Synthesis and biological activity of simplified belactosin C analogues

Article abstract of DOI:10.1039/c2ob25586c

Successful biochemical studies of the natural products belactosin A and C and their acylated congeners have shown a β-lactonecarboxamide moiety to be a possible core structure of powerful proteasome inhibitors. As a part of further investigations, variously decorated simplified β- lactonecarboxamides have been synthesized in order to understand structure-biological activity relations in detail, to find ways of improving their biological activity and stability and to reduce the complexity of their preparation. Biological tests showed that the best compounds possess a high potential against phytopathogenic fungi in the greenhouse. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25586c

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PAPER
Synthesis and biological activity of simplified belactosin C analogues†‡
Armin de Meijere,*a Vadim S. Korotkov,a Alexander V. Lygin,a Oleg V. Larionov,b Viktor V. Sokolov,c
Tine Graefa and Mazen Es-Sayedd
Received 19th March 2012, Accepted 13th June 2012
DOI: 10.1039/c2ob25586c
Successful biochemical studies of the natural products belactosin A and C and their acylated congeners
have shown a β-lactonecarboxamide moiety to be a possible core structure of powerful proteasome
inhibitors. As a part of further investigations, variously decorated simplified β-lactonecarboxamides have
been synthesized in order to understand structure–biological activity relations in detail, to find ways of
improving their biological activity and stability and to reduce the complexity of their preparation.
Biological tests showed that the best compounds possess a high potential against phytopathogenic fungi
in the greenhouse.
belactosin structure that were considered for modification are
marked in Fig. 1.
Introduction
Since their isolation, members of the belactosin family of protea-
some inhibitors have attracted interest from both biological and
synthetic points of view. In order to better understand their pro-
teasome inhibitory power and the important structure–activity
relationships, the prevailing interactions between such molecules
and the proteasome should be known. Towards this end, a pro-
tected homobelactosin C was initially cocrystallized with the
20S proteasome from Saccharomyces cerevisiae, and the struc-
ture of this crystal was elucidated by X-ray diffraction.1 On the
basis of this analysis, it was decided which groups in the inhibi-
tor should be modified in order to improve potentially favorable
interactions with the proteasome. With this in mind, several
belactosin C congeners bearing various protective groups have
meanwhile been synthesized, some of them cocrystallized with
the 20S proteasome, and their structures investigated.2 As those
compounds showed interesting biological activities against HeLa
cells, further modifications on the β-lactone nucleus and the side
chain were initiated in order to achieve two purposes: to increase
the biological activity of the molecules and simultaneously
decrease the complexity of their preparation. Those parts of the
The most drastic modification would be to replace the whole
dipeptide residue on the β-lactonecarboxamide by a relatively
small and easily accessible amide moiety which, at the same
time, would considerably decrease the cost of the synthesis. Of
course, any such modification should not go along without a
loss, but with an increase of biological activity. Looking at the
structure of the first cocrystallisate, a 3,5-dimethoxybenzylamido
group should be a good mimic of the dipeptide residue. There-
fore, the [2R,3S(1S)]-N-(3,5-dimethoxybenzyl)-3-(1-sec-butyl)-
4-oxooxetane-2-carboxamide (2) was targeted, and in order to
determine the influence of the sec-butyl group on the biological
activity, the analogue 3 bearing an isopropyl residue at the C-4
position of the β-lactone moiety was considered as well (Fig. 2).
As detailed investigations of the biological activities of salino-
sporamide A had shown, an additional substituent at the C-2
position of the β-lactone unit can be favorable.3 Accordingly,
simplified belactosin analogues bearing an additional methyl
group at C-2 were targeted as well. For a better understanding of
the structure–activity relationship, the β-lactonecarboxamides
both with the (2R,3S)-configuration as in the natural product as
well as the derivatives (2S,3R)-4 and 5–7 with the opposite
configuration (Fig. 2) were approached.
aInstitut für Organische und Biomolekulare Chemie der Georg-August-
Universität Göttingen, Tammannstrasse 2, D-37077 Göttingen,
Germany. E-mail: Armin.deMeijere@chemie.uni-goettingen.de
bDepartment of Chemistry, The University of Texas at San Antonio,
78249 San Antonio, USA
cDepartment of Chemistry, Saint-Petersburg State University,
Universitetskii pr. 26, 198504 Saint-Petersburg, Russia
dBayer SAS, BayerCropScience, 14 Impasse Pierre Baizet, BP 99163,
F-69263 Lyon Cedex 09, France
†Dedicated to Professor Francois Diederich on the occasion of his 60th
birthday.
‡Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob25586c
Fig. 1 Structure of belactosin C with possible modifications.
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Fig. 2 Targeted belactosin analogues.
Another worthwhile simplification of the belactosin molecule
would be the removal of the stereogenic center in the side chain
of the β-lactone moiety, and this would be achieved by intro-
ducing a 1-ethylcyclopropyl residue instead of the sec-butyl
group. This looked particularly promising as the structure of the
cocrystallized homobelactosin C1 revealed enough space for a
sterically more demanding side chain.
Since the yields of 22 and 38%, respectively, were unsatisfac-
tory, another approach to 2 was developed. Towards that, the free
malic acid 12 was prepared by a two-step hydrolysis of the
phenylthio ethyl ester 11, and the resulting 12 was regioselectively
converted to its 3,5-dimethoxybenzylamide by treatment with tri-
chloroacetic acid anhydride, then 3,5-dimethoxybenzylamine
and finally sodium hydroxide. The monoamide of the β-hydroxy
acid was eventually lactonized by treatment with benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP)
in the presence of triethylamine. The overall yield of 2 in this
sequence was 41% (Scheme 2).
Results and discussion
Synthesis of simplified belactosin analogues
The belactosin analogues 4–7 bearing a methyl group in the
second position of the β-lactone ring were synthesized from the
readily accessible (S)-3-methylpentanethioic acid S-phenyl ester
(13). After an aldol reaction with tert-butyl pyruvate with
immediately ensuing lactone ring closure of the intermediate 14,
The β-lactonecarboxamides 2 and 3 were prepared adopting the
established protocol for the condensation of the malic acid
derivatives 9 and 10 with the belactosin dipeptide fragment1 to
the condensation with 3,5-dimethoxybenzylamine, which also
occurred with immediately ensuing β-lactonization (Scheme 1).
a
mixture of all four possible diastereomeric tert-butyl
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Scheme 1 Synthesis of 4-oxooxetane-2-carboxamides 2 and 3. Reagents and conditions: (a) 3,5-Dimethoxybenzylamine, EDC, HOAt, TMP,
−30 to 0 °C.
Scheme 2 An alternative preparation of the oxooxetanecarboxamide 2. Reagents and conditions: (a) CF3CO2Ag, THF–H2O (4 : 1), 55 °C, 16 h,
89%. (b) 10% aq. HCl, dioxane (1 : 6), 60 °C, 51 h, 90%. (c) (CCl3CO)2O, dioxane, 75 °C, 3 h. (d) 3,5-Dimethoxybenzylamine, THF, 0 °C, 1 h.
(e) aq. NaOH, 16 h, r.t. (f) Benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), Et3N, CH2Cl2, 0 °C to r.t., 3 h, 51%
over 3 steps.
2-oxooxetane-4-carboxylates 15 was obtained with the diastereo-
mer (2S,3R)-15 predominating. The pure (2S,3R)-15 could be
obtained by crystallization of the mixture from hexane–diethyl
ether. The second diastereomer, (2R,3S)-15 with a trans-
orientation of the tert-butoxycarbonyl and the sec-butyl substitu-
ent on the β-lactone ring, was isolated in pure form from the
mixture by repeated column chromatography and recrystalliza-
tions. Subsequent cleavage of the tert-butyl ester groups in both
trans-isomers led to the diastereomeric acids (2S,3R)-16 and
(2R,3S)-16, which were condensed with the corresponding
amines to give the oxooxetanecarboxamides (2S,3R)-4, (2R,3S)-
4 and 5–7 (Scheme 3).
