Synthesis of 1,2,3-Triazoles Bearing a 4-Hydroxyisoxazolidine Moiety from 4,5-Unsubstituted 2,3-Dihydroisoxazoles

Article abstract of DOI:10.1002/ejoc.202000737

A synthetic approach towards new 1,2,3-triazoles bearing the 3-hydroxymethylated 4-hydroxyisoxazolidine moiety has been described. The strategy has relied on dihydroxylation and epoxidation reactions of 4,5-unsubstituted 2,3-dihydroisoxazoles, allowing the introduction of the hydroxy group at the isoxazolidine ring in a trans stereoselective manner with respect to the substituent at C-3 carbon atom. The requisite 5-azidoisoxazolidines have been prepared from activated isoxazolidines possessing a good leaving group at C-5 carbon atom by treatment with trimethylsilyl azide and Lewis acid (isoxazolidinyl benzoates) or with sodium azide (chloroisoxazolidines). The 1,2,3-triazole moiety has been synthesized through copper(I)-catalyzed azide-alkyne cycloaddition.

Full text of DOI:10.1002/ejoc.202000737

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N,O-Heterocycles Synthesis
Synthesis of 1,2,3-Triazoles Bearing a 4-Hydroxyisoxazolidine
Moiety from 4,5-Unsubstituted 2,3-Dihydroisoxazoles
Radka Štadániová,[a] Michal Sahulčík,[a] Jana Doháňošová,[b] Ján Moncol,[c] Ľuboš Janotka,[d]
Kristína Šimoničová,[d] Lucia Messingerová,*[d,e] and Róbert Fischer*[a]
Abstract: A synthetic approach towards new 1,2,3-triazoles site 5-azidoisoxazolidines have been prepared from activated
bearing the 3-hydroxymethylated 4-hydroxyisoxazolidine moi- isoxazolidines possessing a good leaving group at C-5 carbon
ety has been described. The strategy has relied on dihydroxyl- atom by treatment with trimethylsilyl azide and Lewis acid
ation and epoxidation reactions of 4,5-unsubstituted 2,3-di- (isoxazolidinyl benzoates) or with sodium azide (chloroisox-
hydroisoxazoles, allowing the introduction of the hydroxy azolidines). The 1,2,3-triazole moiety has been synthesized
group at the isoxazolidine ring in a trans stereoselective manner through copper(I)-catalyzed azide-alkyne cycloaddition.
with respect to the substituent at C-3 carbon atom. The requi-
Introduction
active structures.[4,5] They have been found to be promising
drug candidates due to their interesting biological properties
including anticancer[6] and antiviral.[7]
Targeted syntheses of functionalized bioactive heterocycles are
continuously of high interest for organic and medicinal chem-
ists. Many of such molecules are potent enzyme inhibitors that
decrease enzyme activity by binding to its active site. Conse-
quently, the preparation of intriguing heterocyclic compounds
and the development of new synthetic methods constitute a
great part of research in medicinal chemistry, e.g. for the per-
formance of general screening,[1] for fragment-based screening
libraries design,[2] or for the synthesis of ligands for decoration
of the lead compound during QSAR studies.[3]
Recently, 1,2,3-triazole-containing isoxazolidines[8] (Figure 1)
have been prepared as base analogs of well-known biologically
effective N,O-nucleosides in which the furanose ring has been
replaced by an isoxazolidine scaffold.[9] Among them, a series of
5-(1H-1,2,3-triazole)isoxazolidines has been synthesized either
through 1,3-dipolar cycloadditions of azidoisoxazolidines with
selected alkyne derivatives (exemplified by compound 1),[8a] or
by the direct nitrone 1,3-dipolar cycloadditions with 1-vinyl tri-
azoles (exemplified by compound 2).[8b] Biological activity of
the phosphonylated isoxazolidinyl triazoles was evaluated in vi-
tro against a variety of DNA and RNA viruses and cancer cells.
No inhibitory activity against any virus was detected for the
Isoxazolidines and their more complex derivatives are unde-
niably an important part of a large group of such biologically
[a] R. Štadániová, M. Sahulčík, Dr. R. Fischer
Institute of Organic Chemistry, Catalysis and Petrochemistry,
Slovak University of Technology in Bratislava,
Radlinského 9, 81237 Bratislava, Slovak Republic
E-mail: robert.fischer@stuba.sk
evaluated compounds at 250 μ
activity, several triazoles were able to inhibit cell proliferation
at concentrations ranging from 40 to 78 μ for HEL cells, and
from 120 to 250 μ for L1210, CEM, and HeLa cells. On the
M. For evaluation of cytotoxic
M
M
http://www.fchpt.stuba.sk/
[b] Dr. J. Doháňošová
other hand, triazolyl isoxazolidines, bearing hydroxymethyl
group, were all able to inhibit proliferation of follicular (FTC-
133) and anaplastic (8305C) human thyroid cancer cell lines,
Central Laboratories, Slovak University of Technology in Bratislava,
Radlinského 9, 81237 Bratislava, Slovak Republic
[c] Prof. J. Moncol
Institute of Inorganic Chemistry, Technology and Materials,
Slovak University of Technology in Bratislava,
Radlinského 9, 81237 Bratislava, Slovak Republic
[d] Ľ. Janotka, K. Šimoničová, Dr. L. Messingerová
Institute of Molecular Physiology and Genetics, Centre of Biosciences,
Slovak Academy of Sciences,
with IC50 values ranging from 3.87 to 8.76 μM.
Dúbravská cesta 9, 84505 Bratislava 4, Slovak Republic
E-mail: lucia.messingerova@stuba.sk
http://www.umfg.sav.sk/obc/
[e] Dr. L. Messingerová
Institute of Biochemistry and Microbiology, Slovak University of Technology
in Bratislava,
Radlinského 9, 81237 Bratislava, Slovak Republic
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.202000737.
Figure 1. Known biologically active isoxazolidinyl triazoles exemplified by
compounds 1[8a] and 2.[8b]
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These beneficial findings inspired us to design modified tri- NMR data with those previously reported for the known com-
azoles 3–5 bearing 3-hydroxymethylated 4-hydroxyisoxazol- pounds 12a,b.[16]
idine moiety (Scheme 1) and evaluate their anticancer activity
using AML (acute myeloid leukemia) MOLM-13 cell line. Despite
a vast number of isoxazolidine analogues of bioactive mol-
ecules built up so far, to the best of our knowledge such struc-
tural modification has not yet been involved. In line with our
ongoing research in the field of reactivity of 4,5-unsubstituted
2,3-dihydroisoxazoles we envisioned that the hydroxy group
could be introduced into the isoxazolidine ring by dihydroxyl-
ation reaction of the double bond.[10] Upon acylation of the
isoxazolidine-4,5-diol the resulting dibenzoate could act as a
powerful electrophile in acid-catalyzed nucleophilic substitu-
tion with trimethylsilyl azide giving 5-azidoisoxazolidine as a
suitable intermediate for triazole ring construction by copper(I)-
catalyzed azide-alkyne cycloaddition with an alkyne. The N-un-
protected isoxazolidine could be obtained in the final stage by
N-debenzylation of the corresponding N-benzyl derivative.
Scheme 2. Synthesis of 5-acetoxyisoxazolidines 12 and 13. Reaction condi-
tions: (a) TBDPSCl, Et3N, DMAP, CH2Cl2, r.t., 12 h, 97 %; (b) K2OsO4·2H2O, NMO
(aq. 50 % w/w), acetone/water (4:1), r.t., 12 h, 95 %; (c) NaIO4, THF/water (3:1),
0 °C, 30 min. then r.t. 12 h, 90 %; (d) CH3NHOH·HCl, NaHCO3, MgSO4, CH2Cl2,
r.t., 12 h, 10 98 %; (e) BnNHOH·HCl, NaHCO3, MgSO4, CH2Cl2, r.t., 12 h, 11
89 %; (f) vinyl acetate, r.t., 4 d, 12a,12b combined yield 88 %, 12a (3,5-cis)/
12b (3,5-trans), 75:25; (g) vinyl acetate, 40 °C, 17 h, 13a,13b combined yield
80 %, 13a (3,5-cis)/13b (3,5-trans), 80:20, diastereomeric ratios were deter-
mined from the 1H NMR spectra of the crude product mixture.
Elimination reactions of 5-acetoxyisoxazolidines 12a,b and
13a,b with a catalytic amount of TMSOTf in the presence of
N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in anhydrous
N-methyl-2-pyrrolidone (NMP) at room temperature afforded
2,3-dihydroisoxazoles 14 and 15 in very good 72 and 89 %
yields, respectively (Scheme 3).[17]
Scheme 1. Retrosynthetic analysis of the 4-hydroxyisoxazolidinyl analogs 3–
5.
Results and Discussion
For the synthesis of N-methyl- and N-benyl-2,3-dihydroisox-
azoles we decided to use 5-acetoxyisoxazolidines 12 and 13
generated by the 1,3-dipolar cycloaddition of known (Z)-config-
ured nitrones 10[11] and 11[12] with vinyl acetate. As depicted
in Scheme 2, nitrones 10 and 11 were readily synthesized in
four steps starting from (Z)-but-2-ene-1,4-diol (6). The reaction
of 6 with tert-butyldiphenylchlorosilane gave silylated alkenol 7
in 97 % yield.[13] Its treatment with a catalytic amount of
K2OsO4·2H2O in the presence of aqueous NMO afforded diol 8
in 95 % yield which was subjected to oxidative cleavage with
sodium periodate in THF/water. Aldehyde 9 was formed in 90 %
yield. Subsequent condensation of 9 with N-methyl- and N-
benzylhydroxylamine hydrochloride under basic conditions in
the presence of anhydrous MgSO4 provided nitrones 10 and 11
in 98 and 89 % yields, respectively.[14,15] The 1,3-dipolar cyclo-
addition of nitrone 10 directly in neat vinyl acetate at room
temperature afforded 5-acetoxyisoxazolidine 12 in 88 % com-
bined yield.[16] More stable nitrone 11 reacted with vinyl acet-
ate at 40 °C faster to give adduct 13 in 80 % combined yield.
Cycloadditions proceeded regioselectively through exo transi-
Scheme 3. Synthesis of benzoylated isoxazolidine-4,5-diols. Reaction condi-
tions: (a) BSTFA, TMSOTf, NMP, r.t., 2 h, 14 72 %, 15 89 %; (b) from 14, K2O-
sO4·2H2O, NMO (aq. 50 % w/w), acetone/water (6:1), 4 °C, 20 h; (c) from 15,
K2OsO4·2H2O, NMO (aq. 50 % w/w), acetone/water (6:1), r.t., 7 h; (d) BzCl,
pyridine, DMAP, CH2Cl2, r.t., 16 h; 18a,b/19a,b combined yield 62 % over two
steps; 20a,b/21a,b combined yield 67 % over two steps.
