Ultrasound assisted synthesis of tetrazole based pyrazolines and isoxazolines as potent anticancer agents via inhibition of tubulin polymerization

Article abstract of DOI:10.1016/j.bmcl.2020.127592

In search of new active molecules against MCF-7, A549 and HepG2, tetrazole based pyrazoline and isoxazoline derivatives under both conventional and ultrasonic irradiation method were designed and efficiently synthesized. Structures of newly synthesized co

Full text of DOI:10.1016/j.bmcl.2020.127592

Bioorganic&MedicinalChemistryLetters30(2020)127592
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Ultrasound assisted synthesis of tetrazole based pyrazolines and isoxazolines
as potent anticancer agents via inhibition of tubulin polymerization
⁎
Vidya S. Dofea, Aniket P. Sarkateb, , Shailee V. Tiwaric, Deepak K. Lokwanid, Kshipra S. Karnikb,
Ishwari A. Kaleb, Suneel Dodamanie, Sunil S. Jalalpuree,f, Prasad V.L.S. Burrag
a Department of Chemistry, Deogiri College, Aurangabad 431 005, Maharashtra, India
b Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431 004, Maharashtra, India
c Department of Pharmaceutical Chemistry, Durgamata Institute of Pharmacy, Dharmapuri, Parbhani 431401, MS, India
d Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education & Research, Shirpur 425405, MS, India
e Dr. Prabhakar Kore Basic Science Research Center, KLE Academy of Higher Education and Research, Nehru Nagar, Belagavi 590010, Karnataka, India
f KLE College of Pharmacy, KLE Academy of Higher Education and Research, Nehru Nagar, Belagavi 590010, Karnataka, India
g Department of Biotechnology, KLEF University, Vaddeswaram, AP 522502, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Tetrazole
Pyrazoline
Isoxazoline
Ultrasound
Anticancer
Tubulin
In search of new active molecules against MCF-7, A549 and HepG2, tetrazole based pyrazoline and isoxazoline
derivatives under both conventional and ultrasonic irradiation method were designed and efficiently synthe-
sized. Structures of newly synthesized compounds 5a-h and 6a-h were characterized by 1H NMR, 13C NMR, MS
and elemental analysis. Several derivatives were found to be excellent cytotoxic against MCF-7, A549 and HepG2
cell lines characterized by lower IC50 values (0.78–3.12 µg/mL). Compounds 5b and 5c demonstrated an an-
tiproliferative effect comparable to that of CA-4. Western blot analysis revealed that, reported compounds ac-
cumulate more tubulin in the soluble fraction. Docking studies suggested that, binding of these compounds
mimics at the colchicine site of tubulin. In vitro study revealed that the tetrazole based pyrazolines and iso-
xazolines may possess ideal structural requirements for further development of novel therapeutic agents.
