Design of a simple and efficient synthesis for bioactive novel pyrazolyl-isoxazoline hybrids

Article abstract of DOI:10.1039/c9nj01532a

A simple and new methodology has been developed for the synthesis of novel bioactive pyrazolyl-isoxazoline hybrids from a pyrazolyl-substituted oxime and various substituted allyloxybenzenes, which are easily available, inexpensive starting materials. All the novel synthesized target hybrids were characterized by NMR, IR, and mass spectroscopic techniques. We have employed some of the hybrid materials in anti-bacterial and anti-fungal activity studies. The hybrid materials 6m and 6p exhibited the best anti-bacterial and anti-fungal activities.

Full text of DOI:10.1039/c9nj01532a

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Design, simple and efficient synthesis of bio active novel pyrazolyl-isoxazoline hybrids
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Balakrishnan Sankar, Muniyasamy Harikrishnan, Renganathan Raja, Velu Sadhasivam, Nelson
Malini, Sepperumal Murugesan, Ayyanar Siva*
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Supramolecular and Organometallic Chemistry Lab, Department of Inorganic Chemistry, School
of Chemistry, Madurai Kamaraj University Madurai-21.
Corresponding author Email (drasiva@gmail.com and siva.chem@mkuniversity.org)
Abstract
A simple and new methodology introduced for the synthesis of novel bioactive pyrazolyl-
isoxazoline hybrids has been described from the reactions of pyrazoline substituted oximes and
various substituted allyoxy benzene through easily available inexpensive starting materials. All
the novel synthesized target hybrids were characterized by NMR, IR, and Mass spectroscopic
techniques. We were applying the some of the hybrid materials for anti-bacterial and anti-fungal
studies. The hybrid materials 6m and 6p having the best anti-bacterial and antifungal activities.
Keywords: Pyrazolyl, isoxazoline hybrids, oximes, anti-bacterial, anti-fungal
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Introduction
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Oxygen and nitrogen atom containing heterocyclic rings compounds are well- regarded
as a privileged structural modify in natural products, medicinal and industrial chemistry. The
infections caused by resistant microbes, often to respond the difenamizole, fluralmes, critotinib,
celecoxib drugs.1-3 These conventional drugs were cured the disease, but still have some side
effects, hence without side effect curing the disease is a great challenging in biochemistry sector.
The development of a simple molecule with different antimicrobial activities for the synthesis of
heterocycles is a worthwhile contribution in heterocyclic chemistry. In now a days, most of the
researchers were trying to develop a new drug which competes with the conventional drug.
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Spirobiisoxazoline4,
naphtho[2,3-d]isoxazole-4,9(3aH,9aH)-diones5,
isoxazoles7,
isoxazoline-functionalized
alkylaminopyrazole
isoquinolines8,
derivatives6,
substituted
Phenylhydrazonopyrazolone9, thiophenyl pyrazoles10 coumarin-pyrazole hybrid11 and the above
mentioned compounds are recently reported for having antibacterial and antifungal activities, but
these compounds have less antibacterial and antifungal activities in the biological studies.
Isoxazoline and their derivatives are very important scaffolds in pharmaceutical chemistry due to
their medicinal and therapeutics applications including insecticidal, antibacterial, antibiotic,
antitumor, antifungal, anti-tuberculosis and anticancer activities etc.,.12-17 On the other hand, the
pyrazole derivatives are also recognized as a significant class of heterocyclic compounds because
of their unique biological activities.18-24 Isoxazole and pyrazole derivatives were carried out the
pharmacological studies, most of the people only report isoxazoline or pyrazoline framework
with other antibacterial agent like phenyl hydrazine, isoquninolines, phenothiazine etc., the
development of a simple molecule with different antimicrobial activities for the synthesis of
heterocycles is a worthwhile contribution in heterocyclic chemistry.25
Moreover, the
construction of hybrid heterocyclic molecules with two or more heterocyclic units in a single
frame from simple and easily accessible starting precursors under facile reaction conditions is
one of the most important targets in organic synthesis. To the best of our knowledge, no one
reported the isoxazole and pyrazole moiety in a single frame. In view of the aforementioned and
pharmacological properties of these distinct heterocycles, it was envisioned that the synthesis of
hitherto more than two novel bioactive hybrid molecules (isoxazoline and pyrazole) can be
designed by a single framework.20 So, we have planned and designed novel materials of
isoxazole and pyrazole moiety in a single framework. The synthesis of these novel hybrid
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materials involves many special features such as inexpensive starting materials, no thermal
condition, no hazardous chemicals used, slightly basic conditions to achieve high yield,
chloramine –T act as both catalyst and base.
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Scheme 1: Synthesis of pyrazolyl-isoxazoline hybrids
(b)
N
1
N
O
O
H
N
EtOH
N
N
+
NH2
OEt
O
Cl
O
2
OH
(c)
N
N
N
N
(d)
O
O
O
N
OH
+
3
4
(e)
R
R-OH
O
5
(f)
N
N
N
O
O
O
19 Examples
6
R
Reaction and condition
(a) EtOH, 0 0C, NEt3 (b) POCl3, DMF, 100oC, (c), Phenol, KOH, DMF (d) hydroxylamine hydrochloride,
EtOH, NaOH, RT, (e) K2CO3, DMF (f) EtOH, Chloramine T
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Result and discussion
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Initially, we synthesized the oxime substituted pyrazole compound from the previous
literature and modified the reaction condition of the previous report for the synthesis of our
desired compound, and also we synthesized an allyoxy benzene compound as shown in the
scheme 1. These two compounds are reacted in a protic polar solvent as well as triethylamine act
as a base, but unfortunately we could not get a hybrid compound, the reaction cannot proceed in
this condition. Hence, we changed the various bases like, pyridine, imidazole,
diisopropylethylamine, benzimidazole and chloramine-T. Among these bases, only chloramine T
forms the hybrid compound. Hence, we decided the chloramine T as a good base for this novel
hybrid materials synthesis, and then we improve the yield of these compounds by changing the
various aprotic solvent likes as THF, DMF, DMSO. Among these solvents, DMF mediated
reaction gave a high yield of the product, this may be due to DMF acts as a base during the
course of reaction, so it easily attracts the oxime proton. (Table 1). Further, in order to obtain the
broad scope, the optimized reaction conditions were established by heterocyclic pyrazoline
oximes with a variety of substituted allyoxy derivatives to afford the respective bio active novel
pyrazolyl-isoxazoline hybrids (Table 2) in an excellent yield (65-85 %) within an hour.
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Table 1. Optimal conditions for the synthesis of hybrid compound 6a.
Yield
S.No.