As for all known 4,5-unsubstituted 2,3-dihydroisoxazoles syn-
tion state. In both cases, two diastereoisomers a (3,5-cis) and b thesized by our group so far, the double bond was character-
(3,5-trans) were formed with good 3,5-cis selectivity (12a/12b, ized by the presence of the signals for the 4-H protons at 4.78–
75:25) and (13a/13b, 80:20). The relative configuration of new 4.85 ppm and for the 5-H protons at 6.37–6.45 ppm with the
adducts 13a and 13b was established by comparison of the small coupling constants (2.6–2.8 Hz). In 13C NMR spectra, the
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1
signals appeared at approximately 96 ppm (C-4) and 142 ppm ucts in 55 and 64 % yields and were fully characterized by H
(C-5). When 2,3-dihydroisoxazoles 14 and 15 were treated with and 13C NMR spectroscopy, including 1D NOESY experiments.
a catalytic amount of K2OsO4·2H2O in the presence of aqueous The relative configuration of the minor isomers 22b, 23a, and
NMO in acetone/water, diastereomeric mixtures of isoxazol- 23b was established by inspection of the NOESY spectra of
idine-4,5-diols 16 and 17 were obtained, consisting of four in- their corresponding mixtures (see the Supporting Information).
separable isomers, which were subsequently transformed with- The obtained results were then applied to determine the rela-
out purification into the corresponding O-benzoylated isoxazol- tive configuration of the corresponding N-benzyl derivatives.
idines (Scheme 3). Both reactions afforded a mixture of four From the stereochemical point of view, we assume that the
stereoisomeric isoxazolidines 18 (3,4-trans), 19 (3,4-cis), and 20 high preference for the formation of 4,5-trans isomers 22a and
(3,4-trans), 21 (3,4-cis), respectively. The ratios and the com- 24a is generally due to neighboring group participation from
bined yields over two steps from 2,3-dihydroisoxazoles are sum- the 4-O protecting benzoyl group. The obtained azidoisoxazol-
marized in Table 1 (Entries 1, 2). To avoid problems associated idines 22a and 24a were further treated with phenylacetylene
with the chromatographic separation of each isomer, that has in the presence of CuSO4·5H2O and L-sodium ascorbate in tert-
emerged during this process, all isomers 18a,b/19a,b and butyl alcohol/water (1:2) at room temperature in 12
20a,b/21a,b were isolated again as product mixtures and used (Scheme 5).[8a]
in the next reaction directly. In order to clarify the stereochemis-
try of previous dihydroxylations, a sample of each mixture was
subjected to repeated preparative thin-layer chromatography
to obtain all isomers in pure form. Their relative configuration
was determined by NMR spectroscopy, including NOESY experi-
ments (see the Supporting Information). The obtained results
suggest that the dihydroxylation reactions of 2,3-dihydroisox-
azoles 14 and 15 proceeded with good trans stereoselectivity
with respect to the siloxymethyl group at C-3 carbon atom,
which is consistent with other dihydroxylations of 2,3-dihydro-
isoxazoles.[10] The O-benzoylated 3,4-trans-isoxazolidine-4,5-ols
18a,b and 20a,b were formed as the major ones with an ap-
proximate ratio of 3,4-trans/3,4-cis, 85:15.
h
Table 1. Synthesis of 5-benzoyloxy- 18–21 and 5-azidoisoxazolidines 22–25.
Scheme 4. Synthesis of 5-azido-substituted isoxazolidines. Reaction condi-
tions: TMSN3, TMSOTf, CH3CN, r.t., 24 h; 22a 55 %; 24a 64 %.
Entry
Substrates
Products
Ratio[a]
Yield [%][b]
1
2
3
4
14
15
18a, 18b, 19a, 19b
20a, 20b, 21a, 21b
13:72:10:5
45:42:6:7
83:8:7:2
62[c]
67[c]
65
18a,b/19a,b 22a, (22b, 23a, 23b)[d]
20a,b/21a,b 24a, (24b, 25a, 25b)[d,e]
84:9:5:2
71
[a] Diastereomeric ratios were determined from the 1H NMR spectra of the
crude product mixture. [b] Isolated combined yield. [c] Yield over two steps
from 2,3-dihydroisoxazole. [d] Products in parenthesis were isolated as a dia-
stereomeric mixture. [e] Diastereomers 25a, 25b were detected by NMR spec-
troscopy, however, the isolation and full characterization attempts failed.
Having benzoates 18–21 with a good leaving group in the
C-5 position, the introduction of the azido group can be
achieved by means of nucleophilic substitution with TMSN3 me-
diated by TMSOTf. Up to now, only a few examples of the prep-
aration of 5-azidoisoxazolidines have been reported in the liter-
ature.[8a] Recently, we reported related cyanation of N-benzyl-
substituted 5-acetoxyisoxazolidines with TMSCN under similar
reaction conditions.[10b] According to this procedure, the reac-
tions of 18a,b/19a,b and 20a,b/21a,b were carried out in
acetonitrile at room temperature for 24 h and afforded dia-
stereomeric mixtures of four isoxazolidines 22a,b/23a,b and
24a,b/25a,b in both cases (Scheme 4). Their combined yields
and ratios are summarized in Table 1 (Entries 3, 4). It is worth
noting that acetonitrile and room temperature were crucial for
successful reaction in terms of reactivity and yield, whereas in
dichloromethane the azidation did not occur at all. For the next
synthetic purpose, only the major isomers 22a and 24a were
Scheme 5. Synthesis of isoxazolidinyl triazoles 3, 4 and 30. Reaction condi-
tions: (a) phenylacetylene, CuSO4·5H2O, sodium L-ascorbate, tBuOH/water
(1:2), r.t., 12 h; 26 93 %; 27 84 %; (b) K2CO3, MeOH, r.t., 30 min; 28 86 %; 29
92 %; (c) TBAF, THF, r.t., 5 h; 3 88 %; 4 95 %; (d) Ac2O, Et3N, DMAP, CH2Cl2, r.t.,
1 h, 97 %.
The reactions yielded the isoxazolidinyl triazoles 26 and 27
isolated by flash column chromatography (FCC) as pure prod- in very good 93 and 84 % yields as single regioisomers. De-
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benzoylation with K2CO3 in methanol gave the isoxazolidin-4- hydrous THF at –78 °C gave isoxazolidin-5-ol 34 in 76 % isolated
ols 28 (86 % yield) and 29 (92 % yield). Finally, desilylation was yield as a mixture of two 3,5-trans and 3,5-cis anomers in an
performed using TBAF in THF and desired 4-hydroxyisoxazol- 80:20 ratio. The relative configuration of the more stable trans
idinyl triazoles 3 and 4 were isolated in 88 and 95 % yields.
In addition, per-O-acetylated isoxazolidine 30 was prepared
in 97 % yield from 3 under common conditions with acetic an-
hydride. To confirm the 3,4-trans-4,5-trans configuration, the
isoxazolidine 3 was subjected to X-ray crystallographic analysis
(see the Supporting Information).
Next, our synthetic efforts were focused on the N-debenzyl-
ation of 4 according to the procedure using Pd-catalyzed hydro-
genolysis in methanol with formic acid as the hydrogen
source.[18,19] Unfortunately, we did not succeed in removing the
benzyl group, observing only a complete decomposition of the
starting material (Scheme 6). Therefore, in order to prepare N-
unprotected isoxazolidinyl triazole 5, we turned our attention
to N-Boc-4-hydroxyisoxazolidines with a readily removable tert-
butyloxycarbonyl group under acidic conditions. Recently, we
have described the stable isoxazolidinyl epoxides, prepared
from 4,5-unsubstituted N-Boc- and N-Cbz-2,3-dihydroisoxazoles,
which were converted into 5-substituted 4-hydroxyisoxazol-
idines by carrying out the regio- and stereospecific epoxide
ring-opening reactions.[20]
isomer 34a was determined by comparison with already re-
ported NMR data of related 5-hydroxyisoxazolidines.[22,24] It is
important to note that the prolonged reaction time as well as
the use of DIBAL-H as a reducing agent definitely caused over-
reduction into the respective primary alcohol.[25] Treatment of
34 with Tf2O in the presence of 2-chloropyridine produced 2,3-
dihydroisoxazole 35 in a moderate 53 % yield. Based on our
experiences with epoxidations of 4,5-unsubstituted 2,3-di-
hydroisoxazoles with in situ generated DMDO,[22] compound 35
was subjected to reaction with Oxone and NaHCO3 in acetone/
water (Scheme 7). The reaction provided isoxazolidinyl epoxide
36 in almost quantitative yield (99 %) and acceptable purity as
a sole trans isomer (dr > 95:5, see the Supporting Information).
1
Spectroscopic H and 13C NMR data were in good agreement
with those previously published for related epoxides.[20,22]
Scheme 7. Synthesis of N-Boc-substituted isoxazolidinyl epoxide 36. Reaction
conditions: (a) DABCO, EtOAc, r.t., 17 h, 80 %; (b) Super-Hydride, THF, –78 °C,
30–50 min, combined yield 76 %, 34a (3,5-trans)/34b (3,5-cis), 80:20; (c) Tf2O,
2-chloropyridine, NMP, –20 °C to r.t., 20 h, 53 %; (d) Oxone, NaHCO3, acetone/
water (3:2), r.t., 30 min, 99 %, (dr > 95:5).
We have recently found out that isoxazolidinyl epoxides can
react with hydrogen chloride or thionyl bromide providing 5-
chloro- and 5-bromo-substituted isoxazolidin-4-ols.[20,22] How-
ever, the reaction of the epoxide 36 led only to a complex
mixture under such reaction conditions. To our delight, the re-
action with lithium chloride in the presence of acetic acid in
THF afforded an anomeric mixture of 5-chloroisoxazolidines
37a,b (4,5-cis-37a/4,5-trans-37b, 70:30) in combined yield of
95 % (Scheme 8).[26] Accordingly, the epoxide 36 was also
briefly tested in reaction with lithium bromide. Unfortunately,
we have only observed the formation of unidentified by-prod-
ucts. Next, the azidation of 37a,b was examined. The reaction
with sodium azide in acetonitrile at 50 °C gave 5-azidoisoxazol-
Scheme 6. Unsuccessful N-debenzylation, and new retrosynthetic analysis of
the N-unsubstituted isoxazolidinyl triazole 5 using N-Boc intermediate.
The starting 2,3-dihydroisoxazoles were prepared from the
corresponding isoxazolidin-5-ols through elimination reactions
using Tf2O/2-chloropyridine couple in anhydrous NMP.[21] Al-
though direct reaction with TMSN3 in the presence of nBu3P
afforded 5-azidoisoxazolidines only in very low yields, this prob-
lem was successfully overcome by preparing 5-bromoisoxazol-
idines which undergo reaction with sodium azide smoothly.[22]
According to the retrosynthetic analysis depicted in idines 38a (4,5-trans) and 38b (4,5-cis) in a ratio of 80:20 with
Scheme 6, we started with isoxazolidin-5-one 33 which was 80 % combined yield. After separation by FCC, their relative
prepared by multicomponent organocatalyzed Knoevenagel- configuration was mainly established on the basis of 1H and
aza-Michael cyclocondensation reaction between N-Boc- 13C NMR spectra. More specifically, narrow doublet for the 5-H
hydroxylamine 31, Meldrum′s acid (32) and aldehyde 9 in 80 % proton of 38a at 5.48 ppm with a small coupling constant
yield (Scheme 7).[23] Reduction of 33 with Super-Hydride in an- 1.4 Hz referred to 4,5-trans isomer (it was later confirmed by
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1D NOESY experiments of compound 40; see the Supporting compounds was assessed by monitoring the NADH/NADPH-
Information). A doublet with a larger coupling constant 5.0 Hz dependent reduction of tetrazolium dye to formazan using the
with chemical shift 5.59 ppm (5-H) denoted 4,5-cis arrangement MTS assay. Mode of cell death was estimated using double
in 38b. The copper-catalyzed azide-alkyne cycloaddition of staining with annexin V/PI. While the metabolic activity of
pure 38a with phenylacetylene afforded isoxazolidinyl triazole MOLM-13 cells after treatment with various concentrations
39 in 90 % yield. Subsequent desilylation of primary hydroxy (0.5–100 μ
group with TBAF in THF gave diol 40 in 80 % yield. Finally, the decrease in metabolic activity was observed after treatment
Boc protecting group was successfully removed using TFA in with 100 μ concentration of compound 4. Unlike other com-
CH2Cl2.[27] Desired N-unprotected isoxazolidine 5 was obtained pounds the highest concentration of compound 40 used for
M
) of compounds 3, 5, and 30 did not change, a
M
in 76 % yield.
cell treatment was 20 μM due to its lower solubility. A higher
concentration of compound 40 would lead to the inhibitory
effect of DMSO per se. A decrease in metabolic activity of
MOLM-13 cells after treatment with 100 μ
M concentration of
compound 4 may indicate some anticancer activity of this com-
pound (Figure 2).
