Mechanochemical synthesis and cycloaddition reactions of fluoro nitrone under solvent-free conditions and potential antimicrobial activities of the cycloadducts

Article abstract of DOI:10.1002/jhet.3736

Synthesis and cycloaddition reactions of fluoro nitrone under solvent-free conditions using ball-milling technique have been reported. Significant change in rate and yields of the cycloadducts have been noticed compared with solvent-free microwave-induced

Full text of DOI:10.1002/jhet.3736

Received: 21 June 2019
DOI: 10.1002/jhet.3736
Revised: 11 August 2019
Accepted: 30 August 2019
N O T E
Mechanochemical synthesis and cycloaddition reactions of
fluoro nitrone under solvent‐free conditions and potential
antimicrobial activities of the cycloadducts
Bhaskar Chakraborty1,2
1 Organic Chemistry Laboratory, Sikkim
Abstract
Government College (NBBDC), Gangtok,
Synthesis and cycloaddition reactions of fluoro nitrone under solvent‐free con-
India
2 Academic visitor, School of Chemistry,
Cardiff University, Cardiff, UK
ditions using ball‐milling technique have been reported. Significant change in
rate and yields of the cycloadducts have been noticed compared with solvent‐
free microwave‐induced reactions of fluoro nitrones. The present study reports
synthesis of N‐benzyl fluoro nitrone and its cycloaddition reactions with
maleimides and few electron deficient alkynes under solvent‐free conditions.
The synthesized fluoro cycloadducts were found to exhibit potential antimicro-
bial activities.
Correspondence
Bhaskar Chakraborty, Organic Chemistry
Laboratory, Sikkim Government College
(NBBDC), Gangtok 737102, Sikkim, India;
or academic visitor, School of Chemistry,
Cardiff University, Park Place, Cardiff
CF10 3AT, Wales, UK.
Email: bhaskargtk@yahoo.com
Funding information
Department of Biotechnology, Govern-
ment of India, New Delhi, Grant/Award
Number: BT/20/NE/2011
1 | INTRODUCTION
challenge nowadays. So, ball‐milling technique may be
used as a lucrative methodology in the synthesis of impor-
Nitrones are excellent reaction intermediates and easily
undergoes 1,3‐dipolar cycloaddition reaction, resulting in
five‐membered heterocyclic compounds (commonly called
isoxazolidine and isoxazolines), which are the core group
of many natural products.[1] Majority of isoxazolines are
found to exhibit medicinal activities such as antibacterial,
anticonvulsant, antibiotic, antituberculer, antifungal, and
anticancer activities.[2,3] Besides green chemistry method-
ologies, majority of the reported procedures for the synthe-
sis of isoxazolidines were found to require drastic
experimental conditions like high temperature and long
reaction times. Moreover, these reactions were found to
suffer from selectivity, and there are possibilities of poor
yields and developments of side products as well.[4] Mech-
anochemical procedures using ball‐milling technique have
become very popular and developed recently in many
aspects. The procedure has also attracted the attention of
organic chemists in recent years.[5] Conducting organic
synthesis under environment‐friendly conditions is a
tant compounds, and this methodology may be considered
as an alternative to conventional procedures like heating,
microwave irradiation, and sonication. Therefore, syn-
thetic organic chemists should apply mechanochemical
procedures like ball‐milling as an effective and greener
technique for various organic transformations.[6]
Fluoro nitrones are a less‐known group of nitrones,
but they are of special interest in organic synthesis for
many reasons. It has been observed from various reported
works that the introduction of fluorine atom into a partic-
ular position of an organic molecule may cause signifi-
cant changes in the stability and biological activities of
the molecules.[7] It could be due to the high electronega-
tivity of the fluorine, the strong C─F bonding, and the
same size of the halogen and hydrogen atoms. Because
of these factors, syntheses of biologically active fluori-
nated compounds are in the focus nowadays.[8] Literature
survey reveals that synthesized fluorinated compounds
not only exhibits excellent biological properties but also
J Heterocyclic Chem. 2019;1–9.
wileyonlinelibrary.com/journal/jhet
© 2019 Wiley Periodicals, Inc.
1

2
CHAKRABORTY
reports how these compounds may be used as medicine
and agrochemicals.[9] It is also to be noted that because
of low polarizability and high lipophilicity of the fluorine
atom, it increases relative metabolic stability and
improves the bioavailability of the new heterocyclic com-
pounds compared with its hydrocarbon analogs.[10,11]
In continuation of our efforts to establish various
green methodologies in nitrone cycloaddition reac-
tions,[12–17] herein, we wish to report the development
of an environment‐friendly mechanochemical route to
isoxazolidine and isoxazoline derivatives using N‐benzyl
fluoro nitrone via 1,3‐dipolar cycloaddition reactions
(Scheme 1, Table 1).