Base
Solvent
(%)
NA
NA
NA
NA
NA
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Triethyl amine
Pyridine
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Methanol
DMSO
Imidazole
Diisopropylethylamine
Benzimidazole
Chloramine-T
Chloramine-T
Chloramine-T
Chloramine-T
Chloramine-T
Chloramine-T
Chloramine-T
Chloramine-T
15
30
THF
N-Methyl THF
DMF
Ethyl acetate
DCM
NA
NA
85
NA
NA
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Table 2. Novel pyrazolyl-isoxazoline hybrids derivatives
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N
N
N
N
O
O
CN
O
O
N
O
N
N
N
O
Br
N
O
N
O
O
N
O
O
O
N
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6c, 78%
O
6a, 85%
6b, 70%
6d, 74%
Cl
O
N
O
O
N
N
O
N
O
O
O
O
N
O
Cl
N
O
N
N
O
N
N
N
N
N
6g, 75%
O
6e, 70%
6f, 79%
6h, 70%
N
O
N
N
O
O
O
O
N
O
O
N
N
N
N
O
N
O
N
N
N
N
N
N
O
O
N
O
N
O
N
Cl
N
6l, 73%
6i, 72%
6k, 70%
6j, 68%
O
N
O
N
O
N
N
O
O
O
O
N
CHO
O
N
N
O
N
N
N
N
N
O
O
O
NO2
O
NO2
6p, 72%
6m, 75%
6o, 76%
6n, 75%
N
OHC
O
O
N
N
O
O
CN
O
N
O
N
O
O
O
N
N
N
N
6r, 65%
6s, 68%
6q, 70%
Under optimized reaction conditions, we carried out the scopes of the substrates for the
synthesis of isoxazole derivatives which are shown in table 2. From the observed results,
aromatic/aliphatic alcohols were treated with compound 2, we got a very good yield of isoxazole
derivatives. When using both electron-donating/withdrawing substrates, the desired products
were obtained in very good yield (Table 2, entries 6a-6s). All the synthesized hybrids 6(a-s) have
been characterized by H, 13C NMR, IR and mass spectral techniques (Fig. S1-S57).
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Furthermore, unambiguously conformed, the X-ray crystal structure (Figure 1) of compound
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Figure. 1 ORTEP diagram of isoxazoline derivative 6n
Mechanism for the formation of isoxazoline
Ts-N-Cl Na
I
Na
R1-CH=N-O-Cl
V
R1-CH=N-O
R1-CH=N-O-H
II
R1-C=N=O
H
-NaCl
-Ts-N-H
Na
VI
III
Ts
Cl
N
Ts-N-H
IV
H
R
R
O
1,3-dipolar
cycloaddition
N
O
O
R1
R1-C
O
N
O
Nitrile oxide
VII
R
R1-C
O
N
VIII
Scheme 2. Plausible reaction pathways for the formation of nitrile oxide using chloramines-T
and 1,3-dipolar cycloaddition of nitrile oxide with dipolarophile to get the cycloadducts 6a-s
The probable mechanism for the making of nitrile oxide using chloramines-T is shown in
scheme 2. The unpaired electron of the nitrogen atom of chloramine-T abstracts a proton from
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=N-OH of substituted aldoxime to form nucleophile, further, which is abstract a chlorine atom
followed by loss of sodium chloride to form the intermediate V. Furthermore, the abstraction of
proton from an intermediate by a mild base TsNH- ion, which is produced in a reaction form
nitrile oxide VII. Then, the probable 1,3-dipolar cycloaddition mechanism for the formation of
cycloadducts (6a-s).
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Biological studies
In the present work, a series of Isoxasole compounds were tested for antimicrobial
activity against Streptococcus mutans, a Gram-positive oral pathogenic bacteria, Salmonella
typhi, a Gram-negative pathogenic bacteria, Candida albicans, an opportunistic pathogenic yeast
and Aspergillus niger fungal culture by well diffusion technique. Ciproflaxacin and Itracanazole
were used as standard drugs for antibacterial and antifungal activity, respectively and distilled
water/DMSO was used as solvent control. Antibacterial activity of test compounds and
ciprofloxacin, taken as positive control, against S. typhi and S. mutans are summarized in Table 3
respectively. The zone of inhibition observed against S.typhi and S. mutans are shown in Fig. 2
and Fig. 5 respectively. Among the test compounds, compound 6b, 6f, 6i, 6j, 6m, 6n, 6p, 6q and
6s shows activity against S.typhi. Compound 6m has shown highest activity with zone of
inhibition of 11±0.0mm. In the Zone of inhibition of active compounds ranged from 6-11mm
are compared to 22mm observed for ciprofloxacin standard. Compounds 6p and 6q shows a
moderate activity with a zone of inhibition of 9mm each. Compound 6b showed less activity
with zone of inhibition of 6mm, and all others compounds 6f, 6i, 6n, and 6s has no activity
against S.typhi. Among the test compounds 6b, 6f, 6i, 6j, 6m, 6n, 6p, 6q and 6s showed
antibacterial activity against S .mutans. The Zone of inhibition of active compounds was in the
range of 7-13mm, the compound 6m has the highest activity with the a zone of inhibition of
13mm compared to 24mm observed against ciprofloxacin standard. Compounds 6n and 6p also
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showed moderate activity with a zone of inhibition of 10 and 11mm respectively. Compound 6b,
6f, 6i, 6j, 6s shows no activity with a zone of inhibition is NA. Antifungal activity of test
compounds and taking Itracanazole, as positive control, against C.albicans and A.niger is
summarized in Table 4 respectively. The zone of inhibition observed against C.albicans and
A.niger is shown in Fig. 3 and Fig. 4 respectively. Among the test compounds, compounds 6b,
6f, 6i, 6j, 6m, 6n, 6p, 6q and 6s shown activity against C.albicans. Compound 6j has shown
highest activity with a zone of inhibition of 13±0.0mm. The zone of inhibition of active
compounds ranged from 7-13mm are compared to 19mm observed for Itracanzole standard.
Compounds 6m, 6p and 6q showed moderate activity with zone of inhibition of 10, 10 and 7mm
each. Compound 6b, 6f, 6i, 6n, 6s has no activity against C.albicans. Among the test compounds,
compounds 6b, 6f, 6i, 6j, 6m, 6n, 6p, 6q and 6s showed antifungal activity against A.niger. The
Zone of inhibition of active compounds was in the range of 6-12mm with high activity,
compound 6m, 6p has a zone of inhibition of 12mm and 10mm compared to 18mm observed
against Itracanazole standard. Compounds 6i, 6j, 6s showed moderate activity with a zone of
inhibition of 6mm, 6mm and 7mm. The compound 6b, 6f, 6n, and 6q showed none antifungal
activity against A.niger. The compound 6m and 6p have good antibacterial and antifungal
behaviors, in both of these compounds has electron withdrawing groups such as -CHO and NO2,
these groups increase the biological activity and furthermore evidence of withdrawing has high
activity such as the compounds 6f has no electron withdrawing groups, but introducing of
withdrawing -CHO group in 6f to 6q, 6q has moderate biological activities.
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Determination of Minimum inhibitory concentration against bacterial cultures.
MIC of active compounds and ciprofloxacin against S.typhi and S. mutans are summarized in
table. 7 and 8 respectively. Standard ciprofloxacin showed a MIC of 1 µg/mL against S.typhi
and S. mutans. MIC of active compounds against S.typhi ranged from 125-250µg/mL with
lowest MIC of 125µg/mL in compounds 6m, 6p and 6q. Compound 6b and 6j showed higher
MIC of 250 µg/mL. MIC of active compounds against S. mutans ranged from 125-250µg/mL
with lowest MIC of 125µg/mL in compounds 6m, 6n and 6p. Compound 6q showed higher MIC
of 250 µg/mL.
