Synthesis and biological evaluation of fluoro-substituted spiro-isoxazolines as potential anti-viral and anti-cancer agents

Article abstract of DOI:10.1039/d0ra06148d

Electrophilic fluorine-mediated dearomative spirocyclization has been developed to synthesize a range of fluoro-substituted spiro-isoxazoline ethers and lactones. The in vitro biological assays of synthesized compounds were probed for anti-viral activity

Full text of DOI:10.1039/d0ra06148d

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PAPER
Synthesis and biological evaluation of fluoro-
substituted spiro-isoxazolines as potential anti-
Cite this: RSC Adv., 2020, 10, 30223
viral and anti-cancer agents†
a
b
Sarah Boone,b Dipanwita Mitra,
Lindsay Turner,c
*
Prasanta Das,
Ritesh Tandon,
b
a
Drazen Raucherc and Ashton T. Hamme, II
*
Electrophilic fluorine-mediated dearomative spirocyclization has been developed to synthesize a range of
fluoro-substituted spiro-isoxazoline ethers and lactones. The in vitro biological assays of synthesized
compounds were probed for anti-viral activity against human cytomegalovirus (HCMV) and cytotoxicity
against glioblastomas (GBM6) and triple negative breast cancer (MDA MB 231). Interestingly, compounds
4d and 4n showed significant activity against HCMV (IC50 ꢀ 10 mM), while 4l and 5f revealed the highest
cytotoxicity with IC50 ¼ 36 to 80 mM. The synthetic efficacy and biological relevance offer an
opportunity to further drug-discovery development of fluoro-spiro-isoxazolines as novel anti-viral and
anti-cancer agents.
Received 14th July 2020
Accepted 6th August 2020
DOI: 10.1039/d0ra06148d
rsc.li/rsc-advances
congeners as potential leads for anti-cancer, anti-HCMV, and
anti-GBM6 agents.11
Introduction
It is worth mentioning that the introduction of uorine in
a biologically active molecules can dramatically modify
several parameters, such as acidity, basicity, and dipole
moments, delivering its drug-like properties, such as lip-
ophilicity, metabolic stability, and bioavailability of mole-
cules.12 As a result, these unique properties of the uorine
atom make it relevant in pharmaceutical,12,13 agrochem-
ical,12,14 and material sciences.15 Hence, inspired by the
medicinal relevance of spiro-isoxazolines and uorine, we
herein report an electrophilic uorine-mediated dearomative
spirocyclization to various uoro-spiro-isoxazolines and
probe their biological activity (Scheme 1). The novelty of the
current method includes the identity of effective uoro-
cyclization, the exploration of the chemistry for several
uoro-spiro-isoxazolines, and their anti-viral and anti-cancer
activities (Scheme 1).
Electrophilic uorination has led to a powerful tool to
access various uorinated hetero- and carbocycles.16 More-
over, electrophilic dearomative-uorination17 of aromatic
compounds has received much attention since they produce
3D-molecular skeletons with two consecutive stereogenic
centers. Though the mechanism of electrophilic uorination
remains controversial to date, isoxazole can act like a glycal
system,16c,18 in which an oxonium ion mediated electrophilic
uorocyclization leads to the desired spirocyclization. If
successful, this strategy would serve as a practical route to
readily access a range of uoro-spiro-isoxazolines in order to
probe biological activity.
Spiro-heterocycles are prevalent in plant and animal domains
and have become the cornerstone of pharmaceutical and
medicinal chemistry.1 While their conformations, chirality,
and structural implications are suitable for biological systems,
a diverse group of spiro-heterocycles has widely been used in
the drug-discovery process.1 Among heterocycles, the signi-
cance of isoxazole2 has been driven by a broader biological
spectrum, including analgesic,3 anti-bacterial,4 anti-depres-
sant,5 anti-cancer,6 and anti-herpetic7 of activities; it is worth
mentioning that the synthesis of isoxazoles and isoxazolines is
therefore receiving attention from organic chemists.2c In this
context, the spiro-architecture represents a potential avenue
for a diverse range of siblings8 for further synthetic and bio-
logical exploration. Moreover, the unique spiro-isoxazoline
core, found in bromotyrosine derived marine natural prod-
ucts, delivering a wide range of biological activities,9,10 has
attracted much synthetic attention. Due to their outstanding
biological prole in combination with an intriguing molecular
structure, we have focussed our research interest toward the
synthesis of spiro-isoxazoline natural products and its
aDepartment of Chemistry and Biochemistry, Jackson State University, Jackson,
Mississippi 39217, USA. E-mail: prasanta.das@jsums.edu; Tel: +1-601-203-5149
bDepartment of Microbiology and Immunology, University of Mississippi Medical
Center, Jackson, Mississippi 39216, USA
cDepartment of Cellular and Molecular Biology, University of Mississippi Medical
Center, Jackson, Mississippi 39216, USA
† Electronic supplementary information (ESI) available: Copies of 1H, 13C, and 19
F
In the line with our continued interest in the synthesis of
spiro-isoxazoline (Scheme 1),11 we herein report a two-step
NMR for synthesized compounds have been included. See DOI:
10.1039/d0ra06148d
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Table 1 Optimization of reaction conditions for (ꢂ)-5aa
Entry
F+-source
Solvent
Timeb (h)
Yieldc (%)
1
2
3
4
5
6
7
8
Selectuor
Selectuor
Selectuor
Selectuor
Selectuor
Selectuor
Selectuor
Selectuor
Selectuor
Selectuor
NFSI
CH2CI2
CHCI3
DCE
THF
Ether
Ethyl acetate
Acetone
CH3CN
MeOH
CF3CH2OH
CH3CN
CH3CN
24 h
24 h
24
12 h
24
24 h
24 h
24 h
24 h
24 h
24 h
24 h
—
—
10
—
—
—
—
90
85
10
—
—
9
10
11
12
NFOBS
Scheme 1 Synthesis of 4-halo-spiro-isoxazoline derivatives.
a
General conditions: isoxazoline acid 3p (0.2 mmol, 1.0 equiv.) and F-
method that includes 1,3-dipolar cycloaddition and electro-
philic uoro-cyclization to access uorinated variants of this
important motifs. The effectiveness of this strategy is also
examined using various substituted nitrile oxides and alkynes
to obtain a diverse group.
b
source (1.5 equiv.) in 2 mL solvent was reuxed at 80 ꢁC. Time
required for the reaction.
c
Based on isolated product aer
purication by chromatography.
the desired reaction was completely absent (entry 10, Table 1).
We speculated that due to the electronegativity of the tri-
uoromethyl group in TFE, the corresponding alcohol exhibits
a stronger acidic character compared to methanol; thereby,
plausible complexation with isoxazole, protonation of alkoxide
or carboxylate anion was detrimental to reactivity. At this point,
based on the reaction time, we chose CH3CN as our optimized
solvent for the desired reaction. To further our investigation, we
used two other uorine sources that are also known for uori-
nation.19 In doing so, we performed a similar reaction but using
1.5 equiv. of N-uorobenzenesulfonimide (NFSI) or N-uoro-o-
benzenedisulfonimide (NFOBS); unpredictably, no desired u-
orolactonizations were observed (entries 11 and 12, Table 1).
Therefore, we decided to use Selectuor (1.5 equiv.) in CH3CN at
80 ꢁC as an optimized reaction condition to achieve the desired
transformation (Table 1).
Results and discussion
Chemistry
The uorolactonization of isoxazoline 3p was selected as
a model reaction to optimize the reaction conditions, including
electrophilic uorine sources, solvents, and temperatures
(Table 1). As Selectuor is inexpensive, commercially available,
and widely used for electrophilic uorocyclization, we chose to
screen Selectuor on 3p while using various protic, aprotic, and
halogenated solvents at room temperature. However, since the
role of the solvents was crucial for the desired transformation,
none of the solvents were found suitable for the uorolactoni-
zation at room temperature. To our surprise, as depicted in
Table 1, when the temperature of the reaction was elevated to
ꢁ
80 C, we observed remarkable changes in reactivity. However,
A plausible mechanistic pathway has been proposed in
Scheme 2. Under the reuxed condition, Selectuor could
dearomatize the isoxazole ring via an oxonium-ion mediated
electrophilic uorination on C4–C5 double bond, regiose-
lectivity, leading to a reactive intermediate A. Subsequently, the
neighboring group participation of the carboxylate anion to the
the chlorinated solvents CH2Cl2, CHCl3, and ClCH2CH2Cl (DCE)
were abortive to bring about the uorolactonization, even at
reuxed temperature. This prompted us to examine the reaction
in aprotic polar and nonpolar solvents, such as THF, Et2O,
acetone, and ethyl acetate (entry 5–7, Table 1). To our disap-
pointment, the desired uorolactonization was unsuccessful,
and we recovered the substrate 3p, quantitatively. In mark
contrast, when we performed the reaction in polar solvents,
such as CH3CN and MeOH at 80 ꢁC (entry 8 and 9, Table 1),
a signicant enhancement in reactivity was observed, and we
were able to isolate 5a in 90% and 85% yields. Notably, when
triuoroethanol (TFE) was used as a uorinated polar solvent,
intermediate
(Scheme 2).
A
produced the uoro-spiro-isoxazoline
Having optimized conditions at hand, we proceeded to
explore the synthetic compatibility, rst by introducing
various substituents on the isoxazole ring, and then, by eval-
uating the feasibility of ve and six-membered ether and
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between alkynol 1 or alkynoic acid 2 and 1,3-dipole precursors
B (Table 2).11a
Although the substrate scope for the desired uorocycliza-
tion was examined at the nal stage, various functionalities
were introduced by using several 1,3-dipole precursors B (Table
2). Therefore, as illustrated in Table 2, electron-donating,
electron-withdrawing, and halo-functionality were introduced
on an aromatic system; a couple of alkyl functionalities were
also incorporated to introduce hydrophobic nature to the
molecule for biological investigation. The 1,3-dipolar reaction
was performed between 1 or 2 and in situ generated nitrile oxide
from the corresponding chloro-oxime B upon treatment with
Scheme 2 Plausible mechanism for 4-fluoro-spiro-isoxazolines.
Table 2 Synthesis of isoxazole precursors 3(a–z)a,b,c
Et3N/CH2Cl2.11a As summarized in Table 2,
a range of
substituted isoxazoles 3(a–z), comprising pendant alcohol 1 and
acid 2 of different chain lengths, were successfully synthesized
in high yields (Table 2). To determine the ester functionality
that could facilitate obtaining an acid and amide functional
group, we conducted a base-catalyzed 1,3-dipolar cycloaddition
reaction, using ethyl-nitroacetate and alkynoic acid 2, under
sealed tube reuxed condition, which provided the desired
product 3z in 82% yield (Table 2).11a It is worth mentioning that
the 1,3-dipolar cycloaddtion produced the 3,5-disubstituted
isoxazole as a major isomers; however, 3,4-disubstituted iso-
xazole was insignicant to be isolated.
Entry
Alkyne
R
3(a–z)
Yieldc (%)
1
2
3
4
5
6
7
8
1a, n ¼ 1, x ¼ H2
1a, n ¼ 1, x ¼ H2
1a, n ¼ 1, x ¼ H2
1a, n ¼ 1, x ¼ H2
1a, n ¼ 1, x ¼ H2
1a, n ¼ 1, x ¼ H2
1a, n ¼ 1, x ¼ H2
1a, n ¼ 1, x ¼ H2
1b, n ¼ 2, x ¼ H2
1b, n ¼ 2, x ¼ H2
1b, n ¼ 2, x ¼ H2
1b, n ¼ 2, x ¼ H2
1b, n ¼ 2, x ¼ H2
1b, n ¼ 2, x ¼ H2
1b, n ¼ 2, x ¼ H2
2a, n ¼ 1, x ¼ O
2a, n ¼ 1, x ¼ O
2a, n ¼ 1, x ¼ O
2a, n ¼ 1, x ¼ O
2a, n ¼ 1, x ¼ O
2a, n ¼ 1, x ¼ O
2a, n ¼ 1, x ¼ O
2a, n ¼ 1, x ¼ O
2a, n ¼ 2, x ¼ O
2a, n ¼ 2, x ¼ O
2a, n ¼ 2, x ¼ O
Ph
4-Me-Ph
4-F-Ph
4-CI-Ph
4-Br-Ph
2,6-Di-CI-Ph
4-CF3-Ph
4-NO2-Ph
Ph
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
96
83
95
95
95
96
94
80
92
94
92
93
94
95
78
96
89
80
82
85
80
92
76
78
80
82
Table 3 Synthesis of 4-fluoro-spiro-isoxazoline-ethers (ꢂ)-4(a–o)a
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26b
4-F-Ph
4-CI-Ph
4-Br-Ph
2,6-Di-CI-Ph
4-CF3-Ph
4-NO2-Ph
Ph
4-Me-Ph
4-F-Ph
4-CI-Ph
4-Br-Ph
2,6-Di-CI-Ph
4-CF3-Ph
Me
n-Pr
2,6-Di-CI-Ph
CO2Et
3s
3t
3u
3v
3w
3x
3y
3z
a
General conditions 1: alkynol 1 (1.0 equiv.), hydroximoyl chloride (1.2
equiv.), triethylamine (1.5 equiv.) in CH2Cl2 (10 mL) at rt for 24 h or
alkynoic acid 2 (1 equiv.), hydroximoyl chloride (2 equiv.), and K2CO3
b
(2.5 equiv.) in deionized water (1 M) at rt for 24 h. General
conditions 2: alkynoic acid 2b (1.0 equiv.), ethylnitroacetate (2.5
equiv.), DABCO (0.2 equiv.), ethanol (10 mL) at 80 ꢁC in sealed vessel
c
for
3
days.