materials was used, but when we tried different ratios of
starting materials, we have found incomplete conversion
to the target molecule. Sodium bicarbonate was added in
the mixture during the synthesis of nitrones because it
was observed that it could activate the hydroxylamine. It
may be due to the fact that because of the addition of
sodium bicarbonate, the reaction mixture becomes faintly
alkaline and also neutralize the liberated hydrochloric
acid. It has been observed under mechanochemistry condi-
tions that pKa data do not make normal sense and actually
weaker bases can be used to deprotonate things. We have
observed that sometimes, in mechanochemistry, we can
form a paste or a gum in the reaction, and things do not
mix well unless we had a solid material to help the mixture
flow more easily as solid. Since dry‐milling procedure has
been found to be more efficient, therefore, solid sodium
bicarbonate was used, and we received best results. We
have also tested the same reaction in absence of sodium
bicarbonate, but at the end of the reaction, a gummy crude
product was recovered by methanol. It was filtered, the sol-
vent was evaporated off, and we have observed that the
2 | RESULTS AND DISCUSSION
According to our proposed plan of work, we have con-
ducted the reactions taking one equivalent each of 2,6‐
difluoro benzaldehyde and N‐benzylhydroxylamine
hydrochloride along with one equivalent of sodium bicar-
bonate for the synthesis of fluoro nitrone 1(Scheme 1). 1H
NMR of the product indicated only the development of
expected product. The nitrone was used for cycloaddition
reactions without further purification. It is to be noted that
during the process of ball‐milling, slight heat was devel-
oped in the reaction vessel and also slight pressure inside
the vessel. It was observed while opening the reaction ves-
sel. We have obtained best results when 1:1 ratio of starting
1
reaction was incomplete from the H NMR spectrum of
the crude product. It has been also observed that if we per-
form the reactions at a lower frequency (10 or 20 Hz) for a
time period between 30 minutes and 2 hours, the aldehyde
and hydroxylamine are still present in the crude product.
This could be probably due to a lesser amount of energy
per impact and indicates that ball‐milling procedure with
a pause results in lower conversion.
SCHEME 1 Synthesis of fluoro
isoxazolidine and isoxazoline derivatives
using ball‐milling technique [Color figure
can be viewed at wileyonlinelibrary.com]

CHAKRABORTY
3
TABLE 1 Physicochemical data of synthesized molecules (2a‐4a; 2b‐4b and 5‐7)
Cycloadducts, m.p.(°C),
Time, h 2a‐4a: cis; 2b‐4b: trans
Cis/trans
ratio, %
c
Entry Nitrone
Dipolarophilea
Yieldb,
%
1
2
3
N‐benzyl fluoro nitrone N‐phenyl
5
5
6
2a: White crystals, 126
2b: White solid, 103
2a: 65 (57) 95 (82)
2b: 30 (25)
Maleimide
N‐benzyl fluoro nitrone N‐methyl
3a: Yellowish solid, 133
3b: Yellowish crystals, 122 3b: 28 (26)
3a: 65 (50) 93 (76)
Maleimide
N‐benzyl fluoro nitrone N‐cyclohexyl
4a: Yellow crystals, 140
4b: Yellow solid, 111
4a: 64 (47) 93 (76)
4b: 29 (29)
Maleimide
4
5
6
N‐benzyl fluoro nitrone Methyl phenyl propiolate
N‐benzyl fluoro nitrone Dimethyl acetylene dicarboxylate
N‐benzyl fluoro nitrone Acetylene dicarboxylic acid
6
7
7
5: Dark red gummy liquid
6: Red liquid
91 (72)
91 (70)
90 (68)
7: White thick liquid
aReaction conditions: nitrone (1 mmol), dipolarophiles (1 equivalent), ball‐milling, RT.
bAll products were characterized by IR,1H NMR, 13C NMR, and MS spectral data.
cIsolated yield after purification. Figures in parentheses indicate yields obtained under MWI & endo/exo ratio.
Since the development of nitrone was fast in this
technique, the intermolecular cycloaddition reactions
of the fluoro nitrone with activated double‐bonded
dipolarophiles (maleimides) and electron‐deficient
dipolarophiles (alkynes) were carried out following the
same technique only. It was observed that intermolecular
cycloaddition reactions of fluoro nitrone with
dipolarophiles have been found to be slower than the for-
mation of nitrone and only 56% of cycloadducts (eg, 2a)
were obtained after 2 hours of ball‐milling (Table 1, entry
1). We have observed that cycloaddition reactions were
completed only after 5 to 7 hours of ball‐milling process.
We have also tested cycloaddition reactions in presence
of polar solvents like acetonitrile and ethanol (1 mL) in
the ball‐milling process, but we did not observe any nota-
ble changes in the rate of cycloaddition reactions. On the
basis of these observations, we have conducted all the
cycloaddition reactions in ball‐milling procedure without
using any solvents.
We have also compared the advantages of the ball‐
milling technology over microwave technology under
solvent‐free conditions[18–20] and found that lower yields
of nitrone and cycloadducts were noted compared with
that of the corresponding ball‐milling techniques. In
microwave technology, the reactions proceeded very
slowly (up to 2 to 3 hours) at a high temperature
(120°C) and also without achieving a total conversion of
the starting material (2,6‐difluoro benzaldehyde). It could
be due to the degradation of N‐benzylhydroxylamine,
which could be the reason for a low yield. High yields
of cycloadducts (up to 90%) could be obtained in dichloro-
methane solution if we use an excess of hydroxylamine
(2 equivalents) under microwave irradiation.[19] Column
chromatography was necessary to purify the products.