Both the cis- and trans-3-(1-ethylcyclopropyl)-2-oxooxetane-
carboxamides cis-8 and trans-8 were synthesized as racemates
from the well-established versatile building block tert-butyl
2-chloro-2-cyclopropylideneacetate (17).4 The sequence started
with a one-pot transformation involving Michael addition of
ethylmagnesium bromide and ensuing aldol reactions with ethyl
glyoxylate that led to an inseparable mixture of two diastereo-
mers of 18 (ratio 4 : 1). Then the chlorine substituent was reduc-
tively removed by treatment of 18 with ammonium formate in
the presence of palladium on charcoal to give 19 (a 4 : 1 mixture
of diastereomers). The ethyl ester was selectively hydrolyzed
with lithium hydroxide in aqueous tetrahydrofuran, and the free
carboxyl group was condensed with 3,5-dimethoxybenzylamine.
The diastereomeric carboxamides (R*,R*)- and (R*,S*)-20 could
be separated by column chromatography, and the individual
diastereomers were converted to the oxooxetanecarbox-
amides cis-8 and trans-8 by acid-catalyzed cleavage of the
tert-butoxycarbonyl groups and subsequent HATU-mediated
cyclization of the β-hydroxy acids (Scheme 4).
Biological activities of simplified belactosin analogues
Since the in vivo biological activity of a given substance in the
greenhouse depends not only on the in vitro biological activity at
the target, but also on such parameters as solubility, cell per-
meability, and others that cannot be easily taken into account, it
was decided to synthesize a library of compounds to be able to
choose the most potent ones. Following the previously expressed
hypothesis that the binding site in the proteasome is structurally
rather flexible, 200 diverse amines were condensed with the
hydroxy acid 9,5 and the activities of the corresponding amides
were determined against phytopathogenic fungi in the green-
house (Table 1). Many of these amides showed activities
comparable to that of the protected belactosine C at the target,
but significantly better activity in the greenhouse. This effect
may be related to beneficial physicochemical properties and is
more strongly affected by the amines used.6 For example, the
target activity appears to be independent of the substitution
pattern on the phenyl ring and the spacer between the phenyl
ring and the amide nitrogen. Compound 23 turned out to be by
far the best compound in the greenhouse.
The α-branched side chain in the 3-position of the lactone
appears to be important for good activity, compare e.g. the
1-methylpropyl analogue 2 with the 2-methylpropyl derivative
(not shown) (Table 2). The isopropyl group in the same position
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Scheme 3 Preparation of the 2-oxooxetanecarboxamides (2R,3S)-4, (2S,3R)-4 and 5–7. Reagents and conditions: (a) (1) LiNiPr2, THF, −78 °C, then
tert-butyl pyruvate −78 to 0 °C, 1.5 h, 36%; (2) crystallization from Et2O–hexane, 22%. (b) CF3CO2H, CH2Cl2, 16 h, 96%. (c) RCH2NH2, EDC.
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Scheme 4 Preparation of the 2-oxooxetanecarboxamides 8. Reagents and conditions: (a) EtMgBr, Et2O–THF, 0 °C to r.t., then ethyl glyoxylate
(50% in toluene), 0 °C to r.t., 15 min, 58%. (b) HCO2NH4, Pd/C, MeOH, 0 °C, 2 h, 88%. (c) LiOH, H2O–THF, r.t., 45 min, 99%. (d) 3,5-Dimethoxy-
benzylamine, EDC, EtNiPr2, DMF, CH2Cl2, −30 °C, 1 h, then r.t., 28 h. (e) (1) CF3CO2H, Et3SiH, CH2Cl2, r.t., 7 h; (2) HATU (2-(7-aza-1H-benzo-
triazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), EtNiPr2, CH2Cl2, 0 °C to r.t., 1 h, 67%.
affects the target activity slightly, but resulted in very weak
greenhouse efficacy. The gem-disubstituted spirocyclopropanated
analogues trans-8 and cis-8 are much weaker even at the target,
with only little activity for the trans-configured lactone trans-8.
Surprisingly weak were also all analogues bearing a methyl
group in the 2-position of the lactone.
developed methodologies, a first structure–activity relationship
has been established. This includes broad variation of the amine
part as well as the side chain in the 3-position and modulation of
the 2-position of the lactone ring. The target activity reacts in a
very flexible way on the variation of the amine part, but has a
strong effect on the greenhouse activity. Smaller residues likely
help to optimize the physico-chemical properties and thereby
improve the transfer of target activity to the greenhouse. Substi-
tuents in the 2-position of the lactone are not allowed, whereas the
side chain in the 3-position requires an α-branching methyl group.
Conclusion
Simplified analogues of the natural dipeptide belactosin show
very good activities against the 20S proteasome,8 which are
translated into promising fungicidal activities against some
phytopathogenic fungi in the greenhouse. This opens up possible
applications in crop protection and underlines once more the
potential of natural products9 for the identification of novel
chemical classes with a novel mode of action. Based on various
Experimental part
General remarks
1H and 13C NMR spectra were recorded at 250, 300, 600 (1H),
and 75.5, 151 MHz (13C), additional APT (attached proton test)
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Table 1 Activities of selected belactosin analogues against the 20S proteasome and phytopathogenic fungi in the greenhouse
aCompounds 21, 22 and 23 were synthesized according to the published procedure.5,7
on Bruker AM 250, Varian AMX 300 and Inova 600 instruments
for CDCl3 solutions if not otherwise specified, chemical shifts
δ in ppm, coupling constants J in Hz. IR: Bruker IFS 66 (FT-IR)
spectrometer, measured as KBr pellets or as films between KBr
plates. MS (ESI): Finnigan MAT 95 spectrometer. Optical
rotations: Perkin-Elmer 241 digital polarimeter, 1 dm cell. Start-
ing materials: CH2Cl2 was distilled from P4O10. (2R,3S,4S)-2-
Hydroxy-4-methyl-3-phenylsulfanylcarbonylhexanoic acid ethyl
ester (11) and (S)-3-methylpentanethioic acid S-phenyl ester (13)
were prepared according to de Meijere and Larionov.5 (2R,3S)-2-
Abbreviations used: BOP: benzotriazole-1-yloxytris(dimethyl-
amino)phosphonium hexafluorophosphate; HOAt: 1-hydroxy-7-
azabenzotriazole; EDC: 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide; TMP: 2,4,6-trimethylpyridine; HOBt: 1-hydroxyben-
zotriazole;
HATU:
O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate.
Ethyl
(2R,3S,4S)-2-hydroxy-4-methyl-3-carboxylhexanoate.
Silver trifluoroacetate (7.7 g, 35 mmol) was added to a solution
of the malic acid derivative 9 (5 g, 17.5 mmol) in THF (20 mL)
and H2O (5 mL). The mixture was stirred at 55 °C for 16 h. The
precipitate was filtered off and washed with THF. Then the vola-
tiles were removed from the filtrate under reduced pressure.
Water (20 mL) was added to the residue, and this mixture was
extracted with CH2Cl2. The solution was dried (Na2SO4), and
the solvent was distilled off to yield 3.0 g (89%) of the product.
Hydroxy-4-methyl-3-phenylsulfanylcarbonylpentanoic
acid
ethyl ester (10) was prepared according to Evans et al.10 tert-
Butyl pyruvate was obtained according to Carpino.11 All other
chemicals were used as commercially available. All reactions
were conducted under an atmosphere of dry nitrogen. If not
stated otherwise, organic extracts were dried over Na2SO4.