Docking
Microtubules composed of α, β-tubulin heterodimers are essential in
a variety of fundamental cellular processes like cell division and
maintenance of cell shape, regulation of motility, cell signalling, in-
tracellular transport, and segregation of chromosomes during mitosis.1
Dynamic equilibrium between microtubule polymerization and depo-
lymerization is central to most of microtubule mediated functions in-
cluding cell division.2 Microtubules possess three ligand-binding sites as
vinca, colchicines and taxol domains. Colchicine (A) and Com-
bretastatin A-4 (CA-4) (B) exhibited cytotoxicity against a broad range
of human cancer cell lines as well as multidrug resistant cell lines by
inhibiting tubulin polymerization and binding to the colchicine binding
site.3 Their distinctive structural features inspired many researchers
worldwide to develop antimitotic agents, as these scaffolds structurally
possess two and three rings with a trimethoxyphenyl group (ring-A), cis-
configuration at the olefinic bridge and (ring-B) modifications. Five
membered heterocyclic compounds as pyrazole,4 imidazole,5 thiazole,6
1,3,4-oxadiazole,7 isoxazole,8 1,2,3-thiadiazole,9 triazole,10 tetrazole,11
oxazole12 and pyrazoline13 are considered as a good surrogate for the
double bond of CA-4 to retain cis-alkene configuration, known for
potent tubulin inhibition with enhanced cytotoxicity. Small molecules
disrupting the microtubule/tubulin dynamics are used widely in cancer
treatment. Thus, to retain the appropriate configuration of the two
adjacent aryl groups required for bioactivity, heterocombretastatin
derivatives with general structure (C) were obtained by replacing the
stilbene core of CA-4 with tetrazole ring.14 Hence, discovery and de-
velopment of newer small molecules with antitubulin activity have
attracted medicinal chemists for past few years.15 Researchers have
reported anticancer, antibacterial and anti-HIV activity of compounds
possessing isoxazoline nucleus.16–18 Isoxazoline linked to tetrazole (D)
significantly reduced the growth of cancer cell lines, and disrupted
tubulin polymerization.19 Molecules containing isoxazolines have been
found to elicit anticancer activities with improved pharmacokinetics
profile like diaryl analogues (E) demonstrated potent cytotoxic activity
by blocking most of the cancer cells in G2 phase.20 Pyrazoline analo-
gues are well known in the area of pharmaceutical research for wide
21–23
24
range of biological potential like cytotoxic,
CNS depressant,
25
antimicrobial
and antimalarial activity.26–27 Pyrazole-oxadiazole
conjugates (F, G) resulted in promising antitumor activity that
⁎ Corresponding author.
E-mail address: dbsaniket09@gmail.com (A.P. Sarkate).
https://doi.org/10.1016/j.bmcl.2020.127592
Received 22 June 2020; Received in revised form 18 September 2020; Accepted 25 September 2020
Availableonline30September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

V.S. Dofe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127592
Fig. 1. Chemical structures of microtubule targeting agents: Colchicine (A), CA-4 (B), Tetrazole (C), Tetrazole-Isoxazoline hybrids (D), Isoxazoline (E), Pyrazole-
Oxadiazole conjugates (F, G), Tetrazole incorporated Pyrazoline and isoxazoline (H, I).
significantly inhibited tubulin polymerization.28 Tetrazole scaffold is a
unique structure, possessing antihypertensive, anti-allergic, anti-
1H-tetrazole derivatives (6a-h) were synthesized from Claisen-Schmidt
condensation of 1-(4-((1H-tetrazol-5-yl)methoxy)-3-methoxyphenyl)
ethanone (3) with substituted benzaldehydes in presence of a base KOH
microbial, anti-inflammatory, anticonvulsant, antiamoebic and antic-
29–36
ancer activity.
Tetrazole moiety is generally accepted to exhibit
gives
substituted
(E)-1-(4-((1H-tetrazol-5-yl)methoxy)-3-methox-
stronger resistance to in vivo metabolization than the carboxylate group,
thus conferring the bioavailability in blood (see Fig. 1).37
yphenyl)-3-phenylprop-2-en-1-one (4a-h) in good yield which on fur-
ther reacted with hydrazine hydrate in ethanol under ultrasound irra-
diation led to novel pyrazolines (5a-h) with excellent yield. Moreover,
substituted (E)-1-(4-((1H-tetrazol-5-yl)methoxy)-3-methoxyphenyl)-3-
phenylprop-2-en-1-one (4a-h) reacted with hydroxyl ammonium hy-
drochloride in ethanol under ultrasound irradiation afforded sub-
stituted isoxazolines (6a-h) with excellent yield (Scheme 2).