Based on isolated product aer purication by
chromatography.
lactone ring formation. The desired precursors 3(a–z), pos-
sessing an isoxazole and pendant alcohol or acid, were initially
synthesized by using a 1,3-dipolar cycloaddition reaction
a
Reaction conditions: isoxazole alcohol 3(a–o) (0.2 mmol), Selectuor
(0.3 mmol, 1.5 equiv.), CH3CN (2 mL) at 80 ꢁC for 12 h, product
isolated aer purication by chromatography.
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To evaluate the feasibility of ve and six-membered ether spiro-isoxazoline-lactones. As depicted in Table 4, regardless
ring formations, the list of alcohols 3(a–o) from Table 2 was of various substitution on the phenyl ring and electronic
executed for the uorocyclization (Table 3), following our opti- properties, the reaction proceeded smoothly and provided the
mized reaction condition. As summarized in Table 3, the desired products 5(a–k) in excellent yields (Table 4). The direct
electron-donating effect of 4-Me was tolerated under our stan- alkyl substituents (Me and Pr) on the isoxazoline also endured
dard reaction conditions to afford 4b in 89% yield. Next, for the the reaction conditions and resulted 5h and 5i in 82% and 80%
halo substitutions, 4-F-Ph, 4-Cl-Ph, 4-Br-Ph, and 4-CF3-Ph yield, respectively (Table 4).
afforded 4(c–e) and 4g in 93%, 95%, 95%, and 92% isolated
Furthermore, an ester functionality that is directly connected
yields, respectively (Table 3). A similar pattern of reactivity was to an isoxazoline system also exhibited good compatibility to
also observed for a six-membered etherication 4(i–l) and 4n provide 5k in 80% yield. To our surprise, spiro-lactones 5(a–k)
with high yields. On the other hand, the spirocyclization of 2,6- were separated well, which delivered a dr 1 : 1 (Table 4).
dichloro substituted isoxazoles 3f and 3m were also successful
to produce 4f and 4m, albeit with decreased efficiencies (75%
and 78%) due to the steric environment generated between 2,6-
di-Cl and F-atoms. The electrophilic uoro-cyclization was also
found to be effective in the presence of an electron-withdrawing
group such as NO2, producing the desired cyclized products 4h
and 4o in 80% and 82% yields, respectively. Therefore, the
reaction conditions were found to be well tolerated for a wide
range of functionalities leading to (ꢂ)-4-uoro-spiro-
isoxazoline-ether 4(a–o) (Table 3). Importantly, the diastereo-
mers were isolated with a 1 : 1 diastereomeric ratio with an
identical chemical shis (d) in NMR for most cases. However,
a well-dened chemical shi for the CHF proton was observed
for few diastereomers.
Biology
Having synthesized diverse uoro-containing spiro-ethers and
-lactones in Tables 3 and 4, we were interested in evaluating
their anti-viral and anti-cancer activity.
Antiviral activity against HCMV. Initial screening for the test
compounds 4(a–o) and 5(a–k) having a potential HCMV inhib-
itory effect was performed using quantitative uorescence
microscopy. HFF-1 cells were pre-treated with the test
compounds (10 mM) or DMSO (control) for 1 hour followed by
infection with HCMV (Towne-BAC-GFP strain) at a multiplicity
of infection (MOI) of 3.0. This engineered HCMV strain contains
a green uorescent protein (GFP) and thus the infection is
associated with the expression of GFP.20 If a compound inhibits
HCMV replication, the expression of GFP, quantied as mean
uorescence intensity (MFI), is reduced. Based on preliminary
screening of test compounds, only ve compounds (4h, 4o, 4i,
4n, and 4d) showed a reduction in GFP levels. On further
analysis, signicant reduction in MFI was evident upon the
Inspired by this success, we next explored the scope of iso-
xazole acids 3 to afford about ve and six-membered uoro-
Table 4 Synthesis of 4-fluoro-spiro-isoxazoline-lactones (ꢂ)-5(a–k)a
Fig. 1 Determination of IC50 of the test compounds. Confluent HFF
cells were pre-treated with serial dilutions (0–20 mM) of test
compounds 4h, 4o, 4i, 4n, and 4d. As a control, cells were pre-treated
with ganciclovir (GCV) followed by infection with HCMV (Towne-BAC-
GFP) strain at a MOI of 3.0. At 5 days post infection, cells were fixed in
3.7% formaldehyde, and relative fluorescence units (RFU) were quan-
tified using a microwell image cytometer (Celigo, Nexcelom Biosci-
ence LLC, Lawrence, MA). Estimation of IC50 for the compounds 4o, 4i,
4n, and 4d as 2.54 mM, 11.2 mM, 10.47 mM, and 9.47 mM, respectively, is
based on non-linear curve fitting on transformed data in Prism
GraphPad software. Error bars represent standard error of mean (SEM)
from three independent experiments. IC50 for GCV was 4.96 mM,
which is close to the reported value of 5.2 mM.21
a
Reaction conditions: isoxazole acid 3(p–z) (0.2 mmol), Selectuor
(0.3 mmol, 1.5 equiv.), CH3CN (2 mL) at reux for 12 h, product
isolated aer purication by chromatography.
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Fig. 2 Effect of the test compounds on cell viability. HFF-1 cells were
treated with the test compounds at 2ꢃ the IC50 concentrations or
untreated (UT). Cells were either infected (I) with HCMV at MOI of 3.0
or left uninfected (UI). Cell viability was quantified by trypan blue
exclusion assay at day 5 post infection. Error bars represent standard
error of mean (SEM) from three independent experiments.
Fig. 4 GBM6 were plated at 1000 cells per well on 96-well format.
After 24 hours, the cells were treated with a range of concentrations
(0.8–100 mM). MTT assay was performed 72 hours after treatment. n ¼
3, bars are S.E.M.
treatment with these compounds 4h, 4o, 4i, 4n, and 4d, indi-
cating that these ve compounds may have potential anti-
HCMV properties.
Finally, we determined the possible cytotoxicity of the test
compounds on HFF cells (Fig. 2). Cells were treated with the test
compounds (4d and 4n) at 2ꢃ the IC50 concentrations or le
untreated (UT). Cells were then either infected (I) with HCMV at
MOI of 3.0 or le uninfected (UI), and cell viability at the end
point was quantied by trypan blue exclusion assay.22 As pre-
sented in Fig. 2, cell viability for all tested compounds remained
near 100%. Most importantly, the compounds 4n and 4d did
not show signicant cytotoxicity even when maintained on cells
for a period of 5 days.
Based on the biological assay, it is depicted that spiro-
lactones 5(a–k) were more inactive against HCMV than the
corresponding ether derivatives. Likewise, the phenyl and
electron-donating groups on uoro-spiro derivatives did not
show an acceptable range of inhibition against HCMV replica-
tion. It is also imperative to highlight that compounds (4h, 4o,
4i, 4n, and 4d) containing electron-withdrawing groups such as
NO2, –Cl, and –CF3 were primarily found to have potential anti-
HCMV properties. The Ph and 4-NO2-Ph substituted uoro-
isoxazoline 4o and 4i proved to be moderately active against
HCMV with IC50 2.54 mM and 11.2 mM. Interestingly,
replacement of functionality to halo substituents such as the 4-
Cl-Ph and 4-CF3-Ph in 4d and 4n lead to substantial increase in
potency (IC50 values 9.47 mM and 10.47 mM). Although, their
potency is comparatively lower than that of the control
Next, we determined the half maximal inhibitory concen-
tration (IC50) for the ve compounds that showed promise in
the preliminary screening against HCMV. Based on the number
of GFP+ cells present in infected cells pre-treated with serial
dilutions of test compounds, the IC50 for the compounds 4o, 4i,
4n, and 4d was calculated as 2.54 mM, 11.2 mM, 10.47 mM, and
9.47 mM, respectively (Fig. 1). IC50 for ganciclovir (GCV), a clin-
ically approved drug for HCMV treatment, was calculated as
4.96 mM, which is very close to the reported IC50 of 5.2 mM,21
proving the validity of this assay. Since IC50 values are inversely
proportional to the potency of a compound, this data showed
that compounds 4d and 4n are the most effective inhibitors of
HCMV in cell culture among the compounds tested (Fig. 1).
The compounds 4o and 4i showed virus inhibition in this
assay; however, much more concentration is required, which
may be impractical to attain physiologically.
Fig. 3 GBM6 were plated at 1000 cells per well on 96-well format.
After 24 hours, the cells were treated at 100 mM concentration of each Fig. 5 MDA MB 231 were plated at 2000 cells per well on 96-well
given compound. The MTT assay was performed 72 hours after format. After 24 hours, the cells were treated with a range of
treatment and the absorbance (570 nm) data from each experimental concentrations. MTT assay was performed 72 hours after treatment. n
group (n ¼ 3) was normalized to control average, bars are S.E.M.
¼ 6, bars are S.E.M.
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Table 5 IC50 Values from Fig. 4 and 5
the uoro-spiro-isoxazolines have the potential to serve as
inhibitors of HCMV infection as well as anti-cancer agents.
4l
5f
IC50 in GBM6
36.08
23.21 to 55.06
68.18
43.21
35.69 to 52.38
79.80
Experimental section
Materials and methods
95% C.I. in GBM6
IC50 in MDA MB 231
95% C.I. in MDA MB 231
64.27 to 72.25
75.58 to 84.95
General consideration. NMR, IR, and HRMS techniques have
been used for the characterization of all new compounds. H
1
NMR and 13C NMR spectra were recorded on Varian 500 MHz
and 125 MHz, respectively. 19F measurements were performed
at 376ꢁMHz on Bruker 400 MHz. All NMR spectra were measured
at 25 C in the indicated deuterated solvents. Proton, carbon,
and uorine chemical shis (d) were reported in parts per
million (ppm) and coupling constants (J) were reported in Hertz
compound ganciclovir (GCV), considering widespread resis-
tance against GCV in clinical strains of HCMV, and the well-
known side effects such as neutropenia, the compounds 4d,
and 4n may be developed into potential future drugs for
HCMV.23–25
Cytotoxicity against glioblastomas (GBM6). The synthesized
compounds were next evaluated against human glioblastoma
cell line GBM6 to determine antiproliferative activity using MTT
assay. Treatment concentration for all compounds was 100 mM,
and these experiments indicated robust anti-proliferative
activity of 4l and 5f (Fig. 3).
The IC50 value was evaluated using an increased concentra-
tion (0.8–100 mM) for 4l and 5f. Survival percentages were
plotted in Fig. 4 and IC50 values predicted based on non-linear
regression curve t, using Graphpad 8. These values are shown
in Table 5.
Cytotoxicity against triple negative breast cancer (MDA MB
231). Similarly, MDA MB 231 cells were treated initially with 100
mM concentration of each compound (data not shown).
Compounds 4l and 5f showed the highest anti-proliferative effect
at this concentration, so these two compounds were chosen for
dose–response experiments (Fig. 5). MDA MB 231 cells were
treated for 72 hours with concentrations ranging from 0.8 mM to
100 mM. Survival percentages were plotted in Fig. 5 and IC50 values
predicted based on non-linear regression curve t, using Graphpad
Prism 8. These values are shown in Table 5.
1
(Hz). The resonance multiplicities in the H NMR spectra are
described as s(singlet), d(doublet), t(triplet), and m(multiplet),
and broad resonances indicated by br. The residual protic
solvent, CDCl3 (1H, d 7.26 ppm; 13C, d 77.0 ppm, the central
resonance of the triplet), was utilized as the internal reference
for 1H and 13C NMR, while CFCl3 (d ¼ 0.00) was utilized as
reference for 19F NMR. Melting points remain uncorrected. IR
spectra of the synthesized compounds were recorded using
Perkin-Elmer-Spectrum where all types of samples (e.g. solids
and liquids) are placed directly on the ATR crystal with
absorptions measurements taken within spectrometer range of
4000–4500 cmꢄ1. High-resolution mass spectrometry (HRMS)
analyses were performed based on positive electrospray ioni-
zation on a Bruker 12 Tesla APEX – Qe FTICR-MS with an Apollo
II ion source. Either protonated molecular ions [M + nH]n+ or
sodium adducts [M + Na]+ were used for empirical formula
conrmation.
The reactions mentioned below are performed in non-inert
atmosphere using HPLC grade CH2Cl2, commercial grade
EtOH, anhydrous DMF, and deionized water as solvent. All
reagents were used as supplied without prior purication unless
otherwise stated. The progress of the reaction is monitored by
As depicted in Table 5, compound 4l shows better activity
than 5f against both GBM6 and MDA MB 231 cells suggesting
that spiro-ether is more effective to show cytotoxicity than the
corresponding spiro-lactone. The Br substitution on 4l, situated
at the periphery of the molecular geometry, shows anti-GBM
activity; however, the dichloro substitutions at 2,6-position are
away from the periphery shows anti-MDA MB 231 activity.
˚
analytical thin-layer chromatography comprised of 60 A silica
gel medium with layer-thickness 250 mm and visualized on
254 nm light, or KMnO4/Na2CO3/NaOH mixture and subse-
quent development with either no or gentle heating. Purica-
tions by ash column chromatography were performed using
˚
ash silica gel (60 A, 0.060–0.200 mm) with the indicated eluent.
