NMR
spectrometer. Optical rotation measurements on purified samples
were recorded using Autopol IV automatic polarimeter (Rudolph
Research Analytical, NJ).
14: RP‐HPLC, method A: tR 11.42 minutes. [α]D = +29.0 (c 0.1,
THF). Accurate mass analysis using ESI‐MS gave peak at: m/z
333.2538 (C15H22N2O5Na) [M
+ = 310.1528
Na]+; calcd. m/z
(C15H22N2O5). 1H NMR (600 MHz, CDCl3): δ 7.32 (2H, t, J = 7.4 Hz),
7.27 to 7.22 (3H, m), 4.84 (1H, d, J = 8.1 Hz), 4.48 to 4.44 (2H, m),
4.31 (1H, ddd, J = 2.2 Hz, 2.9 Hz, 6.1 Hz), 3.84 (1H, q, J = 7.7 Hz),
3.40 (1H, bs), 2.97 to 2.93 (2H, m), 1.42 (9H, s) ppm. 13C NMR
(600 MHz, CDCl3): δ 155.93, 137.19, 129.20, 128.70, 126.81, 80.28,
79.20, 68.43, 53.89, 38.13, 28.22 ppm.
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2.1.1
tert‐Butyl N‐((2R, 3R)‐3‐hydroxy‐4‐nitro‐1‐
phenylbutan‐2‐yl)‐carbamate (14)/tert‐butyl N‐((2R, 3S)‐3‐
hydroxy‐4‐nitro‐1‐phenylbutan‐2‐yl)‐carbamate (15)
27
15: RP‐HPLC, method A: tR 10.84 minutes. [α]D = +6.00 (c 0.1,
To a suspension of NaH (36.1 g, 904 mmol; 60% dispersion in mineral
oil) in hexanes (350 mL) at 0°C was added nitromethane (44.5 mL,
822 mmol) followed by addition of diethyl ether (1450 mL),
Boc‐D‐Phe‐H17,18 (102.5 g, 411 mmol), and 15‐crown‐5 (80 mL,
411 mmol) in 30‐minute intervals of each. The reaction mixture was
stirred at 0°C for 1 hour and allowed to proceed at ambient tempera-
ture overnight. Reaction was monitored by HPLC. After 22 hours,
reaction mixture was cooled to 0°C and acidified with 1 N aq. HCl
(1050 mL) to pH 4. The resulting mixture was diluted with ethyl
acetate (2000 mL), and the 2 layers were separated. The organic layer
was washed with 0.5 N aq. HCl (2 × 750 mL), brine (1 × 750 mL),
saturated aq. NaHCO3 (2 × 750 mL), and brine (2 × 750 mL). The
organic layer was dried over Na2SO4 and evaporated at reduced
pressure to afford a yellowish residue, which upon trituration with
hexane gave a precipitate. Filtration of the precipitate yielded a
mixture of 14 and 15 as slightly off‐white solid (81.7 g, 64%). A small
sample of the above mixture was purified by semi‐prep HPLC to
isolate 14 and 15 as white solids for analytical purposes.
THF). Accurate mass analysis using ESI‐MS gave peak at: m/z
349.2300 (C15H22N2O5K) [M
+ = 310.1528
K]+; calcd. m/z
(C15H22N2O5). 1H NMR (600 MHz, CDCl3): δ 7.32 (2H, t, J = 7.6 Hz),
7.26 to 7.25 (1H, m), 7.21 (2H, d, J = 7.6 Hz), 4.58 (1H, d, J = 7.0 Hz),
4.50 (1H, d, J = 12.9 Hz), 4.43 to 4.41 (1H, m), 4.31 (1H, bs), 3.90
(1H, bs), 3.01 to 2.98 (1H, m), 2.88 to 2.85 (1H, m), 1.37 (9H, s) ppm.
13C NMR (600 MHz, CDCl3): δ 156.15, 136.44, 129.29, 128.82,
127.02, 80.62, 78.48, 71.12, 54.23, 36.19, 28.18 ppm.