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Table 2 SAR for analogues of belactosin C with modifications in the side chain in the 3-position and substituents in the 2-position of the lactone
Compound
Side chain
Pharmacophore
pI50 (proteasome)
5.3
trans-8
cis-8
4.5
3
6.1
Not numbered
(2R,3S)-4
4.7
<4.0
(2S,3R)-4
<4.0
23
7.1
6
<4.0
5
7
4.4
4.7
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1H NMR (300 MHz): 0.92 (3 H, t, J = 7 Hz), 1.03 (3 H, d, J = 7
Hz), 1.20–1.30 (1 H, m), 1.27 (3 H, t, J = 7 Hz), 1.44–1.56
(1 H, m), 1.98–2.09 (1 H, m), 2.74 (1 H, dd, J = 8, 3 Hz),
4.17–4.29 (2 H, m), 4.38 (1 H, d, J = 3 Hz) ppm. 13C NMR
(125.7 MHz): 11.2 (CH3), 14.0 (CH3), 16.1 (CH3), 27.3 (CH2),
33.5 (CH), 53.1 (CH), 62.0 (CH2), 69.3 (CH), 173.8 (C), 178.2
(C) ppm. IR (KBr): ν˜ = 2967 cm−1, 2937, 1734, 1465, 1386,
1202, 1141, 1096, 1027. [α]2D0 = +0.0° (c 1.0, CHCl3). MS
(ESI): positive ion mode: m/z = 241 ([M + Na+], 100). Negative
ion mode: m/z = 217 ([M − H+], 100). HRMS (ESI) [M + H+]
To a solution of the crude malic acid monoamide (0.5 g,
1.5 mmol) in CH2Cl2 (40 mL) was added at 0 °C first Et3N
(0.45 g, 4.5 mmol), then BOP (0.92 g, 2.1 mmol). The cooling
bath was removed, and the reaction mixture was stirred at r.t. for
3 h. Then the solvent was distilled off in vacuo and the residue
subjected to column chromatography (eluent pentane–diethyl
ether 1 : 1) to give 2 as a colorless solid. Yield 240 mg (51%),
Rf = 0.16 [pentane–diethyl ether (1 : 1)], m.p. 104–5 °C.
1H NMR (300 MHz): δ = 0.94 (3 H, t, J = 7 Hz), 1.07 (3 H, d,
J = 7 Hz), 1.22–1.40 (1 H, m), 1.57–1.72 (1 H, m), 1.91–2.06
(1 H, m), 3.61 (1 H, dd, J = 4, 8 Hz), 3.77 (6 H, s), 4.29–4.50
(2 H, m), 4.63 (1 H, d, J = 4 Hz), 6.35–6.43 (3 H, m), 6.70–6.78
(1 H, m) ppm. 13C NMR (125.7 MHz): δ = 11.0 (CH3), 16.3
(CH3), 26.7 (CH2), 33.8 (CH), 43.4 (CH2), 55.4 (CH3), 63.0
(CH), 70.7 (CH), 99.6 (CH), 105.8 (CH), 139.3 (C), 161.1 (C),
167.8 (C), 169.1 (C) ppm. IR (KBr): ν˜ = 3302 cm−1, 2958,
1828, 1651, 1601, 1473, 1206, 1151, 915, 838. [α]2D0 = +2.2°
(c 1.0, CHCl3). MS (ESI) m/z = 366 ([M + HCOO−], 68), 320
([M − H+], 100). Calcd for C17H23NO5: C 63.54%, H 7.21%,
N 4.36%; found: C 63.34%, H 7.05%, N 4.24%.
+
calcd for C10H19O5 : 219.1233; found: 219.1227.
[2R,3S(1S)]-3-sec-Butyl-2-hydroxybutanedioic acid (12). A
solution of ethyl (2R,3S,4S)-2-hydroxy-4-methyl-3-carboxylhex-
anoate (1.6 g, 7.3 mmol) in a mixture of dioxane (95 mL) and
10% aq. HCl (15 mL) was stirred at 60 °C for 50 h. Then the
volatiles were removed under reduced pressure, and the solid
residue was recrystallized from hexane–EtOAc 1 : 2 to yield
1
1.2 g (86%) of 12, m.p. 140–1 °C. H NMR (300 MHz, [d6]
DMSO): δ = 0.84 (3 H, t, J = 7 Hz), 0.93 (3 H, d, J = 7 Hz),
1.02–1.21 (1 H, m), 1.39–1.53 (1 H, m), 1.66–1.80 (1 H, m),
2.48–2.55 (1 H, m), 4.16 (1 H, d, J = 6 Hz), 12.20–12.40 (2 H,
br m) ppm. 13C NMR (125.7 MHz, [d6] DMSO): δ = 11.2
(CH3), 116.3 (CH3), 26.7 (CH2), 33.8 (CH), 43.4 (CH2), 55.4
(CH3), 63.0 (CH), 70.7 (CH), 99.6 (CH), 105.8 (CH), 139.3 (C),
[2R,3S(1S)]-3-sec-Butyl-N-(4-tert-butylbenzyl)-4-oxooxetane-2-
carboxamide (23). Compound 23 was prepared from 4-tert-
butylbenzylamine, (2R,3S,4S)-2-hydroxy-4-methyl-3-phenylsul-
fanylcarbonylhexanoic acid (9), TMP, EDC·HCl and HOAt
adopting the established procedure for belactosin C.5 Yield:
14%. 1H NMR (300 MHz, CD3CN): δ = 0.91 (3 H, t, J = 7 Hz),
1.01 (3 H, d, J = 7 Hz), 1.29 (9 H, s), 1.50–1.65 (1 H, m),
1.84–1.97 (3 H, m), 3.66 (1 H, dd, J = 4, 8 Hz), 4.25–4.47 (2 H,
m), 4.69 (1 H, d, J = 4 Hz), 7.21 (2 H, d, J = 8 Hz), 7.38 (2 H,
d, J = 8 Hz) ppm. MS (ESI) m/z = 318 ([M + H], 100).
161.1 (C), 167.8 (C), 169.1 (C) ppm. IR (KBr): ν˜ = 2964 cm−1
,
1702, 1289, 1090, 914. [α]2D0 = −11.5° (c 1.0, CHCl3). MS
(ESI): positive ion mode: m/z = 235 ([M − H+ + 2Na+], 100),
213 ([M + Na+], 40). Negative ion mode: m/z = 189 ([M − H+],
100). Calcd for C8H14O5: C 50.52%, H 7.42%; found: C
50.35%, H 7.55%.
(2R,3S)-2-Hydroxy-4-methyl-3-phenylsulfanylcarbonylpenta-
noic acid (10). To a solution of ethyl (2R,3S)-2-hydroxy-4-
methyl-3-phenylsulfanylcarbonylpentanoate5 (2.90 g, 9.8 mmol)
in dioxane (70 mL) was added 10% aq. HCl (11.6 mL), and the
mixture was stirred at 55–60 °C for 50 h. It was then concen-
trated to dryness under reduced pressure at 45 °C, and the solid
residue was recrystallized from EtOAc (10 mL) and hexane
(30 mL) to give the acid 10 (2.05 g, 78%) as a colorless solid.
[2R,3S(1S)]-3-sec-Butyl-N-(3,5-dimethoxybenzyl)-4-oxooxetane-
2-carboxamide (2)
Method A. Compound 2 was prepared from 3,5-dimethoxy-
benzylamine (0.84 g, 5.00 mmol), (2R,3S,4S)-2-hydroxy-
4-methyl-3-phenylsulfanylcarbonylhexanoic acid (9) (1.40 g,
4.96 mmol), TMP (2.40 g, 19.83 mmol), EDC·HCl (1.90 g,
9.92 mmol) and HOAt (0.70 g, 5.15 mmol) in a mixture of
DMF (17 mL) and CH2Cl2 (66 mL) adopting the established
procedure for belactosin C.4 Yield: 366 mg (22%).
1
[α]2D0 = +11.9° (c = 0.62, CHCl3). H NMR (300 MHz): δ =
1.10 (3 H, d, J = 6.5 Hz), 1.12 (3 H, d, J = 6.6 Hz), 2.25–2.41
(1 H, m), 2.88 (1 H, dd, J = 9.4, 3.0 Hz), 4.50 (1 H, d, J =
3.0 Hz), 7.27–7.30 (2 H, m), 7.35–7.39 (3 H, m), 10.7 (1 H, br s)
ppm. 13C NMR (75.5 MHz): δ = 20.2 (CH3), 21.1 (CH3), 28.3
(CH), 61.9 (CH), 70.2 (CH), 126.5 (C), 129.3 (CH), 129.8 (CH),
134.3 (CH), 177.5 (C), 200.5 (C) ppm. IR (KBr): ν˜ =
3437 cm−1, 3020, 2085, 1676, 1215, 756. MS (ESI): m/z =
842.8 (38) [M + K+], 574.9 (100) [2M + K+], 290.9 (16)
[M + Na+]; negative ion mode: 841.0 (57) [3M + K+ − 2H+],
534.8 (100), 266.8 (9) [M − H+]. C13H16O4S (268.33) calcd
C 58.19, H 6.01; found C 57.99, H 5.79.