Scheme 8. Synthesis of N-unsubstituted isoxazolidinyl triazole 5. Reaction
conditions: (a) LiCl, AcOH, THF, r.t., 2 h, 37a, 37b combined yield 95 %, 37a
(4,5-cis)/37b (4,5-trans), 70:30; (b) NaN3, CH3CN, 50 °C, 18 h; 38a, 38b com-
bined yield 80 %, 38a (4,5-trans)/38b (4,5-cis), 80:20; (c) phenylacetylene,
CuSO4·5H2O, sodium L-ascorbate, tBuOH/water (1:2), r.t., 17 h, 90 %; (d) TBAF,
THF, 0 °C, 1 h, 80 %; (e) TFA/CH2Cl2 (1:5), 0 °C, 30 min then, r.t., 1 h, 76 %.
Figure 2. Cell line MOLM-13 was cultured in absence or in presence of differ-
As mentioned above, the reaction between epoxide 36 and
LiCl in the presence of acetic acid provided the mixture of two
anomers 37a,b in favor of the 4,5-cis isomer 37a (determined
on the basis of a doublet at δ = 6.28 ppm with J4,5 = 4.3 Hz for
5-H proton) which was in accordance with our previous re-
sults.[20,22] However, we have noticed that both anomeric 5-
chloroisoxazolidines were formed in nearly equimolar amounts
at the early stage of the reaction. The predominance of the
thermodynamically more stable 4,5-cis isomer was observed
with progressing reaction time and graduated even after the
conversion completion (for example when keeping the com-
pound in solution for a longer time). We assume that the spon-
taneous isomerization of the trans isomer takes place probably
through anomeric effect (the 37a/37b ratio changes from 45:55
to 90:10, see the Supporting Information).
ent concentrations of prepared isoxazolidines (0; 0.5; 1; 5; 10; 20; 100 μM) for
48 h. After cultivation, metabolic activity of the cells was assessed by an MTS
assay. Metabolic activity of control cells was set as 100 %. The data represent
results of three independent measurements SD. DMSO column represents
the metabolic activity of cells after treatment with the highest corresponding
concentration of DMSO. Compounds 3, 5 and 30 were prepared as 10 m
stock solutions in 1 % DMSO, compound 4 as 5 m stock solution in 30 %
DMSO and compound 40 as 2.5 m stock solution in 50 % DMSO.
M
M
M
Subsequently, these results were further confirmed using
double staining with annexin V (marker of cell apoptosis)/pro-
pidium iodide (cell necrosis factor). Measurement of cell death
was conducted after treatment of MOLM-13 cells with 0.5–
100 μM
concentration of compounds 3–5, 30 and 0.5–20 μM of
compound 40. Results for cells treated with the highest concen-
tration used are summarized in Figure 3. Treatment with com-
pounds 3, 5, 30 and 40 did not change the proportion of viable
(unstained) cells compared to control. Induction of cell death
was observed after treatment with 100 μ
compound 4.
M concentration of
Biological Assay
Isoxazolidinyl triazoles 3–5, 30, and 40 were evaluated for
Lower metabolic activity of MOLM-13 cells after treatment
inhibitory activity against acute myeloid leukemia cell line with compound 4 was also confirmed by a higher portion of
MOLM-13. The cell metabolic activity after treatment with these cells stained with annexin V and/or PI.
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The 2,3-dihydroisoxazoles have been obtained from 5-acetoxy-
isoxazolidines by using a catalytic amount of TMSOTf in
the presence of N,O-bis(trimethylsilyl)trifluoroacetamide. The
requisite 5-azidoisoxazolidines have been prepared either by
TMSOTf-mediated nucleophilic substitution of the isoxazolidinyl
benzoates with TMSN3 or by the reaction of 5-chloro derivatives
with sodium azide with 4,5-trans selectivity being favored in
both cases. Finally, the 1,2,3-triazole unit has been synthesized
by copper(I)-catalyzed azide-alkyne cycloaddition.
Additionally, the significance of this method lies in its varia-
bility and applicability to a concise synthesis of various other 5-
substituted 4-hydroxyisoxazolidine derivatives via nucleophilic
substitution reactions at the C-5 position of the isoxazolidine
ring, starting from easily synthesized 4,5-unsubstituted 2,3-di-
hydroisoxazoles.
Isoxazolidinyl triazoles 3–5, 30, and 40 were evaluated for
inhibitory activity against the MOLM-13 cell line. After treat-
ment with compounds 3, 5, 30, and 40 no inhibitory activity
was observed. Moderate inhibition of metabolic activity and
induction of cell death was observed after treatment with com-
pound 4. To confirm the anticancer activity of compound 4
further research is necessary.
To prove the anticancer activity of isoxazolidinyl triazoles 3–
5, 30, and 40 in this study we used AML cell line. We did not
detect the relevant anticancer activity of compounds 3, 5, 30,
and 40 in the treatment of this cell line, but these compounds
may be active in the treatment of other cell lines (solid tumors).
Further research in this field is necessary.
Experimental Section
General Methods: Flash column chromatography (FCC) was carried
out with a Büchi system (Pump Manager C-615 and Fraction Collec-
tor C-660) using Normasil 60 silica gel (0.040–0.063 mm; VWR). Thin
Layer Chromatography (TLC) analysis was carried out using TLC sil-
ica gel 60 F254 (aluminium sheets, Merck), and plates were visual-
ized with UV light or by treatment with permanganate solution
followed by heating. Infrared (IR) spectra were recorded as neat
samples with a Nicolet 5700 FTIR spectrometer with an ATR Smart
Orbit Diamond adapter (Thermo Electron Corporation). NMR spec-
tra were recorded with a Varian INOVA-300 spectrometer (1H,
299.95 MHz, and 13C, 75.42 MHz) and a Varian VNMRS-600 instru-
ment (1H, 599.75 MHz, and 13C, 150.81 MHz) in CDCl3 (using tetra-
methylsilane as the internal standard) and in CD3OD (residual
[D3]methanol, δH = 3.31, 4.87 ppm, δC = 49.00 ppm). Data are pre-
sented as follows: chemical shift, multiplicity (s = singlet, d = dou-
blet, t = triplet, q = quartet, dd = doublet of doublets, ddd = dou-
blet of doublet of doublets, td = triplet of doublets, dt = doublet
of triplets, m =multiplet, bs = broad singlet), coupling constants
(J/Hz) and integration. HRMS analysis was carried out with an Orbi-
trap Velos Pro spectrometer (Thermo Fisher Scientific). All solvents
used were dried and distilled according to conventional methods.
Figure 3. Pie charts represent proportion of viable (unstained), FITC-annexin
V, FITC-annexin V/propidum iodide and propidium iodide stained cells after
treatment with 100 μ
M concentration of compounds 3–5, 30 and 20 μM of
compound 40. Chart for DMSO represents induction of cell death after treat-
ment with the highest concentration of DMSO used.
Conclusion
In summary, we have synthesized new 1,2,3-triazoles bearing
3-hydroxymethylated 4-hydroxyisoxazolidine moiety. A hydroxy
group at the C-4 position of the isoxazolidine ring has been
introduced by dihydroxylation and epoxidation of 4,5-unsubsti-
tuted 2,3-dihydroisoxazoles via the reactions with potassium
osmate/NMO and Oxone/NaHCO3/acetone with good trans se-
Typical Procedure for the Synthesis of 3-[(tert-Butyldiphenyl-
silyloxy)methyl]-2-methyl-2,3-dihydroisoxazole (14): The reac-
tion flask with 5-acetoxyisoxazolidines 12a,b (1.1 g, 2.66 mmol) was
sealed with a rubber septum, evacuated, and filled with argon. An-
hydrous NMP (27 mL) was added followed by BSTFA (850 μL,
3.20 mmol). The stirred solution was cooled down to 0 °C, and
lectivity with respect to the substituent at C-3 carbon atom. TMSOTf (95 μL, 0.52 mmol) was added dropwise. The reaction mix-
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ture was warmed to room temperature and stirred for 2 h. After
this time, TLC showed that the reaction was complete (hexanes/
EtOAc, 1:1). The reaction mixture was cooled in an ice/water-bath,
quenched by the addition of a sat. aq. NaHCO3 (10 mL), and water
was added (200 mL). The resulting mixture was extracted with Et2O
(3 × 30 mL). The combined organic layers were dried with MgSO4,
and the solvent was evaporated in vacuo. The residue was purified
by FCC (hexanes/EtOAc, 95:5) to give 2,3-dihydroisoxazole 14
(680 mg, 1.92 mmol, 72 %) as a yellowish oil. Rf = 0.57 (hexanes/
ture was subjected to repeated preparative TLC (hexanes/EtOAc,
9:1) to give three pure isomers 18a,18b and 19a. Unfortunately, the
fourth isomer (3,4-cis-4,5-cis)-19b was not isolated, and therefore
was not characterized. Data for (3,4-trans-4,5-trans)-18a: Colour-
less oil. Rf = 0.23 (hexanes/EtOAc, 9:1). IR (ATR): νmax = 3070, 2930,
˜
2856, 1722, 1256, 1105, 1065, 1023, 939, 700, 502, 488 cm–1
.
1H
NMR (600 MHz, CDCl3): δ = 8.05–8.01 (m, 4 H, H-Ph), 7.71–7.68 (m,
4 H, H-Ph), 7.62–7.32 (m, 12 H, H-Ph), 6.49 (s, 1 H, 5-H), 5.72 (d, J =
5.1 Hz, 1 H, 4-H), 4.06–4.01 (m, 2 H, CH2OSi), 3.11–3.09 (m, 1 H, 3-
EtOAc, 7:3). IR (ATR): νmax = 2957. 2930, 2856, 1619, 1427, 1100, H), 2.96 (s, 3 H, N-CH3), 1.05 (s, 9 H, tBu) ppm. 13C NMR (150 MHz,
˜
1
1080, 823, 739, 698, 612, 502, 487 cm–1. H NMR (300 MHz, CDCl3): CDCl3): δ = 165.4, 165.3, 135.6 (2 ×), 133.6, 133.3, 132.9 (2 ×), 130.1,
δ = 7.70–7.65 (m, 4 H, H-Ph), 7.44–7.34 (m, 6 H, H-Ph), 6.41–6.37 (m,
1 H, 5-H), 4.78 (pseudo t, J = 2.8, 2.6 Hz, 1 H, 4-H), 3.83–3.77 (m, 1
H, 3-H), 3.66 (dd, AB, J = 10.0, 6.8 Hz, 1 H, CH2OSi), 3.55 (dd, AB, J =
10.0, 5.7 Hz, 1 H, CH2OSi), 2.76 (d, J = 0.7 Hz, 3 H, N-CH3), 1.06 (s, 9
129.9, 129.8 (2 ×), 129.5, 129.0, 128.5, 128.3, 127.8 (2 ×), 98.2, 83.7,
74.9, 62.8, 46.2, 26.8, 19.2 ppm. HRMS (ESI): calcd. for C35H38NO6Si
[M + H]+ 596.2463, found 596.2455. Data for (3,4-trans-4,5-cis)-
18b: Colourless oil. R = 0.19 (hexanes/EtOAc, 9:1). IR (ATR): ν
=
˜
f
max
H, tBu) ppm. 13C NMR (75 MHz, CDCl3): δ = 142.4, 135.7, 135.6, 3070, 2930, 2857, 1728, 1272, 1257, 1111, 1066, 1020, 975, 700, 503,
1
133.7, 133.5, 129.6 (2 ×), 127.6 (2 ×), 96.4, 73.7, 67.3, 47.5, 26.8,
487 cm–1. H NMR (600 MHz, CDCl3): δ = 7.97–7.91 (m, 4 H, H-Ph),
19.3 ppm. HRMS (ESI): calcd. for C21H28NO2Si [M + H]+ 354.1884, 7.70–7.66 (m, 4 H, H-Ph), 7.56–7.53 (m, 2 H, H-Ph), 7.42–7.34 (m, 10
found 354.1877.