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2.1.2
tert‐Butyl (4R, 5R)‐4‐benzyl‐2,2‐dimethyl‐5‐
nitromethyl‐1,3‐oxazolidine‐3‐carboxylate (16)
To a solution of the mixture of 14 and 15 (81.5 g, 263 mmol) in dry
acetone (1200 mL) and 2,2‐dimethoxypropane (1200 mL) at 0°C was
added boron trifluoride etherate (3 mL) dropwise. The reaction was
stirred for 1 hour at 0°C and overnight at ambient temperature. The
HPLC showed formation of 71.6% product as
a single peak
(tR = 19.49 minutes, method A). The reaction was quenched with
triethyl amine (3.5 mL). The volatiles were removed under reduced

SHANG ET AL.
3 of 6
pressure, and the resulting viscous residue was diluted with diethyl
ether (2500 mL) and partitioned with saturated aq. NaHCO3
(1000 mL). The organic layer was separated and washed with saturated
aq. NaHCO3 (1 × 300 mL), brine (1 × 300 mL), 0.25 N HCl (2 × 300 mL),
and brine (4 × 300 mL). The organic layer was dried over Na2SO4 and
evaporated under reduced pressure to afford the crude product
(86.0 g, 93%), which was purified by silica gel column chromatography
using a gradient of ethyl acetate (0%–6%) in hexane to afford 16
reaction mixture was partitioned with ethyl acetate (2500 mL),
followed by washing with 0.25 N aq. HCl (3 × 200 mL), saturated aq.
NaHCO3 (3 × 200 mL), and brine (3 × 200 mL). The organic layer was
dried over Na2SO4 and evaporated to afford a viscous residue
(44.9 g, 89.2%), which after flash silica gel column chromatography in
hexane/ethyl acetate (0%–6%) afforded a crystalline white solid
(34.9 g, 69.4% over 2 steps). RP‐HPLC, method B: tR 12.39 minutes.
[α]D26 = −8.4 (c 0.5, CHCl3). Accurate mass analysis using ESI‐MS gave
peak at: m/z 505.4783 (C28H45N2O6) [M + H]+; calcd. m/z = 504.3199
(C28H44N2O6). 1H NMR (600 MHz, CDCl3): δ 7.27 (2H, t, J = 7.7 Hz),
7.24 to 7.19 (3H, m), 6.77 (1H, d, J = 8.1 Hz), 4.45 (2H, dd,
J = 8.1 Hz, 14.0 Hz), 4.29 (1H, d, J = 4.8 Hz), 3.15 (2H, d,
J = 10.6 Hz), 1.66 to 1.61 (3H, m), 1.60 (3H, s), 1.52 (9H, s), 1.45 (9H,
s), 1.25 (3H, bs), 0.95 (6H, d, J = 5.5 Hz) ppm. 13C NMR (600 MHz,
CDCl3): δ 171.55, 170.37, 151.64, 136.93, 130.23, 128.48, 128.37,
126.63, 96.01, 81.94, 80.34, 77.32, 60.57, 51.26, 42.13, 28.47,
27.99, 25.09, 22.55, 22.30 ppm.
23
(46.28 g, 54%) as a white solid, [α]D = +12.8 (c 0.5, CHCl3) and its
23
cis isomer (2.79 g, 3.26%), [α]D = +22 (c 0.5, CHCl3).
Accurate mass analysis using ESI‐MS gave peak at: m/z 373.2966
(C18H26N2O5Na) [M + Na]+; calcd. m/z = 350.1841 (C18H26N2O5). 1H
NMR (600 MHz, CDCl3): δ 7.35 (2H, t, J = 7.5 Hz), 7.29 to 7.24 (3H,
m), 4.65 (1H, dt, J = 4.0, 4.4 Hz), 4.34 to 4.31 (1H, m), 4.01 to 3.96
(2H, m), 3.43 to 3.41 (1H, m), 2.81 to 2.78 (1H, m), 1.64 (3H, s), 1.57
(9H, s), 1.50 (3H, s) ppm. 13C NMR (600 MHz, CDCl3): δ 151.67,
136.82, 129.30, 128.93, 127.14, 95.60, 80.80, 78.01, 75.73, 61.41,
39.05, 28.48, 27.26 ppm.