Considering the reported literature and in view of need for new
anticancer agents, it was of interest to synthesize and evaluate tetrazole
based pyrazoline and isoxazoline (H, I) for the most common types of
cancer found in males (lung cancer) and females (breast cancer). In our
previous studies, a series of quinoline based oxadiazole and thiazoli-
dinone derivatives were synthesized for targeting MCF-7 cell line.38
Ultrasound irradiation, which accelerates chemical reactions via
formation and adiabatic collapse of transient cavitation bubbles, has
gained popularity in organic synthesis over traditional methods, be-
cause of efficiency and convenience.39 In continuation of our earlier
work of tetrazole derivatives,40–41 we would like to report synthesis of
novel tetrazole based pyrazoline and isoxazoline derivatives using ul-
trasonic irradiation, evaluation of anticancer activity and molecular
docking studies.
Compounds 4a-h, 5a-h and 6a-h were synthesized with conven-
tional method and ultrasonic irradiation in shorter time, high yield and
simple work-up procedure (Table 1). All newly synthesized compounds
4a-h, 5a-h and 6a-h were characterized by spectroscopic techniques
viz. 1H NMR, 13C NMR, MS and elemental analysis.43
MTT assay44 was performed to evaluate the cytotoxic effects of all
newly synthesized analogues of 5-((4-(4,5-dihydro-5-phenyl-1H-pyr-
azol-3-yl)-2-methoxyphenoxy)methyl)-1H-tetrazoles and 5-((4-(4,5-di-
hydro-5-phenylisoxazol-3-yl)-2-methoxyphenoxy)methyl)-1H-tetra-
zoles against selected human cancer cell lines viz., lung
adenocarcinoma (A549), liver (HepG2) and breast (MCF-7), using CA-4
as reference compound. IC50 (µM) values are presented in Table 2.
Most of the compounds from these series displayed potent broad
spectrum IC50 values against all the tested cancer cell lines. In structure
activity relationship (SAR) studies, it was considered to investigate the
presence of pyrazolines and isoxazolines influence on activity along
with tetrazole ring. It was observed that compounds 5a-h containing
pyrazoline ring showed relatively excellent IC50 values than iso-
xazolines with IC50 values in range of 0.85–3.16 µM against some
Starting
material
1-(4-((1H-tetrazol-5-yl)methoxy)-3-methox-
yphenyl)ethanone (3) was synthesized from 1-(4-hydroxy-3-methox-
yphenyl)ethanone (1) alkylated with chloroacetonitrile in presence of
K2CO3 in N,N’-dimethyl formamide (DMF) afforded 1-(4-phenox-
yacetonitrile-3-methoxyphenyl)ethanone (2) in 90% yield, which on
further reaction with sodium azide and zinc bromide in water at 100 °C
give desired compound (3) in 87% yield (Scheme 1).42
Target compounds 5-((4-(4,5-dihydro-5- phenyl-1H-pyrazol −3-yl)
−2-methoxyphenoxy) methyl)-1H-tetrazole derivatives (5a-h) and 5-
((4-(4,5-dihydro-5-phenylisoxazol-3-yl)-2-methoxyphenoxy)methyl)-
2

V.S. Dofe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127592
Scheme 1. Reagents and conditions: (i)
chloroacetonitrile, DMF, K2CO3 (ii) sodium
azide, ZnBr2, H2O, 100 °C.
representative cancer cell lines. Among synthesized derivatives, com-
pound 5c having fluoro group and 5g having methyl group exhibited
excellent activity with IC50 value of 0.94 and 0.99 µM against the lung
cancer cell line. Moreover compounds 5b having chloro and 5c having
fluoro group showed significant activity with IC50 value of 0.96 and
0.85 µM against the HepG2 and compound 5c also exhibited excellent
activity with IC50 value of 0.92 µM against the MCF-7. Tetrazole based
isoxazoline compounds 6a-h resulted in moderate activity and com-
pound 6g having electron donating methyl group were the most potent
one among synthesized derivatives exhibiting IC50 values of 0.78 µM
against A549. We then proceeded to explore the influence of sub-
stituents on phenyl ring with respect to antiproliferative activity.