H, H-Ph), 6.74 (d, J = 4.5 Hz, 1 H, 5-H), 5.59 (dd, J = 7.9, 4.5 Hz, 1 H,
4-H), 3.98 (dd, AB, J = 10.7, 6.8 Hz, 1 H, CH2OSi), 3.91 (dd, AB, J =
10.7, 4.6 Hz, 1 H, CH2OSi), 3.49–3.44 (m, 1 H, 3-H), 3.08 (s, 3 H, N-
CH3), 1.05 (s, 9 H, tBu) ppm. 13C NMR (150 MHz, CDCl3): δ = 165.0,
164.9, 135.6 (2 ×), 133.5, 133.4, 132.9 (2 ×), 129.8 (3 ×), 129.7, 129.6,
128.9, 128.4 (2 ×), 127.8 (2 ×), 93.8, 76.7, 69.9, 64.0, 49.5, 26.8,
19.2 ppm. HRMS (ESI): calcd. for C35H38NO6Si [M + H]+ 596.2463,
found 596.2458. Data for (3,4-cis-4,5-trans)-19a: Colourless oil.
2-Benzyl-3-[(tert-butyldiphenylsilyloxy)methyl]-2,3-dihydrois-
oxazole (15): The typical procedure described above was applied
using 5-acetoxyisoxazolidines 13a,b (1.3 g, 2.65 mmol), BSTFA
(0.84 mL, 3.16 mmol), TMSOTf (95 μL, 0.52 mmol), and anhydrous
NMP (26 mL). TLC monitoring (hexanes/EtOAc, 1:1). FCC (hexanes/
EtOAc, 95:5) gave 2,3-dihydroisoxazole 15 (1.02 g, 2.37 mmol, 89 %)
as a yellowish oil. Rf = 0.52 (hexanes/EtOAc, 8:2). IR (ATR): νmax
=
˜
R = 0.14 (hexanes/EtOAc, 9:1). IR (ATR): ν
= 3070, 2929, 2856,
˜
f
max
3068, 2929, 2856, 1620, 1427, 1111, 1065, 823, 800, 737, 613, 502,
1724, 1243, 1091, 1051, 1023, 929, 700, 503, 486 cm–1 1H NMR
.
1
487 cm–1. H NMR (300 MHz, CDCl3): δ = 7.68–7.62 (m, 4 H, H-Ph),
(600 MHz, CDCl3): δ = 8.08–7.99 (m, 4 H, H-Ph), 7.67–7.23 (m, 16 H,
H-Ph), 6.51 (s, 1 H, 5-H), 5.98 (d, J = 4.5 Hz, 1 H, 4-H), 4.09 (dd, AB,
J = 10.3, 7.1 Hz, 1 H, CH2OSi), 3.90 (dd, AB, J = 10.3, 6.5 Hz, 1 H,
CH2OSi), 3.53–3.44 (m, 1 H, 3-H), 2.96 (s, 3 H, N-CH3), 1.02 (s, 9 H,
tBu) ppm. 13C NMR (150 MHz, CDCl3): δ = 165.4, 164.7, 135.7, 135.6,
133.8, 133.7, 133.1, 132.8, 130.2, 130.1, 130.0 (2 ×), 129.5, 129.1,
128.7, 128.6, 128.0, 127.9, 98.2, 80.7, 69.8, 60.9, 49.7, 26.9, 19.2 ppm.
HRMS (ESI): calcd. for C35H38NO6Si [M + H]+ 596.2463, found
596.2454.
7.41–7.26 (m, 11 H, H-Ph), 6.45–6.41 (m, 1 H, 5-H), 4.85 (pseudo t,
J = 2.8, 2.6 Hz, 1 H, 4-H), 4.16 (d, AB, J = 13.1 Hz, 1 H, N-CH2Ph),
4.08–4.02 (m, 1 H, 3-H), 3.91 (dd, AB, J = 13.1 Hz, 1 H, N-CH2Ph),
3.63 (dd, AB, J = 10.0, 6.8 Hz, 1 H, CH2OSi), 3.54 (dd, AB, J = 10.0,
5.7 Hz, 1 H, CH2OSi), 1.03 (s, 9 H, tBu) ppm. 13C NMR (75 MHz,
CDCl3): δ = 142.9, 137.0, 135.8, 135.7, 133.8, 133.7, 129.7 (2 ×), 129.3,
128.5, 127.8 (2 ×), 127.5, 97.0, 71.1, 67.4, 63.7, 27.0, 19.4 ppm. HRMS
(ESI): calcd. for C27H32NO2Si [M + H]+ 430.2197, found 430.2191.
Typical Procedure for the Synthesis of 3-[(tert-Butyldiphenyl-
silyloxy)methyl]-2-methylisoxazolidine-4,5-diyl Dibenzoates
(18, 19): 2,3-Dihydroisoxazole 14 (1.73 g, 4.89 mmol) was dissolved
in acetone/water (49 mL, 6:1 v/v), and NMO (2.03 mL, 9.78 mmol,
50 % w/w in water) was added at 0 °C followed by K2OsO4·2H2O
(92 mg, 0.25 mmol). The reaction mixture was warmed to room
temperature and stirred for 7 h. When TLC showed that the reaction
was complete (hexanes/EtOAc, 1:1), the mixture was diluted with
water (200 mL) and extracted with EtOAc (3 × 30 mL). The com-
bined organic layers were washed with a sat. aq. NaCl (50 mL), dried
with MgSO4, and the solvent was evaporated in vacuo. The resulting
crude diols 16 were used for the next step without further purifica-
tion.
2-Benzyl-3-[(tert-butyldiphenylsilyloxy)methyl]isoxazolidine-
4,5-diyl Dibenzoates (20, 21): The typical procedure described
above was applied using 2,3-dihydroisoxazole 15 (965 mg,
2.25 mmol), NMO (0.94 mL, 4.53 mmol, 50 % w/w in water), K2O-
sO4·2H2O (41 mg, 0.11 mmol), and acetone/water (21 mL, 6:1 v/v).
TLC monitoring (hexanes/EtOAc, 1:1). The resulting crude diols 17
were directly used for the next step after evaporation of the solvent.
The typical procedure described above was applied using diols 17,
DMAP (35 mg, 0.29 mmol), pyridine (0.47 mL, 5.84 mmol), benzoyl
chloride (0.51 mL, 4.39 mmol), and anhydrous CH2Cl2 (15 mL). TLC
monitoring (hexanes/EtOAc, 1:1). FCC (hexanes/EtOAc, 95:5) gave
the mixture of four isomers 20a,b and 21a,b (1.01 g, 1.50 mmol,
67 % over two steps, 20a/20b/21a/21b, 46:43:5:6) as a colourless
oil. With the aim to completely characterize the new compounds, a
sample of the mixture was subjected to repeated preparative TLC
(hexanes/EtOAc, 9:1) to give two pure isomers 20a and 20b and a
mixture of two isomers 21a and 21b. Data for (3,4-trans-4,5-
trans)-20a: Colourless oil. Rf = 0.25 (hexanes/EtOAc, 9:1). IR (ATR):
To a solution of diols 16 in anhydrous CH2Cl2 (30 mL) at room
temperature were added sequentially DMAP (80 mg, 0.65 mmol),
pyridine (1.08 mL, 13.35 mmol) and benzoyl chloride (1.17 mL,
10.08 mmol). The reaction mixture was stirred for 16 h. After this
time, TLC showed that the reaction was complete (hexanes/EtOAc,
1:1). The reaction mixture was diluted with H2O (200 mL), and the
organic layer was separated. The aqueous layer was extracted with
CH2Cl2 (2 × 20 mL). The combined organic layers were dried with
MgSO4, and the solvent was evaporated in vacuo. The residue was
ν
= 3068, 2930, 2856, 1723, 1258, 1105, 1065, 1024, 939, 698,
˜
max
503, 488 cm–1
.
1H NMR (300 MHz, CDCl3): δ = 8.04–7.96 (m, 4 H,
H-Ph), 7.68–7.64 (m, 4 H, H-Ph), 7.62–7.26 (m, 17 H, H-Ph), 6.60 (s, 1
purified by FCC (hexanes/EtOAc, 8:2) to give a mixture of four iso- H, 5-H), 5.82 (dd, J = 3.6, 0.5 Hz, 1 H, 4-H), 4.40, (d, AB, J = 14.2 Hz,
mers 18a,b and 19a,b (1.81 g, 3.04 mmol, 62 % over two steps,
18a/18b/19a/19b, 13:72:10:5) as a colourless oil. With the aim to
1 H, N-CH2Ph), 4.25 (d, AB, J = 14.2 Hz, 1 H, N-CH2Ph), 4.03 (dd, AB,
J = 10.7, 5.9 Hz, 1 H, CH2OSi), 3.92 (dd, AB, J = 10.7, 6.8 Hz, 1 H,
completely characterize the new compounds, a sample of the mix- CH2OSi), 3.43 (ddd, J = 6.8, 5.9, 3.6 Hz, 1 H, 3-H), 1.03 (s, 9 H, tBu)
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ppm. 13C NMR (75 MHz, CDCl3): δ = 165.4, 165.3, 136.5, 135.7 (2 ×), 5-Azido-2-benzyl-3-[(tert-butyldiphenylsilyloxy)methyl]isox-
133.7, 133.4, 133.1 (2 ×), 130.1, 130.0, 129.9 (2 ×), 129.6, 129.2 (2 ×), azolidin-4-yl Benzoate (24, 25): The typical procedure described
128.6, 128.5, 128.4, 127.9 (2 ×), 127.5, 98.4, 83.5, 71.5, 63.2, 62.8, above was applied using isoxazolidines 20a,b and 21a,b (690 mg,
26.9, 19.3 ppm. HRMS (ESI): calcd. for C41H42NO6Si [M + H]+
1.03 mmol), TMSN3 (275 μL, 2.07 mmol), TMSOTf (95 μL, 0.52 mmol),
and anhydrous CH3CN (4 mL). TLC monitoring (hexanes/EtOAc, 9:1).
FCC (CH2Cl2/hexanes, 85:15) gave one single isomer 24a and the
.
1H mixture of 24a and 24b. The other minor diastereomers 25a and
672.2776, found 672.2767. Data for (3,4-trans-4,5-cis)-20b: Colour-
less oil. Rf = 0.18 (hexanes/EtOAc, 9:1). IR (ATR): νmax = 3069, 2930,
˜
2857, 1728, 1271, 1105, 1065, 1022, 698, 613, 503, 488 cm–1
NMR (300 MHz, CDCl3): δ = 7.97–7.93 (m, 4 H, H-Ph), 7.69–7.53 (m, 25b were detected by NMR spectroscopy, however, the isolation
6 H, H-Ph), 7.44–7.22 (m, 15 H, H-Ph), 6.78 (d, J = 4.6 Hz, 1 H, 5-H),
5.71 (dd, J = 7.0, 4.6 Hz, 1 H, 4-H), 4.43 (d, AB, J = 14.2 Hz, 1 H, N-
CH2Ph), 4.37 (d, AB, J = 14.2 Hz, 1 H, N-CH2Ph), 3.90 (dd, AB, J =
10.6, 6.6 Hz, 1 H, CH2OSi), 3.84 (dd, AB, J = 10.6, 5.2 Hz, 1 H, CH2OSi),
3.73–3.67 (m, 1 H, 3-H), 1.02 (s, 9 H, tBu) ppm. 13C NMR (75 MHz,
CDCl3): δ = 165.2, 164.9, 136.6, 135.8, 135.7, 133.6, 133.5, 133.0 (2
and characterization attempts failed. Data for (3,4-trans-4,5-
trans)-24a: Yield: 390 mg (0.66 mmol, 64 %). White solid. Rf = 0.52
(CH2Cl2/hexanes, 9:1). M.p. 85–86 °C. IR (ATR): νmax = 3068.2960,
˜
2865, 2109, 1728, 1264, 1106, 1024, 737, 695, 516, 483 cm–1
.