We have also tried a few of these cycloaddition reactions
under microwave irradiation in the presence of N,N‐
dimethyl formamide (DMF) and noted that the yields
were 76% to 72% (which were much lower than that of
the ball‐milling process).
Significant diastereoselectivity has been observed from
1
the H NMR analysis of the crude products when the
cycloaddition reaction was conducted between fluoro
nitrone 1 with maleimides. The mixture of diastereomers
2a‐4a and 2b‐4b were obtained in almost 65:35 ratios.
The ratio was calculated on the basis of the intensities
of two well‐separated double doublets at an average value
of 6.20 and 2.25 δ ppm, respectively, in the maleimide
cycloadducts. These signals are due to 4‐H protons of
the isoxazolidine moiety of the two isomers. This is also
to be noted that three asymmetric centers were generated
in a single step of this cycloaddition reaction. It could be
due to the exo approach of Z nitrone towards maleimides
for the development of major cycloadducts 2a‐4a (transi-
tion state 1, Figure 1), while the endo approach of Z
nitrone towards maleimides develop minor cycloadducts
2b‐4b (transition state 2, Figure 1). Usual separation of
the diastereomers were carried out using column chroma-
tography, and the diastereomers were identified consider-
ing the multiplicity of the proton signals at 3‐H and 4‐H
protons along with their coupling constant values.[21,22]
In isoxazolidines 2a‐4a, the values of coupling constant
between 3‐H and 4‐H protons have been measured in
the range of 6.00 to 6.26 Hz, implying a cis relationship
between 3‐H and 4‐H protons, while in the case of
isoxazolidines 2b‐4b, the coupling constant has been
measured in the range of 2.00 to 2.26 Hz, which implies
a trans relationship between 3‐H and 4‐H protons.[21,22]
Electron‐deficient alkynes like dimethyl acetylene
dicarboxylate, phenyl methyl propiolate, and acetylene
dicarboxylic acid (but‐2‐ynedioic acid) furnishes

4
CHAKRABORTY
FIGURE
1
Exo/endo approach of the Z‐nitrone to the
maleimides in cycloaddition reactions [Color figure can be viewed
at wileyonlinelibrary.com]
regioselective isoxazoline derivatives (5‐7) in 1,3‐dipolar
cycloaddition reaction with N‐benzyl‐fluoro nitrone 1.
These results can be rationalized by an exo approach of
the Z nitrone 1 for the dipolarophiles (alkynes) in the
development of fluoro isoxazoline derivatives (5‐7; transi-
tion state 3).[21,23]
FIGURE 2 Conformations of fluoro isoxazoline derivatives
According to Huisgen's theory,[24] acetylenic
dipolarophiles are less reactive then expected on the basis
of their ionization potentials. Since alkynes have larger
highest occupied molecular orbital (HOMO)–lowest
unoccupied molecular orbital (LUMO) gap than alkenes,
it is expected that during interactions with alkyne, LUMO
plays the most significant part and hence alkynes are less
reactive than expected. However, the reactivity of
nitrones with electron‐deficient alkynes are actually
determined by dipole (HOMO)‐dipolarophiles (LUMO)
interactions and the regiochemistry.[25] In case of alkyne,
the dipole (HOMO)‐dipolarophile (LUMO) interactions
become very important and dominate the reaction for
the formation of regioselective adducts. Stereochemistry
of the fluoro isoxazoline derivatives (5‐7) could not be
determined in detail since two most important protons
are absent at 4‐H and 5‐H positions and the lone singlet
signal due to 3‐H proton is unable to predict it. The major
and minor conformers of the novel isoxazoline ring sys-
tems (5‐7) may be represented as follows (Figure 2).
The isolated isoxazolidine and isoxazoline derivatives
are stable, and mass spectral analysis shows the presence
of all the desired fragmentation peaks like molecular ion
peak and base peak. The maleimide cycloadducts (2‐4)
have shown a common base peak (B.P), which is due to
the fragmentation of benzyl and 2,6‐difluoro phenyl ring.
Prominent carbonyl group and aromatic C─H absorp-
tions have been noticed in the IR spectral studies. The
fragmentation pattern of isoxazoline derivatives (5‐7) are
of much attraction as they are found to develop aziridine
derivatives. Prominent base peaks are observed because of
the fragmentation of PhCO for phenyl methyl propiolate,
COOCH3 for dimethyl acetylene dicarboxylate, and
[Color figure can be viewed at wileyonlinelibrary.com]
COOH for acetylene dicarboxylic acid. Therefore, we
can confirm that the isoxazoline derivatives underwent
rearrangement to aziridine derivatives during mass frag-
1
mentation. On the basis of the spectral data of H NMR,
13C NMR, MS, and FT‐IR, structures of all the synthesized
isoxazolidine and isoxazoline derivatives (2‐7) have been
confirmed. We have also obtained satisfactory elemental
analysis data for all the cycloadducts.