|
2.1.5
(2S)‐2‐((2S, 3R)‐3‐amino‐2‐hydroxy‐4‐
|
2.1.3
(4R, 5S)‐4‐benzyl‐3‐[(tert‐butoxy)carbonyl]‐2,2‐
phenylbutanamido)‐4‐methylpentaoic acid hydrochloride (1)
dimethyl‐1,3‐oxazolidine‐5‐carboxylic acid (17)
Compound 18 (10.0 g, 19.84 mmol) was dissolved in a mixture of
trifluoroacetic acid (190 mL) and water (10 mL) precooled to 0°C. After
2 hours, an additional amount of water (10 mL) was added. After
stirring for additional 30 minutes, the volatiles were removed under
reduced pressure. The residue was precipitated with acetonitrile/
diisopropyl ether to afford a white solid (8.0 g). The solid was dissolved
in 20% acetonitrile/water and passed through Dowex Cl− form
ion‐exchange resin and lyophilized to yield 1 (6.4 g, 99.7%) as a white
To a solution of 16 (30 g, 85.7 mmol) in methanol (750 mL) precooled
to 0°C was added a solution of KOH (14.4 g, 257 mmol) in methanol
(500 mL). Five minutes later, a solution of KMnO4 (41 g, 257 mmol)
and Na2HPO4 (36 g, 257 mmol) in water (250 mL) was added, and
the resulting mixture was stirred vigorously. After 2 hours, the reaction
was quenched by adding a slurry prepared from Na2SO3 (54 g,
343 mmol), NaCl (65 g, 1111 mmol), and 1 N HCl (900 mL, 900 mmol).
The resulting mixture was diluted with ethyl acetate (3000 mL), stirred
for 5 minutes, and filtered to remove the brown precipitate that
formed. The 2 layers were separated, and the organic layer was
washed with 0.2 N aq. HCl (2 × 300 mL) and brine (3 × 300 mL). It
was dried over Na2SO4 and evaporated under reduced pressure to
afford 17 (28.8 g, 100%) as an off‐white solid, which was 94.45% pure
25
powder. RP‐HPLC, method C: tR 7.32 minutes. [α]D = −14.2 (c 1,
1.0 N HCl). Accurate mass analysis using ESI‐MS gave peak at: m/z
309.2910 (C16H25N2O4) [M
+ = 308.3733
H]+; calcd. m/z
(C16H24N2O4). 1H NMR (600 MHz, DMSO‐d6): δ 12.69 (1H, bs), 8.21
(1H, d, J = 7.7 Hz), 8.12 (3H, bs), 7.33 (4H, bs), 7.25 (1H, bs), 6.82
(1H, bs), 4.21 to 4.19 (1H, m), 4.00 (1H, s), 3.51 (1H, s), 2.98 to 2.93
(2H, m), 1.69 to 1.64 (2H, m), 1.63 to 1.51 (1H, m), 0.88 (3H, d,
J = 6.6 Hz), 0.86 (3H, d, J = 6.6 Hz) ppm. 13C NMR (600 MHz,
DMSO‐d6): δ 173.65, 171.02, 136.57, 129.5, 128.60, 126.87, 68.22,
54.42, 50.54, 34.48, 34.33, 22.75, 21.66. Anal. Calcd for
24
by RP‐HPLC, method A: tR 14.29 minutes. [α]D = 30.0 (c 0.1, THF).
Accurate mass analysis using ESI‐MS gave peak at: m/z
336.2961(C18H26NO5) [M + H]+; calcd. m/z = 335.1732(C18H25NO5).
1H NMR (600 MHz, CDCl3): δ 7.31 (2H, t, J = 7.4 Hz), 7.29 to 7.22
(3H, m), 5.93 to 5.91 (2H, b), 4.51 (1H, d, J = 8.8 Hz), 4.39 (1H, d,
J = 2.6 Hz), 3.23 (1H, dd, J = 2.6 Hz, 13.6 Hz), 3.01 to 2.98 (1H, b),
1.62 (3H, s), 1.54 (9H, s), 1.41 (3H, s) ppm. 13C NMR (600 MHz, CDCl3):
δ 174.67, 151.59, 136.97, 129.80, 128.60, 126.83, 96.46, 80.70,
76.47, 61.13, 38.75, 28.49, 27.38 ppm.
C
16H24N2O4.HCl. 0.25H2O: C, 54.93; H, 6.87; N, 8.01; Cl, 10.16.