Method B. A mixture of [2R,3S(1S)]-3-sec-butyl-2-hydroxy-
butanedioic acid (11) (0.7 g, 3.7 mmol), trichloroacetic acid
anhydride (2.3 g, 7.4 mmol) and dioxane (2 mL) was stirred at
75 °C for 3 h. Then the volatiles were removed under reduced
pressure at r.t., the residue was dissolved in THF (10 mL) and
the solution cooled to 0 °C. 3,5-Dimethoxybenzylamine (1.4 g,
8.4 mmol) was added, and the reaction mixture was stirred first
at 0 °C for 1 h, then at r.t. for 16 h. The volatiles were removed
in vacuo, the residue was taken up in an aq. solution of NaOH
(1.5 mL, 6 N), and the mixture was stirred at r.t. for 16 h. Then
the solution was neutralized with conc. HCl, dichloromethane
(20 mL) was added, and the mixture was washed with 1 N HCl
(2 × 5 mL). The organic phase was dried (Na2SO4). The solvent
was distilled off, the residue was purified by flash chromato-
graphy on silica; by-products were removed by eluting with
pentane–diethyl ether (1 : 4), and the desired product was eluted
with MeOH. The solvent was distilled off in vacuo, and the
crude malic acid monoamide was directly used for the next step.
(2R,3S)-N-(3,5-dimethoxybenzyl)-3-isopropyl-4-oxooxetane-2-
carboxamide (3). A solution of the acid 10 (161 mg, 0.6 mmol)
in CH2Cl2 (8 mL) was added to a solution of 3,5-dimethoxy-
benzylamine (100.2 mg, 0.6 mmol) in CH2Cl2 (2 mL) at −30 °C.
HOAt (0.085 g, 0.603 mmol), TMP (0.146 g, 1.2 mmol), and
EDC (186 mg, 1.2 mmol) were then added, the reaction mixture
was stirred at −30 °C for 6 h, then at r.t. for 30 h. It was then
6370 | Org. Biomol. Chem., 2012, 10, 6363–6374
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concentrated under reduced pressure. The oily residue was
purified by column chromatography [hexane–EtOAc (2 : 1)] to
give the desired product 3 (70 mg, 38%) as a colorless solid.
Rf = 0.30 (hexane–EtOAc 2 : 1). [α]2D0 = +0.4° (c = 0.8,
(CH3), 26.3 (CH), 32.3 (CH2), 67.3 (CH), 78.9 (C), 83.6 (C),
168.5 (C), 168.9 (C) ppm. IR (KBr): 2982 cm−1, 1831, 1743,
1375, 1310, 1153, 1050, 880. MS (ESI) m/z = 506.9 (90)
[2M + Na+], 264.9 (100) [M + Na+]. HRMS (ESI) [M + Na+]
calcd for C13H22O4Na+: 265.14103; found: 265.14110.
1
CHCl3). H NMR (300 MHz): δ = 1.11 (3 H, d, J = 6.7 Hz),
1.13 (3 H, d, J = 6.7 Hz), 2.15–2.26 (1 H, m), 3.50 (1 H, dd, J =
8.3, 4.5 Hz), 3.78 (6 H, s), 4.34 (1 H, dd, J = 14.6, 5.6 Hz), 4.47
(1 H, dd, J = 14.6, 6.2 Hz), 4.63 (1 H, d, J = 4.5 Hz), 6.38 (1 H,
m), 6.42 (2 H, m), 6.75 (1 H, br s) ppm. 13C NMR (75.5 MHz):
δ = 19.4 (CH3), 20.0 (CH3), 27.9 (CH), 43.3 (CH2), 55.3 (CH3),
64.4 (CH), 71.2 (CH), 99.5 (CH), 105.8 (CH), 139.3 (C), 161.1
(C), 167.7 (C), 168.7 (C) ppm. IR (film): ν˜ = 3056 cm−1, 2963,
2882, 1838, 1611, 1521, 1465, 1386, 1206, 1153, 1066. MS
(ESI): m/z = 637.2 (85) [2M + Na+], 330.1 (100) [M + Na+].
HRMS (ESI) [M + Na+] calcd for C16H21NO5Na: 330.1312;
found: 330.1319.
[2S,3R(1S)]-3-sec-Butyl-2-methyl-4-oxooxetane-2-carboxylic
acid ((2S,3R)-16). To a cooled solution of the β-lactonecarboxy-
late (2S,3R)-15 (667 mg, 2.76 mmol) in dichloromethane
(18.7 mL) was added dropwise at 0 °C trifluoroacetic acid
(18.7 mL). The mixture was stirred at this temperature for 16 h,
then the solvents were removed in vacuo, and the residue azeo-
troped from anhydrous toluene (20 mL). After further concen-
tration, the mixture became a colorless solid, which was used
without further purification.
(2S,3R)-16. m.p. 69–70 °C, [α]2D0 = −5.6° (c = 0.7, CHCl3).
1H (CDCl3, 300 MHz): δ = 0.96 (3 H, t, J = 7.3 Hz), 0.98 (3 H,
d, J = 6.6 Hz), 1.29 (1 H, m), 1.80 (3 H, s), 1.94 (1 H, m), 2.05
tert-Butyl [2S,3R(1S)]-3-sec-Butyl-2-methyl-4-oxooxetane-2-
carboxylate ((2S,3R)-15) and tert-butyl [2R,3S(1S)]-3-sec-butyl-
(m, 1 H), 3.56 (d, J = 11.7 Hz, 1 H), 10.17 (br s, 1 H) ppm. 13
C
(CDCl3, 125 MHz): δ = 10.4 (CH3), 16.9 (CH3), 17.6 (CH3),
26.2 (CH), 31.1 (CH2), 63.5 (CH), 77.1 (C), 168.1 (C), 176.2
(C) ppm. IR (KBr): 2975 cm−1, 1828, 1734, 1467, 1381, 1309,
1194, 1101, 1042, 1016, 933, 881, 819, 733. MS (ESI) m/z =
187.1 (6) [M + H+], 393.1 (100) [2M − 2H + Na−], 371.0(50)
[2M − H−], 185.0 (21) [M − H−]. HRMS (ESI) [M + Na+]
calcd for C9H14O4Na+: 209.07843; found: 209.07851.
2-methyl-4-oxooxetane-2-carboxylate
((2R,3S)-15). n-Butyl-
lithium (2.5 M solution in hexane, 4.2 mL, 10.5 mmol) was
added dropwise at 0 °C over 5 min to a solution of diisopropyl-
amine (1.55 mL, 11.0 mmol) in THF (50 mL). After 15 min, the
ice bath was replaced with a dry ice–acetone bath (−78 °C), and
a solution of phenyl (S)-3-methylpentanethioate (13) (2.08 g,
10.0 mmol) in THF (2 mL) was added dropwise at −78 °C over
15 min. After 30 min, a pre-cooled (−78 °C) solution of tert-
butyl pyruvate (1.44 g, 10.0 mmol) in THF (15 mL) was added
dropwise within 20 min. The reaction mixture was stirred at
−78 °C for 40 min and then allowed to warm to 0 °C over 1.5 h.
A half-saturated solution of NH4Cl (50 mL) was then added, and
the resulting mixture was partitioned between water (100 mL)
and diethyl ether (100 mL). The organic phase was extracted
with a 10% solution of K2CO3 (2 × 150 mL) and brine
(100 mL), dried, filtered and concentrated to afford a yellow oil.