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Determination of Minimum inhibitory concentration against fungal cultures
MIC of active compounds and Itracanazole against C.albicans and A.niger is summarized
in table 9 and 10 respectively. Standard Itracanazole showed a MIC of 16 µg/mL against
C.albicans and A.niger. MIC of active compounds against C.albicans and A.niger ranged from
125-250µg/mL with lowest MIC of 125µg/mL in compounds 6m and 6p. Compound 6q showed
higher MIC of 250 µg/mL.
Figure 2: Sample activity against Salmonella typhi. S- Standard, C- Control.
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Figure 3: Sample activity against Candida albicans. S- Standard, C- Control.
Figure 4: Sample activity against Aspergillus niger. S- Standard, C- Control.
Figure 5: Sample activity against Streptococcus mutans. S- Standard, C- Control
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Table 3. Inhibitory activity and determination of Minimum Inhibitory concentration
(MIC) of hybrid compound against Salmonella typhi and Streptococcus mutans
6
7
Compounds
Salmonella typhi
Streptococcus mutans
8
9
MIC (µg/ml)
Zone of
MIC (µg/ml)
Zone of
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inhibition (mm)
inhibition (mm)
Control
Ciproflaxine
NA
1
NA
22
6
NA
NA
7.5
11
NA
9
NA
1
NA
24
6b
6f
6i
250
NA
NA
250
125
NA
125
125
NA
NA
NA
NA
NA
125
125
125
250
NA
NA
NA
NA
NA
13
10
11
7
NA
6j
6m
6n
6p
6q
6s
9
NA
Table 4. Inhibitory activity and determination of Minimum Inhibitory concentration of
hybrid compound against Candida albicans and Aspergillus niger
Compounds
Candida albicans
Aspergillus niger
MIC (µg/ml)
Zone of
inhibition (mm)
MIC (µg/ml)
Zone of
inhibition (mm)
Control
Itraconazole
NA
16
NA
19
NA
NA
NA
13
10
NA
10
NA
1
NA
18
NA
NA
6
6b
6f
6i
250
NA
NA
125
125
NA
125
250
NA
NA
NA
NA
NA
125
125
125
250
NA
6j
6
6m
6n
6p
6q
6s
12
NA
10
NA
7
7
NA
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Conclusion
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A facile and efficient synthesis of bioactive novel pyrazolyl-isoxazoline hybrids has been
accomplished via easily available, inexpensive starting materials from the reactions of diverse
oximes and allyoxy benzene derivatives in excellent yields. The compounds 6m and 6p are
having an excellent antibacterial and anti-fungal behavior. Further, the main advantage of our
work is an two active biological compounds such as pyrazolyl and isoxazoles are made up of in a
single platform, which is the novelty of our work and also we have many advantages in the
synthetic conditions such as, reaction occurs under mild reaction condition, used inexpensive
starting materials, simplicity and achieve a high yield of the product, transition metal free
methodology, DMF acts as both base as well as solvent.
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Experimental Section
Materials and Methods
All the chemicals and reagents were purchased from commercial reagent suppliers, used
without further purification as commercially available unless otherwise noted. All the solvents
were obtained from laboratory reagent grades. The 1H and 13C NMR spectra were recorded on a
Bruker (Avance) 300 and 400 MHz NMR instrument using TMS as an internal standard and
CDCl3 and DMSO-d6 as solvent. Chemical shifts are given in ppm (δ-scale) and the coupling
constants are given in Hz. The FT-IR spectra were recorded from a JASCO FT/IR-410
spectrometer. The melting points were measured in open capillary tubes and are uncorrected.
Electronspray ionization mass spectrometry (ESI-MS) analyses were recorded in LCQ Fleet,
Thermo Fisher Instrument in negative or positive ion mode. The collision voltage and ionization
voltage were +35 V and +5 kV, respectively, using nitrogen as atomization and desolvation gas.
The desolvation temperature was set at 300 °C. The scan range of mass spectrum was 300−2000
m/z. Column chromatography was carried out on silica gel (60-120 mesh) using n-hexanes and
ethyl acetate as an eluent. The single crystal X-ray data set for compounds 6n was collected on
Bruker AXS KAPPA APEX-2 diffractometer equipped with graphite monochromator.26 The
structure was solved by direct methods and refined by fullmatrix least-squares calculations using
SHELXL−2014.
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Synthesis of 1, 3-dimethyl-1H-pyrazol-5(4H)-one (1)
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6
7
8
To a solution of methylhydrazine sulfuric acid (5.47 g, 37.95 mmol) in ethanol (100 mL)
at 0°C was added triethylamine (30.7 g, 303.39 mmol) dropwise. The resulting solution was
stirred for about 30 min at 0°C before ethyl-3-oxobutanoate (5 g, 38.42 mmol) was added
dropwise followed by the portion wise addition of magnesium sulfate (9.12 g, 75.6 mmol). The
above solution was allowed to stirring, for an additional 2 days at room temperature. The solid
were removed by filtration and the filtrate was concentrated. The resulting residue was purified
by flash column chromatography (6% v/v of methanol in dichloromethane) provide the desired
product as a yellow solid (8.0 g).
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Synthesis of 5-Chloro-1,3-dimethyl-1H-pyrazole-4-carbaldehyde27 (2)
To a stirred solution of N, N-dimethylformamide (20 mmol) was added dropwise POCl3
(30 mmol) 2,5-dimethyl-2,4-dihydro-3H-pyrazol-3-one (20 mmol) was added to the above
reaction mixture in portions. The resulting mixture was stirred at 0 °C for 30 min and then heated
to 110 °C and stirred for 10 h at 110 °C. The reaction solution was cooled to room temperature
and poured into ice-water. The pH value was adjusted to 7–8 by sodium hydroxide solution, and
the yellow solid was combined by filtration. The filtrate was continuously extracted with ethyl
acetate (3 × 80 mL). The combined organic layer was dried over anhydrous Na2SO4, and
concentrated in a rotatory evaporator to afford yellow solid. The crude solid was recrystallized
from petroleum ether/ethyl acetate to produce 5-chloro-1,3-dimethyl-1H-pyrazole-4-
carbaldehyde with yield of 80%, m.p. 76–78 °C.
Synthesis of 1,3-Dimethyl-5-phenoxy-1H-pyrazole-4-carbaldehyde28
To a well stirred solution of phenol (30 mmol) in N,N-dimethylformamide (50 mL) was
added potassium hydroxide (40 mmol) at room temperature. The resulting mixture was warmed
to 40 °C for about 6 h and the 5-chloro-1,3-dimethyl-1H-pyrazole-4-carbaldehyde (20 mmol)
was added in to the reaction mixture. The resulting mixture was heated to 100 °C for about 10-15
h. The reaction solution was cooled to room temperature, poured into ice water (80 mL). The
reaction mixture was extracted with ethyl acetate (3x30 mL) and the organic layer was dried over
anhydrous sodium sulfate. The solvent was removed under reduced pressure to provide 1,3-
dimethyl-5-phenoxy-1H-pyrazole-4-carbaldehyde with 82 % yield.