Found: C, 54.89; H, 7.03; N, 7.99; Cl, 10.08 ppm.
|
3
RESULTS
|
2.1.4
tert‐Butyl‐(4R, 5S)‐4‐benzyl‐5‐(((2S)‐1‐(tert‐butoxy)‐
As shown in Scheme 1, Boc‐D‐Phe‐OH was converted into
Boc‐D‐Phe‐H (13) via Weinreb amide by reacting with N,O‐
dimethylhydroxylamine followed by reduction with lithium aluminum
hydride in 76% yield (over 2 steps) according to the reported proce-
dures.17,18 The aldehyde (13) was reacted with nitromethane in the
presence of NaH in diethyl ether/hexane containing 15‐crown‐5 to
afford a mixture of nitroaldols (14/15).19,20 The mixture was not
separated because it had the tendency to precipitate on the silica gel
column due to poor solubility in ethyl acetate/hexane solvent mixture,
but carried to the next step without purification. However, in our
initial studies, we isolated small amounts of nitroaldols, 14 and 15, by
4‐methyl‐1‐oxopentan‐2‐yl)‐carbamoyl)‐2,2‐dimethyl‐1,3‐
oxazolidine‐3‐carboxylate (18)
To a solution of 17 (27.88 g, 83.22 mmol) in dry THF (560 mL) was
added N‐methylmorpholine (9.1 mL) at 0°C. After vigorous stirring
for 10 minutes at the same temperature, the mixture was cooled to
−12 2°C, and isobutyl chloroformate (10.8 mL, 83.22 mmol) was
added dropwise. After 30 minutes, a suspension of H‐Leu‐OtBu.HCl
(22.35 g, 100 mmol) in dimethyl formamide (56 mL) and
N‐methylmorpholine (9.3 mL) was added. After 90 minutes at the same
temperature, the reaction was quenched with 1 N HCl (200 mL). The

4 of 6
SHANG ET AL.
SCHEME 1 Reagents and conditions: a)
nitromethane (2 equiv.), NaH (2.2 equiv.), 15‐
crown‐5 (1.0 equiv.), Et2O/hexane (8:2), then
0.25 N aq. HCl; 64%; b) 2,2‐dimethoxypropane,
BF3.OEt2, then Et3N; c) silica gel column
chromatography, 54%; d) KMnO4 (3 equiv.),
KOH (3 equiv.), Na2HPO4 (3 equiv.), MeOH/
H2O (5:1), quant.; e) iBuOCOCl/NMM,
30 minutes, then H‐Leu‐OtBu.HCl/NMM,
DMF, 90 minutes; f) silica gel column
chromatography, 69.4% over 2 steps; g) TFA/
H2O (9:1), 99%; h) Cl− ion‐exchange resin, then
lyophilization, 99%
RP‐HPLC and carried each through the entire synthesis separately in
order to confirm the retention times and determine stereochemistry.
Bestatin derived from anti‐isomer 15 showed optical rotation of
and passed through a short Dowex ion‐exchange column to afford the
HCl salt of bestatin (1) in an overall yield of 24.7% after lyophilization.
Its purity was 99.7% as determined by RP‐HPLC, and its optical
rotation was [α]D −14.2 (c = 1, 1.0 N HCl); lit.24 [α]D −14.3
26
25
25
[α]D + 3.8 (c 1, 1 N HCl; cf. vide supra).
The mixture of nitroaldols (14/15) was treated with 2,2‐
dimethoxypropane in the presence of boron trifluoride etherate to
afford a mixture of dimethyl oxazolidines in 72% yield.21 The two
isomers, ie, trans and cis were formed resulting from syn and anti
nitroaldols, respectively, and were easily separated by silica gel column
chromatography (Rf values: trans‐isomer 16: 0.42, cis‐isomer 0.51,
solvent: 20% ethyl acetate/Hexane, visualization: ninhydrin). The
isolated yield of the desired trans‐isomer‐16 was 54%. Its cis‐isomer
was also isolated but in 3.26% yield only.
(c = 0.5, 1.0 N HCl).
|
4
DISCUSSION
As shown in Table 1, the nitroaldol reaction of Boc‐D‐Phe‐H with
nitromethane was tried using different bases, including 1,8‐
diazabicyclo[5.4.0]undec‐7‐ene (DBU) in toluene, tetramethyl guani-
dine (TMG) in toluene, sodium hydride (NaH) in THF, and NaH in
diethyl ether/hexane/15‐crown‐5. The isolated yields of nitroaldols
14/15 varied from 57% to 73% and the ratios of 14 to 15 ranged from
3.54 to 5.76 as determined by RP‐HPLC. We also tried LDA inTHF as a
base, but the yield was poor (39%).25 Because NaH in diethyl ether/
hexane/15‐crown‐5 gave the best ratio of the desired nitroaldol (14),
we were able to increase the scale of reaction to 100 g using these
conditions. The optimized reaction conditions utilized 2.2 equiv. of
NaH and 2 equiv. of nitromethane in diethyl ether/hexane (5:1)
containing 1 equiv. of 15‐crown‐5 at 0°C overnight. Under these
experimental conditions, an inseparable mixture of 14/15 was
obtained in 68% yield as an off‐white solid. The other impurities were
either volatile or easily washed off by precipitation of product with
ethyl acetate/hexane solvent mixture.