Column chromatography on silica gel (pentane–diethyl ether
7 : 1) gave 1.38 g (57%) of a mixture of four diastereomers as a
colorless oil (TLC – hexane–diethyl ether 7 : 1, Rf = 0.57). After
crystallization from pentane at −24 °C, 560 mg of a colorless
solid consisting predominantly of two diastereomers (according
[2R,3S(1S)]-3-sec-Butyl-2-methyl-4-oxooxetane-2-carboxylic
acid
((2S,3R)-16). [2R,3S(1S)]-3-sec-Butyl-2-methyl-4-oxo-
oxetane-2-carboxylic acid was synthesized according to the
same procedure as (2S,3R)-16.
(2R,3S)-16. m.p. 92–93 °C, [α]2D0 = −94.3° (c = 0.28, CHCl3).
1H (CDCl3, 300 MHz): δ = 0.93 (3 H, t, J = 7.3 Hz), 0.95 (3 H,
d, J = 6.6 Hz), 1.28 (1 H, m), 1.86 (3 H, s), 1.89 (1 H, m), 1.99
(1 H, m), 3.36 (1 H, d, J = 11.0 Hz), 9.58 (1 H, br s) ppm.
13C (CDCl3, 125 MHz): δ = 10.4 (CH3), 16.9 (CH3), 22.9
(CH3), 26.4 (CH), 32.6 (CH2), 68.0 (CH), 78.5 (C), 167.9 (C),
175.0 (C) ppm. IR (KBr): 2970 cm−1, 1828, 1732, 1468, 1384,
1311, 1195, 1101, 1043, 1016, 936, 880, 821, 734. MS (ESI)
m/z = 187.1 (16) [M + H+], 393.1 (100) [2M − 2H + Na−],
371.0 (55) [2M − H−], 185.0 (14) [M − H−]. HRMS (ESI)
1
+
Na+] calcd for C9H14O4Na+: 209.07843; found:
to a H NMR spectrum of this mixture) was collected. Repeated
[M
column chromatography (pentane–diethyl ether 7 : 1) and crys-
tallization of two fractions from pentane at −24 °C afforded two
diastereomers in a pure form.
209.07849.
[2S,3R(1S)]-3-sec-Butyl-N-(3,5-dimethoxybenzyl)-2-methyl-4-
oxooxetane-2-carboxamide ((2S,3R)-4). To a cooled solution of
the acid (2S,3R)-16 (89 mg, 0.48 mmol) in CH2Cl2 (4 mL), kept
at 0 °C, was added distilled water (4 mL), then HOBt × 2H2O
(328 mg, 1.92 mmol) and EDC (183 mg, 0.96 mmol). The
biphasic mixture was then stirred rapidly at 0 °C for 10 min, and
the organic phase was transferred to a cooled solution (0 °C) of
3,5-dimethoxybenzylamine (80 mg, 0.48 mmol) and TMP
(203 mg, 1.44 mmol) in CH2Cl2 (4 mL). The water phase was
extracted with CH2Cl2 (2 × 2 mL), and the combined extracts
were added to the same solution with 3,5-dimethoxybenzyl-
amine. The mixture was then further stirred at 0 °C for 2 h, and
the solvents were removed in vacuo. The product was isolated by
column chromatography (pentane–diethyl ether 5 : 1. TLC:
pentane–diethyl ether 1 : 1, Rf = 0.59) to give 75 mg (47%) of
(2S,3R)-4 as a colorless oil. [α]2D0 = −0.8° (c = 0.78, CHCl3).
Diastereomer (2S,3R)-15. m.p. 46 °C, [α]2D0 = −11.6° (c =
1
0.25, CHCl3). H (CDCl3, 300 MHz): δ = 0.95 (3 H, t, J = 7.3
Hz), 0.96 (3 H, d, J = 6.6 Hz), 1.26 (1 H, m), 1.52 (9 H, s), 1.71
(3 H, s), 1.96 (2 H, m), 3.41 (1 H, d, J = 11.7 Hz) ppm. 13C
(CDCl3, 75.5 MHz): δ = 10.4 (CH3), 16.9 (CH3), 17.8 (CH3),
26.1 (CH), 27.8 (CH3), 31.0 (CH2), 62.8 (CH), 77.9 (C), 83.2
(C), 169.1 (C), 169.7 (C) ppm. IR (KBr): 2979 cm−1, 1831,
1741, 1371, 1312, 1151, 1047, 880. MS (ESI) m/z = 506.9 (90)
[2M + Na+], 264.9 (100) [M + Na+]. HRMS (ESI) [M + Na+]
calcd for C13H22O4Na+: 265.14103; found: 265.14113.
Diastereomer (2R,3S)-15. m.p. 51–52 °C, [α]2D0 = −63.0° (c =
1.07, CHCl3). 1H (CDCl3, 300 MHz): δ = 0.92 (3 H, t, J =
7.3 Hz), 0.92 (3 H, d, J = 6.6 Hz), 1.23–1.27 (1 H, m), 1.53 (9 H,
s), 1.76 (3 H, s), 1.91 (2 H, m), 3.21 (1 H, d, J = 11.0 Hz) ppm.
13C (CDCl3, 75.5 MHz): δ = 10.4 (CH3), 17.0 (CH3), 23.2
This journal is © The Royal Society of Chemistry 2012
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1H (CDCl3, 300 MHz): δ = 0.94 (3 H, t, J = 7.3 Hz), 1.04 (3 H,
d, J = 6.2 Hz), 1.26 (1 H, m), 1.75 (3 H, s), 1.92 (1 H, m), 2.04
(1 H, m), 3.35 (1 H, d, J = 11.7 Hz), 3.78 (6 H, s), 4.32 (dd, 1
H, J = 14.6, 5.6 Hz), 4.45 (dd, 1 H, J = 14.6, 6.1 Hz), 6.40 (3 H,
m), 6.72 (1 H, br s) ppm. 13C (CDCl3, 75 MHz): δ = 10.4
(CH3), 16.9 (CH3), 18.2 (CH3), 26.1 (CH), 31.1 (CH2), 43.4
(CH), 55.3 (CH3), 63.3 (CH2), 79.5 (C), 99.6 (CH), 105.7 (CH),
139.5 (C), 161.1 (C), 168.9 (C), 170.9 (C) ppm. IR (film):
3082 cm−1, 2968, 2867, 1835, 1615, 1528, 1469, 1388, 1210,
1158, 1066, 734. MS (ESI) m/z = 693.3 (46) [2M + Na+], 432.2
(64), 358.2 (100) [M + Na+]. HRMS (ESI) [M + Na]+ calcd for
C18H25O5NNa+: 358.1625; found: 358.1629.
64). HRMS (ESI) [M + H]+ calcd for C15H20ClN2O3 :
311.11570; found: 311.11559.
+
[2S,3R(1S)]-3-sec-Butyl-N-(4-tert-butylbenzyl)-2-methyl-4-
oxooxetane-2-carboxamide (6). To a cooled solution of the acid
(2S,3R)-16 (82 mg, 0.44 mmol) in CH2Cl2 (4 mL), kept at 0 °C,
was added distilled water (4 mL), then HOBt·2H2O (302 mg,
1.76 mmol) and EDC (169 mg, 0.88 mmol). The biphasic
mixture was stirred rapidly at 0 °C for 10 min, followed by drop-
wise transfer of the organic phase to a cooled solution (0 °C) of
tert-butylbenzylamine (72 mg, 0.44 mmol) and TMP (187 mg,
1.32 mmol) in CH2Cl2 (4 mL). The mixture was then further
stirred at this temperature for 2 h, followed by removal of all sol-
vents in vacuo. The crude material was directly purified by
column chromatography (eluent pentane–diethyl ether 5 : 1) to
give 69 mg (47%) of the product 6 as a colorless solid. Rf = 0.59
(pentane–diethyl ether 1 : 1). [α]2D0 = −12.8° (c = 0.4, CHCl3).