1H
NMR (300 MHz, CDCl3): δ = 8.01–7.96 (m, 2 H, H-Ph), 7.70–7.57 (m,
5 H, H-Ph), 7.50–7.27 (m, 13 H, H-Ph), 5.37 (s, 1 H, 5-H), 5.32 (dd, J =
×), 130.0, 129.9 (3 ×), 129.7, 129.5, 129.1, 128.6 (2 ×), 128.5, 127.9 4.1, 0.7 Hz, 1 H, 4-H), 4.47 (d, AB, J = 14.4 Hz, 1 H, N-CH2Ph), 4.09
(2 ×), 127.7, 94.6, 77.0, 67.7, 65.6, 64.4, 26.9, 19.3 ppm. HRMS (ESI): (d, AB, J = 14.4 Hz, 1 H, N-CH2Ph), 3.98 (dd, J = 10.8, 5.4 Hz, 1 H,
calcd. for C41H42NO6Si [M + H]+ 672.2776, found 672.2768. Data CH2OSi), 3.93 (dd, J = 10.8, 6.8 Hz, 1 H, CH2OSi), 3.29 (ddd, J = 6.8,
for 21a, 21b: Colourless oil. (21a/21b, 10:90). Rf (21a+21b) = 0.25
5.4, 4.1 Hz, 1 H, 3-H), 1.03 (s, 9 H, tBu) ppm. 13C NMR (75 MHz,
(hexanes/EtOAc, 9:1). Partial 1H NMR data extracted from the mix- CDCl3): δ = 165.7, 136.7, 135.7 (2 ×), 133.8, 133.0, 132.9, 130.0 (3 ×),
ture for (3,4-cis-4,5-trans)-21a: 1H NMR (300 MHz, CDCl3): δ = 6.54
(s, 0.1 H, 5-H), 6.05 (d, J = 4.7 Hz, 0.1 H, 4-H), 3.74 (td, J = 7.1, 4.6 Hz,
129.1, 128.8, 128.6, 128.4, 127.9 (2 ×), 127.5, 91.9, 84.4, 72.4, 63.3,
62.6, 26.9, 19.3 ppm. HRMS (ESI): calcd. for C34H37N4O4Si [M + H]+
593.2579, found 593.2569. Data for 24a, 24b: Yield: 45 mg
0.1 H, 3-H) ppm. Partial 1H NMR data extracted from the mixture
1
for (3,4-cis-4,5-cis)-21b: H NMR (300 MHz, CDCl3): δ = 6.75 (d, J = (0.08 mmol, 8 %, 24a/24b, 15:85). Colourless oil. Partial 1H NMR
5.2 Hz, 0.9 H, 5-H), 5.85 (dd, J = 7.9, 5.2 Hz, 0.9 H, 4-H), 3.48 (td, J =
data extracted from the mixture for 24b: 1H NMR (300 MHz, CDCl3):
7.2, 5.5 Hz, 0.9 H, 3-H) ppm. MS (ESI + APCI): m/z = 672.2 δ = 5.71 (d, J = 5.2 Hz, 1 H, 5-H), 5.40 (dd, J = 6.8, 5.2 Hz, 1 H, 4-H),
(C41H42NO6Si) [M + H]+.
4.39 (d, AB, J = 13.4 Hz, 1 H, N-CH2Ph), 4.33 (d, AB, J = 13.4 Hz, 1 H,
N-CH2Ph), 3.87 (dd, AB, J = 10.7, 6.4 Hz, 1 H, CH2OSi), 3.78 (dd, AB,
J = 10.7, 5.1 Hz, 1 H, CH2OSi), 3.57 (td, J = 6.6, 5.1 Hz, 1 H, 3-H)
ppm. MS (ESI + APCI): m/z = 593.2 (C34H37N4O4Si) [M + H]+.
Typical Procedure for the Synthesis of 5-Azido-3-[(tert-butyldi-
phenylsilyloxy)methyl]-2-methylisoxazolidin-4-yl Benzoate (22,
23): The reaction flask was charged with isoxazolidines 18a,b and
19a,b (1.2 g, 2.01 mmol), sealed with a rubber septum, evacuated,
and filled with argon. Anhydrous CH3CN (8 mL) was added followed
by TMSN3 (535 μL, 4.03 mmol). The stirred solution was cooled
down to 0 °C, and TMSOTf (185 μL, 1.02 mmol) was added dropwise.
After 5 min, the reaction mixture was warmed up to room tempera-
ture and stirred for 24 h. When TLC showed that the reaction was
complete (hexanes/EtOAc, 9:1), the reaction was cooled down to
0 °C and quenched with sat. aq. NaHCO3 (5 mL). The mixture was
diluted with water (50 mL) and extracted with CH2Cl2 (3 × 25 mL).
The combined organic layers were dried with MgSO4, and the sol-
vent was evaporated in vacuo. The residue was purified by FCC
(hexanes/EtOAc, 95:5) to give one single isomer 22a along with a
mixture of three isomers 22b, 23a and 23b. Data for (3,4-trans-
4,5-trans)-22a: Yield: 570 mg (1.10 mmol, 55 %). Colourless oil. Rf =
Typical Procedure for the Synthesis of 3-[(tert-Butyldiphenyl-
silyloxy)methyl]-2-methyl-5-(4′-phenyl-1H-1,2,3-triazol-1-yl)-
isoxazolidin-4-yl Benzoate (26): To a stirred solution of azidoisox-
azolidine 22a (450 mg, 0.87 mmol) in tBuOH (1.5 mL) at room tem-
perature were added sequentially a solution of CuSO4·5H2O (22 mg,
0.09 mmol) in H2O (3 mL), sodium L-ascorbate (35 mg, 0.18 mmol)
and phenylacetylene (0.1 mL, 0.91 mmol). The reaction mixture was
stirred at room temperature for 12 h. When TLC showed that the
reaction was complete (hexanes/EtOAc, 7:3), the mixture was di-
luted with H2O (5 mL) and extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were dried with MgSO4, and the solvent
was evaporated under reduced pressure. The residue was purified
by FCC (hexanes/EtOAc, 8:2) to give isoxazolidinyl triazole 26
(500 mg, 0.81 mmol, 93 %) as a white solid. M.p. 53–56 °C. Rf = 0.41
(hexanes/EtOAc, 7:3). IR (ATR): νmax = 3070, 2930, 2857, 1725, 1428,
˜
0.31 (hexanes/EtOAc, 9:1). IR (ATR): νmax = 3071, 2958, 2930, 2857,
˜
1264, 1105, 1068, 1025, 971, 764, 692, 503, 487 cm–1
.
1H NMR
2108, 1725, 1266, 1238, 1105, 1069, 700, 503, 488 cm–1
.
1H NMR
(600 MHz, CDCl3): δ = 8.23 (s, 1 H, 5′-H), 8.06–8.03 (m, 2 H, H-Ph),
7.73–7.61 (m, 7 H, H-Ph), 7.49–7.47 (m, 2 H, H-Ph), 7.41–7.29 (m, 9
H, H-Ph), 6.39 (d, J = 1.2 Hz, 1 H, 5-H), 6.13 (dd, J = 5.8, 1.2 Hz, 1 H,
4-H), 4.05 (dd, AB, J = 11.4, 5.7 Hz, 1 H, CH2OSi), 4.01 (dd, AB, J =
11.4, 3.4 Hz, 1 H, CH2OSi), 3.08 (td, J = 5.7, 3.5 Hz, 1 H, 3-H), 2.94 (s,
3 H, N-CH3), 1.03 (s, 9 H, tBu) ppm. 13C NMR (150 MHz, CDCl3): δ =
165.4, 148.4, 135.7, 135.6, 133.9, 132.9, 132.7, 130.7, 130.1 (3 ×),
128.8 (2 ×), 128.7, 128.2, 128.0 (2 ×), 126.1, 118.9, 90.6, 83.7, 75.8,
61.6, 45.0, 27.0, 19.4 ppm. HRMS (ESI): calcd. for C36H39N4O4Si [M +
H]+ 619.2736, found 619.2732.
(600 MHz, CDCl3): δ = 8.01–7.98 (m, 2 H, H-Ph), 7.69–7.59 (m, 5 H,
H-Ph), 7.48–7.34 (m, 8 H, H-Ph), 5.35 (s, 1 H, 5-H), 5.23 (d, J = 4.9 Hz,
1 H, 4-H), 3.96–3.91 (m, 2 H, CH2OSi), 3.00–2.98 (m, 1 H, 3-H), 2.94
(s, 3 H, N-CH3), 1.04 (s, 9 H, tBu) ppm. 13C NMR (150 MHz, CDCl3):
δ = 165.6, 135.6 (2 ×), 133.6 (2 ×), 132.8 (2 ×), 129.9, 129.8, 128.9,
128.5, 127.8 (2 ×), 92.1, 84.7, 74.8, 62.9, 45.7, 26.7, 19.1 ppm. HRMS
(ESI): calcd. for C28H33N4O4Si [M + H]+ 517.2266, found 517.2261.
Data for 22b, 23a, 23b: Yield: 105 mg (0.20 mmol, 10 %, 22b/23a/
23b, 40:50:10). Colourless oil. Partial 1H NMR data extracted from
the mixture for 22b: 1H NMR (600 MHz, CDCl3): δ = 5.65 (d, J =
5.2 Hz, 0.4 H, 5-H), 5.32 (dd, J = 7.5, 5.2 Hz, 0.4 H, 4-H), 3.31 (ddd,
2-Benzyl-3-[(tert-butyldiphenylsilyloxy)methyl]-5-(4′-phenyl-
1H-1,2,3-triazol-1-yl)isoxazolidin-4-yl Benzoate (27): The typical
1
J = 7.5, 6.7, 4.5 Hz, 0.4 H, 3-H) ppm. Partial H NMR data extracted
from the mixture for 23a: 1H NMR (600 MHz, CDCl3): δ = 5.57 (d, procedure described above was applied using azidoisoxazolidine
J = 4.5 Hz, 0.5 H, 4-H), 5.47 (s, 0.5 H, 5-H), 3.41–3.34 (m, 0.5 H, 3-H)
24a (380 mg, 0.64 mmol), CuSO4·5H2O (17 mg, 0.07 mmol), H2O
-ascorbate (25 mg, 0.13 mmol), phenylacetylene
ppm. Partial 1H NMR data extracted from the mixture for 23b: 1H (1.5 mL), sodium
L
NMR (600 MHz, CDCl3): δ = 5.71 (dd, J = 7.4, 5.9 Hz, 0.1 H, 4-H), 5.50
(d, J = 5.8 Hz, 0.1 H, 5-H), 3.02–2.99 (m, 0.1 H, 3-H) ppm. MS (ESI +
APCI): m/z = 517.2 (C28H33N4O4Si) [M + H]+.