3 | EXPERIMENTAL
Mechanochemical procedure (ball‐milling) was per-
formed in Retsch MM500 digital mixer mill, Retsch
GmbH, 42781, Hann, Germany. Bruker DRX 300 spec-
trometer (300 MHz, FT NMR) was used for recording
1H NMR spectra and TMS was used as internal standard.
Same instrument was used for recording 13C NMR spec-
tra at 75 MHz. The coupling constants (J) are expressed
in Hz. Perkin‐Elmer RX 1–881 machine was used for
recording IR spectra as film or as KBr pellets for all the
molecules. Jeol SX‐102 (FAB) instrument was used for
recording MS spectra. Perkin‐Elmer 2400 series CHN
analyzer was used for elemental analyses (C,H,N).
Progress of all the reactions were monitored by TLC
using 0.25 mm silica gel plates (Merck 60F254 UV
indicator). Column chromatography was performed with
silica gel (E.Merck Germany) with 60–200 mesh.
The starting materials and reagents used in the reactions
(N‐benzylhydroxylamine hydrochloride and 2,6‐difluoro
benzaldehyde) were obtained commercially from Aldrich,

CHAKRABORTY
5
Lancaster and were used without further purification
unless otherwise indicated. All other reagents and sol-
vents were purified before starting reactions or column
chromatography.
3.2.1 | Spectroscopic data of fluoro
isoxazolidine derivatives (2‐4)
(3S)‐2‐Benzyl‐3‐(2,6‐difluorophenyl)‐dihydro‐5‐phenyl‐
2H‐pyrrolo[3,4‐d]isoxazole‐4,6(5H,6 a‐H)‐dione, 2a
White crystals. Yield 65%; R f = 0.66; IR (KBr): υmax 3025
(m), 2924 (m), 2830 (m), 1758 (s), 1686 (s), 1482 (m),
1342 (m), 800 (s), 772 (s) cm−1 1H NMR (CDCl3): δ
;
3.1 | General procedure for the synthesis
of N‐benzyl fluoro nitrone (1) in
ball‐milling procedure
7.73–7.65 (m, 3H, C6H3F2), 7.14–6.80 (m, 2X5H, C6H5
protons), 5.80 (d, 1H, J = 6.68 Hz, C5H), 3.43 (dd, 1H,
J = 6.06, 6.16 Hz, C4H), 3.50 (s, 2H, C6H5CH2), 2.92 (d,
1H, J = 6.30 Hz, C3H); 13C NMR (CDCl3): δ 172.38,
172.24 (carbonyl carbons), 138.24, 138.17, 138.10,
137.90, 136.87, 136.68, 136.54, 136.40 (phenyl carbons),
134.40, 134.35, 134.20, 133.90, 133.80 (2,6‐difluoro phenyl
carbons), 86.17 (C5), 76.90 (C3), 58.83 (C4), 39.88
(CH2C6H5); FAB‐MS: m/z 420 (M+,100%), 343, 329, 306,
252, 216 (B.P), 113, 91, 77; Anal. Calcd. for
C24H18O3N2F2: C, 68.57; H, 4.28; N, 6.66%. Found: C,
68.40; H, 4.20; N, 6.54.
N‐benzylhydroxylamine hydrochloride (1 mmol), 2,6‐
difluoro benzaldehyde (1 equivalent), and NaHCO3
(1 equivalent) were mixed together and ball‐milled at
30 Hz for 1 hour in a 10‐mL steel vessel and 15‐mm diam-
eter balls at room temperature (16°C). After the comple-
tion of reaction, the reaction mixture was taken in
CH2Cl2. It was filtered on cotton for the removal of NaCl.
The filtrate was evaporated under vacuum to afford N‐
benzyl fluoro nitrone 1 as white crystalline solid with
high purity (84%, m.p; 82°C).
(3R)‐2‐Benzyl‐3‐(2,6‐difluorophenyl)‐dihydro‐5‐phenyl‐
2H‐pyrrolo[3,4‐d]isoxazole‐4,6(5H,6 a‐H)‐dione, 2b
White solid. Yield 30%; R f = 0.56; IR (KBr): υmax 3015
(m), 2916 (m), 2828 (m), 1766 (s), 1684 (s), 1482 (m),
3.1.1 | Spectroscopic data for nitrone 1
1340 (m), 862 (s), 778 (s) cm−1 1H NMR (CDCl3): δ
;
UV λmax 235 nm; IR (KBr): υmax 3022 (m), 2233 (m), 1684
1
(m), 1624 (s), 1442 (m), 1150 (m), 786 (s) cm−1. H NMR
7.72–7.62 (m, 3H, C6H3F2), 7.30–7.14 (m, 2X5H, C6H5
protons), 5.74 (d, 1H, J = 2.26 Hz, C5H), 3.62 (dd, 1H,
J = 2.20, 2.08 Hz, C4H), 3.32 (s, 2H, C6H5CH2), 3.02 (d,
1H, J = 3.00 Hz, C3H); 13C NMR (CDCl3): δ 172.26,
172.17 (carbonyl carbons), 137.46, 137.36, 137.23, 137.20,
137.14, 136.48, 136.30, 136.25 (phenyl carbons), 134.67,
134.48, 134.30, 134.25, 134.10 (2,6‐difluoro phenyl car-
bons), 82.60 (C5), 76.58 (C3), 56.66 (C4), 43.20 (CH2C6H5);
FAB‐MS: m/z 420 (M+,100%), 343, 329, 306, 216 (B.P),
113, 91, 77; Anal. Calcd. for C24H18O3N2F2: C, 68.57; H,
4.28; N, 6.66%. Found: C, 68.40; H, 4.14; N, 6.56.