The trans‐isomer 16 was subjected to Nef reaction using a
modified procedure.22 The optimized conditions required 3 equiva-
lents each of potassium permanganate, potassium hydroxide, and
disodium hydrogen phosphate in methanol/water (5:1) for 2 hours.
Upon completion of reaction, 0.25 N aq. HCl was added to quench
the reaction, and the precipitated manganese dioxide was filtered.
Upon work up, fully protected AHPBA (17) was obtained in >95%
yield. No purification was required as the reaction was very clean.
The fully protected acid thus obtained was reacted with
H‐Leu‐OtBu.HCl using mixed anhydride method generated from the
reaction of 17 with isobutyl chloroformate and N‐methylmorpholine
(NMM). The coupling reaction was also tried using EDAC/HOBt as
activator. NMM was used as a base to neutralize the bound HCl. Both
reactions afforded quantitative yield of the coupled product (18).23
Cleavage of the fully protected bestatin (16) was performed with
90% TFA/H2O, which afforded bestatin (1) in quantitative yield as
theTFA salt. TheTFA salt of 1 was dissolved in 20% acetonitrile/water
Based on the observation of syn to anti ratios in Figure 2, addition
of a nucleophile to N‐protected D‐Phe‐H preferentially occurs via a
chelation‐controlled transition state.26 However, in the present study,
there was no chelating metal present. Perhaps, hydrogen‐bonding
TABLE 1 Reaction conditions and products of nitroaldol reactiona
Entry
Solvent
Base (Equiv.)
Temp/Time
14:15
Yieldc
1
2
3
4
5
Toluene
Toluene
THF
DBU (2)
0°C, 1 hour; rt, 12 hours
0°C, 1 hour; rt, 2 hours
0°C, 1 hour; rt, 3 hours
0°C, 1 hour; rt, 22 hours
−72‐0°C, 30 minutes
4.11:1
4.12:1
3.54:1
5.76:1
n.d.
71%
57%
73%
64%
39%
TMG (2)
NaH (2)
Et2O/Hexaneb
NaH (2.2)
LDA (1.05)
THF
aReaction performed with 2 equiv. of nitromethane.
bReaction contained 1 equiv. of 15‐crown‐5 ether.
cIsolated yield.

SHANG ET AL.
5 of 6
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19
4. Bourinbaiar A, Lee‐Huang S, Krasinski K, Borkowsky W. Inhibitory
effect of the oral immune response modifier, bestatin, on cell‐mediated
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FIGURE 3 Plausible hydrogen‐bonded transition state
5. Feske BD. Bestatin: three decades of synthetic strategies. Curr Org
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between the Boc‐NH‐ and C=O of the aldehyde group, as depicted by
19 (Figure 3) stabilizes transition state, much in the way a chelation‐
controlled transition state would operate, albeit of weaker nature.
The fact that bases DBU (pKa 12.0), TMG (pKa 13.6), and nitromethane
(pKa ~10 in water; 17.2 in DMSO) are not sufficiently strong bases to
deprotonate the carbamate (pKa ~20.8), nor Na+ is a chelator, lends
some credence to the stabilization afforded in transition state due to
H‐bonding.27 The enhanced ratio of syn to anti upon use of crown
ether in a relatively nonpolar solvent mixture further supports the
statements above (dielectric constants: THF 7.52, Et2O 4.267, toluene
2.38, hexane 1.89).28
6. Herranz R, Castro‐Pichel J, Vinuesa S, Garcia‐Lopez MT. An improved
one‐pot method for the stereoselective synthesis of the (2S,3R)‐3‐
amino‐2‐hydroxy acids: key intermediates for bestatin and amastatin.