(70 μL, 0.64 mmol), and tBuOH (1.0 mL). TLC monitoring (hexanes/
EtOAc, 7:3). FCC (hexanes/EtOAc, 8:2) gave isoxazolidinyl triazole 27
(380 mg, 0.54 mmol, 84 %) as a colourless oil. Rf = 0.41 (hexanes/
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EtOAc, 7:3). IR (ATR): νmax = 3070, 2930, 2857, 1726, 1263, 1105, combined organic layers were dried with MgSO4, and the solvent
˜
1069, 1026, 761, 739, 593, 503, 488 cm–1. 1H NMR (600 MHz, CDCl3): was evaporated in vacuo. The residue was purified by FCC (EtOAc/
δ = 8.10 (s, 1 H, 5′-H), 8.07–8.04 (m, 2 H, H-Ph), 7.71–7.60 (m, 7 H,
H-Ph), 7.51–7.46 (m, 2 H, H-Ph), 7.40–7.26 (m, 14 H, H-Ph), 6.38 (s, 1 a white solid. M.p. 133–135 °C. Rf = 0.25 (EtOAc). IR (ATR): νmax
hexanes, 7:3) to give isoxazolidine 3 (200 mg, 0.72 mmol, 88 %) as
=
˜
H, 5-H), 6.20 (dd, J = 5.2, 1.1 Hz, 1 H, 4-H), 4.52 (d, AB, J = 14.6 Hz,
1 H, N-CH2Ph), 4.11–4.00 (m, 3 H, N-CH2Ph, CH2OSi), 3.37 (td, J =
5.6, 3.6 Hz, 1 H, 3-H), 1.01 (s, 9 H, tBu) ppm. 13C NMR (150 MHz,
CDCl3): δ = 165.4, 148.1, 136.5, 135.7, 135.6, 133.9, 132.9, 132.7,
130.7, 130.1 (3 ×), 128.9, 128.8, 128.7 (2 ×), 128.6, 128.2, 128.0 (2 ×),
127.7, 126.0, 118.8, 90.6, 83.5, 73.5, 62.1, 61.7, 26.9, 19.4 ppm. HRMS
(ESI): calcd. for C42H43N4O4Si [M + H]+ 695.3049, found 695.3042.
3471, 3151, 2882, 2837, 2568, 1357, 1237, 1159, 1079, 1032, 984,
1
763, 692, 658, 519 cm–1. H NMR (600 MHz, CD3OD): δ = 8.53 (s, 1
H, 5′-H), 7.84–7.82 (m, 2 H, H-Ph), 7.45–7.33 (m, 3 H, H-Ph), 6.10 (d,
J = 1.9 Hz, 1 H, 5-H), 4.97 (dd, J = 6.5, 1.9 Hz, 1 H, 4-H), 3.90 (dd,
AB, J = 12.2, 2.8 Hz, 1 H, CH2OH), 3.81 (dd, AB, J = 12.2, 5.2 Hz, 1 H,
CH2OH), 2.89 (s, 3 H, N-CH3), 2.77 (ddd, J = 6.5, 5.2, 2.8 Hz, 1 H, 3-
H) ppm. 13C NMR (150 MHz, CD3OD): δ = 149.1, 131.6, 130.0, 129.4,
126.7, 120.9, 94.6, 83.3, 79.1, 59.9, 45.0 ppm. HRMS (ESI): calcd. for
C13H17N4O3 [M + H]+ 277.1296, found 277.1299.
Typical Procedure for the Synthesis of 3-[(tert-Butyldiphenyl-
silyl)oxymethyl]-2-methyl-5-(4′-phenyl-1H-1,2,3-triazol-1-yl)is-
oxazolidin-4-ol (28): Isoxazolidinyl triazole 26 (560 mg, 0.91 mmol)
2-Benzyl-3-(hydroxymethyl)-5-(4′-phenyl-1H-1,2,3-triazol-1-yl)-
was dissolved in anhydrous methanol (9 mL), K2CO3 (40 mg, isoxazolidin-4-ol (4): The typical procedure described above was
0.29 mmol) was added, and the reaction mixture was stirred at
room temperature for 30 min. After this time, TLC showed that the
applied using isoxazolidine 29 (250 mg, 0.42 mmol), TBAF (0.63 mL,
0.63 mmol, 1 solution in THF), and anhydrous THF (4 mL). Reac-
M
starting isoxazolidine disappeared (hexanes/EtOAc, 7:3). The mix- tion time: 1.5 h. TLC monitoring (EtOAc). FCC (hexanes/EtOAc, 1:1)
ture was diluted with water (25 mL) and extracted with EtOAc
(4 × 10 mL). The combined organic layers were dried with MgSO4,
and the solvent was evaporated in vacuo. The residue was purified
by FCC (hexanes/EtOAc, 75:25) to give isoxazolidine 28 (400 mg,
gave isoxazolidine 4 (140 mg, 0.40 mmol, 95 %) as a white solid.
M.p. 156–157 °C. Rf = 0.42 (EtOAc). IR (ATR): νmax = 3091, 3051, 2861,
˜
1
2496, 1057, 1014, 961, 792, 767, 740, 693 cm–1. H NMR (300 MHz,
CD3OD): δ = 8.31 (s, 1 H, 5′-H), 7.78–7.73 (m, 2 H, H-Ph), 7.45–7.23
0.78 mmol, 86 %) as a white solid. M.p. 151–153 °C. Rf = 0.26 (hex- (m, 8 H, H-Ph), 6.09 (d, J = 1.7 Hz, 1 H, 5-H), 4.97 (dd, J = 6.2, 1.8 Hz,
anes/EtOAc, 7:3). IR (ATR): νmax = 3169, 2935, 2873, 1425, 1329, 1129, 1 H, 4-H), 4.50 (d, AB, J = 14.5 Hz, 1 H, N-CH2Ph), 4.02 (d, AB, J =
1H NMR (600 MHz, 14.5 Hz, 1 H, N-CH2Ph), 3.94 (dd, AB, J = 12.1, 3.3 Hz, 1 H, CH2OH),
˜
1106, 1082, 954, 763, 703, 646, 493, 459 cm–1
.
CDCl3): δ = 8.12 (s, 1 H, 5′-H), 7.68–7.63 (m, 6 H, H-Ph), 7.42–7.27 3.83 (dd, AB, J = 12.1, 5.6 Hz, 1 H, CH2OH), 3.06 (td, J = 5.8, 3.3 Hz,
(m, 9 H, H-Ph), 6.07 (d, J = 1.6 Hz, 1 H, 5-H), 5.18 (d, J = 4.7 Hz, 1 H,
OH), 4.89 (ddd, J = 6.6, 4.7, 1.6 Hz, 1 H, 4-H), 3.91 (dd, AB, J = 11.3,
3.4 Hz, 1 H, CH2OSi), 3.87 (dd, AB, J = 11.3, 6.0 Hz, 1 H, CH2OSi),
2.94 (td, J = 6.3, 3.4 Hz, 1 H, 3-H), 2.91 (s, 3 H, N-CH3), 1.00 (s, 9 H,
tBu) ppm. 13C NMR (150 MHz, CDCl3): δ = 147.7, 135.5 (2 ×), 132.8,
132.7, 130.1, 129.9 (2 ×), 128.8, 128.2, 127.9 (2 ×), 125.7, 118.6, 93.6,
83.4, 77.2, 61.8, 45.1, 26.8, 19.2 ppm. HRMS (ESI): calcd. for
1 H, 3-H) ppm. 13C NMR (75 MHz, CD3OD): δ = 148.8, 138.4, 131.6,
130.0 (2 ×), 129.4, 129.2, 128.4, 126.6, 120.7, 94.6, 83.2, 76.5, 62.3,
60.5 ppm. HRMS (ESI): calcd. for C19H21N4O3 [M + H]+ 353.1609,
found 353.1608.
[4-Acetoxy-2-methyl-5-(4′-phenyl-1H-1,2,3-triazol-1-yl)isoxaz-
olidin-3-yl]methyl Acetate (30): To a stirred solution of isoxazol-
idine 4 (90 mg, 0.33 mmol) in anhydrous CH2Cl2 (3 mL) at 0 °C
were added sequentially DMAP (9 mg, 0.07 mmol), Et3N (0.185 mL,
1.33 mmol) and Ac2O (95 μL, 1.00 mmol). The reaction mixture was
warmed up to room temperature and stirred for 1 h. After this time,
TLC showed that the reaction was complete (hexanes/EtOAc, 1:1).
C
29H35N4O3Si [M + H]+ 515.2473, found 515.2478.
2-Benzyl-3-[(tert-butyldiphenylsilyloxy)methyl]-5-(4′-phenyl-
1H-1,2,3-triazol-1-yl)isoxazolidin-4-ol (29): The typical procedure
described above was applied using isoxazolidinyl triazole 27
(180 mg, 0.26 mmol), K2CO3 (12 mg, 0.09 mmol), and anhydrous The mixture was quenched with sat. aq. NaHCO3 (3 mL), diluted
methanol (3 mL). TLC monitoring (hexanes/EtOAc, 7:3). FCC (hex- with water (5 mL) and extracted with CH2Cl2 (3 × 5 mL). The com-
anes/EtOAc, 8:2) gave isoxazolidine 29 (140 mg, 0.24 mmol, 92 %)
as a white solid. M.p. 165–166 °C. Rf = 0.25 (hexanes/EtOAc, 7:3). IR
bined organic layers were dried with MgSO4, and the solvent was
evaporated in vacuo. The residue was purified by FCC (hexanes/
EtOAc, 7:3) to give isoxazolidine 30 (115 mg, 0.32 mmol, 97 %) as a
(ATR): νmax = 3171, 2927, 2872, 1428, 1095, 958, 693, 603, 505,
˜
487 cm–1
.
1H NMR (300 MHz, CDCl3): δ = 7.91 (s, 1 H, 5′-H), 7.64– white solid. M.p. 141–143 °C. Rf = 0.25 (hexanes/EtOAc, 1:1). IR (ATR):
7.59 (m, 6 H, H-Ph), 7.41–7.26 (m, 14 H, H-Ph), 6.05 (d, J = 0.6 Hz, 1
H, 5-H), 5.25 (bs, 1 H, OH), 4.94 (d, J = 6.1 Hz, 1 H, 4-H), 4.52 (d, AB,
J = 14.6 Hz, 1 H, N-CH2Ph), 4.03 (d, AB, J = 14.6 Hz, 1 H, N-CH2Ph),
3.95 (dd, AB, J = 11.2, 3.5 Hz, 1 H, CH2OSi), 3.88 (dd, AB, J = 11.2,
6.1 Hz, 1 H, CH2OSi), 3.23 (td, J = 6.1, 3.6 Hz, 1 H, 3-H), 0.98 (s, 9 H,
tBu) ppm. 13C NMR (75 MHz, CDCl3): δ = 147.4, 137.1, 135.6 (2 ×),
132.8 (2 ×), 130.3, 130.1 (2 ×), 129.0, 128.8, 128.5, 128.3, 128.0 (2 ×),
127.7, 125.7, 118.7, 93.7, 83.4, 75.1, 62.3, 61.9, 26.9, 19.3 ppm. HRMS
(ESI): calcd. for C35H39N4O3Si [M + H]+ 591.2786, found 591.2782.
νmax = 3131, 2988, 1739, 1369, 1235, 1222, 1025, 969, 964, 596,
˜
548 cm–1 1H NMR (600 MHz, CDCl3): δ = 8.14 (s, 1 H, 5′-H), 7.86–
.
7.84 (m, 2 H, H-Ph), 7.44–7.33 (m, 3 H, H-Ph), 6.26 (d, J = 0.9 Hz, 1
H, 5-H), 5.95 (dd, J = 5.1, 1.1 Hz, 1 H, 4-H), 4.49 (dd, AB, J = 12.1,
3.3 Hz, 1 H, CH2OAc), 4.29 (dd, AB, J = 12.1, 5.1 Hz, 1 H, CH2OAc),
3.03 (td, J = 5.1, 3.3 Hz, 1 H, 3-H), 2.93 (s, 3 H, N-CH3), 2.18 (s, 3 H,
COCH3), 1.99 (s, 3 H, COCH3) ppm. 13C NMR (150 MHz, CDCl3): δ =
170.3, 169.8, 148.1, 130.4, 128.8, 128.3, 125.8, 118.6, 90.2, 83.4, 73.3,
60.5, 44.5, 20.7, 20.6 ppm. HRMS (ESI): calcd. for C17H21N4O5 [M +
H]+ 361.1507, found 361.1500.