(CDCl3): δ 7.95–7.77 (m, 3H, C6H3F2), 7.64–7.34 (m, 5H,
C6H5CH2), 6.98 (s, 1H,─CH═N+), 3.37 (s, 2H,
C6H5CH2); 13CNMR (CDCl3):
δ
141.12 (CH═N+),
134.82, 134.33, 134.15, 133.97 (phenyl carbons), 131.72,
130.23, 129.46, 129.25, 128.76, 128.42 (2,6‐difluoro phenyl
carbons).
3.2 | General procedure of synthesis of
diastereomeric fluoro isoxazolidine
derivatives (2‐4) in ball‐milling procedure
(3S)‐2‐Benzyl‐3‐(2,6‐difluorophenyl)‐dihydro‐5‐methyl‐
2H‐pyrrolo[3,4‐d]isoxazole‐4,6(5H,6 a‐H)‐dione, 3a
Yellowish solid. Yield 65%; R f = 0.60; IR (KBr): υmax
3010 (m), 2938 (m), 2822 (m), 1767 (s), 1672 (s), 1466
N‐benzyl fluoro nitrone 1 (1 equivalent) and N‐phenyl
maleimide (1 equivalent) was mixed together and ball‐
milled at 30 Hz for 5 hours in a 10‐mL steel vessel and
15‐mm diameter balls at room temperature (16°C). After
the completion of reaction, the reaction mixture was
taken in CH2Cl2. It was filtered on cotton for the removal
of NaCl. The filtrate was evaporated under vacuum to
afford mixture of crude fluoro isoxazolidines as white
crystals (95%). The crude product was directly charged
on silica gel column and eluted with a mixture of ethyl
acetate:n‐hexane (1:8) resulting in pure fluoro
isoxazolidines 2a and 2b (entry 1, Table 1, Scheme 1).
The same methodology was adopted for the synthesis of
other fluoro isoxazolidine derivatives (entries 2 and 3).
1
(s), 1342 (m), 810 (s), 776 (s) cm−1; H NMR (CDCl3):
δ 7.90–7.84 (m, 3H, C6H3F2), 7.65–7.42 (m, 5H, C6H5
protons), 6.53 (d, 1H, J = 6.12 Hz, C5H), 3.84 (s, 2H,
C6H5CH2), 3.76 (dd, 1H, J = 6.04, 5.98 Hz, C4H), 3.46
(s, 3H, N─CH3), 2.96 (d, 1H, J = 6.74 Hz, C3H); 13C
NMR (CDCl3): δ 170.48, 170.35 (carbonyl carbons),
136.36, 136.28, 136.20, 136.16 (phenyl carbons), 132.67,
132.60, 132.53, 132.40, 132.25 (2,6‐difluoro phenyl car-
bons), 83.90 (C5), 75.57 (C3), 59.80 (C4), 39.52
(CH2C6H5), 37.59 (N─CH3); FAB‐MS: m/z 358
(M+,100%), 345, 267, 252, 244, 154 (B.P), 113, 91; Anal.

6
CHAKRABORTY
Calcd. for C19H16O3N2F2: C, 63.68; H, 4.46; N, 7.82%.
Found: C, 63.37; H, 4.28; N, 7.50.
128.47, 128.43, 128.40, 128.27 (2,6‐difluoro phenyl car-
bons), 82.58 (C5), 77.60 (C3), 58.36 (C4), 36.17 (CH2C6H5),
26.80, 26.58, 26.37, 26.25, 24.80, 24.77 (cyclohexyl car-
bons); FAB‐MS: m/z 426 (M+,100%), 343, 312, 252, 222
(B.P), 113, 91, 83, 77; Anal. Calcd. for C24H24O3N2F2: C,
67.60; H, 5.63; N, 6.57%. Found: C, 67.40; H, 5.28; N, 6.22.