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Synthesis of 1, 3-Dimethyl-5-phenoxy-1H-pyrazole-4-carbaldehyde oxime28
5
6
7
8
To a solution of 1,3-dimethyl-5-phenoxy-1H-pyrazole-4-carbaldehyde (7.0 g, 32.37
mmol) and hydroxylamine hydrochloride (2.5 g, 36 mmol) in ethanol (20 mL) was added slowly
aqueous sodium hydroxide solution (1.44 g, 36 mmol, 40%) at below 30°C. After 30 mins of
stirring at room temperature, the resulting precipitate was filtered to produce 1,3-dimethyl-5-
phenoxy-1H-pyrazole-4-carbaldehyde oxime.
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Synthesis of pyrazolyl-isoxazoline hybrids (6)
To a solution of 1,3-dimethyl-5-phenoxy-1H-pyrazole-4-carbaldehyde oxime (200 mg,
0.8654 mmol) in ethanol (2.0 mL) was added chloramine-T (216.7 mg, 0.9519 mmol) and
allyloxy benzene (127.6 mg, 0.9519 mmol) at room temperature. The resulting reaction mass
was stirred for 5-7 h at reflux temperature and the reaction was monitored by TLC. After
completion of the reaction, the solvent was removed by distillation, add water (2.0 mL) and
extracted in dichloromethane (2.0 mL). The solvent was removed by distillation and crude
purified by column chromatography to provide desired product as white solid.
3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-5-(phenoxymethyl)-4,5-dihydroisoxazole (6a)
White solid; Mp. 156.5-157.8 °C. Fig. S1. 1H NMR (300 MHz, DMSO-d6): δ 7.41-7.37 (m, 2H),
7.28-7.24 (m, 2H), 7.14 (t, J=7.6, 7.2 Hz ,1H,), 6.93 (d, J=8.4 Hz, 3H), 6.84 (d, J=8.0 Hz, 2H),
4.84-4.77 (m, 1H), 3.96-3.81 (m, 2H), 3.51 (s, 3H), 3.17 (dd, J=10.8, 10.4, 1H), 2.99 (dd, J=6.8,
7.2, 1H), 2.33 (s, 3H). Fig. S2.13C NMR (75 MHz, DMSO-d6): δ 158.61, 156.86, 149.88, 146.95,
145.95, 130.85, 129.87, 124.16, 121.23, 115.29, 114.90, 98.34, 77.34, 68.92, 38.27, 34.62, 15.59.
IR (KBr) cm-1: 3418, 3050, 2932, 2871, 2486, 1739.95, 1597, 1544, 1486, 1455, 1395, 1324,
1298, 1275, 1249, 887, 756, 692; Fig. S3. Mass: m/z: [M+2] Cal for C21H21N3O3; 363.16; Found
365.1.
5-((3-Bromophenoxy)methyl)-3-(1,3-dimethyl-5-phenoxy-1H-pyrazol-4-yl)-4,5-dihydro
isoxazole (6b)
1
White solid; Mp. 90-91 °C. Fig. S4. H NMR (300 MHz, DMSO-d6): δ 7.39 (t, J=8.4, 7.6 Hz,
2H), 7.21 (t, J=8.0 Hz, 1H), 7.15-7.11 (m, 2H), 7.06-7.05 (m, 1H), 6.93 (d, J=8.0 Hz, 2H), 6.86
(dd, J=1.6, 2.0 Hz, 1H), 4.81-4.78 (m, 1H), 3.99-3.90 (m, 2H), 3.50 (s, 3H), 3.17 (dd, J =10.8 Hz,
13
1H), 2.97 (dd, J =7.2 Hz, 1H, 2.32 (s, 3H). Fig. S5. C NMR (75 MHz, DMSO-d6): δ 159.6,
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156.8, 149.9, 146.9, 145.9, 131.5, 130.8 124.1, 122.4, 117.2, 115.7, 115.3, 98.3, 77.1, 69.3, 38.1,
34.6, 15.5. IR (KBr) cm-1: 3747, 3409, 3044, 2939, 1589, 1546, 1479, 1454, 1396, 1290, 1249,
1226, 1200, 918, 886, 860, 763. Fig. S6. Mass: m/z: [M+1] Calcd for C21H20BrN3O3: 441.07 ;
Found 441.80.
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3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-5-((o-tolyloxy)methyl)-4,5-dihydroisoxazole
(6c)
1
White solid; Mp. 91-92 °C. Fig. S7. H NMR (300 MHz, DMSO-d6): δ 7.37 (t, J=8.4 Hz, 7.2,
2H), 7.14-7.08 (m, 3H), 6.92 (d, J=8.0 Hz, 2H), 6.85-6.80 (m, 2H), 4.85-4.79 (m, 1H), 3.97-3.89
(m, 2H), 3.51 (s,3H), 3.18 (J=10.8, 11.2, 1H), 3.04 (J=7.2, 6.8, 1H), 2.33 (s, 3H), 1.97 (s, 3H).
Fig. S8. 13C NMR (75 MHz, DMSO-d6): δ 156.91, 156.69, 149.77, 146.92, 145.96, 130.82,
130.77, 127.33, 126.18, 124.13, 120.92, 115.20, 111.86, 98.39, 77.32, 69.13, 38.16, 34.63, 16.01,
15.59. IR (KBr) cm-1: 3782, 3066, 2941, 1725, 1676, 1596, 1547, 1489, 1454, 1414, 1394, 1249,
1199, 1124, 915, 860, 754. Fig. S9. Mass m/z: [M+1] Calcd for C22H23N3O3: 377.2; Found
379.2.
4-((3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-4,5-dihydroisoxazol-5-yl)methoxy)
benzonitrile (6d)
1
White solid; Mp. 132.0-133.4 °C. Fig. S10. H NMR (300 MHz, DMSO-d6): δ 7.74 (d, J=8.8
Hz, 2H), 7.39 (t, J=8.0 Hz, 2H), 7.13 (t, J=7.6 Hz, 7.2 Hz, 1H), 7.02 (d, J=8.8 Hz, 2H), 6.93 (d,
J=8.0 Hz, 2H), 4.87-4.80 (m, 1H), 4.09- 3.99 (m, 2H), 3.50 (s, 3H), 3.19 (dd, J=10.8 Hz, 1H)
2.98 (dd, J=6.8 Hz, 1H), 2.32 (S, 3H). Fig. S11. 13C NMR (75 MHz, DMSO-d6): δ 164.27,
159.02, 152.10, 149.15, 148.15, 136.76, 133.03, 126.35, 121.68, 118.20, 117.46, 105.71, 100.44,
79.21. 71.63, 40.39, 36.81, 17.75. IR (KBr) cm-1: 3868, 3840, 3675, 3359, 3264, 3049, 2595,
2332, 1918, 1805, 1596, 1527, 1392, 1301, 1158, 1098, 905, 814, 703. Fig. S12. Mass: m/z:
[M+1] Calcd for C22H20N4O3: 388.1; Found 388.8.