Further, all synthesized compounds allowed to investigate SAR ef-
fects of electron-withdrawing (F, Cl) as well as electron-releasing sub-
stituents like (OMe, Me), on phenyl ring attached to pyrazoline and
isoxzoline ring. It was observed that among different substituents stu-
died on phenyl ring attached to pyrazoline ring, fluoro group at para
position exhibited significant increase in activity compared to other
substituents (H, Me, OMe, OH) irrespective of pyrazoline and isoxazo-
line rings. Based on these findings, some other substituents were in-
troduced on phenyl ring attached to pyrazoline and isoxazoline to study
the effect on antiproliferative activity. Initially, a fluoro group in-
troduced at para position and resulting compound 5c showed pro-
nounced cytotoxic activity on HepG2 cell line compared to MCF-7 and
A549 cell line with IC50 value of 0.85 µM having tetrazole incorporated
pyrazoline ring. Compound 5g with IC50 values of 0.99 µM and 6g with
IC50 values of 0.78 µM having electron donating methyl group at para
position exhibited more significant activity against A549 cell line.
Introduction of methoxy group in pyrazoline 5f show moderate activity
with IC50 values of 2.98–3.12 µM as compared to isoxazoline deriva-
tives 6f against all cell lines. It is important to notice that, an additional
methoxy group viz. disubstitution at meta and para position 5d having
pyrazoline ring, produced better antiproliferative activity with IC50
values of 2.82–3.06 µM against all cancer cell lines. Surprisingly, when
electron withdrawing substituents fluoro and chloro are incorporated at
para position on the phenyl attached to pyrazoline 5c and 5b and
isoxazoline ring 6b and 6c, a substantial increase in cytotoxic potency
was observed in all derivatives. On other hand, unsubstituted phenyl
ring (R = H) of pyrazoline ring has relatively a stronger influence on
activity compared to compounds having isoxazoline moiety. Moreover,
introduction of electron–releasing hydroxyl group at meta position on
phenyl ring of pyrazoline 5e showed significant activity with IC50 va-
lues in range of 1.16–2.88 µM against all cell lines except HepG2 cell
line. Compound having hydroxyl group at para position of phenyl ring
of pyrazoline 5h shown excellent activity with IC50 values in the range
of 1.16–2.02 µM against all cell lines. Interestingly, compounds 5b and
5c exhibited promising antiproliferative activity against HepG2 cancer
cell line. Effect of heterocyclic rings revealed that, replacement of
pyrazoline by isoxazoline ring resulted in lowering the antiproliferative
activity.
Overall, SAR studies of these tetrazole based pyrazolines and iso-
xazolines suggest that, superior activity of pyrazolines compared to
Scheme 2. Reagents and conditions: (i) substituted benzaldehyde, EtOH, NaOH, 15–25 min (ii) NH2NH2·H2O, EtOH, 10–15 min (iii) NH2OH.HCl, EtOH, 20–25 min.
3

V.S. Dofe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127592
Table 1
Table 3
Synthesis of substituted chalcones 4a-h, pyrazolines 5a-h and isoxazolines 6a-h
using conventional and ultrasonic irradiation methods.
Inhibition of tubulin polymerization (IC50) of 5b, 5c and CA-4.
a
Compound
IC50
SD (in µM)
% of Tubulin inhibition
Entry
Conventional
Ultrasonic Irradiation
5b
2.94
2.16
1.62
0.08
0.12
0.18
62.56
65.44
74.24
Time (h)
Yielda (%)
Time (min)
Yield (%)
5c
CA-4
4a
4b
4c
4d
4e
4f
4
3
3
4
4
4
3
4
5
5
5
4
5
5
5
4
3
3
3
4
3
4
3
3
69
75
73
64
68
70
60
72
92
95
79
82
78
82
95
83
85
94
92
81
47
52
80
71
17
23
20
22
18
21
25
23
10
12
12
14
12
14
12
14
22
20
22
24
20
24
22
22
88
90
84
89
81
76
81
85
98
97
96
95
96
95
93
94
98
97
98
96
70
67
97
96
a
Concentration of drug to inhibit 50% of tubulin assembly. Values indicated
are the mean
SD of two different experiments performed in triplicates.