(3R)‐2‐Benzyl‐3‐(2,6‐difluorophenyl)‐dihydro‐5‐methyl‐
2H‐pyrrolo[3,4‐d]isoxazole‐4,6(5H,6 a‐H)‐dione, 3b
Yellowish crystals. Yield 28%; R f = 0.54; IR (KBr): υmax
3018 (m), 2900 (m), 2835 (s), 1763 (s), 1680 (s), 1455 (s),
1350 (m), 820 (s), 780 (s) cm−1 1H NMR (CDCl3): δ
;
3.3 | General procedure of synthesis of
fluoro isoxazoline derivatives (5‐7) in
ball‐milling procedure
7.86–7.80 (m, 3H, C6H3F2), 7.64–7.47 (m, 5H, C6H5 pro-
tons), 6.53 (d, 1H, J = 3.20 Hz, C5H), 3.86 (s, 2H,
C6H5CH2), 3.70 (dd, 1H, J = 1.94, 2.10 Hz, C4H), 3.46
(s, 3H, N─CH3), 2.90 (d, 1H, J = 1.94 Hz, C3H); 13C
NMR (CDCl3): δ 171.40, 171.34 (carbonyl carbons),
135.90, 135.78, 135.67, 135.60 (phenyl carbons), 133.38,
133.26, 132.20, 132.18, 132.15 (2,6‐difluoro phenyl car-
bons), 84.77 (C5), 73.46 (C3), 54.80 (C4), 41.36 (CH2C6H5),
39.20 (N─CH3); FAB‐MS: m/z 358 (M+,100%), 345, 267,
252, 154 (B.P), 113, 91, 77; Anal. Calcd. for
C19H16O3N2F2: C, 63.68; H, 4.46; N, 7.82%. Found: C,
63.37; H, 4.30; N, 7.55.
N‐benzyl fluoro nitrone 1 (1 equivalent) and methyl phe-
nyl propiolate (1 equivalent) was mixed together and ball‐
milled at 30 Hz for 6 hours in a 10‐mL steel vessel and
15‐mm diameter balls at room temperature (16°C). After
the completion of reaction, the reaction mixture was
taken in CH2Cl2. It was filtered on cotton for the removal
of NaCl. The filtrate was evaporated under vacuum to
afford crude fluoro isoxazoline as dark red gummy liquid
(91%). The crude product was directly charged on silica
gel column and eluted with a mixture of ethyl acetate:
n‐hexane (1:8) to afford pure fluoro isoxazoline 5 (entry 4,
Table 1, Scheme 1). The same methodology was adopted
for the synthesis of other fluoro isoxazoline derivatives
(entries 5 and 6).
(3S)‐2‐Benzyl‐5‐cyclohexyl‐3‐(2,6‐difluorophenyl)‐
dihydro‐2H‐pyrrolo[3,4‐d]isoxazole‐4,6(5H,6 a‐H)‐
dione, 4a
Yellow crystals. Yield 64%, R f = 0.62; IR (KBr): υmax 3014
(m), 2910 (s), 2836 (m), 1765 (s), 1666 (br, s), 1470 (s),
1330 (m), 800 (s), 780 (s) cm−1 1H NMR (CDCl3): δ
;
7.64–7.55 (m, 3H, C6H3F2), 7.18–7.04 (m, 5H, C6H5 pro-
tons), 6.30 (d, 1H, J = 6.76 Hz, C5H), 3.66 (s, 2H,
C6H5CH2), 3.44 (dd, 1H, J = 6.20, 6.20 Hz, C4H), 2.80
(d, 1H, J = 6.74 Hz, C3H), 1.94–1.50 (m, 11H, cyclohexyl
protons); 13C NMR (CDCl3): δ 168.40, 168.34 (carbonyl
carbons), 131.60, 131.54, 131.50, 131.42 (phenyl carbons),
129.48, 129.30, 128.26, 128.12, 128.05 (2,6‐difluoro phenyl
carbons), 85.60 (C5), 75.72 (C3), 57.20 (C4), 38.80
(CH2C6H5), 27.60, 27.54, 26.40, 26.26, 26.16, 26.06
(cyclohexyl carbons); FAB‐MS: m/z 426 (M+,100%), 343,
335, 312, 252, 222 (B.P), 113, 91, 83; Anal. Calcd. for
C24H24O3N2F2: C, 67.60; H, 5.63; N, 6.57%. Found: C,
67.37; H, 5.43; N, 6.30.
3.3.1 | Spectroscopic data of fluoro
isoxazoline derivatives (5‐7)
(S)‐Methyl‐2‐benzyl‐3‐(2,6‐difluorophenyl‐2,3‐dihydro‐
5‐phenylisoxazole‐4‐carboxylate, 5
Dark red gummy liquid. Yield 91%; R f = 0.70; IR (KBr):
υmax 3020 (m), 2246 (m), 1748 (s), 1714 (s), 1688 (s),
1616 (s), 1482 (s), 1324 (s), 1214 (s), 816 (m), 787 (s)
1
cm−1; H NMR (CDCl3): δ 7.85–7.78 (m, 3H, C6H3F2),
7.65–7.30 (m, 2x5H, C6H5), 3.34 (s, 3H, ─COOCH3),
2.65 (s,2H, C6H5CH2), 1.23 (s,1H, C3H); 13C NMR
(CDCl3): δ 169.50 (─COOCH3), 136.28, 136.25, 136.20,
136.15, 135.10, 135.05, 134.73, 134.48 (aromatic carbons),
133.60, 133.52, 133.47, 133.38, 133.28, 133.17 (2,6‐difluoro
phenyl carbons), 87.44 (C5), 74.90 (C3), 59.66 (C4), 45.28
(─COOCH3), 35.25 (benzylic carbon); FAB‐MS (m/z):
407 (M+), 330, 294, 211 (B.P), 203, 113, 105, 91, 77. Anal.