5-((4-Chlorophenoxy)methyl)-3-(1,3-dimethyl-5-phenoxy-1H-pyrazol-4-yl)-4,5-dihydro
isoxazole (6e)
1
White solid; Mp.87-88 °C. Fig. S13. H NMR (300 MHz, DMSO-d6): δ 7.39 (t, J=7.2, 8.4 Hz,
2H), 7.29 (d, J=9.2 Hz, 2H), 7.14 (t, J =7.6, 7.2 Hz, 1H), 6.93 (d, J=8.4 Hz, 2H), 6.87 (d, J=8.8
Hz, 2H), 4.81-4.79 (m, 1H), 3.96-3.87 (m, 2H), 3.50 (s,3H), 3.17 (dd, J=10.4 Hz, 1H), 2.97 (dd,
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J=7.2 Hz, 1H), 2.32 (s, 3H). Fig. S14. 13C NMR (75 MHz, DMSO-d6): δ 157.50, 156.86, 149.88,
146.95, 145.96, 130.84, 129.61, 124.94, 124.16, 116.71, 115.28, 98.30, 77.22, 69.39, 38.21,
34.61, 15.58. IR (KBr) cm-1: 3822, 3750, 3645, 3465, 3067, 2927, 2529, 1937, 1876, 1592,
1548, 1489, 1453, 1282, 1234, 1192, 1028, 916, 862, 826, 742. Fig. S15. Mass: m/z: [M] Calcd.
for C21H20ClN3O3: 397.12; Found 397.7.
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3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-5-((naphthalen-1-yloxy)methyl)-4,5-
dihydroisoxazole (6f)
1
White solid; Mp.138-139 °C. Fig. S16. H NMR (300 MHz, DMSO-d6): δ 7.92 (d, J=8.4 Hz,
1H), 7.86 (d, J=8.0 Hz, 1H), 7.53-7.46 (m, 2H), 7.43-7.36 (m, 2H), 7.31-7.27 (m, 2H), 7.07 (t,
J=6.8 Hz, 7.6 Hz, 1H), 6.92-6.89 (m, 3H), 4.99-4.93 (m, 1H), 4.18-4.09 (m, 2H), 3.52 (s, 3H),
13
3.27 (dd, J=10.8, 1H), 3.11 (dd, J=6.8, 6.4 Hz, 1H), 2.38 (s, 1H). Fig. S17. C NMR (75 MHz,
DMSO-d6): δ 156.88, 154.08, 149.93, 146.98, 146.01, 134.39, 130.76, 127.87, 126.90, 126.56,
125.60, 125.20, 124.10, 121.71, 120.64, 115.21, 105.73, 98.43, 77.22, 69.60, 38.31, 34.64, 15.65.
IR (KBr) cm-1: 3937, 3854, 3661, 3521, 3054, 2937, 2512, 2401, 2098, 1971, 1809, 1737, 1677,
1585, 1543, 1484, 1394, 1325, 1273, 1245, 1167, 1103, 1076, 916, 887, 824, 772. Fig. S18.
Mass: m/z: [M+1] Calcd for C25H23N3O3: 413.17 ; Found 413.9.
N-((3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-4,5-dihydroisoxazol-5-yl)methyl)-
1,4,4a,8a-tetrahydronaphthalen-1-amine (6g)
1
White solid; Mp.121-122 °C. Fig. S19. H NMR (300 MHz, DMSO-d6): δ 7.41-7.36 (m, 2H),
7.14 (t, J=7.6, 7.2 Hz, 1H), 6.90 (d, J=7.6 Hz, 3H), 6.86-6.83 (m, 1H), 6.51 (d, J=8.4 Hz, 1H),
6.48-6.44 (m, 1H), 4.72-4.68 (m, 1H), 3.50 (s, 3H), 3.24-3.19 (m, 3H), 3.12-3.08 (m, 2H), 2.78
13
(dd, J=7.6 Hz, 1H), 2.64-2.59 (m, 2H), 2.31 (s, 3H), 1.76-1.68 (m, 2H). Fig. S20. C NMR (75
MHz, DMSO-d6): δ 156.87, 150.11, 146.92, 145.94, 145.34, 130.83, 129.32, 127.26, 124.13,
122.03, 115.89, 115.23, 111.05, 98.39, 77.56, 54.34, 50.58, 39.37, 34.59, 27.97, 22.04, 15.60. IR
(KBr) cm-1: 3063, 3013, 2936, 2847, 2606, 2393, 1947, 1889, 1859, 1790, 1598, 1548, 1505,
1414, 1394, 1336, 1250, 1200, 1118, 1072, 997, 909, 860, 799. Fig. S21. Mass: m/z: [M+1]
Calcd for C24H26N4O2: 402.21; Found 403.0.
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5-(((5-Chloroquinolin-8-yl)oxy)methyl)-3-(1,3-dimethyl-5-phenoxy-1H-pyrazol-4-yl)-4,5-
5
6
dihydroisoxazole (6h)
7
8
1
White solid; Mp.135-137 °C. Fig. S22. H NMR (300 MHz, DMSO-d6): δ 8.93-8.92 (m, 1H),
9
8.50 (dd, J=1.2 Hz, 1H), 7.74-7.71 (m, 1H), 7.66 (d, J=8.4 Hz, 1H), 7.32 (t, J=8.4, 7.6 Hz, 2H),
7.16 (d, J=8.4 Hz, 1H), 7.07 (t, J=7.2 Hz, 1H), 6.92 (d, J=8.4 Hz, 2H), 4.98-4.94 (m, 1H), 4.15
(d, J=5.2 Hz, 2H), 3.50 (s, 3H), 3.30-3.21 (m, 1H), 3.14-3.08 (m, 1H), 2.33 (s, 3H). Fig. S23. 13C
NMR (75 MHz, DMSO-d6): δ 156.81, 154.04, 150.22, 150.07, 146.97, 145.99, 140.60, 132.57,
130.76, 127.13, 126.55, 124.09, 123.46, 121.55, 115.30, 110.64, 98.41, 77.36, 70.17, 38.54,
34.63, 15.59. IR (KBr) cm-1: 3069, 2928, 2878, 2619, 2406, 1971, 1847, 1613, 1592, 1547,
1500, 1461, 1413, 1385, 1311, 1286, 1236, 1204, 1100, 1072, 1010, 967, 930, 872, 780. Fig.
S24. Mass: m/z: [M+1] Calcd for C24H21ClN4O3: 448.13; Found 448.7.
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5-(((6-Chloroquinoxalin-2-yl)oxy)methyl)-3-(1,3-dimethyl-5-phenoxy-1H-pyrazol-4-yl)-4,5-
dihydroisoxazole (6i)
White solid; Mp. 120.8-121.8 °C. Fig. S25. 1H NMR (300 MHz, DMSO-d6): δ 8.56 (s, 1H), 8.10
(s, 1H), 7.80 (s, 2H), 7.34 (t, J=7.6, 8.0 Hz, 2H), 7.04 (t, J=7.2 Hz, 1H), 6.91 (d, J= 8.0 Hz, 2H),
4.94-4.92 (m, 1H), 4.41-4.37 (m, 2H), 3.51 (s, 3H), 3.23 (dd, J=10.8 Hz, 1H), 3.04 (dd, J=7.2 Hz,
13
1H), 2.33 (s, 3H). Fig. S26. C NMR (75 MHz, DMSO-d6): δ 157.29, 156.83, 149.99, 146.99,
145.99, 141.30, 139.17, 138.58, 131.44, 131.31, 130.76, 129.05, 127.96, 124.04, 115.25, 98.21,
76.72, 67.27, 38.38, 34.61, 15.58. IR (KBr) cm-1: 3043, 2970, 2371, 1799, 1658, 1594, 1548,
1488, 1449, 1410, 1306, 1264, 1213, 1072, 1034, 965, 926, 869, 934, 750. Fig. S27. Mass: m/z:
[M+1] Calcd for C23H20ClN5O3: 449.13; Found 450.2.