Biological assay for antiviral activity against human
cytomegalovirus (HCMV)
Conclusion
In conclusion, we have developed an efficient and practical route
Cell culture. Human foreskin broblasts (HFF-1) (Catalog
to access various 4-uoro-substituted spiro-isoxazolines. The #SCRC1041, American Type Culture Collection, Manassas, VA)
efficacy of this strategy mainly relies on 1,3-dipolar cycloaddition cells were maintained in Dulbecco's modied Eagle's medium
and electrophilic uoro-etherication or -lactonization mediated (DMEM) (Cellgro, Manassas, VA) supplemented with 4.5 g mLꢄ1
by commercially available Selectuor. The protocol has been glucose, 10% fetal bovine serum (FBS) (Atlanta Biologicals,
shown to be useful for accessing spirocyclic compound Flowery Branch, GA), 1 mM sodium pyruvate, 2 mM L-gluta-
comprising alkyl, aromatic, and ester substituted isooxazoline mine, and 100 U mLꢄ1 penicillin-streptomycin (Cellgro) at 37 ꢁC
and ve and six membered cyclic-ethers and lactones. Addition- with 5% CO2.
ally, few synthetic compounds showed selective and moderate
Cytotoxic assessment. HFF-1 cells were grown in 12 well
anti-HCMV, anti-GBM6, and anti-MDA MB 231 activity. The tissue-culture plates. At conuency, cells were treated with the
structural implication along with biological activity validates that test compounds at 2ꢃ the IC50 concentrations (control) or le
30228 | RSC Adv., 2020, 10, 30223–30237
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untreated and incubated further. At ve days post treatment,
cell viability was determined by trypan blue exclusion assay as
described earlier (PMID: 26529666) using a TC20 automated cell
counter (Bio-Rad Laboratories, Hercules, CA). In brief, medium
was removed, and cells were harvested using 0.025% Trypsin-
EDTA (ThermoFisher Scientic, Waltham, MA) diluted in
phosphate-buffered saline (PBS) and neutralized with FBS-
supplemented DMEM. Ten mL of cell suspension and 10 mL of
0.4% trypan blue dye (Hyclone Laboratories, Logan, UT) were
mixed, and 10 mL of this sample was loaded immediately onto
the outer chamber of the counting slide. The slide was inserted
into TC20, which automatically determined the percentage of
viable cells.
Virus titers and IC50 determination. HFF-1 cells were grown
to conuency in 96-well tissue culture plates and pre-treated
with serial dilutions (0–20 mM) of the test compounds for 1
hour. As a control, cells were pre-treated with ganciclovir (GCV).
The cells were infected with HCMV (Towne-BAC-GFP) strain at
a MOI of 3.0. At 5 days post infection, cells were xed in 3.7%
Proliferation assay. MDA-MB-231 proliferation was assessed
aer 72 hours of treatment. Briey, 2000 cells per well were
plated on 96-well plates. Aer overnight incubation, medium
was removed and replaced with treatment. Aer 72 hours 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT,
5 mg mLꢄ1) was added, and plates were incubated for 4 hours at
ꢁ
37 C. Then all media was removed and replaced with 100 mL
DMSO per well to dissolve formazan. Absorbance was measured
at 570 nm with a reference wavelength of 630 nm with a Synergy
4 plate reader (Biotek, Winooski, VT).
IC50 determination. Survival percentages from proliferation
experiments (treatment concentrations ranging from 0.78 mM to
100 mM) were plotted using Graphpad Prism 8. Non-linear
regression curve t analysis was used to predict the concen-
tration at which cell survival would have been 50%.
General procedure for 1,3-dipolar cycloaddition of the alkynol
(condition 1)
formaldehyde and relative uorescence units (RFU) were A solution of the alkynol (1 equiv.) and the hydroximoyl chloride
quantied using a microwell image cytometer (Celigo, Nexce- (1.2 equiv.) in 10 mL of dichloromethane was treated with
lom Bioscience LLC, Lawerence, MA). Ganciclovir (GCV), triethylamine (1.5 equiv.). The reaction mixture was stirred at rt
a known inhibitor of HCMV (PMID: 8393055) was used as for 24 h until complete consumption of starting material as
a positive control in this assay.
monitored by TLC analysis. Aer the reaction was complete,
a minimum amount of silica gel was added, and the solvent was
evaporated under reduced pressure. The crude products were
puried by column chromatography over silica gel using
hexanes/ethyl acetate as eluent to provide the desired products.
3-(3-(p-Tolyl)isoxazol-5-yl)propan-1-ol (3b). Following the general
procedure for 1,3-dipolar cycloaddition (condition 1), alkynol 1a
(0.14 g, 1.66 mmol), (Z)-N-hydroxy-4-methylbenzimidoyl chloride
(0.34 g, 1.99 mmol), and Et3N (0.35 mL, 2.49 mmol) provided 3b
(0.30 g, 83%) as a white solid aer column chromatography
(hexanes/EtOAc, 4/1); Rf 0.4 (1 : 1 ethyl acetate : hexanes); mp: 48–
50 ꢁC; IR nmax 3261, 3018, 2922, 2871, 2853, 1606, 1430, 1062, 1040,
798 cmꢄ1; 1H NMR (500 MHz, CDCl3): d 7.65 (d, J ¼ 8.1 Hz, 2H),
7.23 (d, J ¼ 8.0 Hz, 2H), 6.28 (s, 1H), 3.71 (t, J ¼ 6.2 Hz, 2H), 2.88 (t,
J ¼ 7.6 Hz, 2H), 2.37 (s, 3H), 2.00–1.95 (m, 2H); 13C{1H}NMR (125
MHz, CDCl3): d 173.4, 162.4, 140.0, 129.6 (2C), 126.6 (2C), 126.3,
99.1, 61.4, 30.3, 23.2, 21.4; HRMS (ESI-TOF) m/z: [M + Na]+ calcd
for C13H15NO2Na: 240.0995; found 240.0995.
Biological assay for cytotoxicity against glioblastomas (GBM6)
Cell culture. GBM6 cells were propagated in Dulbecco's
Modied Eagles Medium (Corning, Manassas, VA) supple-
mented by 10% fetal bovine serum (Atlanta Biologicals, Flowery
Branch, GA) and 100 U mLꢄ1 penicillin-streptomycin (Cellgro,
Manassas, VA). Cells were incubated at 37 C and 5% CO2. To
maintain log-phase, cells were trypsinized and split every 2–3
days.
Proliferation assay. GBM6 proliferation was assessed aer 72
hours of treatment. Briey, 1000 cells per well were plated on
96-well plates. Aer overnight incubation, medium was
removed and replaced with treatment. Aer 72 hours 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
5 mg mLꢄ1) was added, and plates were incubated for 4 hours at
37 ꢁC.26 Then all media was removed and replaced with 100 mL
DMSO per well to dissolve formazan. Absorbance was measured
at 570 nm with a reference wavelength of 630 nm with a Synergy
4 plate reader (Biotek, Winooski, VT).
IC50 determination. Survival percentages from proliferation
experiments (treatment concentrations ranging from 0.78 mM to
100 mM) were plotted using Graphpad Prism 8. Non-linear
regression curve t analysis was used to predict the concen-
tration at which cell survival would have been 50%.
ꢁ
3-(3-(4-Fluorophenyl)isoxazol-5-yl)propan-1-ol (3c). Following the
general procedure for 1,3-dipolar cycloaddition (condition 1), alky-
nol 1a (0.20 g, 2.37 mmol), (Z)-4-uoro-N-hydroxybenzimidoyl
chloride (0.49 g, 2.85 mmol), and Et3N (0.5 mL, 3.55 mmol)
provided 3c (0.50 g, 95%) as a white solid aer column chroma-
tography (hexanes/EtOAc, 4/1); Rf 0.4 (1 : 1 ethyl acetate : hexanes);
ꢁ
mp: 70–72 C; IR nmax 3241, 3122, 2931, 2870, 1612, 1524, 1432,
1236, 1035, 842, cmꢄ1; 1H NMR (500 MHz, CDCl3): d 7.76–7.72 (m,
2H), 7.11 (t, J ¼ 8.7 Hz, 2H), 6.27 (s, 1H), 3.72 (t, J ¼ 6.2 Hz, 2H), 2.90
(t, J ¼ 7.6 Hz, 2H), 2.26 (br s, 1H, OH), 1.98 (tt, J ¼ 7.3, 6.2 Hz, 2H);
Biological assay for MDA MB 231 breast carcinoma cells
Cell culture. MDA MB 231 cells were propagated in Dulbecco's 13C{1H}NMR (125 MHz, CDCl3): d 173.7, 163.7 (d, J ¼ 249.6 Hz),
Modied Eagles Medium (Corning, Manassas, VA) supplemented 161.5, 128.6 (d, J ¼ 8.4 Hz, 2C), 125.4 (d, J ¼ 3.4 Hz), 115.9 (d, J ¼
by 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, 21.8 Hz, 2C), 99.0, 61.4, 30.3, 23.2; HRMS (ESI-TOF) m/z: [M + Na]+
GA) and 100 U mLꢄ1 penicillin-streptomycin (Cellgro, Manassas, calcd for C12H12Cl2FNO2Na: 244.0744; found 244.0743.
VA). Cells were incubated at 37 ꢁC and 5% CO2. To maintain log-
3-(3-(4-Bromophenyl)isoxazol-5-yl)propan-1-ol (3e). Following the
phase, cells were trypsinized and split every 3–4 days.
general procedure for 1,3-dipolar cycloaddition (condition 1),
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alkynol 1a (0.20 g, 2.37 mmol), (Z)-4-bromo-N-hydroxybenzimidoyl 1), alkynol 1b (0.30 g, 3.06 mmol), (Z)-4-bromo-N-hydrox-
chloride (0.67 g, 2.84 mmol), and Et3N (0.5 mL, 3.55 mmol) ybenzimidoyl chloride (0.86 g, 3.67 mmol), and Et3N (0.64 mL,
provided 3e (0.62 g, 93%) as a white solid aer column chroma- 4.59 mmol) provided 3l (0.84 g, 93%) as a pale yellow solid aer
tography (hexanes/EtOAc, 4/1); Rf 0.55 (1 : 1 hexanes/ethyl acetate); column chromatography (hexanes/EtOAc, 3/1); Rf 0.5 (2 : 3 ethyl
mp 80–82 ꢁC; IR nmax 3240, 3150, 2931, 2873, 1605, 1426, 1061, 1041, acetate : hexanes); mp: 60–62 ꢁC; IR nmax 3468, 3132, 2945, 2923,
833, 801 cmꢄ1; 1H NMR (500 MHz, CDCl3): d 7.63 (d, J ¼ 8.6 Hz, 2H), 2853, 1601, 1417, 1343, 1054, 902, 818, 721, 518 cmꢄ1; 1H NMR
7.56 (d, J ¼ 8.6 Hz, 2H), 6.29 (s, 1H), 3.73 (t, J ¼ 6.2 Hz, 2H), 2.91 (t, J (500 MHz, CDCl3): d 7.62 (d, J ¼ 8.5 Hz, 2H), 7.54 (d, J ¼ 8.5 Hz,
¼ 7.6 Hz, 2H), 2.07 (br s, 1H, OH), 1.98 (dd, J ¼ 14.3, 6.9 Hz, 2H); 13
C
2H), 6.26 (s, 1H), 3.66 (t, J ¼ 6.4 Hz, 2H), 2.8 (t, J ¼ 7.6 Hz, 2H),
{1H}NMR (125 MHz, CDCl3): d 173.8, 161.5, 132.1 (2C), 128.2 (2C), 2.12 (br s, 1H, OH), 1.81 (dt, J ¼ 15.4, 7.6 Hz, 2H), 1.67–1.61 (m,
128.1, 124.1, 99.0, 61.4, 30.2, 23.2; HRMS (ESI-TOF) m/z: [M + Na]+ 2H); 13C{1H}NMR (125 MHz, CDCl3): d 174.2, 161.4, 132.1 (2C),
calcd for C12H12BrNO2Na: 303.9943; found 303.9942.
128.2 (2C), 128.1, 124.1, 98.8, 62.1, 31.8, 26.5, 23.8; HRMS (ESI-
3-(3-(4-(Triuoromethyl)phenyl)isoxazol-5-yl)propan-1-ol (3g). TOF) m/z: [M + Na]+ calcd for C13H14BrNO2Na: 318.0100; found
Following the general procedure for 1,3-dipolar cycloaddition 318.0099.
(condition 1), alkynol 1a (0.30 g, 3.57 mmol), (Z)-N-hydroxy-4-
4-(3-(4-(Triuoromethyl)phenyl)isoxazol-5-yl)butan-1-ol (3n).
(triuoromethyl)benzimidoyl chloride (0.96 g, 4.28 mmol), and Following the general procedure for 1,3-dipolar cycloaddition
Et3N (0.75 mL, 5.35 mmol) provided 3g (0.91 g, 94%) as a white (condition 1), alkynol 1b (0.32 g, 3.26 mmol), (Z)-N-hydroxy-4-
solid aer column chromatography (hexanes/EtOAc, 4/1); Rf 0.5 (triuoromethyl)benzimidoyl chloride (0.87 g, 3.91 mmol),
(1 : 1 ethyl acetate : hexanes); mp: 50–52 ꢁC; IR nmax 331, 2955, and Et3N (0.68 mL, 4.89 mmol) provided 3n (0.88 g, 95%) as
2936, 2874, 1603, 1438, 1462, 1324, 1164, 1111, 1063, 848 cmꢄ1; 1H a white solid aer column chromatography (hexanes/EtOAc, 3/
ꢁ
NMR (500 MHz, CDCl3): d 7.90 (d, J ¼ 8.1 Hz, 2H), 7.70 (d, J ¼ 1); Rf 0.5 (2 : 3 ethyl acetate : hexanes); mp: 58–60 C; IR nmax
9.1 Hz, 2H), 6.37 (s, 1H), 3.75 (t, J ¼ 6.1 Hz, 2H), 2.95 (t, J ¼ 7.6 Hz, 3400, 3137, 2947, 2922, 2871, 1601, 1461, 1321, 1175, 1123,
1
2H), 2.04–1.99 (m, 2H), 1.80 (br s, 1H); 13C{1H}NMR (125 MHz, 1111, 1060, 1015, 912, 851 cmꢄ1; H NMR (500 MHz, CDCl3):
CDCl3): d 174.2, 161.3, 132.6, 131.7 (q, J ¼ 32.7 Hz), 127.1 (d, J ¼ d 7.88 (d, 8.3 Hz, 2H), 7.68 (d. J ¼ 8.3 Hz, 2H), 6.34 (s, 1H), 3.69
8.2 Hz, 2C), 125.8 (q, J ¼ 3.8 Hz, 2C), 123.8 (q, J ¼ 272.3 Hz), 99.2, (t, J ¼ 6.4 Hz, 2H), 2.84 (t, J ¼ 7.6 Hz, 2H), 1.97 (br s, 1H, OH),
61.4, 30.2, 23.2; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C13H12- 1.97–1.81 (m, 2H), 1.66 (dt, J ¼ 13.3, 6.4 Hz, 2H); 13C{1H}NMR
F3NO2Na: 294.0712; found 294.0711.