4g
4h
5a
5b
5c
5d
5e
5f
5g
5h
6a
6b
6c
6d
6e
6f
Fig. 2. A549 cells were treated with 1 µM of 5b and 5c for 48 h. CA-4 was used
as reference standard. Levels of tubulin were detected by western blot analysis
(S: Solubilised fraction; P: Polymerised fraction).
6g
6h
fraction. As expected, cells treated with 5b and 5c significantly in-
creased the tubulin content in soluble fraction. Specifically, 5c treated
cells showed a more distinct shift in tubulin balance, with almost all
tubulin present in the soluble fraction similar to that of positive control.
Therefore, increased tubulin in soluble fraction of cells treated by these
hybrids corroborated with inhibition of tubulin assembly as shown in
Table 3 and Fig. 2.
a
Isolated yield.
Table 2
aIC50 (µM) values of pyrazoline and isoxazoline derivatives.
Cytotoxicity of synthesized compounds
Entry
MCF-7b
A549c
HepG2d
To investigate and clarify binding mode of tetrazole based pyr-
azolines and isoxazoline analogues, molecular docking studies were
performed to colchicine binding site of α,β-tubulin (PDB: 1SA0) by
using Glide XP docking mode.46 The result was shown in (Fig. 3 and
Fig. 4). Superimposition of all compounds over colchicine in tubulin
structure showed that all compounds firmly occupied the colchicine
binding site of α,β-tubulin mostly buried in the β subunit (Fig. 3).
Docking scores of compounds varied from −9.66 to −6.38. Relative
binding free energies of docked compounds were also calculated, varied
from −72.95 to −48.14. Tetrazole ring is seen as coplanar to amino
acid residue Val 238 and thus could act as hydrogen bond donor in all
compounds. 2-methoxy group of centre phenyl ring is a promising hy-
drogen bond acceptor of the thiol proton of Cys 241 (Fig. 4). Docking
results also revealed that pyrazoline/isoxazoline and substituted phenyl
group of all compound was bound into the hydrophobic pocket of tu-
bulin consisting of the amino acid residues; Asn 258, Met 259, Val 315,
Ala316, Val318, Asn 350, Val 351 and Lys 352. In some compounds,
substituted phenyl group showed aromatic hydrogen bonding with Asn
258 and Asn 350 (Fig. 4A).
5a
5b
5c
5d
5e
5f
3.16
1.04
0.92
2.94
2.16
3.10
1.76
1.94
12.5
6.25
3.12
25
2.98
1.11
0.94
2.82
2.24
3.12
0.99
1.16
6.25
6.25
3.12
12.5
25
3.15
0.96
0.85
3.06
2.88
2.98
1.08
2.02
6.25
12.5
3.12
3.12
1.56
12.5
6.25
6.25
0.009
5g
5h
6a
6b
6c
6d
6e
6f
6.25
12.5
12.5
25
6.25
0.78
3.12
0.05
6g
6h
CA-4
0.04
a50% Inhibitory concentration; bBreast cancer cell line; cLung cancer cell line;
dLiver cancer cell line.
isoxazolines is in agreement with exhibited excellent activity.
In conclusion, an efficient and convenient route for synthesis of new
tetrazole based pyrazoline and isoxazoline derivatives has been re-
ported. The process also exhibits significant functional group tolerance
and allows for the preparation of number of substituted tetrazole ana-
logues in good to excellent yields. Importance of substituted tetrazole
analogues would render this protocol attractive for both synthetic and
medicinal chemistry. Among synthesized derivatives, compounds 5b,
5c and 6g were significantly cytotoxic against MCF-7, A549 and HepG2
cancer cell lines at micromolar concentrations. Compound 5c inhibited
tubulin polymerization with IC50 value of 2.16. In silico docking studies
of synthesized compounds showed binding affinity toward colchicine
binding site of α,β-tubulin with highest score of −9.66. Consequently,
reported derivatives may serve as lead scaffold for development of
novel anticancer agents.