Calcd. for C24H19O3F2N: C, 70.76; H, 4.66; N, 3.43.
Found: C, 70.48; H, 4.53; N, 3.31%.
(3R)‐2‐Benzyl‐5‐cyclohexyl‐3‐(2,6‐difluorophenyl)‐
dihydro‐2H‐pyrrolo[3,4‐d]isoxazole‐4,6(5H,6 a‐H)‐
dione, 4b
Yellow solid. Yield 29%, R f = 0.54; IR (KBr): υmax 3008
(m), 2900 (s), 2842 (m), 1758 (s), 1672 (s), 1460 (s), 1334
1
(m), 813 (s), 783 (s) cm−1; H NMR (CDCl3): δ 7.55–7.47
(m, 3H, C6H3F2), 7.26–7.15 (m, 5H, C6H5 protons), 6.14
(d, 1H, J = 2.20 Hz, C5H), 3.50 (s, 2H, C6H5CH2), 3.52
(dd, 1H, J = 2.08, 2.08 Hz, C4H), 2.85 (d, 1H,
J = 2.75 Hz, C3H), 1.93–1.40 (m, 11H, cyclohexyl protons);
13C NMR (CDCl3): δ 168.84, 168.74 (carbonyl carbons),
132.50, 132.30, 130.26, 130.22 (phenyl carbons), 128.58,
(S)‐Dimethyl‐2‐benzyl‐3‐(2,6‐difluorophenyl)‐2,3‐
dihydroisoxazole‐4,5‐dicarboxylate, 6
Red liquid. Yield 91%; R f = 0.72; IR (KBr): υmax 3015 (m),
2254 (m), 1726 (s), 1682 (s), 1610 (s), 1445 (s), 1264 (s),
1226 (s), 800 (m), 783 (s) cm−1 1H NMR (CDCl3): δ
;
7.44–7.37 (m, 3H, C6H3F2), 7.12–6.95 (m, 5H, C6H5),

CHAKRABORTY
7
3.37 (s, 3H, ─COOCH3), 3.28 (s, 3H, ─COOCH3), 2.60
(s, 2H, C6H5CH2), 1.74 (s,1H, C3H); 13C NMR (CDCl3):
δ 170.66, 170.56 (─COOCH3, carbonyl carbons of the
ester group), 136.70, 136.65, 136.47, 136.36 (aromatic car-
bons), 132.70, 132.48, 132.40, 132.18, 132.05, 132.00 (2,6‐
difluoro phenyl carbons), 86.20 (C5), 75.68 (C3), 57.80
(C4), 47.38, 47.26 (─COOCH3, methyl carbons of the ester
methyl group), 39.57 (benzylic carbon); FAB‐MS (m/z):
389 (M+), 358, 330, 302, 276, 271 (B.P), 185, 113, 91, 77;
Anal. Calcd. for C20H17O5F2N: C, 61.69; H, 4.37; N, 3.59.
Found: C, 61.52; H, 4.21; N, 3.34%.
1H NMR (CDCl3): δ 10.10 (s, 2H, 2XCOOH), 7.92–7.84
(m, 3H, C6H3F2), 7.68–7.40 (m, 5H, C6H5), 2.93 (s,2H,
C6H5CH2), 2.85 (s,1H, C3H); 13C NMR (CDCl3): δ
172.80, 172.74 (carboxyl carbons), 137.68, 137.60, 137.53,
137.45 (aromatic carbons), 134.38, 134.27, 134.23, 134.17,
134.06, 134.00 (2,6‐difluoro phenyl carbons), 87.80 (C5),
75.90 (C3), 57.53 (C4), 34.22 (benzylic carbon); FAB‐MS
(m/z): 361 (M+), 344, 316, 288, 271 (B.P), 248, 157, 113,
91, 77. Anal. Calcd. for C18H13O5F2N: C, 59.83; H, 3.60;
N, 3.87. Found: C, 59.72; H, 3.33; N, 3.55%.