(125 MHz, CDCl3): d 174.5, 161.2, 132.7, 131.6 (q, J ¼ 32.6 Hz),
3-(3-(4-Nitrophenyl)isoxazol-5-yl)propan-1-ol (3h). Following 127.0 (2C), 125.8 (q, J ¼ 3.8 Hz, 2C), 123.8 (q, J ¼ 272.2 Hz), 99.0,
the general procedure for 1,3-dipolar cycloaddition (condition 62.1, 31.8, 26.5, 23.8; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
1), alkynol 1a (0.10 g, 1.19 mmol), (Z)-N-hydroxy-4-
C
14H14F3NO2Na: 308.0868; found 308.0867.
nitrobenzimidoyl chloride (0.29 g, 1.43 mmol), and Et3N (0.25
4-(3-(4-Nitrophenyl)isoxazol-5-yl)butan-1-ol (3o). Following
mL, 1.78 mmol) provided 3h (0.24 g, 80%) as a brown oil aer the general procedure for 1,3-dipolar cycloaddition (condition
column chromatography (hexanes/EtOAc, 5/2); Rf 0.4 (1 : 1 ethyl 1), alkynol 1b (0.21 g, 2.14 mmol), (Z)-N-hydroxy-4-
acetate : hexanes); IR nmax 3350, 3130, 2960, 2928, 2850, 1590, nitrobenzimidoyl chloride (0.52 g, 2.57 mmol), and Et3N (0.45
1
1514, 1460, 1340, 1175, 1105, 1003, cmꢄ1; H NMR (500 MHz, mL, 3.21 mmol) provided 3o (0.44 g, 78%) as a white solid aer
CDCl3): d 8.30 (d, J ¼ 8.7 Hz, 2H), 7.96 (d, J ¼ 8.7 Hz, 2H), 6.41 (s, column chromatography (hexanes/EtOAc, 3/1); Rf 0.4 (1 : 1 ethyl
1H), 3.76 (t, J ¼ 5.9 Hz, 2H), 2.97 (t, J ¼ 7.6 Hz, 2H), 2.05–2.00 (m, acetate : hexanes); mp: 62–64 ꢁC; IR nmax 3349, 3132, 2955, 2923,
2H); 13C{1H}NMR (125 MHz, CDCl3): d 174.6, 160.6, 148.5, 135.4, 2854, 1593, 1514, 1460, 1342, 1175, 1105, 1003, 862, 809 cmꢄ1
;
126.6 (2C), 124.2 (2C), 99.3, 61.4, 30.2, 23.2; HRMS (ESI-TOF) m/ 1H NMR (500 MHz, CDCl3): d 8.31 (d, J ¼ 8.9 Hz, 2H), 7.97 (d, J ¼
z: [M + Na]+ calcd for C12H12N2O4Na: 271.0689; found 271.0688. 8.9 Hz, 2H), 6.4 (s, 1H), 3.72 (t, J ¼ 6.3 Hz, 2H), 2.89 (t, J ¼ 7.6 Hz,
4-(3-(4-Fluorophenyl)isoxazol-5-yl)butan-1-ol (3j). Following 2H), 1.88 (dt, J ¼ 15.4, 7.6 Hz, 2H), 1.69 (dt, J ¼ 13.5, 6.4 Hz, 2H),
the general procedure for 1,3-dipolar cycloaddition (condition 1.34 (br s, 1H, OH); 13C{1H}NMR (125 MHz, CDCl3): d 175.0,
1), alkynol 1b (0.31 g, 3.16 mmol), (Z)-4-uoro-N-hydrox- 160.5, 148.5, 135.4, 127.6 (2C), 124.1 (2C), 99.2, 62.2, 31.8, 26.5,
ybenzimidoyl chloride (0.66 g, 3.79 mmol), and Et3N (0.66 mL, 23.8; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C13H14N2O4Na:
4.74 mol) provided 3j (0.70 g, 94%) as a white solid aer column 285.0845; found 285.0844.
chromatography (hexanes/EtOAc, 3/1); Rf 0.4 (1 : 1 ethyl aceta-
te : hexanes); mp: 58–60 ꢁC; IR nmax 3443, 3405, 3132, 2940, noic acid (condition 1).
2929, 2861, 1606, 1591, 1524, 1433, 1346, 1231, 910, 842 cmꢄ1
Deionized water (10 mL) was added into a round-bottomed
General procedure for 1,3-dipolar cycloaddition of the alky-
;
1H NMR (500 MHz, CDCl3): d 7.74–7.72 (m, 2H), 7.10 (t, J ¼ ask containing the hydroximoyl chloride (2 equiv.) with stir-
8.9 Hz, 2H), 6.24 (s, 1H), 3.66 (t, J ¼ 6.4 Hz, 2H), 2.79 (t, J ¼ ring. The alkynoic acid 2a (1 equiv.) was added into this mixture.
7.8 Hz, 2H), 2.61 (br s, 1H OH), 1.83–1.77 (m, 2H), 1.66–1.623 Potassium carbonate (2.5 equiv.) was then added in small
(m, 2H); 13C{1H}NMR (125 MHz, CDCl3): d 174.1, 163.6 (d, J ¼ portions. The reaction mixture was stirred at rt until the reac-
249.7 Hz), 161.4, 128.6 (d, J ¼ 8.4 Hz, 2C), 125.4 (d, J ¼ 3.3 Hz), tion was complete as evidenced by TLC/NMR analysis. The
115.9 (d, J ¼ 21.8 Hz, 2C), 98.9, 62.0, 31.8, 26.5, 23.8; HRMS (ESI- reaction mixture was acidied with 4 N HCl and treated with
TOF) m/z: [M + Na]+ calcd for C13H14FNO2Na: 258.0900; found diethyl ether and water (1 : 1, 20 mL). Sodium hydroxide was
258.0900.
added to this mixture until the mixture was basic (litmus paper).
4-(3-(4-Bromophenyl)isoxazol-5-yl)butan-1-ol (3l). Following The mixture was extracted with diethyl ether (3 ꢃ 10 mL), and
the general procedure for 1,3-dipolar cycloaddition (condition 4 M HCl was then added to the aqueous layer until it was acidic
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(litmus paper). The acidied aqueous layer was extracted with 929, 846, 815 cmꢄ1; 1H NMR (500 MHz, CD3COCD3): d 8.1 (d, J ¼
ethyl acetate (3 ꢃ 15 mL), and the resulting organic layer was 8.0 Hz, 2H), 7.85 (d, J ¼ 8.4 Hz, 2H), 6.82 (s, 1H), 3.15 (t, J ¼
dried over MgSO4, ltered, and concentrated under reduced 6.3 Hz, 2H), 2.83 (t, J ¼ 6.4 Hz, 2H); 13C{1H}NMR (125 MHz,
pressure to provide the crude product, which was puried via CD3COCD3): d 173.7, 172.3, 161.0, 133.3, 130.9 (q, J ¼ 32.3 Hz),
column chromatography over silica gel using an appropriate 127.2 (2C), 125.9 (d, J ¼ 3.5 Hz, 2C), 124.2 (q, J ¼ 271.4 Hz), 99.3,
hexanes–ethyl acetate ratio as an eluent system.
3-(3-(p-Tolyl)isoxazol-5-yl)propanoic acid (3q). Following the NO3Na: 308.0505; found 308.0504.
30.8, 21.9; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C13H10F3-
general procedure for 1,3-dipolar cycloaddition (condition 1),
3-(3-Methylisoxazol-5-yl)propanoic acid (3w). Following the
alkynoic acid 2a (0.25 g, 2.54 mmol), (Z)-N-hydroxy-4- general procedure for 1,3-dipolar cycloaddition (condition 2),
methylbenzimidoyl chloride (0.86 g, 5.08 mmol), and K2CO3 alkynoic acid 2a (0.26 g, 2.65 mmol), (Z)-N-hydroxyacetimidoyl
(0.88 g, 6.35 mmol) provided 3q (0.52 g, 89%) as a white solid chloride 3-(3-methylisoxazol-5-yl)propanoate (0.46 g, 5.30
aer column chromatography (hexanes/EtOAc, 1 : 1); Rf 0.4 mmol), and K2CO3 (0.92 g, 6.63 mmol) provided 3v (0.31 g, 76%)
(1 : 1 ethyl acetate : hexanes); mp: 162–164 ꢁC; IR nmax 3122, as a white solid aer column chromatography (hexanes/EtOAc,
2912, 2856, 1693, 1601, 1421, 1243, 1206, 951, 809 cmꢄ1 1H 1 : 1); Rf 0.4 (1 : 1 ethyl acetate : hexanes); mp: 80–82 ꢁC; IR nmax
;
NMR (500 MHz, CD3COCD3): d 7.74 (d, J ¼ 8.1 Hz, 2H), 7.30 (d, J 3129, 3033, 2993, 2927, 2866, 1690, 1600, 1436, 1412, 1309,
1
¼ 7.9 Hz, 2H), 6.64 (s, 1H), 3.11 (t, J ¼ 7.4 Hz, 2H), 2.80 (d, J ¼ 1196, 1001, 924, 881, 827, 671 cmꢄ1; H NMR (500 MHz, CD3-
7.4 Hz, 2H), 2.37 (s, 3H); 13C{1H}NMR (125 MHz, CD3COCD3): COCD3): d 6.01 (s, 1H), 3.00 (t, J ¼ 7.4 Hz, 2H), 2.71 (t, J ¼ 7.4 Hz,
d 172.7, 172.3, 162.0, 139.8, 129.5 (2C), 126.7, 126.4 (2C), 98.9, 2H), 2.2 (s, 3H); 13C{1H}NMR (125 MHz, CD3COCD3): d 172.3,
30.9, 21.9, 20.4; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for 171.6, 159.4, 101.5, 30.8, 21.7, 10.3; HRMS (ESI-TOF) m/z: [M +
C
13H13NO3Na: 254.0787; found 254.0787.
Na]+ calcd for C7H9NO3Na: 178.0474; found 178.0474.
3-(3-(4-Fluorophenyl)isoxazol-5-yl)propanoic
acid
(3r).
3-(3-Propylisoxazol-5-yl)propanoic acid (3x). Following the
Following the general procedure for 1,3-dipolar cycloaddition general procedure for 1,3-dipolar cycloaddition (condition 2), alky-
(condition 2), alkynoic acid 2a (0.22 g, 2.24 mmol), (Z)-4-uoro- noic acid 2a (0.22 g, 2.24 mmol), (Z)-N-hydroxybutyrimidoyl chloride
N-hydroxybenzimidoyl chloride (0.78 g, 4.48 mmol), and K2CO3 3-(3-propylisoxazol-5-yl)propanoate (0.54 g, 4.48 mmol), and K2CO3
(0.77 g, 5.60 mmol) provided 3r (0.42 g, 80%) as a white solid (0.77 g, 5.60 mmol) provided 3x (0.32 g, 78%) as a white solid aer
aer column chromatography (hexanes/EtOAc, 1 : 1); Rf 0.4 column chromatography (hexanes/EtOAc, 1 : 1); Rf 0.4 (1 : 1 ethyl
(1 : 1 ethyl acetate : hexanes); mp: 160–162 ꢁC; IR nmax 3117, acetate : hexanes); mp: 98–100 ꢁC; IR nmax 3124, 2958, 2931, 2871,
2927, 2848, 1691, 1605, 1589, 1524, 1428, 1233, 1204, 1233, 1690, 1598, 1431, 1414, 1309, 1227, 1215, 1000, 924, 828, 878 cmꢄ1
;
1204, 1156, 903, 815 cmꢄ1
;
1H NMR (500 MHz, CD3COCD3): 1H NMR (500 MHz, CD3COCD3): d 6.07 (s, 1H), 3.01 (t, J ¼ 7.4 Hz,
d 7.92 (dd, J ¼ 8.4, 5.6 Hz, 2H), 7.27 (t, J ¼ 8.7 Hz, 2H), 6.69 (s, 2H), 2.72 (t, J ¼ 7.4 Hz, 2H), 2.56 (t, J ¼ 7.5 Hz, 2H), 1.69–1.61 (m,
1H), 3.12 (t, J ¼ 7.3 Hz, 2H), 2.81 (t, J ¼ 7.3 Hz, 2H); 13C{1H}NMR 2H), 0.94 (t, J ¼ 7.4 Hz, 3H); 13C{1H}NMR (125 MHz, CD3COCD3):
(125 MHz, CD3COCD3): d 173.1, 172.2, 163.6 (d, J ¼ 247.4 Hz), d 172.3, 171.5, 163.4, 100.4, 30.9, 27.6, 21.8, 21.3, 13.1; HRMS (ESI-
161.2, 128.7 (d, J ¼ 8.6 Hz, 2C), 125.9 (d, J ¼ 2.8 Hz), 115.8 (d, J ¼ TOF) m/z: [M + Na]+ calcd for C9H13NO3Na: 206.0787; found
22.0 Hz, 2C), 99.0, 30.8, 21.9; HRMS (ESI-TOF) m/z: [M + Na]+ 206.0789.
calcd for C12H10FNO3Na: 258.0537; found 258.0536.