5-(((1H-Benzo[d][1,2,3]triazol-1-yl)oxy)methyl)-3-(1,3-dimethyl-5-phenoxy-1H-pyrazol-4-
yl)-4,5-dihydroisoxazole (6j)
White solid; Mp.98-99 °C. Fig. S28. 1H NMR (300 MHz, DMSO-d6): δ 8.06 (d, J=9.2 Hz, 1H),
7.70 (d, J=8.4 Hz, 1H), 7.62 (t, J=6.8, 8.4 Hz, 1H), 7.47 (t, J=7.6 Hz, 1H), 7.38 (t, J=8.4, 7.6 Hz,
2H), 7.13 (t, J=7.2 Hz, 1H), 6.93 (d, J=8.8 Hz, 2H), 4.92-4.86 (m, 1H), 4.62 (dd, J=2.8 Hz, 1H),
4.53 (dd, J=6.8 Hz, 7.2 Hz, 1H), 3.49 (s, 3H), 3.23 (dd, J=11.2 Hz, 1H), 3.02 (dd, J= 7.6, 7.2 Hz,
13
1H) ,2.32 (s, 1H). Fig. S29. C NMR (75 MHz, DMSO-d6): δ 156.79, 150.03, 147.05, 145.99,
143.15, 130.85, 128.89, 127.36, 125.43, 124.19, 120.13, 115.33, 109.66, 98.12, 81.46, 76.30,
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38.14, 34.67, 15.61. IR (KBr) cm-1: 3736, 3646, 2931, 2363, 1655, 1593, 1549, 1488, 1393,
1330, 1239, 1204, 1096, 974, 904, 857, 742. Fig. S30. Mass: m/z: [M+1] Calcd for C21H20N6O3:
404.16; Found 405.0.
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Methyl 4-((3-(1,3-dimethyl-5-phenoxy-1H-pyrazol-4-yl)-4,5-dihydroisoxazol-5-yl)methoxy)
benzoate (6k)
White solid; Mp. 111-112.8 °C. Fig. S31. 1H NMR (300 MHz, DMSO-d6): δ 7.88 (d, J=8.8 Hz,
2H), 7.39 (t, J=8.8, 7.6 Hz, 2H), 7.13 (t, J=7.6, 6.8 Hz, 1H), 6.94 (t, J=9.2, 7.6 Hz, 4H), 4.85-4.80
(m, 1H), 4.07-3.97 (m ,2H), 3.81 (s, 3H), 3.50 (s, 3H), 3.19 (dd, J=10.8, 11.2 Hz, 1H), 2.99 (dd,
13
J=7.2 Hz, 1H, 2.32 (s, 3H). Fig. S32. C NMR (75 MHz, DMSO-d6): 166.26, 162.49, 156.86,
149.91, 146.98, 145.97, 131.60, 130.85, 124.17, 122.53, 115.30, 114.95, 98.29, 77.11, 69.32,
52.26, 38.21, 34.62, 23.23, 15.58. IR (KBr) cm-1: 3875, 3643, 3406, 3052, 2949, 2563, 1956,
1713, 1604, 1548, 1487, 1287, 1194, 1109, 1058, 1011, 968, 924, 754. Fig. S33. Mass: m/z:
[M+1] Calcd for C23H23N3O5: 421.16; Found 421.7.
3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-5-((quinoxalin-2-yloxy)methyl)-4,5-
dihydroisoxazole (6l)
White solid; Mp.101.5 102.4 °C. Fig. S34. 1H NMR (300 MHz, DMSO-d6): δ 8.51 (s, 1H), 8.02
(d, J=8.4 Hz, 1H), 7.81-7.74 (m, 2H), 7.68-7.64 (m, 1H), 7.33 (t, J=8.4, 7.6 Hz, 2H), 7.03 (t,
J=7.6, 7.2 Hz, 1H), 6.91 (d, J=7.6 Hz, 2H), 4.96-4.90 (m, 1H), 4.42-4.40 (m, 2H), 3.51 (s, 3H),
13
3.23 (dd, J=10.8 Hz, 1H), 3.04 (dd, J=7.2 Hz, 1H), 2.33 (s, 3H). Fig. S35. C NMR (75 MHz,
DMSO-d6): δ 157.03, 156.84, 148.99, 147.01, 146.00, 140.02, 139.78, 138.89, 130.99, 130.76,
129.13, 127.44, 127.30, 124.06, 115.25, 98.22, 76.81, 67.07, 38.39, 34.61, 15.59. IR (KBr) cm-1:
3048, 2968, 2374, 1796, 1660, 1597, 1550, 1488, 1447, 1408, 1309, 1254, 1218, 1066, 1034,
965, 926, 934, 750. Fig. S36. Mass: m/z: [M+1] Calcd for C23H21N5O3: 415.16; Found 416.2.
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4-((3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-4,5-dihydroisoxazol-5-yl)methoxy)
5
6
benzaldehyde (6m)
7
8
1
White solid; Mp.94-95 °C. Fig. S37. H NMR (300 MHz, DMSO-d6): δ 9.86 (s,1H), 7.84 (d,
9
J=8.8 Hz, 2H), 7.39 (t, J=8.4, 7.6 Hz, 2H), 7.13 (t, J=7.6 Hz, 1H), 7.04 (d, J=8.4 Hz, 2H), 6.93
(d, J=7.6 Hz, 2H), 4.87-4.82 (m, 1H), 4.12-4.01 (m, 2H), 3.50 (s, 3H), 3.20 (dd, J=10.8 Hz, 1H),
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2.99 (dd, J=6.8, 7.2 Hz, 1H), 2.32 (s, 3H). Fig. S38. C NMR (75 MHz, DMSO-d6): δ 191.79,
163.57, 156.86, 149.93, 146.97, 145.96, 132.19, 130.87, 130.26, 124.18, 115.42, 115.30, 98.29,
77.07, 69.46, 38.21, 34.65, 15.59. IR (KBr) cm-1: 3078, 2935, 2838, 2753, 2522, 2326, 2014,
1686, 1597, 1547, 1509, 1489, 1457, 1313, 1263, 1197, 1163, 1027, 841.02, 752.41. Fig. S39.
Mass: m/z: [M+1] Calcd for C22H21N3O4: 391.15; Found 392.2.