1H NMR (CDCl3, 300 MHz): δ = 0.94 (3 H, t, J = 7.5 Hz), 1.03
(3 H, d, J = 6.5 Hz), 1.18–1.28 (1 H, m), 1.31 (s, 9 H), 1.73 (s,
3 H), 1.95–1.87 (m, 1 H), 2.05–1.95 (m, 1 H), 3.34 (d, J = 11.8
Hz, 1 H), 4.31 (1 H, dd, J = 14.5, 5.6 Hz), 4.40 (1 H, dd, J =
14.5, 5.9 Hz), 6.69 (br s, 1 H), 7.19 (d, J = 8.1 Hz, 2 H), 7.36
(d, J = 8.4 Hz, 2 H) ppm. 13C NMR (CDCl3, 75 MHz): δ =
10.35 (CH3), 16.82 (CH3), 18.20 (CH3), 26.08 (CH), 31.09
(CH2), 31.23 (CH3), 34.48 (C), 43.01 (CH2), 63.24 (CH), 79.53
(C), 125.74 (CH), 127.55 (CH), 134.05 (C), 150.84 (C), 168.97
(C), 170.80 (C) ppm. IR (film): 3339 cm−1, 2965, 1831, 1671,
1534, 1464, 1192, 1016, 865, 823. MS (DCI) m/z = 680.8
[2R,3S(1S)]-3-sec-Butyl-N-(3,5-dimethoxybenzyl)-2-methyl-4-
oxooxetane-2-carboxamide ((2R,3S)-4). The compound (2R,3S)-
4 was prepared according to the same procedure as (2S,3R)-4
starting from (2R,3S)-16 (89 mg, 0.48 mmol). Yield: 78 mg
1
(49%). [α]2D0 = −7.1° (c = 0.99, CHCl3). H (CDCl3, 300 MHz):
δ = 0.86 (3 H, t, J = 7.3 Hz), 1.04 (3 H, d, J = 6.2 Hz), 1.22 (1
H, m), 1.77 (2 H, m), 1.82 (3 H, s), 3.29 (1 H, d, J = 9.9 Hz),
3.78 (6 H, s), 4.36 (1 H, dd, J = 14.5, 5.9 Hz), 4.44 (1 H, dd, J =
14.6, 6.0 Hz), 6.41 (3 H, m), 6.86 (1 H, br s) ppm. 13C (CDCl3,
75 MHz): δ = 10.4 (CH3), 17.4 (CH3), 23.9 (CH3), 25.9 (CH),
32.9 (CH2), 43.5 (CH), 55.3 (CH3), 66.9 (CH2), 80.9 (C), 99.6
(CH), 105.8 (CH), 139.6 (C), 161.1 (C), 168.7 (C), 168.8 (C)
ppm. IR (film): 3084 cm−1, 2960, 2861, 1834, 1614, 1530,
1472, 1388, 1211, 1159, 1063, 733. MS (ESI) m/z = 693.3 (46)
[2M + Na+], 432.2 (64), 358.2 (100) [M + Na+]. HRMS (ESI)
+
+
([2M + NH4 ], 28), 663.7 ([2M + H+], 2), 349.5 ([M + NH4 ],
100), 332.4 ([M + H+], 10). HRMS (ESI) [M + H]+ calcd for
C20H30O3N: 332.22192; found: 332.22192.
[M
+
Na+] calcd for C18H25O5NNa+: 358.1625; found:
358.1628.
[2S,3R(1S)]-3-sec-Butyl-N-(2-chloropyrid-4-ylmethyl)-2-methyl-
4-oxooxetane-2-carboxamide (5). To a cooled solution of the
acid (2S,3R)-16 (100 mg, 0.54 mmol) in CH2Cl2 (5 mL) at 0 °C
was added distilled water (5 mL), then HOBt·2H2O (368 mg,
2.15 mmol) and EDC (206 mg, 1.08 mmol). The biphasic
mixture was stirred rapidly at 0 °C for 10 min, followed by drop-
wise transfer of the organic phase to a cooled solution (0 °C) of
(2-chloropyridylmethyl)amine (76 mg, 0.54 mmol) and TMP
(228 mg, 1.61 mmol) in CH2Cl2 (5 mL). The mixture was then
further stirred at this temperature for 2 h, followed by removal of
all solvents in vacuo. The crude product was directly purified by
column chromatography (pentane–diethyl ether 2 : 1) to give
120 mg (72%) of the product 5 as a colorless solid. Rf = 0.60
(diethyl ether). [α]2D0 = +8.6° (c = 0.7, CHCl3). 1H NMR
(CDCl3, 300 MHz): δ = 0.91 (3 H, t, J = 7.5 Hz), 0.98 (d, J =
6.5 Hz, 3 H), 1.30–1.13 (1 H, m), 1.69 (3 H, s), 1.90–1.80 (1 H,
m), 1.93–2.05 (1 H, m), 3.28 (1 H, d, J = 11.8 Hz), 4.34 (1 H,
dd, J = 15.0, 5.9 Hz), 4.44 (1 H, dd, J = 15.0, 6.0 Hz), 7.08 (1
H, br s), 7.28 (1 H, d, J = 8.1 Hz), 7.59 (1 H, dd, J = 8.4 Hz, J =
2.5 Hz), 8.30 (1 H, d, J = 2.4 Hz) ppm. 13C NMR (CDCl3,
75 MHz): δ = 10.33 (CH3), 16.78 (CH3), 18.12 (CH3), 26.04
(CH), 31.04 (CH3), 40.02 (CH2), 63.33 (CH), 79.45 (C), 124.33
(CH), 132.20 (C), 138.49 (CH), 149.07 (CH), 150.80 (C),
168.81 (C), 171.31 (C) ppm. IR (film): 3262 cm−1, 2971, 1824,
1670, 1541, 1463, 1387, 1200, 1108, 1036, 862, 811, 739. MS
(ESI): positive ion mode: m/z = 311.1 (100) [M + H+]. Negative
ion mode: m/z = 309.1 ([M − H], 100), 355.0 ([M + HCOO−],
[2S,3R(1S)]-3-sec-Butyl-N-(chroman-2-ylmethyl)-2-methyl-4-
oxooxetane-2-carboxamide (7). To a cooled solution of the acid
(2S,3R)-16 (100 mg, 0.54 mmol) in CH2Cl2 (5 mL) at 0 °C was
added distilled water (5 mL), then HOBt·2H2O (368 mg,
2.15 mmol) and EDC (206 mg, 1.08 mmol). The biphasic
mixture was stirred rapidly at 0 °C for 10 min, followed by drop-
wise transfer of the organic phase to a cooled solution (0 °C) of
(chroman-2-ylmethyl)amine (88 mg, 0.54 mmol) and TMP
(228 mg, 1.61 mmol) in CH2Cl2 (5 mL). The mixture was then
further stirred at this temperature for 2 h, followed by removal of
all solvents in vacuo. The crude material was directly purified by
column chromatography (pentane–diethyl ether 2 : 1. TLC:
diethyl ether, Rf = 0.78) to give 144 mg (80%) of the product 7
as a colorless oil. [α]2D0 = −15.3° (c = 1, CHCl3). 1H NMR
(CDCl3, 300 MHz): δ = 0.94 (3 H, t, J = 7.5 Hz), 1.02 (3 H, d,
J = 6.4 Hz), 1.30–1.13 (2 H, m), 1.73 (3 H, s), 1.85–2.05 (3 H,
m), 2.94–2.72 (2 H, m), 3.31–3.51 (1 H, m), 3.34 (1 H, d, J =
11.7 Hz), 3.83–3.67 (1 H, m), 4.05–4.15 (1 H, m), 6.81 (1 H, d,
J = 7.9 Hz), 6.87 (1 H, d, J = 7.2 Hz), 6.89 (1 H, br s), 7.04 (1
H, d, J = 7.2 Hz), 7.09 (1 H, t, J = 7.2 Hz) ppm. 13C NMR
(CDCl3, 75 MHz): δ = 10.35 (2 CH3), 16.83 (CH3), 16.84
(CH3), 18.22 (CH3), 18.26 (CH3), 24.27 (CH2), 24.31 (CH2),
24.84 (CH2), 25.04 (CH2), 26.10 (CH), 26.11 (CH), 31.08
(CH2), 31.10 (CH2), 43.25 (CH2), 43.39 (CH2), 63.32 (CH),
63.34 (CH), 74.23 (CH), 74.36 (CH), 79.49 (C), 79.52 (C),
116.71 (CH), 116.76 (CH), 120.61 (CH), 120.63 (CH), 121.48
(C), 121.51 (C), 127.35 (2 CH), 129.48 (CH), 129.50 (CH),
6372 | Org. Biomol. Chem., 2012, 10, 6363–6374
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154.02 (C), 154.08 (C), 168.92 (C), 168.93 (C), 171.27 (C),
171.36 (C) ppm. IR (film): 3351 cm−1, 2965, 1830, 1677, 1583,
1534, 1489, 1458, 1384, 1234, 1108, 1033, 817, 756. MS (ESI):
positive ion mode: m/z = 386.3 ([M − H + 2Na+], 100), 354.2
([M + Na+], 74). Negative ion mode: m/z = 330.3 ([M − H],
100). HRMS (ESI) [M + Na]+ calcd for C19H25O4NNa+:
354.16758; found: 354.16750.