3-(3-(4-Bromophenyl)isoxazol-5-yl)propanoic
Following the general procedure for 1,3-dipolar cycloaddition
acid
(3t).
General procedure for uoro-etherication 4(a–o)
(condition 2), alkynoic acid 2a (0.31 g, 3.16 mmol), (Z)-4-bromo- A solution of isoxazole alcohol 3(a–o) (0.2 mmol) and Selectuor
N-hydroxybenzimidoyl chloride (1.48 g, 6.32 mmol), and K2CO3 (0.11 g, 0.3 mmol) in CH3CN (2 mL) was reuxed under N2 for
(1.09 g, 7.90 mmol) provided 3t (0.79 g, 85%) as a white solid 24 h. Then the reaction mixture was cooled, and water was
aer column chromatography (hexanes/EtOAc, 1 : 1); Rf 0.4 added and extracted with ethyl acetate. The organic phase was
(1 : 1 ethyl acetate : hexanes); mp: 180–182 ꢁC; IR nmax 3120, washed several times with water and nally with brine. The
2928, 2849, 1690, 1600, 1590, 1525, 1425, 1230, 1200, 1155, combined organic layer was dried over anhydrous MgSO4 and
900 cmꢄ1; 1H NMR (500 MHz, CD3COCD3): d 7.82 (d, J ¼ 8.6 Hz, ltered. The ltrate was evaporated under reduced pressure to
2H), 7.68 (d, J ¼ 8.6 Hz, 2H), 6.72 (s, 1H), 3.13 (t, J ¼ 7.3 Hz, 2H), give the crude product which was puried over silica gel column
2.81 (t, J ¼ 7.3 Hz, 2H); 13C{1H}NMR (125 MHz, CD3COCD3): chromatography using hexane/ethyl acetate (9 : 1) as eluent.
d 173.3, 172.2, 161.2, 132.1 (2C), 128.7, 128.4 (2C), 123.5, 99.0,
4-Fluoro-3-phenyl-1,6-dioxa-2-azaspiro[4.4]non-2-ene
30.8, 21.9; HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C12H10
-
((ꢂ)-4a/4a0). Oil; 39.8 mg (90%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl ace-
BrNO3Na: 317.9736; found 317.9736.
tate : hexanes); IR nmax 3063, 2990, 2957, 2926, 1447, 1375, 1275,
3-(3-(4-(Triuoromethyl)phenyl)isoxazol-5-yl)propanoic acid 1260, 1060, 1039, 907, 750 cmꢄ1; H NMR (500 MHz, CDCl3):
1
0
(3v). Following the general procedure for 1,3-dipolar cycload- d4a/4a 7.8–7.76 (m, 2H), 7.44 (d, J ¼ 3.5 Hz, 3H), 5.76 (d, J ¼
dition (condition 2), alkynoic acid 2a (0.25 g, 2.55 mmol), (Z)-N- 53.2 Hz, 1H), 4.17 (dd, J ¼ 14.8, 7.4 Hz, 1H), 4.09 (dd, J ¼ 14.8,
hydroxy-4-(triuoromethyl)benzimidoyl chloride (1.13 g, 5.10 7.4 Hz, 1H), 2.52–2.45 (m, 1H), 2.34–2.22 (m, 2H), 2.13–2.05 (m,
mmol), and K2CO3 (0.88 g, 6.38 mmol) provided 3v (0.67 g, 92%) 1H); 13C{1H}NMR (125 MHz, CDCl3) d4a/4a 162.2 (d, J ¼ 16.0 Hz),
0
as a white solid aer column chromatography (hexanes/EtOAc, 130.6, 128.9 (2C), 127.8, 126.7 (2C), 116.2 (d, J ¼ 28.3 Hz), 96.7
1 : 1); Rf 0.4 (1 : 1 ethyl acetate : hexanes); mp: 173–175 C; IR (d, J ¼ 187.6 Hz), 69.7, 28.8 (d, J ¼ 5.6 Hz), 23.9; 19F NMR (376
ꢁ
nmax 3129, 2929, 2851, 1702, 1599, 1441, 1320, 1166, 1133, 1062, MHz, CDCl3): d4a ꢄ185.4 (d, J ¼ 53.1 Hz); 19F NMR (376 MHz,
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0
CDCl3): d4a ꢄ201.6 (d, J ¼ 52.5 Hz); HRMS (ESI-TOF) m/z: [M + MHz, CDCl3): d4e 7.66–7.62 (m, 2H), 7.58–7.56 (m, 2H), 5.72 (d, J
Na]+ calcd for C12H12FNO2Na: 244.0744; found 244.0745.
¼ 53.3 Hz, 1H), 4.23–4.14 (m, 1H), 4.13–4.07 (m, 1H), 2.56–2.51
4-Fluoro-3-(p-tolyl)-1,6-dioxa-2-azaspiro[4.4]non-2-ene
(m, 1H), 2.33–2.20 (m, 2H), 2.13–2.06 (m, 1H); 13C{1H}NMR (125
((ꢂ)-4b/4b0). Oil; 41.8 mg (89%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl ace- MHz, CDCl3) d4e 157.1 (d, J ¼ 16.5 Hz), 132.2 (2C), 132.1, 128.1
tate : hexanes); IR nmax 3036, 2987, 2957, 2922, 2894, 1611, 1456, (2C), 125.1, 101.6 (d, J ¼ 23.1 Hz), 96.4 (d, J ¼ 188.1 Hz), 69.8,
1372, 1275, 1260, 1059, 036, 906, 782 cmꢄ1; 1H NMR (500 MHz, 28.9 (d, J ¼ 5.6 Hz), 23.9; 19F NMR (376 MHz, CDCl3): d4e ꢄ186.1
1
0
0
CDCl3): d4b/4b 7.66 (d, J ¼ 7.5 Hz, 2H), 7.24 (d, J ¼ 7.9 Hz, 2H), (d, J ¼ 53.8 Hz); H NMR (500 MHz, CDCl3): d4e 7.66 (d, J ¼
5.74 (d, J ¼ 53.2 Hz, 1H), 4.16 (dd, J ¼ 14.9, 7.3 Hz, 1H), 4.08 (dd, 8.3 Hz, 2H), 7.59 (d, J ¼ 6.8 Hz, 2H), 5.86 (d, J ¼ 52.7 Hz, 1H),
J ¼ 14.3 Hz, 7.4 Hz, 1H), 2.50–2.44 (m, 1H), 2.39 (s, 3H), 2.32– 4.25–4.22 (m, 1H), 4.18–4.10 (m, 1H), 2.47–2.43 (m, 1H), 2.36–
2.19 (m, 2H), 2.13–2.04 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3) 2.22 (m, 2H), 2.17–2.08 (m, 1H); 19F NMR (376 MHz, CDCl3): d4e
0
d4b/4b 155.6 (d, J ¼ 15 Hz), 141.0, 129.6 (2C), 126.6 (2C), 125.0, ꢄ190.4 (d, J ¼ 52.2 Hz); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
0
116.0 (d, J ¼ 27.8 Hz), 96.8 (d, J ¼ 187.6 Hz), 69.6, 28.8 (d, J ¼ 5.4
C
12H11BrFNO2Na: 321.9849; found 321.9851.
Hz), 23.9, 21.5; 19F NMR (376 MHz, CDCl3): d4b ꢄ185.52 (d, J ¼
3-(2,6-Dichlorophenyl)-4-uoro-1,6-dioxa-2-azaspiro[4.4]
53.2 Hz); 19F NMR (376 MHz, CDCl3): d4b ꢄ201.7 (d, J ¼ 52.5 non-2-ene ((ꢂ)-4f/4f0). Oil; 43.5 mg (75%, dr 1 : 1); Rf 0.4 (1 : 3
0
Hz); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C13H14FNO2Na: ethyl acetate : hexanes); IR nmax 2988, 2956, 2850, 2920, 2890,
1
258.0900; found 258.0901.
2595, 1494, 1400, 1350, 1272, 1090, 1060, 1010, 833 cmꢄ1; H
0
4-Fluoro-3-(4-uorophenyl)-1,6-dioxa-2-azaspiro[4.4]non-2-ene
NMR (500 MHz, CDCl3): d4f/4f 7.40 (dd, J ¼ 7.7, 3.3 Hz, 2H), 7.31
((ꢂ)-4c/4c0). Oil; 44.5 mg (93%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl aceta- (ddd, J ¼ 8.9, 7.4, 2.7 Hz, 1H), 6.13 (d, J ¼ 10.1 Hz, 1H), 4.23 (t, J
te : hexanes); IR nmax 2990, 2962, 2896, 1602, 1512, 1415, 1371, ¼ 6.3 Hz, 1H), 3.21 (t, J ¼ 7.5 Hz, 1H), 2.95 (t, J ¼ 7.5 Hz, 1H),
1229, 1059, 1033, 907, 887 cmꢄ1; H NMR (500 MHz, CDCl3): d4c 2.84 (t, J ¼ 7.5 Hz, 1H), 2.36–2.31 (m, 1H), 2.17–2.11 (m, 1H); 13
C
1
7.76 (dd, J ¼ 8.0, 5.8 Hz, 2H), 7.13 (t, J ¼ 8.5 Hz, 2H), 5.73 (d, J ¼ {1H}NMR (125 MHz, CDCl3) d4f/4f 158.8, 135.5 (2C), 130.9, 128.3,
53.3 Hz, 1H), 4.17 (dd, J ¼ 14.6, 7.4 Hz, 1H), 4.1 (dd, J ¼ 14.2, 128.2 (2C), 102.8 (d, J ¼ 12.42 Hz), 97.3 (d, J ¼ 154.9 Hz), 63.6,
7.5 Hz, 1H), 2.50–2.44 (m, 1H), 2.33–2.20 (m, 2H), 2.14–2.05 (m, 31.7, 23.6; 19F NMR (376 MHz, CDCl3): d4f ꢄ187.2 (d, J ¼ 52.7
0
1H); 13C{1H}NMR (125 MHz, CDCl3) d4c 164.0 (d, J ¼ 251.6 Hz), Hz); 19F NMR (376 MHz, CDCl3): d4f ꢄ202.4 (d, J ¼ 53.4 Hz);
0
154.7 (d, J ¼ 15.1 Hz), 128.7 (d, J ¼ 8.6 Hz, 2C), 124.1 (d, J ¼ 3.4 Hz), HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C12H10Cl2FNO2Na:
116.3 (d, J ¼ 27.7 Hz), 116.1 (d, J ¼ 22.1 Hz, 2C), 96.7 (d, J ¼ 187.9 311.9965; found 311.9967.
Hz), 69.7, 28.8 (d, J ¼ 5.5 Hz), 23.9; 19F NMR (376 MHz, CDCl3): d4c
4-Fluoro-3-(4-(triuoromethyl)phenyl)-1,6-dioxa-2-azaspiro
ꢄ108 (m), ꢄ185.8 (d, J ¼ 53.2 Hz); 1H NMR (500 MHz, CDCl3): d4c [4.4]non-2-ene ((ꢂ)-4g/4g0). Oil; 53.2 mg (92%, dr 1 : 1); Rf 0.4
7.79 (ddd, J ¼ 8.8, 5.3, 1.1 Hz, 2H), 7.13 (t, J ¼ 8.7 Hz, 2H), 5.86 (d, J (1 : 3 ethyl acetate : hexanes); IR nmax 3005, 2954, 2928, 2850,
0
¼ 52.6 Hz, 1H), 4.28–4.22 (m, 1H), 4.13–4.09 (m, 1H), 2.47–2.41 (m, 1460, 1325, 1270, 1265, 1170, 1130, 1070, cmꢄ1; H NMR (500
1
1H), 2.35–2.22 (m, 2H), 2.17–2.09 (m, 1H); 13C{1H}NMR (125 MHz, MHz, CDCl3): d4g 7.89 (d, J ¼ 8.1 Hz, 2H), 7.70 (d, J ¼ 8.2 Hz, 2H),
0
CDCl3) d4c 164.2 (d, J ¼ 251.9 Hz), 155.3 (d, J ¼ 16.2 Hz), 128.9 (d, J 5.77 (d, J ¼ 53.3 Hz, 1H), 4.19 (dd, J ¼ 14.6, 7.3 Hz, 1H), 4.12 (dd,
¼ 8.6 Hz, 2C), 123.9 (d, J ¼ 2.2 Hz), 116.1 (d, J ¼ 22.1 Hz, 2C), 112.7 J ¼ 14.5, 7.4 Hz, 1H), 2.53–2.46 (m, 1H), 2.35–2.30 (m, 1H), 2.29–
(d, J ¼ 13.1 Hz), 93.6 (d, J ¼ 201.7 Hz), 70.1, 34.1, 24.2; 19F NMR 2.22 (m, 1H), 2.15–2.08 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3)
0
(376 MHz, CDCl3): d4c ꢄ108 (tt, J ¼ 8.4, 5.3 Hz), ꢄ202.5 (d, J ¼ 52.6 d4g 154.6 (d, J ¼ 15.2 Hz), 131.2, 128.2, 126.9 (2C), 125.9 (dd, J ¼
Hz); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C12H11F2NO2Na: 7.5, 3.9 Hz, 2C), 123.7 (q, J ¼ 276.6 Hz), 116.8 (d, J ¼ 27.8 Hz),
1
262.0650; found 262.0651.