3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-5-((4-methoxyphenoxy)methyl)-4,5-dihydro
isoxazole (6n)
White solid; Mp.103-104 °C. Fig. S40. 1H NMR (300 MHz, DMSO-d6): δ 7.39 (t, J=8.4, 7.2 Hz,
2H), 7.14 (t, J=7.2, 7.6 Hz, 1H), 6.93 (d, J=8.0 Hz, 2H), 6.82, 6.77 (dd, J=9.2 Hz, 4H), 4.81-4.74
(m, 1H), 3.89-3.81 (m, 2H), 3.68 (s, 3H), 3.50 (s, 3H), 3.16 (dd, J=10.8 Hz, 1H), 2.97 (dd, J=7.2
Hz, 1H), 2.32 (s, 3H). Fig. S41. 13C NMR (75 MHz, DMSO-d6): δ 156.87, 153.96, 152.66,
149.86, 146.95, 145.94, 130.87, 124.18, 115.91, 115.30, 114.95, 98.35, 77.45, 69.62, 55.76,
38.26, 34.63, 15.58. IR (KBr) cm-1: 3058, 2936, 2871, 2837, 2542, 2396, 2058, 2006, 1880,
1813, 1748, 1620, 1590, 1552, 1508, 1390, 1301, 1235, 1200, 1105, 1062, 1031, 965, 923, 886,
758. Fig. S42. Mass: m/z: [M+2] Calcd for C22H23N3O4: 393.17; Found 395.0.
3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-5-((2-methyl-4-nitrophenoxy)methyl)-4,5-
dihydroisoxazole (6o):
1
White solid; Mp.100-101 °C. Fig. S43. H NMR (300 MHz, DMSO-d6): δ 8.08-8.04 (m, 2H),
7.38 (t, J=7.6, 8.4 Hz, 2H), 7.13-7.07 (m, 2H), 6.93 (d, J=8.0 Hz, 2H), 4.93-4.87 (m, 1H), 4.18-
4.08 (m, 2H), 3.52 (s, 3H), 3.23 (dd, J=10.8 Hz, 1H), 3.06 (dd, J=6.4 Hz, 1H), 2.34 (s, 3H), 2.06
(s, 3H). Fig. S44. 13C NMR (75 MHz, DMSO-d6): δ 162.08, 156.87, 149.81, 146.91, 145.97,
140.85, 130.81, 127.75, 125.88, 124.11, 124.01, 115.16, 111.66, 98.24, 76.86, 70.05, 38.09,
34.60, 15.86, 15.60. IR (KBr) cm-1: 3747, 3616, 3441, 3060, 2944, 2352, 1715, 1591, 1550,
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1492, 1393, 1337, 1270, 1202, 1093, 1009, 963, 925, 885, 755. Fig. S45. Mass: m/z: [M-1]
Calcd for C22H22N4O5: 406.4; Found 409.6
7
8
3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-5-((2-methyl-3-nitrophenoxy)methyl)-4,5-
9
dihydroisoxazole (6p):
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1
White solid; Mp.119-120 °C. Fig. S46. H NMR (300 MHz, DMSO-d6): δ 7.45 (d, J=8.0 Hz,
1H), 7.36 (t, J=7.2, 8.8 Hz, 3H), 7.24 (d, J=8.0 Hz, 1H), 7.09 (t, J=7.2 Hz, 1H), 6.91 (d, J=8.0
Hz, 2H), 4.90-4.84 (m, 1H), 4.09-4.01 (m, 2H), 3.51 (s, 3H), 3.21 (dd, J=10.8, 11.2 Hz, 1H),
13
3.03 (dd, J=6.8 Hz, 1H), 2.32 (s, 3H), 2.07 (s, 3H). Fig. S47. C NMR (75 MHz, DMSO-d6): δ
157.32 156.88, 151.01, 149.82, 146.95, 145.97, 130.79, 127.85, 124.10, 120.89, 116.44, 116.19,
115.19, 98.26, 77.01, 70.34, 38.19, 34.62, 15.57, 11.33. IR (KBr) cm-1: 3744, 3619, 3438, 3064,
2939, 2355, 1717, 1589, 1553, 1489, 1391, 1333, 1272, 1202, 1098, 999, 966, 928, 882, 759.
Fig. S48. Mass: m/z: [M+2] Calcd for C22H22N4O5: 422.16; Found 422.6.
1-((3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-4,5-dihydroisoxazol-5-yl)methoxy)-2-
naphthaldehyde (6q):
1
White solid; Mp. 176-178 °C. Fig. S49. H NMR (300 MHz, DMSO-d6): δ 10.57 (s, 1H),
9.09 (d, J=8.4 Hz, 1H), 8.26 (d, J=9.2 Hz, 1H), 7.94 (d, J=8.0 Hz, 1H), 7.65 (t, J=8.4, 7.2 Hz,
1H), 7.52-7.46 (m, 2H), 7.33 (t, J=7.6, 8.4 Hz, 2H), 7.05 (t, J=7.2, 7.6 Hz, 1H), 6.91 (d, J=8.4
Hz, 2H), 4.94-4.91 (m, 1H), 4.36-4.26 (m, 2H), 3.51 (s, 3H), 3.26 (dd, J=10.8, 11.2 Hz, 1H),
13
3.07 (dd, J= 6.4, 6.8 Hz, 1H), 2.32 (s, 3H). Fig. S50. C NMR (75 MHz, DMSO-d6): δ 191.53,
163.73, 156.86, 149.87, 146.96, 145.93, 138.38, 130.97, 130.80, 130.26, 129.05, 128.71, 125.23,
124.32, 124.11, 116.17, 115.23, 115.18, 98.19, 77.06, 71.07, 38.24, 34.64, 15.59. IR (KBr) cm-1:
3059, 2934, 2881, 2801, 1945, 1731, 1668, 1618, 1591, 1551, 1508, 1415, 1340, 1270, 1244,
1201, 1152, 1075, 1022, 920, 862, 820, 755. Fig. S51. Mass: m/z: [M+1] Calcd for C26H23N3O4:
441.17; Found 441.8.
3-((3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-4,5-dihydroisoxazol-5-
yl)methoxy)benzonitrile (6r):
1
White solid; Mp. 119.5-121 °C. Fig. S52. H NMR (300 MHz, DMSO-d6): δ 7.48-7.44 (m, 1H),
7.41-7.37 (m, 3H), 7.33 (s, 1H), 7.21-7.18 (m, 1H), 7.15-7.11 (m, 1H), 6.93 (d, J=8.0 Hz, 2H),
4.86-4.79 (m, 1H), 4.06-3.96 (m, 2H), 3.50 (s, 3H), 3.19 (dd, J=10.8 Hz, 1H), 2.97 (dd, J=7.2 Hz,
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1H), 2.32 (s, 3H). Fig. S53. C NMR (75 MHz, DMSO-d6): δ 158.77, 156.86, 149.91, 146.96,
145.96, 131.24, 130.85, 125.23, 124.15, 120.89, 119.01, 117.80, 115.29, 112.59, 98.29, 77.08,
69.50, 38.21, 34.64, 15.58. IR (KBr) cm-1: 3949, 3868, 3336, 2952, 2892, 2228, 1773, 1581,
1547, 1487, 1395, 1293, 1258, 1197, 1045, 971, 930, 875, 784, 747. Fig. 54. Mass: m/z: [M+1]
Calcd for C22H20N4O3: 388.15; Found 388.7.