Typical Procedure for the Synthesis of 3-(Hydroxymethyl)-2-
methyl-5-(4′-phenyl-1H-1,2,3-triazol-1-yl)isoxazolidin-4-ol (3):
The reaction flask with isoxazolidine 28 (420 mg, 0.82 mmol) was
sealed with a rubber septum, evacuated, and filled with argon. An-
hydrous THF (8 mL) was added, the stirred solution was cooled
tert-Butyl 3-[(tert-Butyldiphenylsilyloxy)methyl]isoxazole-
2(3H)-carboxylate (35): 5-Hydroxyisoxazolidine 34 (1.32 g,
2.88 mmol) was placed in a reaction flask, which was subsequently
sealed with a rubber septum, evacuated, and filled with argon. An-
hydrous NMP (30 mL) was added, the resulting solution was cooled
down to –20 °C (ice/NaCl-bath). 2-Chloropyridine (1.91 mL,
20.2 mmol) and Tf2O (0.63 mL, 3.74 mmol) were added. The reaction
mixture was warmed to room temperature. When the starting mate-
rial disappeared (20 h; TLC, hexanes/EtOAc; 8:2), the reaction mix-
down to 0 °C, and TBAF (1.23 mL, 1.23 mmol, 1
M in solution THF)
was added. The reaction mixture was warmed up to room tempera-
ture and stirred for 5 h. After this time, TLC showed that the reaction
was complete (hexanes/EtOAc, 1:1). The mixture was diluted with
sat. aq. NaHCO3 (10 mL) and extracted with EtOAc (4 × 15 mL). The
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ture was diluted with H2O (300 mL), and sat. aq. NaHCO3 (20 mL),
and the product was extracted into Et2O (3 × 50 mL). The combined
organic layers were dried with MgSO4, and the solvent was evapo-
rated in vacuo. The residue was purified by FCC (hexanes/EtOAc,
(m, 1 H, 3-H), 2.41 (d, J = 10.4 Hz, 1 H, OH), 1.47 (s, 9 H, tBuO), 1.06
(s, 9 H, tBuSi) ppm. 13C NMR (75 MHz, CDCl3): δ = 135.8, 135.6,
133.3, 132.9, 130.0 (2 ×), 127.9 (2 ×), 97.5, 83.1, 76.5, 63.6, 62.3, 28.2,
26.9, 19.4 ppm (the signal concerning to the carbonyl carbon is
95:5) to give 2,3-dihydroisoxazole 35 (670 mg, 1.52 mmol, 53 %) as missing). MS (ESI + APCI): m/z = 492.2 (C25H3535ClNO5Si) [M + H]+
a colourless oil. Rf = 0.43 (hexanes/EtOAc, 8:2). IR (ATR): νmax = 3074,
492.2.
˜
2931, 2857, 1712, 1367, 1111, 1079, 822, 741, 699, 613, 502,
1
487 cm–1. H NMR (300 MHz, CDCl3): δ = 7.69–7.64 (m, 4 H, H-Ph),
tert-Butyl 5-Azido-3-[(tert-butyldiphenylsilyloxy)methyl]-4-
hydroxyisoxazolidine-2-carboxylate (38): The reaction flask with
5-chloroisoxazolidines 37a,b (525 mg, 1.07 mmol) and NaN3
(210 mg, 3.23 mmol) was sealed with a rubber septum, evacuated,
and filled with argon. Anhydrous CH3CN (11 mL) was added, and
the resulting mixture was heated at 50 °C and stirred for 18 h. When
TLC showed that the reaction was complete (hexanes/EtOAc, 8:2),
the mixture was diluted with water (50 mL) and sat. aq. NaHCO3
(10 mL) and extracted with CH2Cl2 (3 × 15 mL). The combined or-
ganic layers were dried with MgSO4, and the solvent was evapo-
rated in vacuo. The residue was purified by FCC (hexanes/EtOAc,
9:1) to give two single 5-azidoisoxazolidines 38a and 38b. Data for
(3,4-trans-4,5-trans)-38a: Yield: 305 mg (0.61 mmol, 57 %). Colour-
7.43–7.36 (m, 6 H, H-Ph), 6.57 (dd, J = 3.1, 2.0 Hz, 1 H, 5-H), 5.09
(dd, J = 3.1, 2.4 Hz, 1 H, 4-H), 5.01–4.96 (m, 1 H, H-3), 3.74 (dd, AB,
J = 9.9, 4.7 Hz, 1 H, CH2OSi), 3.64 (dd, AB, J = 9.9, 6.0 Hz, 1 H,
CH2OSi), 1.48 (s, 9 H, tBuO), 1.05 (s, 9 H, tBuSi) ppm. 13C NMR
(75 MHz, CDCl3): δ = 156.6, 142.9, 135.7 (2 ×), 133.6 (2 ×), 129.8 (2
×), 127.8 (2 ×), 99.2, 82.7, 66.2, 65.7, 28.3, 26.9, 19.5 ppm. HRMS
(ESI): calcd. for C25H34NO4Si [M + H]+ 440.2252, found 440.2244.
tert-Butyl 4-[(tert-Butyldiphenylsilyloxy)methyl]-2,6-dioxa-3-
azabicyclo[3.1.0]hexane-3-carboxylate (36): Solid NaHCO3
(1.25 g, 14.98 mmol) was placed in a reaction flask, and water was
added (4.5 mL), followed by acetone (7 mL). The resulting mixture
was cooled down to 0 °C and stirred for 20 min. Oxone (1.32 g,
2.14 mmol) was added in one portion, and the suspension was
stirred at 0 °C for further 15 min. 2,3-Dihydroisoxazole 35 (470 mg,
1.07 mmol) was then added in one portion. The mixture was
warmed up to room temperature and stirred for additional 30 min.
After this time, TLC showed that the reaction was complete (hex-
anes/EtOAc, 8:2). The reaction mixture was diluted with water
(50 mL) and extracted with CH2Cl2 (3 × 10 mL). The combined or-
ganic layers were dried with MgSO4, and the solvent was evapo-
rated in vacuo, to give isoxazolidinyl epoxide 36 (485 mg,
1.06 mmol, 99 %) as a colourless oil of satisfactory purity. Rf = 0.38
less oil. Rf = 0.16 (hexanes/EtOAc, 8:2). IR (ATR): νmax = 3401,
˜
2931, 2858, 2112, 1709, 1248, 1111, 1056, 823, 739, 700, 613, 502,
1
487 cm–1. H NMR (300 MHz, CDCl3): δ = 7.69–7.64 (m, 4 H, H-Ph),
7.44–7.36 (m, 6 H, H-Ph), 5.48 (d, J = 1.4 Hz, 1 H, 5-H), 4.49 (bs, 1 H,
4-H), 4.12 (ddd, J = 9.2, 6.0, 1.4 Hz, 1 H, 3-H), 3.93 (dd, AB, J = 10.2,
6.0 Hz, 1 H, CH2OSi), 3.76 (dd, AB, J = 10.2, 9.3 Hz, 1 H, CH2OSi),
2.57 (bs, 1 H, OH), 1.44 (s, 9 H, tBuO), 1.07 (s, 9 H, tBuSi) ppm. 13C
NMR (75 MHz, CDCl3): δ = 156.9, 135.7 (2 ×), 133.2 (2 ×), 130.0 (2
×), 127.9 (2 ×), 95.6, 83.1, 79.4, 69.4, 62.5, 28.2, 27.0, 19.4 ppm. HRMS
(ESI): calcd. for C25H35N4O5Si [M + H]+ 499.2372, found 499.2364.
Data for (3,4-trans-4,5-cis)-38b: Yield: 125 mg (0.25 mmol, 23 %).
(hexanes/EtOAc, 8:2). IR (ATR): ν
= 2931, 2858, 1715, 1320, 1111,
˜
max
Colourless oil. Rf = 0.22 (hexanes/EtOAc, 8:2). IR (ATR): νmax = 3403,
˜
1057, 822, 738, 700, 612, 503, 488 cm–1. H NMR (600 MHz, CDCl3):
δ = 7.66–7.63 (m, 4 H, H-Ph), 7.46–7.37 (m, 6 H, H-Ph), 5.38 (d, J =
1.5 Hz, 1 H, 5-H), 4.63 (bs, 1 H, 3-H), 3.91 (d, J = 1.8 Hz, 1 H, 4-H),
3.81 (dd, AB, J = 10.7, 5.1 Hz, 1 H, CH2OSi), 3.67 (dd, AB, J = 10.7,
1
2931, 2857, 2118, 1709, 1255, 1111, 978, 740. 700, 609, 502,
1
487 cm–1. H NMR (600 MHz, CDCl3): δ = 7.68–7.65 (m, 4 H, H-Ph),
7.44–7.35 (m, 6 H, H-Ph), 5.58 (d, J = 5.1 Hz, 1 H, 5-H), 4.64 (dt, J =
9.3, 5.3 Hz, 1 H, 4-H), 4.00–3.78 (m, 3 H, 3-H, CH2OSi), 2.24 (d, J =
9.5 Hz, 1 H, OH), 1.50 (s, 9 H, tBuO), 1.06 (s, 9 H, tBuSi) ppm. 13C
NMR (150 MHz, CDCl3): δ = 158.0, 135.6, 135.5, 133.2, 132.9, 129.8
(2 ×), 127.8, 127.7, 92.0, 82.7, 76.0, 65.1, 63.2, 28.0, 26.8, 19.3 ppm.
HRMS (ESI): calcd. for C25H35N4O5Si [M + H]+ 499.2372, found
499.2368.
7.2 Hz, 1 H, CH2OSi), 1.48 (s, 9 H, tBuO), 1.07 (s, 9 H, tBuSi) ppm. 13
C
NMR (150 MHz, CDCl3): δ = 135.7 (2 ×), 133.0 (2 ×), 130.1 (2 ×),
128.0 (2 ×), 82.8, 80.3, 62.3, 62.0, 58.4, 28.2, 26.9, 19.3 ppm (the
signal corresponding to the carbonyl carbon is missing). HRMS (ESI):
the calcd. value for C25H33NO5+H+: 456.2201 was not found, how-
ever, the NMR spectroscopic data are in good agreement with those
reported earlier for related isoxazolidinyl epoxides.[20,22]
tert-Butyl 3-[(tert-Butyldiphenylsilyloxy)methyl]-4-hydroxy-5-
(4-phenyl-1H-1,2,3-triazol-1-yl)isoxazolidine-2-carboxylate (39):
To a stirred solution of azidoisoxazolidine 38a (315 mg, 0.63 mmol)
tert-Butyl 3-[(tert-Butyldiphenylsilyloxy)methyl]-5-chloro-4-
hydroxyisoxazolidine-2-carboxylate (37): To a stirred solution of
epoxide 36 (485 mg, 1.06 mmol) in THF (11 mL) at room tempera- in tBuOH (0.7 mL) at room temperature were added sequentially
ture were added sequentially LiCl (72 mg, 1.70 mmol) and acetic
acid (0.188 mL, 3.23 mmol). The reaction mixture was stirred at
room temperature for 2 h. After this time, TLC showed that the
a solution of CuSO4·5H2O (17 mg, 0.07 mmol) in H2O (1.4 mL), so-
dium -ascorbate (25 mg, 0.13 mmol) and phenylacetylene (70 μL,
0.64 mmol). The reaction mixture was stirred at room temperature
L
reaction was complete (hexanes/EtOAc, 8:2). The mixture was di- for 17 h. After this time, TLC showed that the reaction was complete
luted with water (80 mL), treated carefully with sat. aq. NaHCO3
(10 mL) and extracted with CH2Cl2 (3 × 15 mL). The combined or-
(hexanes/EtOAc, 7:3). The mixture was diluted with water (15 mL)
and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers
ganic layers were dried with MgSO4, and the solvent was evapo- were dried with MgSO4, and the solvent was evaporated under re-
rated under reduced pressure, to give inseparable anomeric mixture
of 5-chloroisoxazolidines 37a,b (495 mg, 1.01 mmol, 95 %, 37a/37b,
70:30) as a colourless oil. The resulting crude product was directly
duced pressure. The residue was purified by FCC (hexanes/EtOAc,
8:2) to give isoxazolidinyl triazole 39 (345 mg, 0.57 mmol, 90 %) as
a white solid. M.p. 121–123 °C. Rf = 0.21 (hexanes/EtOAc, 7:3). IR
used for the next step without further purification. Rf = 0.29 (hex- (ATR): ν
= 3419, 2931, 2857, 1712, 1306, 1115, 1038, 765, 692,
˜
max
anes/EtOAc, 8:2). NMR data of the thermodynamically more stable
product (3,4-trans-4,5-cis)-37a extracted from the corresponding
anomeric mixture: 1H NMR (300 MHz, CDCl3): δ = 7.69–7.65 (m, 4
H, H-Ph), 7.43–7.36 (m, 6 H, H-Ph),6.28 (d, J = 4.3 Hz, 1 H, 5-H), 4.88
(ddd, J = 10.4, 7.3, 4.3 Hz, 1 H, 4-H), 4.01 (dd, AB, J = 10.6, 3.1 Hz, 1
H, CH2OSi), 3.90 (dd, AB, J = 10.6, 4.3 Hz, 1 H, CH2OSi), 3.84–3.78
612, 505, 488 cm–1 1H NMR (300 MHz, CDCl3): δ = 7.88 (s, 1 H,
.