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8. Velmourougane G, Harbut MB, Dall S, et al. Synthesis of new (−)‐
bestatin‐based inhibitor libraries reveals a novel binding mode in the
S1 pocket of the essential malaria M1 metalloaminopeptidase. J Med
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9. Manickam G, Nogami H, Kanai M, Groger H, Shibasaki M. Anti‐ and syn‐
selective cyanosilylation reactions promoted by a sugar‐based bifunc-
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for HIV protease inhibitors and bestatin. Synlett. 2001;617:
The practicality of this synthetic strategy was observed in N,O‐
acetal formation with 2,2‐dimethoxpropane in the presence of
boron trifluoride etherate leading to oxazolidine 16. It was found that
the syn‐diastereoisomer 14 reacted approximately 3 times faster
than the anti‐isomer 15 as monitored by RP‐HPLC. Thus, the
reaction mixture was further enriched in the desired stereoisomer
(dr > 16:1), which was isolated in good yield after silica gel column
chromatography. Our research corroborates the earlier observation
of enhancement in diastereoisomeric ratio upon cyclization of an
allylic acetate (12) to oxazoline from 1.1:1 (syn/anti) to >14:1 (trans/
cis) by Goerge et al.12
10. Matsumoto K, Misumi Y. Diastereoselective asymmetric nitro‐aldol
reaction of α‐aminoaldehydes under high pressure without a catalyst.
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11. Lee BW, Lee JH, Jang KC, et al. Diastereoselective synthesis of syn‐
aminoalcohols via contributing CH‐π interaction: simple synthesis of
(−)‐bestatin. Tetrahedron Lett. 2003;44:5905
12. George S, Suryavanshi GS, Sudalai A. A short enantioselective synthesis
of (−)‐bestatin via L‐proline‐catalyzed α‐amination of an aldehyde.
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13. Reetz MT. Synthesis and diastereoselective reactions of N,N‐
dibenzylamino aldehydes and related compounds. Chem Rev.
1999;99:1121
The reaction conditions of Nef reaction were modified due to
heterogeneity of the reaction mixture. A mixture of methanol water
(5:1) was used instead of tBuOH.22 Furthermore, the ratio of MeOH
to water was critical. Use of excess water led to isolation of very little
or no product.
14. Andres JM, Martinez MA, Pedrosa R, Perez‐Encabo A. Stereoselective
cyanation of chiral α‐amino aldehydes by reaction with Nagata's
reagent: a route to enantiopure β‐amino‐α‐hydroxy acids. Tetrahedron:
Asymmetry. 2001;12:347
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dibenzyl α‐amino aldehydes with nitromethane catalyzed by
enantiopure guanidines. Tetrahedron Lett. 2002;43:9401
The reaction of fully protected acid (17) with H‐Leu‐OtBu.HCl
occurred in quantitative yield using either mixed anhydride method
or EDAC/HOBt as activator. Acidolytic cleavage of all the protecting
groups followed by counter‐ion exchange afforded the final product
as HCl salt.
16. Noboro O. The Nitro Group in Organic Synthesis. New York: Wiley‐
VCH; 2001:30.
17. Nahm S, Weinreb SM. N‐methoxy‐N‐methylamides as effective
acylating agents. Tetrahedron Lett. 1981;22:3815
Thus, we have developed a convenient stereoselective synthesis
of a fully protected AHPBA (17) intermediate by utilizing nitroaldol
and Nef reactions, which after coupling with H‐Leu‐OtBu.HCl
and subsequent deprotection of the acid‐labile groups in a single
step, afforded (−)‐bestatin. The 8‐step synthesis starting from
Boc‐D‐Phe‐OH is robust, scalable, and required only 2 silica gel
column chromatography purifications.
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a rigid chiral quaternary ammonium salt: a highly
stereoselective synthesis of the HIV protease inhibitor amprenavir
(Vertex 478). Angew Chem Int Ed. 1999;38:1931
20. Sasai H, Kim W‐S, Suzuki T, et al. Diastereoselective catalytic
asymmetric nitroaldol reaction utilizing rare earth‐Li‐(R)‐BINOL
complex. A highly efficient synthesis of norstatine. Tetrahedron Lett.
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ORCID
Satendra S. Chauhan http://orcid.org/0000-0001-8890-8563

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How to cite this article: Shang S, Willems AV, Chauhan SS. A
practical diastereoselective synthesis of (−)‐bestatin. J Pep Sci.
2018;e3067. https://doi.org/10.1002/psc.3067
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reaction. Tetrahedron: Asymmetry. 2006;17:3315