96.2 (d, J ¼ 188.1 Hz), 69.9, 28.9 (d, J ¼ 5.6 Hz), 23.9; H NMR
0
3-(4-Chlorophenyl)-4-uoro-1,6-dioxa-2-azaspiro[4.4]non-2-
(500 MHz, CDCl3): d4g 7.90 (d, J ¼ 8.1 Hz, 2H), 7.74 (d, J ¼
ene ((ꢂ)-4d/4d0). Oil; 48.6 mg (95%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl 8.3 Hz, 2H), 5.94 (d, J ¼ 52.2 Hz, 1H), 3.00–2.85 (m, 3H), 2.79–
acetate : hexanes); IR nmax 2987, 2957, 2851, 2922, 2896, 2597, 2.73 (m, 1H), 2.62–2.57 (m, 1H), 2.36–2.31 (m, 1H); 19F NMR
1494, 1405, 1355, 1275, 1091, 1062, 1014, 833 cmꢄ1; H NMR (376 MHz, CDCl3): d4g ꢄ62.9 (s), ꢄ191.6 (d, J ¼ 52.4 Hz); HRMS
1
(500 MHz, CDCl3): d4d 7.72 (d, J ¼ 8.5 Hz, 2H), 7.41 (d, J ¼ 8.4 Hz, (ESI-TOF) m/z: [M + Na]+ calcd for C26H20F8N2O4Na: 599.1187;
2H), 5.72 (d, J ¼ 53.3 Hz, 1H), 4.20–4.15 (m, 1H), 4.10 (dd, J ¼ found 599.1200.
14.9, 7.5 Hz, 1H), 2.55–2.41 (m, 2H), 2.31–2.20 (m, 1H), 2.13–
4-Fluoro-3-(4-nitrophenyl)-1,6-dioxa-2-azaspiro[4.4]non-2-
2.05 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3) d4d 156.9, 136.7, ene ((ꢂ)-4h/4h0). Oil; 42.6 mg (80%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl
129.2 (2C), 128.2 (2C), 125.8, 110.0, 96.5 (d, J ¼ 187.9 Hz), 70.2, acetate : hexanes); IR nmax 3008, 2987, 2950, 2921, 2852, 1607,
31.2, 23.9; 19F NMR (376 MHz, CDCl3): d4d ꢄ185.9 (d, J ¼ 52.6 1518, 1458, 1345, 1275, 1260, 1055, 914, 853 cmꢄ1; 1H NMR (500
0
Hz); 1H NMR (500 MHz, CDCl3): d4d 7.71 (d, J ¼ 8.7 Hz, 2H), 7.41 MHz, CDCl3): d4h/4h 8.30 (d, J ¼ 8.9 Hz, 2H), 7.94 (d, J ¼ 8.0 Hz,
(d, J ¼ 8.7 Hz, 2H), 5.86 (d, J ¼ 52.6 Hz, 1H), 4.33–4.26 (m, 1H), 2H), 5.79 (d, J ¼ 53.3 Hz, 1H), 4.20 (dd, J ¼ 14.5, 7.4 Hz, 1H), 4.13
4.14–4.09 (m, 1H), 2.47–2.42 (m, 1H), 2.35–2.27 (m, 2H), 2.18– (dd, J ¼ 14.7, 7.5 Hz, 1H), 2.53–2.48 (m, 1H), 2.37–2.25 (m, 2H),
0
0
2.12 (m, 1H); 19F NMR (376 MHz, CDCl3): d4d ꢄ202.3 (d, J ¼ 52.5 2.17–2.12 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3) d4h/4h 167.6,
Hz); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C12H11ClFNO2Na: 148.7, 133.8, 127.4 (2C), 124.2 (2C), 117.2 (d, J ¼ 28.0 Hz), 95.9
0
0
278.0354; found 278.0356.
(d, J ¼ 188.3 Hz), 70.1, 28.9 (d, J ¼ 5.5 Hz), 23.8; 19F NMR (376
MHz, CDCl3): d4h ꢄ186.1 (d, J ¼ 53.9 Hz); HRMS (ESI-TOF) m/z:
3-(4-Bromophenyl)-4-uoro-1,6-dioxa-2-azaspiro[4.4]non-2-
ene ((ꢂ)-4e/4e0). Oil; 57.0 mg (95%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl [M + Na]+ calcd for C12H11FN2O4Na: 289.0595; found 289.0594.
acetate : hexanes); IR nmax 3005, 2987, 2960, 2927, 2899, 2858,
4-Fluoro-3-phenyl-1,6-dioxa-2-azaspiro[4.5]dec-2-ene ((ꢂ)-4i/
1594, 1491, 1457, 1275, 1260, 1067, 912, 754 cmꢄ1; 1H NMR (500 4i0). Oil; 42.3 mg (90%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl
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acetate : hexanes); IR nmax 3061, 3003, 2954, 2886, 2853, 1446, ethyl acetate : hexanes); IR nmax 3000, 2955, 2925, 2853, 1578,
1
1377, 1275, 1260, 1203, 905, 889, 764, 750 cmꢄ1; H NMR (500 1560, 1430, 1333, 1275, 1196, 876, 780 cmꢄ1; 1H NMR (500 MHz,
0
0
MHz, CDCl3): d4i/4i 7.78–7.76 (m, 2H), 7.43 (dd, J ¼ 5.2, 1,8 Hz, CDCl3): d4m/4m 7.41–7.39 (m, 2H), 7.33 (dd, J ¼ 8.9, 7.1 Hz, 1H),
3H), 5.5 (d, J ¼ 53.1 Hz, 1H). 4.08 (td, J ¼ 12.3, 2.7 Hz, 1H), 3.76 5.54 (d, J ¼ 53.2 Hz, 1H), 4.14 (td, J ¼ 12.2, 2.7 Hz, 1H), 3.84 (dd,
(dd, J ¼ 11.4, 4.4 Hz, 1H), 2.03–1.89 (m, 4H), 1.81–1.74 (m, 1H), J ¼ 11.6, 4.5 Hz, 1H), 2.06–1.89 (m, 4H), 1.82–1.76 (m, 1H), 1.72–
1.69–1.66 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3) d4i/4i 155.9 1.68 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3) d4m/4m 153.1 (d, J
(d, J ¼ 15.2 Hz), 130.6, 130.9 (2C), 127.8, 126.7 (2C), 107.4 (d, J ¼ ¼ 16.2 Hz), 135.9, 131.5 (2C), 128.4, 128.3 (2C), 108.0 (d, J ¼ 25.8
26.4 Hz), 98.01 (d, J ¼ 188.8 Hz), 63.6, 26.2 (d, J ¼ 7.3 Hz), 24.8, Hz), 99.1 (d, J ¼ 191.1 Hz), 63.9, 26.2 (d, J ¼ 7.6 Hz), 24.8, 18.6 (d,
18.6 (d, J ¼ 2.2 Hz); 19F NMR (376 MHz, CDCl3): d4i ꢄ185.5 (d, J J ¼ 2.1 Hz); 19F NMR (376 MHz, CDCl3): d4m ꢄ188.1 (d, J ¼ 53.5
0
0
¼ 53.2 Hz); 19F NMR (376 MHz, CDCl3): d4i ꢄ201.8 (d, J ¼ 52.5 Hz); 19F NMR (376 MHz, CDCl3): d4m ꢄ193.3 (d, J ¼ 53.7 Hz);
Hz); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C13H14FNO2Na: HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C13H12Cl2FNO2Na:
0
0
258.0900; found 258.0901.
326.0121; found 326.0120.
4-Fluoro-3-(4-uorophenyl)-1,6-dioxa-2-azaspiro[4.5]dec-2-
4-Fluoro-3-(4-(triuoromethyl)phenyl)-1,6-dioxa-2-azaspiro
ene ((ꢂ)-4j/4j0). Oil; 46.1 mg (91%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl [4.5]dec-2-ene ((ꢂ)-4n/4n0). Oil; 57.6 mg (95%, dr 1 : 1); Rf 0.4
acetate : hexanes); IR nmax 3003, 2950, 2927, 2889, 2851, 1603, (1 : 3 ethyl acetate : hexanes); IR nmax 3003, 2952, 2924, 2853,
1513, 1375, 1233, 1160, 1078, 0146, 905, 838, 750 cmꢄ1; 1H NMR 1459, 1324, 1275, 1260, 1169, 1130, 1070, 879, 764 cmꢄ1 1H
;
0
0
(500 MHz, CDCl3): d4j/4j 7.76 (d, J ¼ 7.8, 5.5 Hz, 2H), 7.12 (t, J ¼ NMR (500 MHz, CDCl3): d4n/4n 7.88 (d, J ¼ 8.4 Hz, 2H), 7.69 (d, J
8.6 Hz, 2H), 5.51 (d, J ¼ 53.2 Hz, 1H), 4.08 (td, J ¼ 12.1, 2.7 Hz, ¼ 8.2 Hz, 2H), 5.55 (d, J ¼ 53.2 Hz, 1H), 4.11–4.05 (m, 1H), 3.78
1H), 3.77 (dd, J ¼ 11.4, 4.3 Hz, 1H), 2.03–1.89 (m, 4H), 1.82–1.73 (dd, J ¼ 11.5, 4.2 Hz, 1H), 2.04–1.91 (m, 4H), 1.80–1.74 (m, 1H),
(m, 1H), 1.69–1.67 (br m, 1H); 13C{1H}NMR (125 MHz, CDCl3) 1.71–1.67 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3) d4n/4n 156.2
0
0
d4j/4j 164.0 (d, J ¼ 251.6 Hz), 155.0 (d, J ¼ 14.1 Hz), 128.7 (d, J ¼ (d, J ¼ 15.1 Hz), 130.9 (q, J ¼ 31.6 Hz), 128.8 (d, J ¼ 3.0 Hz), 126.9
8.6 Hz, 2C), 124.1 (d, J ¼ 3.4 Hz), 116.1 (d, J ¼ 22.1 Hz, 2C), 107.5 (2C), 125.87 (dd, J ¼ 6.7, 3.4 Hz, 2C), 123.7 (q, J ¼ 265.0 Hz),
(d, J ¼ 26.3 Hz), 98.0 (d, J ¼ 188.8 Hz), 63.6, 26.1 (d, J ¼ 7.2 Hz), 108.1 (d, J ¼ 25.9 Hz), 97.6 (d, J ¼ 189.9 Hz), 63.7, 29.7, 26.1 (d, J
24.8, 18.6 (d, J ¼ 2.2 Hz); 19F NMR (376 MHz, CDCl3): d4j ꢄ109.1 ¼ 7.2 Hz), 18.5 (d, J ¼ 2.2 Hz); 19F NMR (376 MHz, CDCl3): d4n
0
(m), ꢄ186.0 (d, J ¼ 53.2 Hz); 19F NMR (376 MHz, CDCl3): d4j ꢄ62.8 (s), ꢄ191.4 (d, J ¼ 52.3 Hz); HRMS (ESI-TOF) m/z: [M +
0
ꢄ112.3 (m), ꢄ206.2 (d, J ¼ 52.5 Hz); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C13H10Cl2FNO3$H2ONa: 358.0019; found
Na]+ calcd for C13H13F2NO2Na: 276.0806; found 276.0807.
358.0021.
3-(4-Chlorophenyl)-4-uoro-1,6-dioxa-2-azaspiro[4.5]dec-2-
4-Fluoro-3-(4-nitrophenyl)-1,6-dioxa-2-azaspiro[4.5]dec-2-
ene ((ꢂ)-4k/4k0). Oil; 47.5 mg (88%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl ene ((ꢂ)-4o/4o0). Oil; 46.0 mg (82%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl
acetate : hexanes); IR nmax 2985, 2958, 2850, 2925, 2898, 2595, acetate : hexanes); 1H NMR (500 MHz, CDCl3): d4o/4o 8.30 (d, J ¼
0
1
1490, 1400, 1352, 1276, 1093, 1060, 1010, 833 cmꢄ1; H NMR 8.8 Hz, 2H), 7.97 (d, J ¼ 8.2 Hz, 2H), 5.56 (d, J ¼ 53.3 Hz, 1H)
0
(500 MHz, CDCl3): d4k/4k 7.70 (d, J ¼ 8.0 Hz, 2H), 7.41 (d, J ¼ 4.11–4.06 (td, J ¼ 12.2, 2.7 Hz, 1H), 3.80 (dd, J ¼ 11.3, 4.2 Hz,
8.0 Hz, 2H), 5.51 (d, J ¼ 53.2 Hz, 1H), 4.07 (td, J ¼ 12.3, 2.7 Hz, 1H), 2.05–1.92 (m, 4H), 1.84–1.79 (m, 1H), 1.72–1.69 (m, 1H);
1H), 3.77 (dd, J ¼ 11.4, 4.4 Hz, 1H), 2.02–1.89 (m, 4H), 1.82–1.73 13C{1H}NMR (125 MHz, CDCl3) d4o/4o 154.5 (d, J ¼ 15.2 Hz),
0
(m, 1H), 1.69–1.67 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3) 148.7, 133.4, 127.5 (2C), 124.2 (2C), 108.6 (d, J ¼ 26.2 Hz), 97.2
0
d4k/4k 155.1 (d, J ¼ 15.1 Hz), 136.6, 129.2 (2C), 128.0 (2C), 126.4, (d, J ¼ 189.4 Hz), 63.9, 26.0 (d, J ¼ 7.3 Hz), 24.7, 18.5 (d, J ¼ 1.9
107.7 (d, J ¼ 26.3 Hz), 97.8 (d, J ¼ 188.9 Hz), 63.7, 26.1 (d, J ¼ 7.3 Hz); 19F NMR (376 MHz, CDCl3): d4o ꢄ185.7 (d, J ¼ 54.0 Hz);
Hz), 24.8, 18.6 (d, J ¼ 2.1 Hz); 19F NMR (376 MHz, CDCl3): d4k HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C13H13Cl2FN2O4Na:
ꢄ185.6 (d, J ¼ 53.6 Hz); 19F NMR (376 MHz, CDCl3): d4k ꢄ192.7 303.0751; found 303.0752.