5′-H), 7.68–7.58 (m, 6 H, H-Ph), 7.41–7.28 (m, 9 H, H-Ph), 6.19 (d, J =
2.7 Hz, 1 H, 5-H), 5.45 (dt, J = 4.5, 2.7 Hz, 1 H, 4-H), 4.74 (bs, 1 H,
OH), 4.29 (ddd, J = 7.2, 5.6, 2.7 Hz, 1 H, 3-H), 3.90 (dd, AB, J = 10.5,
5.6 Hz, 1 H, CH2OSi), 3.71 (dd, AB, J = 10.5, 7.2 Hz, 1 H, CH2OSi),
1.46 (s, 9 H, tBuO), 1.00 (s, 9 H, tBuSi) ppm. 13C NMR (75 MHz,
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CDCl3): δ = 157.0, 148.1, 135.6 (2 ×), 133.1, 133.0, 130.0 (2 ×), 129.7,
0.0180) which were used in all calculations. The final R1 was 0.0320
129.0, 128.7, 127.9 (2 ×), 125.9, 118.9, 94.1, 83.6, 80.1, 69.8, 62.5, [I > 2σ(I)] and wR2 was 0.0847 (all data).
28.2, 26.9, 19.4 ppm. HRMS (ESI): calcd. for C33H41N4O5Si [M + H]+
601.2841, found 601.2835.
Deposition Number 1997748 (for 3) contains the supplementary
crystallographic data for this paper. These data are provided free of
charge by the joint Cambridge Crystallographic Data Centre and
Fachinformationszentrum Karlsruhe Access Structures service
www.ccdc.cam.ac.uk/structures.
tert-Butyl 4-Hydroxy-3-(hydroxymethyl)-5-(4-phenyl-1H-1,2,3-
triazol-1-yl)isoxazolidine-2-carboxylate (40): The reaction flask
with isoxazolidine 39 (185 mg, 0.31 mmol) was sealed with a rubber
septum, evacuated, and filled with argon. Anhydrous THF (3 mL)
was added, the resulting solution was cooled down to 0 °C, and
Hirshfeld surface analysis: CrystalExplorer[31] was used to calcu-
late Hirshfeld surface and associated fingerprint plots.[32]
TBAF (0.47 mL, 0.47 mmol, 1
M solution in THF) was added. The
Cell Culture and Cultivation Conditions
reaction mixture was stirred at 0 °C for 1 h. After this time, TLC
showed that the reaction was complete (hexanes/EtOAc, 1:1). The
mixture was diluted with water (25 mL) and sat. aq. NaHCO3 (5 mL)
and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers
were dried with MgSO4, and the solvent was evaporated in vacuo.
The residue was purified by FCC (hexanes/EtOAc, 1:1) to give isox-
azolidine 40 (90 mg, 0.25 mmol, 80 %) as a white solid. M.p. 164–
In this study, AML cell line MOLM-13 (ACC 554) was used. This cell
line was derived from the peripheral blood of a 20-year-old patient
with AML developed from myelodysplastic syndromes supplied by
Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH, Germany). Cell line was cultured in 5 mL
of RPMI medium (5 × 105 cells) containing 12 % fetal bovine serum
(Biotech, SR),100 000 units/L penicillin and 50 mg/L streptomycin
(both from Sigma-Aldrich, USA) for one or two days at 37 °C in a
humidified atmosphere containing 5 % CO2.
166 °C. Rf = 0.11 (hexanes/EtOAc, 1:1). IR (ATR): νmax = 3215, 2986,
˜
2917, 1733, 1464, 1294, 1158, 1030, 963, 836, 771, 698 cm–1
.
1H
NMR (600 MHz, CD3OD): δ = 8.58 (s, 1 H, 5′-H), 7.86–7.84 (m, 2 H,
H-Ph), 7.46–7.35 (m, 3 H, H-Ph), 6.20 (d, J = 2.9 Hz, 1 H, 5-H), 5.22
(dd, J = 2.9, 2.2 Hz, 1 H, 4-H), 4.20 (ddd, J = 6.7, 6.2, 2.2 Hz, 1 H, 3-
H), 3.84 (dd, AB, J = 11.5, 6.2 Hz, 1 H, CH2OH), 3.77 (dd, AB, J = 11.5,
6.7 Hz, 1 H, CH2OH), 1.54 (s, 9 H, tBuO) ppm. 13C NMR (150 MHz,
CD3OD): δ = 159.3, 149.3, 131.2, 130.0, 129.6, 126.8, 122.1, 95.6, 84.5,
80.3, 72.4, 61.5, 28.3 ppm. HRMS (ESI): calcd. for C17H23N4O5 [M +
H]+ 363.1663, found 363.1668.
Estimation of Metabolic Activity Using MTS Assay
We cultured the cell line MOLM-13 in absence or in presence of
different concentrations of prepared isoxazolidines (0; 0.5; 1; 5; 10;
20; 100 μ
10 m stock solutions in 1 % DMSO, compound 4 as 5 m
solution in 30 % DMSO and compound 40 as 2.5 m stock solution
M) for 48 h. Compounds 3, 5 and 30 were prepared as
M
M
stock
M
in 50 % DMSO. After this time, cell metabolic activity was assessed
using CellTiter 96® AQueous One Solution Cell Proliferation Assay
(MTS, ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium inner salt])) (Promega, USA) accord-
ing to the manufacturer′s protocol.
3-Hydroxymethyl-5-(4-phenyl-1H-1,2,3-triazol-1-yl)isoxazolidin-
4-ol (5): To a solution of isoxazolidine 40 (90 mg, 0.25 mmol) in
CH2Cl2 (2 mL) at 0 °C was added TFA (0.39 mL, 5.06 mmol), and the
reaction mixture was stirred at 0 °C for 30 min and then stirred for
additional 1 h allowing to reach room temperature. When TLC
showed that the reaction was complete (hexanes/EtOAc, 2:3), the
mixture was cooled down to 0 °C, diluted with water (10 mL) and
treated carefully with sat. aq. NaHCO3 (10 mL). The organic layer
was separated, and the aqueous layer was extracted with EtOAc
(3 × 10 mL). The combined organic layers were dried with MgSO4,
and the solvent was evaporated in vacuo. The residue was purified
by FCC (CH2Cl2/MeOH, 95:5) to give N-unprotected isoxazolidine 5
(50 mg, 0.19 mmol, 76 %) as a white solid. M.p. 144–146 °C. Rf =
Detection of Cell Death in MOLM-13 Cells After Treatment with
Isoxazolidines Using Double Staining with FITC-Annexin V and
Propidium Iodide
After treatment with 0; 0.5; 1; 5; 10; 20; 100 μ
M concentration of
individual isoxazolidines, cells (1 × 106 cells/mL) were incubated for
48 h under standard culture conditions. Subsequently, the propor-
tion of viable, apoptotic, and necrotic cells was detected using an
annexin V/propidium iodide kit (Calbiochem, USA). Cells were
washed twice with PBS and gently re-suspended in binding buffer
containing 0.5 μg/mL FITC-labelled annexin V. The mixtures were
incubated for 15 min at room temperature in the dark and then
centrifuged (2500 rpm, 15 min). The resulting sediments were re-
suspended in binding buffer, and propidium iodide (final concentra-
tion 0.6 μg/mL) was added to each sample after which the samples
were analyzed by flow cytometry using an Accuri C6 flow cytometer
(BD Bioscience, USA).
0.17 (EtOAc). IR (ATR): νmax = 3193, 2937, 2881, 2370, 1434, 1328,
˜
1154, 1083, 964, 803, 759, 688, 518, 495 cm–1
.
1H NMR (300 MHz,
CD3OD): δ = 8.59 (s, 1 H, 5′-H), 7.86–7.82 (m, 2 H, H-Ph), 7.47–7.32
(m, 3 H, H-Ph), 6.22 (d, J = 2.3 Hz, 1 H, 5-H), 5.13 (dd, J = 5.4, 2.3 Hz,
1 H, 4-H), 3.89 (d, J = 3.6 Hz, 2 H, CH2OH), 3.50 (dd, J = 10.0, 4.9 Hz,
1 H, 3-H) ppm. 13C NMR (150 MHz, CD3OD): δ = 149.0, 131.4, 130.0,
129.5, 126.7, 121.6, 97.9, 82.4, 71.2, 58.8 ppm. HRMS (ESI): calcd. for
C12H15N4O3 [M + H]+ 263.1139, found 263.1141.
Crystallography: Data collection and cell refinement for 3 was car-
ried out using Eulerian-crandle diffractometer Stoe StadiVari using
Pilatus3R 300K HPAD detector at 100 K, and using CuKα radiation
(λ = 1.54186 Å, microfocus source Xenocs Genix3D Cu HF) was used
for the measurement. The diffraction intensities were corrected for
Lorentz, polarization, and absorption effects. The structure was
solved by the direct method using SHELXT,[28] refined by a full-
matrix least-squares procedure with the SHELXL (ver. 2018/3),[29]
and drawn with the OLEX2 package.[30]
Acknowledgments
This work was supported by the Slovak Grant Agency for
Science VEGA (project nos. 1/0552/18, 2/0057/18), and the Re-
search and Development Operational Programmes funded by
the ERDF (ITMS project nos. 26240120001 and 26240120025).
This article was also created with the support of the MŠVVaŠ
of the Slovak Republic within the Research and Development
Operational Programme for the project “University Science Park
of STU Bratislava” (ITMS project no. 26240220084) co-funded by
the European Regional Development Fund.
Crystal Data for 3: C13H16N4O3 (M = 276.30 g/mol): monoclinic,
space group P21/n (no. 14), a = 10.8835(5) Å, b = 10.2458(3) Å, c =
12.3935(6) Å, ꢀ = 110.306(4)°, V = 1296.11(10) Å3, Z = 4, T = 100 K,
μ(CuKα) = 0.859 mm–1, Dcalc = 1.416 g/cm3, 14173 reflections meas-
ured (9.338° ≤ 2Θ ≤ 142.736°), 2469 unique (Rint = 0.0306, Rsigma
=
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Keywords: Diastereoselectivity · Heterocycles · Synthetic
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Received: May 25, 2020
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