171.75 (C), 173.28 (C), 178.58 (C) ppm. MS (DCI) m/z = 304
([M + NH4 ], 100).
+
3-(tert-Butoxycarbonyl)-3-(1-ethylcyclopropyl)-2-hydroxy-
propionic acid. A solution of LiOH (154 mg, 6.4 mmol) in H2O
(10 mL) was added to a solution of 19 (920 mg, 3.21 mmol) in
THF (15 mL) at r.t. Within 45 min the reaction was complete.
Then H2O (20 mL) was added, and the THF was removed from
the reaction mixture under reduced pressure. The residue was
washed with Et2O, acidified with 1 N KHSO4, extracted with
diethyl ether, washed with saturated NaCl solution and dried
over MgSO4. The volatiles were removed under reduced pressure
to give 820 mg (99%) of the title compound as a colorless oil.
1H NMR (250 MHz): 0.41–0.50 (3 H, m), 0.60–0.68 (1 H, m),
0.75–0.85 (3 H, m), 1.41–1.42 (9 H, m), 1.40–1.50 (1 H, m),
1.60–1.67 (1 H, m), 2.38–2.53 (1 H, m), 4.41–4.63 (1 H, m),
4.18–4.30 (2 H, m), 4.39 (0.2 H), 4.58 (0.8 H) ppm. MS (DCI)
Ethyl 3-(tert-butoxycarbonyl)-3-chloro-3-(1-ethylcyclopropyl)-
2-hydroxypropionate (18). Ethylmagnesium bromide (2.8 mL,
6.9 mmol, 2.45 M solution in diethyl ether) was added at 0 °C
over 10 min to a solution of tert-butyl 2-chlorocyclopropylide-
neacetate4c (17) (1.44 g, 6.9 mmol) in THF. This mixture was
stirred at 0 °C for 5 min and then allowed to warm up to r.t. It
was cooled to 0 °C again, and a solution of ethyl glyoxylate
(1.41 g, 50% in toluene, 6.9 mmol) in THF (10 mL) was added.
The reaction mixture was stirred at 0 °C for 5 min and then at r.t.
for 10 min. It was cooled to 0 °C again, and the reaction was
quenched by addition of saturated NH4Cl. The precipitate was
filtered off, the phases were separated, and the aqueous phase
was extracted with diethyl ether. The combined organic phases
were dried over MgSO4. The volatiles were removed under
reduced pressure, and the residue was purified by column chrom-
atography (diethyl ether–hexane 1 : 3) to give the product 18 as a
colorless oil. According to its NMR spectrum, it was a
4 : 1 mixture of two diastereomers. Yield (1.29 g, 58%), Rf =
0.27 (diethyl ether–hexane 1 : 3). 1H NMR (250 MHz):
0.35–0.45 (1 H, m), 0.52–0.57 (1 H, m), 0.74 (3 H, t, J = 7 Hz),
0.80–0.84 (1 H, m), 1.03–1.06 (1 H, m), 1.26 (3 H, d, J = 7 Hz),
1.51 (9 H, s), 1.79–1.84 (2 H, m), 3.22 (0.8 H, d, J = 7 Hz),
3.67 (0.2 H, d, J = 10 Hz), 4.21–4.29 (2 H, m), 4.87 (0.2 H, d,
J = 10 Hz), 4.98 (0.8 H, d, J = 7 Hz) ppm. 13C NMR
(62.5 MHz): 5.86 (CH2), 7.69 (CH2), 9.90 (CH2), 9.99 (CH3),
14.04 (CH3), 23.95 (CH2), 25.81 (C), 27.78 (CH3), 62.53 (CH2),
74.23 (CH), 80.27 (C), 83.13 (C), 166.70 (C), 171.43 (C) ppm.
+
m/z = 276 ([M + NH4 ], 100).
3-(tert-Butoxycarbonyl)-N-(3,5-dimethoxybenzyl)-3-(1-ethyl-
cyclopropyl)-2-hydroxypropionamide (20). HOAt (380 mg,
2.79 mmol) was added at −30 °C to a solution of 3-(tert-butyl-
oxycarbonyl)-3-(1-ethylcyclopropyl)-2-hydroxypropionic acid
(720 mg, 2.79 mmol) in CH2Cl2 (30 mL) followed by DMF
(5 mL), EDC (660 mg, 4.25 mmol) and a solution of 3,5-
dimethoxybenzylamine (500 mg, 2.99 mmol) as well as ethyl-
diisopropylamine (550 mg, 4.25 mmol) in CH2Cl2 (10 mL). The
reaction mixture was stirred at a temperature from −30 to
−20 °C for 5 h, and then at r.t. for 28 h. CH2Cl2 (60 mL) was
added to this mixture, and it was subsequently washed with 1 M
aq. KHSO4, H2O, aq. NaHCO3, aq. NaCl solution and dried
over MgSO4. The volatiles were removed under reduced
pressure, and the residue was purified by column chromato-
graphy (eluent diethyl ether–hexane 4 : 1) to give 1.13 g (99%)
of a mixture of two diastereomers that were separated by
repeated column chromatography (eluent diethyl ether–hexane
4 : 1).
+
+
MS (DCI) m/z = 338 ([M + NH4 ], 100)/340 ([M + NH4 ], 35).
Ethyl 3-(tert-butoxycarbonyl)-3-(1-ethylcyclopropyl)-2-hydroxy-
propionate (19). Palladium on carbon (170 mg, 5%) was added
to a solution of 18 (435 mg, 1.36 mmol) and ammonium
formate (427 mg, 6.78 mmol) in methanol (20 mL) at 0 °C. The
mixture was stirred at r.t. for 2 h and after that filtered through a
pad of Celite. The Celite was washed with diethyl ether and
water. The filtrate was washed with saturated NaHCO3, and the
volatiles were removed under reduced pressure. The residue was
dissolved in Et2O, the solution washed with saturated NaHCO3
and NaCl solutions. The ethereal solution was dried over
MgSO4, and the volatiles were removed under reduced pressure
to give, after column chromatography (diethyl ether–hexane
1 : 3), 340 mg (88%) of the product 19 as a colorless solid. Rf =
0.15 (diethyl ether–hexane 1 : 3). 1H NMR (250 MHz): δ =
0.38–0.42 (3 H, m), 0.58–0.62 (1 H, m), 0.80 (3 H, t, J = 7 Hz),
1.26–1.29 (3 H, m), 1.42–1.44 (9 H, m), 1.44–1.70 (2 H, m),
2.30–2.39 (2 H, m), 3.16 (0.8 H, m), 3.41 (0.2 H, m), 4.18–4.30
(2 H, m), 4.39 (0.2 H, m), 4.58 (0.8 H, m) ppm. 13C NMR
(62.5 MHz): δ = 9.25 (CH2), 10.15 (CH3), 11.23 (CH2), 14.07
(CH3), 20.01 (C), 21.04 (C), 26.61 (CH2), 26.81 (CH2), 28.00
(CH3), 54.54 (CH), 54.88 (CH), 61.52 (CH2), 61.63 (CH2),
70.62 (CH), 71.43 (CH), 81.17 (C), 81.56 (C), 171.67 (C),
Major isomer (R*,R*)-16: Rf = 0.24 (diethyl ether–hexane
4 : 1). H NMR (250 MHz): δ = 0.32–0.38 (2 H, m), 0.48–0.52
1
(1 H, m), 0.61–0.65 (1 H, m), 0.76 (3 H, t, J = 7 Hz), 1.30–1.40
(1 H, m), 1.43 (9 H, s), 1.67–1.80 (1 H, m), 2.39 (1 H, d, J =
5 Hz), 3.76 (6 H, s), 3.96 (1 H, d, J = 3 Hz), 4.33 (1 H, dd, J =
15, 6 Hz), 4.45 (1 H, dd, J = 15, 7 Hz), 4.56 (1 H, dd, J = 5,
3 Hz), 6.33 (1 H, t, J = 2 Hz), 6.42 (1 H, d, J = 2 Hz), 7.05
(1 H, br s) ppm. 13C NMR (62.5 MHz): 9.56 (CH2), 10.20
(CH3), 11.22 (CH2), 20.46 (C), 27.52 (CH2), 27.92 (CH3), 43.18
(CH2), 52.79 (CH), 55.21 (CH3), 72.16 (CH), 81.57 (C), 99.26
(CH), 105.66 (CH), 140.15 (C), 160.90 (C), 171.36 (C), 174.55
(C) ppm. MS (DCI) m/z = 408 ([M + H+], 100).