0
(d, J ¼ 53.2 Hz); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C13-
H10Cl2FNO3$H2ONa: 358.0019; found 358.0021.
General procedure for uoro-lactonization 5(a–k)
3-(4-Bromophenyl)-4-uoro-1,6-dioxa-2-azaspiro[4.5]dec-2-
ene ((ꢂ)-4l/4l0). Oil; 59.1 mg (94%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl A solution of isoxazole acid 3(p–z) (0.2 mmol) and Selectuor
acetate : hexanes); IR nmax 3010, 2950, 2927, 2886, 2851, 1588, (0.11 g, 0.3 mmol) in CH3CN (2 mL) was reuxed under N2 for
1492, 1401, 1267, 1079, 1046, 1010, 908, 878, 764 cmꢄ1; 1H NMR 24 h. Then, the reaction mixture was cooled, and water was
0
(500 MHz, CDCl3): d4l/4l 7.63 (d, J ¼ 7.7 Hz, 2H), 7.56 (d, J ¼ added and extracted with ethyl acetate. The organic phase was
8.6 Hz, 2H), 5.5 (d, J ¼ 53.2 Hz, 1H), 4.10–4.04 (m, 1H), 3.78–3.75 washed several times with water and nally with brine. The
(m, 1H), 2.02–1.91 (m, 4H), 1.81–1.72 (m, 1H), 1.69–1.67 (m, combined organic layers were dried over anhydrous MgSO4 and
1H); 13C{1H}NMR (125 MHz, CDCl3) d4l/4l 155.37 (d, J ¼ 15.1 ltered. The ltrate was evaporated under reduced pressure to
0
Hz), 132.3 (2C), 128.8, 128.3 (2C), 125.1, 107.8 (d, J ¼ 26.2 Hz), give the crude product which was puried over silica gel column
97.9 (d, J ¼ 188.9 Hz), 63.8, 26.3 (d, J ¼ 7.3 Hz), 24.9, 18.7 (d, J ¼ chromatography using hexane/ethyl acetate (4 : 1) as eluent.
2.1 Hz); 19F NMR (376 MHz, CDCl3): d4l ꢄ190.5 (d, J ¼ 52.1 Hz);
4-Fluoro-3-phenyl-1,6-dioxa-2-azaspiro[4.4]non-2-en-7-one
19F NMR (376 MHz, CDCl3): d4l ꢄ206.0 (d, J ¼ 52.5 Hz); HRMS ((ꢂ)-5a/5a0). Oil; 45.6 mg (97%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl ace-
0
(ESI-TOF) m/z: [M + Na]+ calcd for C13H13BrFNO2Na: 336.0005; tate : hexanes); IR nmax 2987, 2950, 2924, 2848, 1798, 1448, 1370,
found 336.0006.
1185, 1108, 1012, 880, 768 cmꢄ1; 1H NMR (500 MHz, CDCl3): d5a
3-(2,6-Dichlorophenyl)-4-uoro-1,6-dioxa-2-azaspiro[4.5]dec- 7.77 (d, J ¼ 7.8 Hz, 2H), 7.51–7.47 (m, 3H), 5.93 (d, J ¼ 52.2 Hz,
2-ene ((ꢂ)-4m/4m0). Oil; 47.4 mg (78%, dr 1 : 1); Rf 0.4 (1 : 3 1H), 2.98–2.83 (m, 2H), 2.76–2.70 (m, 1H), 2.59–2.54 (m, 1H);
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13C{1H}NMR (125 MHz, CDCl3): d5a 173.5, 156.1 (d, J ¼ 14.8 Hz), 30.2 Hz), 96.56 (d, J ¼ 192.1 Hz), 27.02 (d, J ¼ 1.87 Hz), 24.81 (d, J
131.5, 129.2 (2C), 127.0 (2C), 126.4, 112.7 (d, J ¼ 30.1 Hz), 96.8 ¼ 7.1 Hz); 19F NMR (376 MHz, CDCl3): d5d ꢄ190.4 (d, J ¼ 52.6
(d, J ¼ 191.8 Hz), 27.1 (d, J ¼ 1.7 Hz), 24.8 (d, J ¼ 7.1 Hz); 19F Hz); 1H NMR (500 MHz, CDCl3): d5d 7.73 (d, J ¼ 8.5 Hz, 2H), 7.45
0
1
NMR (376 MHz, CDCl3): d5a ꢄ190.2 (d, J ¼ 51.6 Hz); H NMR (d, J ¼ 8.5 Hz, 2H), 6.1 (d, J ¼ 51.5 Hz, 1H), 2.95–2.86 (m, 1H),
(500 MHz, CDCl3): d5a 7.79 (d, J ¼ 7.4 Hz, 2H), 7.49 (dt, J ¼ 14.6, 2.78–2.71 (m, 3H); 13C{1H}NMR (125 MHz, CDCl3): d5d 172.8,
6.8 Hz, 3H), 6.08 (d, J ¼ 51.4 Hz, 1H), 2.95–2.85 (m, 1H), 2.77– 156.2 (d, J ¼ 16.5 Hz), 137.9, 129.4 (2C), 128.3 (d, J ¼ 2.2 Hz, 2C),
0
0
2.71 (m, 3H); 13C{1H}NMR (125 MHz, CDCl3): d5a 173.0, 157.1 124.6 (d, J ¼ 2.2 Hz), 109.0 (d, J ¼ 14.1 Hz), 95.5 (d, J ¼ 207.7 Hz),
0
(d, J ¼ 16.4 Hz), 137.7, 129.0 (2C), 127.1 (d, J ¼ 2.1 HZ, 2C), 126.1 28.7, 27.7; 19F NMR (376 MHz, CDCl3): d5d ꢄ206.4 (d, J ¼ 51.4
0
(d, J ¼ 2.0 Hz), 108.9 (d, J ¼ 14.0 Hz), 95.7 (d, J ¼ 207.5 Hz), 28.7, Hz); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C12H9ClFNO3Na:
27.7; 19F NMR (376 MHz, CDCl3): d5a ꢄ205.9 (d, J ¼ 51.4 Hz); 292.0147; found 292.0146.
0
HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C12H10FNO3Na:
258.0537; found 258.0536.
3-(4-Bromophenyl)-4-uoro-1,6-dioxa-2-azaspiro[4.4]non-2-
en-7-one ((ꢂ)-5e/5e0). Oil; 59.0 mg (94%, dr 1 : 1); Rf 0.4 (1 : 3
ethyl acetate : hexanes); IR nmax 3025, 2957, 2927, 2848, 1793,
4-Fluoro-3-(p-tolyl)-1,6-dioxa-2-azaspiro[4.4]non-2-en-7-one
((ꢂ)-5b/5b0). White solid; 44.8 mg (90%, dr 1 : 1); Rf 0.4 (1 : 3 1591, 1487, 1412, 1269, 1134, 1009, 886 cmꢄ1
;
1H NMR (500
ethyl acetate : hexanes); mp: 112–114 ꢁC; IR nmax 3028, 2990, MHz, CDCl3): d5e 7.66–7.60 (m, 4H), 5.89 (d, J ¼ 52.3 Hz, 1H),
2955, 2922, 2848, 1795, 1608, 1452, 1371, 1278, 1247, 1176, 3.00–2.83 (m, 2H), 2.77–2.69 (m, 1H), 2.60–2.54 (m, 1H); 13C{1H}
1128, 1005, 873, 820 cmꢄ1; 1H NMR (500 MHz, CDCl3): d5b 7.65 NMR (125 MHz, CDCl3): d5e 173.5, 155.3 (d, J ¼ 15.9 Hz), 132.5,
(d, J ¼ 8.0 Hz, 2H), 7.27 (d, J ¼ 8.0 Hz, 2H), 5.91 (d, J ¼ 52.2 Hz, 132.3 (2C), 128.4 (2C), 126.1, 112.8 (d, J ¼ 30.1 Hz), 96.5 (d, J ¼
1H), 2.98–2.82 (m, 2H), 2.75–2.70 (m, 1H), 2.59–2.53 (m, 1H), 192.1 Hz), 27.0 (d, J ¼ 1.8 Hz), 24.8 (d, J ¼ 7.1 Hz); 19F NMR (376
1
2.41 (s, 3H); 13C{1H}NMR (125 MHz, CDCl3): d5b 173.6, 156.0 (d, MHz, CDCl3): d5e ꢄ190.4 (d, J ¼ 52.4 Hz); H NMR (500 MHz,
0
J ¼ 14.7 Hz), 142.1, 129.9 (2C), 125.9 (2C), 123.5, 112.6 (d, J ¼ CDCl3): d5e 7.66 (d, J ¼ 8.3 Hz, 2H), 7.61 (d, J ¼ 8.4 Hz, 2H), 6.1
30.1 Hz), 96.9 (d, J ¼ 191.8 Hz), 27.1 (d, J ¼ 1.8 Hz), 24.8 (d, J ¼ (d, J ¼ 51.5 Hz, 1H), 2.95–2.89 (m, 1H), 2.76–2.74 (m, 3H); 13C
7.0 Hz), 21.6; 19F NMR (376 MHz, CDCl3): d5b ꢄ190.1 (d, J ¼ 51.9 {1H}NMR (125 MHz, CDCl3): d5e 172.8, 156.3 (d, J ¼ 16.7 Hz),
0
Hz); 1H NMR (500 MHz, CDCl3): d5b 7.67 (d, J ¼ 7.1 Hz, 2H), 7.26 132.4 (2C), 131.7 (d, J ¼ 29.1 Hz), 128.4 (d, J ¼ 2.3 Hz, 2C), 126.4,
0
(d, J ¼ 7.6 Hz, 2H), 6.1 (d, J ¼ 51.5 Hz, 1H), 2.93–2.86 (m, 1H), 109.0 (d, J ¼ 14.2 Hz), 95.5 (d, J ¼ 207.6 Hz) 28.7, 27.6; 19F NMR
2.79–2.70 (m, 3H), 2.40 (s, 3H); 19F NMR (376 MHz, CDCl3): d5b (376 MHz, CDCl3): d5e ꢄ206.4 (d, J ¼ 51.5 Hz); HRMS (ESI-TOF)
ꢄ205.8 (d, J ¼ 51.5 Hz); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for m/z: [M + Na]+ calcd for C12H9BrFNO3Na: 335.9642; found
0
0
C
13H12FNO3Na: 272.0693; found 272.0692.
335.9645.
4-Fluoro-3-(4-uorophenyl)-1,6-dioxa-2 azaspiro[4.4]-non-2-
3-(2,6-Dichlorophenyl)-4-uoro-1,6-dioxa-2-azaspiro[4.4]
en-7-one ((ꢂ)-5c/5c0). Oil; 48.1 mg (95%, dr 1 : 1); Rf 0.4 (1 : 3 non-2-en-7-one ((ꢂ)-5f/5f0). Oil; 48.6 mg (80%, dr 1 : 1); Rf 0.4
ethyl acetate : hexanes); IR nmax 3076, 3013, 2952, 2924, 2852, (1 : 3 ethyl acetate : hexanes); IR nmax 3010, 2955, 2924, 2854,
1
1
1796, 1602, 1513, 1416, 1376, 1232, 1022, 882 cmꢄ1; H NMR 1710, 1560, 1432, 1275, 1260, 1194 cmꢄ1; H NMR (500 MHz,
(500 MHz, CDCl3): d5c 7.77 (dd, J ¼ 8.0, 5.5 Hz, 2H), 7.16 (t, J ¼ CDCl3): d5f 7.48–7.37 (m, 3H), 5.89 (d, J ¼ 52.1 Hz, 1H), 3.00–2.91
8.5 Hz, 2H), 5.90 (d, J ¼ 52.5 Hz, 1H), 2.90 (ddt, J ¼ 22.6, 19.4, (m, 1H), 2.90–2.81 (m, 1H), 2.78–2.71 (m, 1H), 2.64–2.59 (m,
9.5 Hz, 2H), 2.73 (ddd, J ¼ 16.4, 9.2, 2.9 Hz, 1H), 2.59–2.54 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3): d5f 173.5, 153.5 (d, J ¼ 16.2
1H); 13C{1H}NMR (125 MHz, CDCl3): d5c 173.4, 164.5 (d, J ¼ Hz), 135.8, 132.2 (2C), 128.4 (2C), 125.2, 112.8 (d, J ¼ 29.8 Hz),
253.3 Hz), 155.2 (d, J ¼ 14.0 Hz), 129.2 (d, J ¼ 8.7 Hz, 2C), 122.6 97.6 (d, J ¼ 94.4 Hz), 27.0 (d, J ¼ 1.8 Hz), 24.8 (d, J ¼ 7.1 Hz); 19
F
1
(d, J ¼ 3.4 Hz), 116.5 (d, J ¼ 22.2 Hz, 2C), 112.7 (d, J ¼ 30.1 Hz), NMR (376 MHz, CDCl3): d5f ꢄ187.6 (d, J ¼ 50.5 Hz); H NMR
96.7 (d, J ¼ 192.0 Hz), 27.1 (d, J ¼ 1.8 Hz), 24.8 (d, J ¼ 7.0 Hz); 19
F
(500 MHz, CDCl3): d5f 7.48–7.37 (m, 3H), 6.1 (d, J ¼ 51.2 Hz, 1H),
0
NMR (376 MHz, CDCl3): d5c ꢄ107.1 (m), ꢄ190.4 (d, J ¼ 52.2 Hz); 2.98–2.88 (m, 1H), 2.83–2.71 (m, 3H); 13C{1H}NMR (125 MHz,
1H NMR (500 MHz, CDCl3): d5c 7.80 (dd, J ¼ 7.7, 5.4 Hz, 2H), CDCl3): d5f 176.1, 153.6, 135.7, 132.3 (2C), 128.3 (2C), 116.2,
7.16 (t, J ¼ 8.6 Hz, 2H), 6.1 (d, J ¼ 51.5 Hz, 1H), 2.95–2.85 (m, 109.1 (d, J ¼ 13.2 Hz), 95.7 (d, J ¼ 209.4 Hz), 28.8, 27.6; 19F NMR
0
0
1H), 2.79–2.71 (m, 3H); 13C{1H}NMR (125 MHz, CDCl3): d5c (376 MHz, CDCl3): d5f ꢄ191.5 (d, J ¼ 51.9 Hz); HRMS (ESI-TOF)
172.9, 164.7 (d, J ¼ 253.5 Hz), 156.1 (d, J ¼ 16.4 Hz), 129.3 (d, J ¼ m/z: [M + Na]+ calcd for C12H8Cl2FNO3Na: 325.9757; found