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3-(1,3-Dimethyl-5-phenoxy-1H-pyrazol-4-yl)-5-((2,5-dimethylphenoxy)methyl)-4,5-
dihydroisoxazole (6s):
1
White solid; Mp.104-105 °C. Fig. S55. H NMR (300 MHz, DMSO-d6): δ 7.37-7.33 (m, 2H),
7.10 (t, J=7.2 Hz, 1H), 6.91 (dd, J=7.6 Hz, 3H), 6.64-6.60 (m, 2H), 4.81-4.77 (m, 1H), 3.92-3.84
(m, 2H), 3.48 (s, 3H), 3.15 (dd, J=10.8 Hz, 1H), 3.00 (dd, J=6.8 Hz, 1H), 2.30 (s, 3H), 2.20 (s,
13
3H), 1.89 (s, 3H). Fig. S56. C NMR (75 MHz, DMSO-d6): δ 156.89, 156.52, 149.76, 146.88,
145.93, 136.54, 130.83, 130.49, 124.14, 122.91, 121.35, 115.19, 112.62, 98.37, 77.29, 69.03,
38.12, 34.63, 21.41, 15.65, 15.62. IR (KBr) cm-1: 3728, 3433, 3407, 3040, 2933, 2737, 2562,
2341, 1961, 1879, 1806, 1616, 1588, 1550, 1507, 1482, 1413, 1329, 1259, 1198, 1160, 1131,
1039, 913, 861, 810,762. Fig. S57. Mass: m/z: [M+1] Calcd for C23H25N3O3: 391.19; Found
392.2.
Biological Activity
Preparation of synthetic compounds
A stock solution of 10 mg of each synthetic compound dissolved in 1 mL of distilled
water/DMSO was prepared. The antimicrobial activity of the synthesized compounds was
evaluated by the well diffusion method and minimum inhibitory concentration (MIC) was
determined by microbroth dilution technique (CLSI, 2000).
Antimicrobial activity
Test microorganisms
A total of 4 reference bacterial and fungal strains was obtained from the Microbial type culture
collections, Chandigarh, India. Bacterial test strains were Streptococcus mutans and Salmonella
typhi. Fungal test cultures were Aspergillus niger and Candida albicans.29
Antimicrobial activity of test compounds using well diffusion assay
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Determination of the prepared synthetic compounds against the Gram-negative and the Gram-
positive bacterial strains were examined by the well diffusion assay (Perez et al., 1990). Bacterial
and fungal cultures were grown in tryptone broth. Bacterial and Candida albicans cell
suspension from overnight (16hrs) grown culture were prepared in peptone water. Spore
suspension from Aspergillus niger culture were prepared in peptone water. The turbidity of
bacterial cell suspension was adjusted to 0.5 McFarland standard units. Fungal spore suspension
was diluted to obtain 1-2 x 105 cells/mL. The cell suspensions from bacteria and fungi (1mL)
were inoculated on Mueller–Hinton agar (MHA, 15mL) by pouring plate technique. Wells
(5mm) were cut on MHA plates using cup borer. The test compounds (25µL) were dispensed to
individual wells on the test organisms inoculated plates. Wells dispensed with Sterile water
(25µL) / DMSO (25 µL), Ciproflaxacin (25µL, 0.1mg/mL) or Itracanazole (25 µL, 4mg/mL)
served as solvent and positive controls. The bacterial plates were incubated at 37°C for 24 hours
and fungal cultures were incubated at 22oC for 48 to 72 Hrs. The inhibition zones around the
wells were measured in millimeter.
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Determination of Minimum inhibitory concentration against test bacteria by microbroth
dilution method
Test samples were serially diluted in MHB to give a final concentration of 7.81, 15.625,
31.25, 62.5, 125, and 250 µg/ml. Similarly Ciproflaxacin standards were serially diluted in MHB
to give a final concentration of 0.25, 0.5, 1, 2, 4, 8, 16 and 32µg/mL. A total of 90 µL of
different test concentration of the compound was added to wells of sterile 96-well microtiter
plates. Bacterial cell suspensions of each test microorganism were adjusted to 0.5 McFarland
standards in Mueller–Hinton broth (MHB). Test bacterial cultures (10 µL) were added to each
well containing test samples or standards, including the wells without any test compounds
(negative control). The plates were incubated at 37°C for 24 hours and the optical density was
measured at 600nm in a multimode plate reader (Tecan, Switzerland). The growth inhibition of
test bacteria was examined visually and MIC was defined as the minimum concentration of test
compounds or standard having 50% inhibition in OD when compared to that of the negative
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Determination of Minimum inhibitory concentration against test fungi by microbroth
dilution method
Test samples were serially diluted in Roswell Park Memorial Institute (RPMI) 1640
medium with L-glutamine, without sodium bicarbonate (RPMI 1640, Gibco, USA) to give a final
concentration of 7.81, 15.625, 31.25, 62.5, 125, and 250 µg/mL. Similarly Itracanzole standards
were serially diluted in RPMI to give a final concentration of 4, 8, 16, 32, 64and 128µg/mL. A
total of 90 µL of different test concentration of the compound was added to wells of sterile 96-
well microtiter plates. Fungal cell suspensions of each test microorganism were adjusted to 1-2 x
105 cfu/mL in RPMI media. Test fungal cell suspension (10 µL) was added to each well
containing test samples or standards, including the wells without any test compounds (negative
control). The plates were incubated at 22°C for 48-72 hrs and optical density was measured at
600nm in multimode plate reader (Tecan, Switzerland). The growth inhibition of test bacteria
was examined visually and MIC was defined as the minimum concentration of test compounds
or standard having 50% inhibition in OD when compared to that of the negative control wells.
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Acknowledgment
We acknowledge the financial support of the Council of Scientific and Industrial
Research, New Delhi, India (Grant No. 01(2540)/11/EMR-II). Department of Science and
Technology, New Delhi, India (Grant No. SR/F/1584/2012-13), University Grants Commission,
New Delhi, India (Grant No.UGC No.41-215/2012 (SR) Department of Science and Technology,
New Delhi, India (Grant No. DST/TM/CERI/C130(G)) and also, we thank DST-PURSE, UPE
program for providing instrumental facilities.
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New Journal of Chemistry
Page 26 of 26
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View Article Online
DOI: 10.1039/C9NJ01532A
Design, simple and efficient synthesis of bio active novel pyrazolyl-isoxazoline hybrids
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Balakrishnan Sankar, Muniyasamy Harikrishnan, Renganathan Raja, Velu Sadhasivam,
Nelson Malini, Sepperumal Murugesan, Ayyanar Siva*
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Supramolecular and Organometallic Chemistry Lab, Department of Inorganic Chemistry,
School of Chemistry, Madurai Kamaraj University Madurai-21.
Corresponding author Email (drasiva@gmail.com and siva.chem@mkuniversity.org)
A simple and new methodology followed for the synthesis of novel bio active
pyrazolyl-isoxazoline hybrids has been described from the reactions of pyrazoline substituted
oximes and various substituted allyoxy benzene. Among the hybrid materials of 6m and 6p
are the best anti-bacterial and antifungal activities.
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OEt
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19 examples