(S)‐2‐Benzyl‐3‐(2,6‐difluorophenyl)‐2,3‐
dihydroisoxazole‐4,5‐dicarboxylic acid, 7
3.4 | Antimicrobial study
White thick liquid. Yield 90%; R f = 0.74; IR (KBr): υmax
3012 (m), 2990 (br), 2245 (m), 1765 (s), 1610 (s), 1470 (s),
All the synthesized fluoro isoxazolidine and isoxazoline
derivatives (2‐7) were screened for antimicrobial activities
against 11 bacterial strains (Table 2) with Gentamycin as
1326 (s), 1210 (s), 1108 (s), 808 (m), 788 (s) cm−1
;
TABLE 2 MIC values (μg/mL) of fluoro isoxazolidine and isoxazoline derivatives (2‐7)
Organisms/Molecules
2a
3b
4a
4b
5
6
7
Escherichia coli ATCC 25930
Salmonella typhi 60
600
600
600
600
800
800
600
800
600
800
600
400
600
400
600
600
600
600
600
400
600
400
600
200
200
_
400
400
600
600
600
400
400
600
600
600
400
_
_
_
_
‐
600
600
_
Vibrio cholerae 21
1000
_
‐
Klebsiella pneumoniae 10030
Shigella dysentery 12
_
_
_
_
_
_
_
_
400
400
400
400
400
400
400
1000
_
_
Pseudomonas AMRI 102
Salmonella typhimurium NTCC 70
Staphylococcus aureus 29735
Bacillus cereus 11770
_
_
_
1000
1000
1000
1000
_
_
Bacillus subtilis 6634
_
Streptococcus epidermidis 1220
_
Note. “‐” represents no antimicrobial activity of the molecules.
TABLE 3 Represents zone of inhibition of molecules (2‐7) and MIC value (in mm)
Organisms/Compounds
2a
3b
4a
4b
5
6
7
Gentamycin
Escherichia coli ATCC 25930
Salmonella typhi 60
12
13
10
14
18
12
18
14
17
16
20
18
22
28
13
23
14
27
18
27
23
18
19
18
16
_
22
16
23
22
20
21
19
23
22
23
20
_
_
23
27
_
38
40
35
21
22
20
25
36
30
40
32
Vibrio cholerae 21
16
_
_
_
_
_
_
_
_
_
_
Klebsiella pneumoniae 10030
Shigella dysentery 12
_
24
18
20
22
22
20
17
24
_
_
Pseudomonas AMRI 102
Salmonella typhimurium NTCC 70
Staphylococcus aureus 29735
Bacillus cereus 11770
_
_
_
20
21
24
22
_
_
Bacillus subtilis 6634
_
Streptococcus epidermidis 1220
_
Note. “‐” represents no measurable zone of diameter at MIC value of molecules under study.

8
CHAKRABORTY
reference.[26] For culture medium, nutrient agar was
used, and the strains were grown at 23°C for 24 hours.
Following McFarland standard,[27] the suspension was
prepared. All the compounds under study were dissolved
in 4% dimethyl sulfoxide (DMSO) solution and sterile dis-
tilled water was added for screening using Agar dilu-
tion.[28] Standard cup plate assay method[29] was used
for finding minimum inhibitory concentration (MIC) of
the organisms to be screened. Bacterial solution
(2 × 106 CFU/mL) of 0.1 mL was transferred to nutrient
agar plates and spread uniformly with a sterile glass
spreader. The clear zone of inhibition on agar surface
around the wells was the area that was measured for eval-
uation of sensitivity after observing for 24 hours of incu-
bation at 23°C.[30] We have observed the MIC values
(Table 2) from antimicrobial study of the synthesized
molecules (2‐7) and also the corresponding zone of inhi-
bition of the molecules using gentamycin as reference
antimicrobial agent (Table 3). Our study shows that com-
pound 7 has no antimicrobial activity and compound 6
has effect on specific organisms like Salmonella typhi 62
and Vibrio cholerae 20. It gives an indication of a new
enteric drug. The molecules 2a, 3b, 4a, 4b, 5, and 6 have
been found to be very effective against both Gram‐
positive and Gram‐negative organisms, and it gives us
an opportunity to develop new “broad‐spectrum antimi-
crobial agents.” Antimicrobial screening study of the mol-
ecules 2b and 3a are going on at present.
the research work at the School of Chemistry, Cardiff
University, Wales, UK.
ORCID
Bhaskar Chakraborty
https://orcid.org/0000-0002-0642-
9794
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4 | CONCLUSION
In summary, fluoro nitrone and fluoro cycloadducts were
synthesized for the first time in a ball‐mill under mecha-
nochemical procedure and solvent‐free conditions with
excellent yield and selectivity. This protocol is promising
and therefore should attract the attention of organic
chemists working in the field of green chemistry. This
methodology also is very simple, cost efficient, and is safe
for the environment as well. Majority of the synthesized
molecules have been found to have potential antimicro-
bial activity against both Gram‐positive and Gram‐
negative organisms, and hence it creates an opportunity
for further biological study (antitumor and anticancer
activity) for these molecules.
ACKNOWLEDGMENTS
The author is grateful to the Department of Biotechnol-
ogy, Government of India, New Delhi for providing Over-
seas Associateship‐Fellowship (grant no BT/20/NE/2011)
and also administrative support and help for conducting

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How to cite this article: Chakraborty B.
Mechanochemical synthesis and cycloaddition
reactions of fluoro nitrone under solvent‐free
conditions and potential antimicrobial activities of
the cycloadducts. J Heterocyclic Chem. 2019;1–9.
https://doi.org/10.1002/jhet.3736
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