Minor isomer (R*,S*)-16: Rf = 0.28 (diethyl ether–hexane
1
4 : 1). H NMR (250 MHz): δ = 0.35–0.55 (3 H, m), 0.75–0.78
(1 H, m), 0.84 (3 H, t, J = 7 Hz), 1.41 (9 H, s), 1.42–1.64 (2 H,
m), 2.91 (1 H, d, J = 2 Hz), 3.77 (6 H, s), 4.23 (1 H, dd, J = 10,
2 Hz), 4.28 (1 H, dd, J = 15, 6 Hz), 4.45 (1 H, dd, J = 15, 7 Hz),
4.75 (1 H, dd, J = 10 Hz), 6.33 (1 H, t, J = 2 Hz), 6.42 (1 H, d,
J = 2 Hz), 7.24 (1 H, br s) ppm. 13C NMR (62.5 MHz): 10.06
(CH2), 10.33 (CH3), 11.16 (CH2), 22.21 (C), 27.88 (CH2), 27.89
(CH3), 43.14 (CH2), 50.08 (CH), 55.23 (CH3), 73.62 (CH),
82.22 (C), 99.21 (CH), 105.35 (CH), 140.45 (C), 160.92 (C),
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6363–6374 | 6373

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173.07 (C), 174.93 (C) ppm. MS (DCI) m/z = 408 ([M + H+],
100).
KHSO4, H2O, aq. NaCl and dried over MgSO4. The solvent was
removed, and the residue was purified by column chromato-
graphy (diethyl ether–hexane 4 : 1) to give 92 mg (33%) of the
trans-N-(3,5-Dimethoxybenzyl)-3-(1-ethylcyclopropyl)-4-oxo-
oxetane-2-carboxamide (trans-8). Trifluoroacetic acid (380 μL,
5.1 mmol) was added to a solution of (R*,S*)-16 (147 mg,
0.361 mmol) and triethylsilane (150 μL, 0.9 mmol) in CH2Cl2
(2 mL). The reaction mixture was stirred at r.t. for 7 h. Then the
solvent was removed under reduced pressure, toluene was added
and removed again under reduced pressure. This operation was
repeated two more times. The residue was dissolved in CH2Cl2
(5 mL), the solution cooled to 0 °C, ethyldiisopropylamine
(170 μL, 1.01 mmol) and HATU (165 mg, 0.99 mmol) were
added and the mixture stirred at r.t. for 1.5 h. Then the mixture
was washed successively with aq. NaHCO3, H2O, aq. KHSO4,
H2O, aq. NaCl, and dried over MgSO4. The solvent was
removed, and the residue was purified by column chromato-
graphy (diethyl ether–hexane 4 : 1) to give 81 mg (67%) of the
1
product cis-8. Rf = 0.29 (diethyl ether–hexane 1 : 4). H NMR
(250 MHz): 0.22–0.38 (2 H, m), 0.42–0.46 (1 H, m), 0.80–0.90
(5 H, m), 2.05–2.07 (1 H), 3.77 (6 H, s), 4.40–4.42 (2 H, m),
4.57 (1 H, d, J = 7 Hz), 4.95 (1 H, d, J = 7 Hz), 6.37 (1 H, t, J =
2 Hz), 6.47 (2 H, d, J = 2 Hz), 6.88 (1 H, br s) ppm. 13C NMR
(62.5 MHz): 7.55 (CH2), 10.06 (CH3), 10.22 (CH2), 17.89 (C),
27.55 (CH2), 43.43 (CH2), 55.23 (2 CH3), 58.13 (CH), 72.66
(CH), 99.46 (CH), 106.17 (2 CH), 139.25 (C), 160.93 (2 C),
166.25 (C), 168.33 (C), 171.43 (C) ppm. IR (KBr): ν˜ =
2962 cm−1, 2840, 1829, 1668, 1596, 1533, 1471. MS (EI) m/z =
333 ([M+], 25), 261 (15), 166 (45), 152 (34), 151 (100).
References
1
product trans-8. Rf = 0.44 (diethyl ether–hexane 1 : 4). H NMR
1 M. Groll, O. V. Larionov, M. Huber and A. de Meijere, Proc. Natl. Acad.
Sci. U. S. A., 2006, 103, 4576–4579.
(250 MHz): δ = 0.45–0.60 (3 H, m), 0.80–0.84 (1 H, m), 0.92
(3 H, t, J = 7 Hz), 1.33–1.37 (1 H, m), 1.62–1.68 (1 H, m), 3.77
(6 H, s), 3.90 (1 H, d, J = 4.6 Hz), 4.02(1 H, d, J = 4.6 Hz), 4.32
(1 H, dd, J = 5.6, 14.6 Hz), 4.44 (1 H, dd, J = 5.6, 14.6 Hz),
6.35–6.40 (3 H, m), 6.78 (1 H, br s) ppm. 13C NMR
(62.5 MHz): δ = 8.83 (CH2), 9.15 (CH3), 10.12 (CH2), 19.37
(C), 28.71 (CH2), 43.24 (CH2), 55.23 (2 CH3), 61.55 (CH),
72.18 (CH), 99.45 (CH), 105.70 (2 CH), 139.30 (C), 161.01 (C),
167.47 (C), 167.92 (C) ppm. IR (KBr): ν˜ = 2967 cm−1, 1837,
1708, 1669, 1598, 1155. MS (EI) m/z = 333 ([M+], 25),
261 (15), 166 (45), 152 (34), 151 (100).
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cis-N-(3,5-Dimethoxybenzyl)-3-(1-ethylcyclopropyl)-4-oxo-
oxetane-2-carboxamide (cis-8). Trifluoroacetic acid (870 μL,
11.6 mmol) was added to a solution of (R*,R*)-16 (337 mg,
0.827 mmol) and triethylsilane (340 μL, 2.1 mmol) in CH2Cl2
(4 mL). The mixture was stirred at r.t. for 7 h. Then the solvent
was removed under reduced pressure, toluene was added and
removed again under reduced pressure. This operation was
repeated two more times. The residue was dissolved in CH2Cl2
(10 mL), the solution cooled to 0 °C, ethyldiisopropylamine
(385 μL, 2.32 mmol) and HATU (377 mg, 0.99 mmol) were
added, and the mixture was stirred at rt for 1.5 h. Then the
mixture was washed successively with aq. NaHCO3, H2O, aq.
10 D. A. Evans, D. W. C. Mac Millan and K. R. Campos, J. Am. Chem.
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6374 | Org. Biomol. Chem., 2012, 10, 6363–6374
This journal is © The Royal Society of Chemistry 2012