8.4 Hz, 2C), 122.4 (d, J ¼ 1.8 Hz), 116.4 (d, J ¼ 22.1 Hz, 2C), 108.9 325.9757.
0
0
(d, J ¼ 14.2 HZ), 95.6 (d, J ¼ 207.5 Hz), 28.6, 27.7; 19F NMR (376
4-Fluoro-3-(4-(triuoromethyl)phenyl)-1,6-dioxa-2-azaspiro
MHz, CDCl3): d5c ꢄ106.7 (m), ꢄ206.2 (d, J ¼ 51.5 Hz); HRMS [4.4]non-2-en-7-one ((ꢂ)-5g/5g0). White solid; 57.6 mg (95%, dr
0
(ESI-TOF) m/z: [M + Na]+ calcd for C12H9F2NO3Na: 276.0443; 1 : 1); Rf 0.4 (1 : 3 ethyl acetate : hexanes); mp: 130–132 C; IR
ꢁ
found 276.0445.
nmax 3005, 2955, 2921, 2851, 1798, 1416, 1325, 1269, 1163, 1115,
3-(4-Chlorophenyl)-4-uoro-1,6-dioxa-2-azaspiro[4.4]non-2-
1025, 883 cmꢄ1
;
1H NMR (500 MHz, CDCl3): d5g 7.90 (d, J ¼
en-7-one ((ꢂ)-5d/5d0). Oil; 50.7 mg (94%, dr 1 : 1); Rf 0.4 (1 : 3 8.2 Hz, 2H), 7.74 (d, J ¼ 8.2 Hz, 2H), 5.94 (d, J ¼ 52.2 Hz, 1H),
ethyl acetate : hexanes); IR nmax 3099, 2957, 2922, 2848, 1798, 3.00–2.85 (m, 2H), 2.75 (d, J ¼ 16.3, 9.1, 2.8 Hz, 1H), 2.62–2.57
1608, 1446, 1275, 1123, 1024, 880, 747 cmꢄ1; 1H NMR (500 MHz, (m, 1H); 13C{1H}NMR (125 MHz, CDCl3): d5g 173.2, 155.1 (d, J ¼
CDCl3): d5d 7.70 (d, J ¼ 8.4 Hz, 2H), 7.45 (d, J ¼ 8.3 Hz, 2H), 5.95 13.6 Hz), 133.1 (q, J ¼ 32.1 Hz), 129.8, 127.3 (2C), 126.2 (d, J ¼
(d, J ¼ 52.1 Hz, 1H), 2.98–2.83 (m, 1H), 2.77–2.69 (m, 1H), 2.59– 3.3 Hz, 2C), 125.5 (q, J ¼ 272.6 Hz), 112.9 (d, J ¼ 30.4 Hz), 96.3 (d,
2.54 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3): d5d 173.4, 155.23 J ¼ 192.4 Hz), 26.9, 24.8 (d, J ¼ 7.2 Hz); 19F NMR (376 MHz,
(d, J ¼ 14.9 Hz), 137.7, 129.5 (2C), 128.2 (2C), 124.8, 112.8 (d, J ¼ CDCl3): d5g ꢄ63.1 (s), ꢄ190.5 (d, J ¼ 52.2 Hz); 1H NMR (500 MHz,
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CDCl3): d5g 7.92 (d, J ¼ 8.3 Hz, 2H), 7.74 (d, J ¼ 8.2 Hz, 2H), 6.1 2.87–2.81 (m, 1H), 2.69–2.63 (m, 1H), 2.35–2.23 (m, 3H), 2.08–
(d, J ¼ 51.4 Hz, 1H), 2.97–2.88 (m, 1H), 2.79–2.73 (m, 3H); 13C 2.04 (m, 1H), 1.40 (t, J ¼ 7.2 Hz, 3H); 13C{1H}NMR (125 MHz,
{1H}NMR (125 MHz, CDCl3): d5g 172.7, 156.0 (d, J ¼ 16.9 Hz), CDCl3) d5k/5k 167.1, 164.7, 160.5 (d, J ¼ 16.0 Hz), 111.9 (d, J ¼
133.3 (dd, J ¼ 63.7, 30.71 Hz), 129.5 (dd, J ¼ 3.7, 2.3 Hz), 127.4 13.8), 95.1 (d, J ¼ 193.7 Hz), 62.9, 29.3, 23.1 (d, J ¼ 7.2 Hz), 15.5,
0
0
(d, J ¼ 2.2 Hz, 2C), 126.0 (dd, J ¼ 7.7, 3.9 Hz, 2C), 125.5 (q, J ¼ 14.0; 19F NMR (376 MHz, CDCl3): d5k ꢄ191.3 (d, J ¼ 55.8 Hz); 19
F
0
272.5 Hz), 109.1 (d, J ¼ 13.9 Hz), 95.3 (d, J ¼ 207 Hz) 28.7, 27.6; NMR (376 MHz, CDCl3): d5k ꢄ198.1 (d, J ¼ 52.7 Hz); HRMS (ESI-
19F NMR (376 MHz, CDCl3): d5g ꢄ63.2 (s), ꢄ206.6 (d, J ¼ 51.5 TOF) m/z: [M + Na]+ calcd for C10H12FNO3$H2ONa: 286.0697;
0
Hz); HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C13H9ClF4NO3Na: found 286.0699.
326.0411; found 326.0413.
4-Fluoro-3-methyl-1,6-dioxa-2-azaspiro[4.4]non-2-en-7-one
Conflicts of interest
((ꢂ)-5h/5h0). Oil; 28.4 mg (82%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl ace-
tate : hexanes); IR nmax 3008, 2957, 2929, 2853, 1797, 1436,1 There are no conicts to declare.
1391, 1260, 1182, 1123, 1033, 905, 839 cmꢄ1; 1H NMR (500 MHz,
CDCl3): d5h 5.37 (d, J ¼ 52.0 Hz, 1H), 2.88 (dt, J ¼ 17.2, 9.5 Hz,
Acknowledgements
1H), 2.79–2.72 (m, 1H), 2.67 (ddd, J ¼ 17.2, 9.8, 2.8 Hz, 1H), 2.45
(ddd, J ¼ 13.4, 9.6, 2.8 Hz, 1H), 2.19 (d, J ¼ 2.9 Hz, 3H); 13C{1H} The project was supported by grants from the National Institute
NMR (125 MHz, CDCl3): d5h 173.7, 154.8 (d, J ¼ 15.1 Hz), 111.9 of General Medicinal Sciences (NIH/NIGMS) (2SC3GM094081-
(d, J ¼ 29.6 Hz), 98.6 (d, J ¼ 191.2 Hz), 27.1, 24.7 (d, J ¼ 6.8 Hz), 08) and partially supported by the National Science Founda-
11.1; 19F NMR (376 MHz, CDCl3): d5h ꢄ189.9 (d, J ¼ 55.9 Hz); 1H tion (HBCU-EiR) (1900127). The Analytical and NMR CORE
0
NMR (500 MHz, CDCl3): d5h 5.57 (d, J ¼ 51.2 Hz, 1H), 2.87–2.79 Facilities were supported by NIH/NIMHD (G12MD007581).
(m, 1H), 2.71–2.63 (m, 3H), 2.14 (s, 3H); 13C{1H}NMR (125 MHz,
0
CDCl3): d5h 173.1, 157.6 (d, J ¼ 17.7 Hz), 108.5 (d, J ¼ 13.5 Hz),
Notes and references
97.1 (d, J ¼ 206.1 Hz), 28.1, 27.7, 10.7; 19F NMR (376 MHz,
0
CDCl3): d5h ꢄ197.9 (d, J ¼ 52.7 Hz); HRMS (ESI-TOF) m/z: [M +
1 (a) P. Saraswat, G. Jeyabalan, M. Z. Hassan, M. U. Rahman
and N. K. Nyola, Synth. Commun., 2016, 46, 1643–1664; (b)
M. Zaki, A. Oukhrib, M. Akssira and S. B. Raboin, RSC
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Na]+ calcd for C7H8FNO3Na: 196.0380; found 196.0380.
4-Fluoro-3-propyl-1,6-dioxa-2-azaspiro[4.4]non-2-en-7-one
((ꢂ)-5i/5i0). Oil; 32.2 mg (80%, dr 1 : 1); Rf 0.4 (1 : 3 ethyl ace-
tate : hexanes); IR nmax 3008, 2965, 2937, 2876, 1796, 1713, 1447,
1275, 1177, 1042, 915, 764 cmꢄ1; 1H NMR (500 MHz, CDCl3): d5i
5.61 (d, J ¼ 51.2 Hz, 1H), 2.87–2.79 (m, 2H), 2.66–2.63 (m, 2H),
2.53–2.41 (m, 2H), 1.77–1.64 (m, 2H), 1.02 (t, J ¼ 7.4 Hz, 3H); 13
C
{1H}NMR (125 MHz, CDCl3) d5i 173.2, 160.6 (d, J ¼ 18.4 Hz),
108.4 (d, J ¼ 13.7 Hz), 96.7 (d, J ¼ 206.5 Hz), 28.1, 27.8, 27.5,
18.8, 13.7; 19F NMR (376 MHz, CDCl3): d5i ꢄ188.8 (d, J ¼ 52.2
1
0
Hz); H NMR (500 MHz, CDCl3): d5i 5.4 (d, J ¼ 51.9 Hz, 1H),
2.92–2.85 (m, 2H), 2.72–2.64 (m, 2H), 2.54–2.48 (m, 2H), 1.77–
1.65 (m, 2H), 1.01 (t, J ¼ 7.4 Hz, 3H); 19F NMR (376 MHz, CDCl3):
+
0
´
d5i ꢄ192.7 (d, J ¼ 50.7 Hz); HRMS (ESI-TOF) m/z: [M + Na] calcd
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3-(2,6-Dichlorophenyl)-4-uoro-1,6-dioxa-2-azaspiro[4.5]dec-
2-en-7-one ((ꢂ)-5j/5j0). Oil; 50.3 mg (79%, dr 1 : 1); Rf 0.4 (1 : 3
ethyl acetate : hexanes); IR nmax 3008, 2957, 2926, 2851, 1707,
1560, 1430, 1275, 1260, 1194, 1052, 782, 750 cmꢄ1; 1H NMR (500
0
MHz, CDCl3): d5j/5j 7.46–7.36 (m, 3H), 5.84 (d, J ¼ 52.5 Hz, 1H),
2.91–2.85 (m, 1H), 2.75–2.62 (m, 2H), 2.35–2.24 (m, 2H), 2.09–
2.03 (m, 1H); 13C{1H}NMR (125 MHz, CDCl3) d5j/5j 168.0, 162.5
0
(d, J ¼ 16.1 Hz), 135.9, 132.1 (2C), 128.4 (2C), 125.3, 110.6 (d, J ¼
26.9 Hz), 98.8 (d, J ¼ 195.4 Hz), 29.3, 23.6 (d, J ¼ 7.8 Hz), 15.4 (d,
J ¼ 1 Hz); 19F NMR (376 MHz, CDCl3): d5j ꢄ187.7 (d, J ¼ 52.3 Hz);
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0
(ESI-TOF) m/z: [M + Na]+ calcd for C13H10Cl2FNO3$H2ONa:
358.0019; found 358.0021.
Ethyl
4-uoro-7-oxo-1,6-dioxa-2-azaspiro[4.5]dec-2-ene-3-
6 (a) K. Kaur, V. Kumar, A. K. Sharma and G. K. Gupta, Eur. J.
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carboxylate ((ꢂ)-5k/5k0). Oil; 39.2 mg (80%, dr 1 : 1); Rf 0.4
(1 : 3 ethyl acetate : hexanes); IR nmax 3005, 2950, 2923, 2853,
1719, 1462, 1275, 1259, 1016, 925, 764 cmꢄ1; 1H NMR (500 MHz,
´
R. S. Compagnone, R. Avila, A. I. Suarez, O. V. Abrams,
0
˜
CDCl3): d5k/5k 5.67 (d, J ¼ 51.8 Hz, 1H), 4.43 (q, J ¼ 7.2 Hz, 2H),
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