Microtubules exhibit dynamic equilibrium with free tubulin mono-
mers in cells. Pharmacological agents exploit this property of micro-
tubules to exert their anticancer effects.45 Since these inhibit tubulin
polymerization and disturbs microtubule dynamics, we elucidated le-
vels of soluble versus polymerized forms of tubulin in A549 cells fol-
lowing treatment with 1 µM of 5b and 5c for 48 h. In addition, cells
were treated with Combretastatin A-4 (CA-4, 1 µM) as positive controls
and DMSO as negative in parallel experiments. Subsequently, these
soluble and polymerized fractions were collected and subjected to
western blot analysis. This analysis reveals that, amount of tubulin
protein in both soluble and polymerized fractions were approximately
same in DMSO treated cells. Combretastatin A-4 treated cells exhibited
a shift of tubulin from the polymerized fraction into the soluble
4

V.S. Dofe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127592
Fig. 3. Structure of α,β-tubulin–ligand complex. (A) Pictorial representation of the overall α,β-tubulin along with superimposition of all compounds over Colchicine
at the Colchicine binding Site. α subunit of tubulin is colored in grey and the β subunit is coloured in white. (B) Close-up view of superimposition of all compounds
(shown in yellow sticks) over Colchicine (shown in green sticks) at the Colchicine binding Site. (C) The interaction mode of Colchicine at active site of α,β-tubulin
Yellow colored dotted lines indicate H-bond interaction.
Fig. 4. The interaction mode of (A) Compound 5b and (B) Compound 5c with α,β-Tubulin at the Colchicine Binding Site. Yellow coloured dotted lines indicate H-
bond interaction and Cyan coloured dotted lines indicate aromatic H-bond interaction between ligand and enzyme.
5

V.S. Dofe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127592
Declaration of Competing Interest
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Bioorganic&MedicinalChemistryLetters30(2020)127592
product as white crystals. Yield: 84 %; mp 146-148 °C; 1H NMR (500 MHz, DMSO-
was added dropwise. The reaction mixture was heated under reflux for 4-5 h and
progress of reaction monitored by TLC. After completion of the reaction, resulting
solution was cooled and poured into crushed ice. Solid pyrazolines 5a-h were filtered
and recrystallized from ethanol. Sonochemical method: To a solution substituted (E)-
1-(4-((1H-tetrazol-5-yl)methoxy)-3-methoxyphenyl)-3-phenylprop-2-en-1-one (0.01
mol) 4a-h in 10 mL of ethanol, (0.05 mol) of hydrazine hydrate (99%) was added
dropwise. Reaction mixture was suspended at centre of the ultrasonic bath and so-
nicated for 10-15 min. After completion of reaction, separated solid was collected by
filtration, washed with water and recrystallized from ethanol. Compound (5a):
yellow solid; mp 131-133 °C; 1H NMR (500 MHz, chloroform-d, δH ppm): 3.21 (dd,
1H, pyrazoline CH2), 3.36 (dd, 1H, pyrazoline CH2), 3.88 (s, 3H, OCH3), 4.84 (t, 1H,
pyrazoline CH), 5.30 (s, 2H, OCH2), 7.07 (d, 1H, ArH), 7.25–7.36 (m, 7H, ArH), 7.86
(d, 1H, ArH). 13C NMR (125 MHz, chloroform-d, δC ppm): 41.7, 56.0, 58.8, 61.5,
107.3, 113.2, 118.7, 122.7, 125.7, 126.7, 127.6, 128.5, 132.2, 141.4, 148.7, 150.5,
151.6, 155.7. HRMS Anal. calcd. for C18H18N6O2 (M+H)+: 351.1569. Found: 351.
1564. Anal. calcd. for C18H18N6O2: C, 61.70; H, 5.18; N, 23.99. Found: C, 61.58; H,
5.46; N, 23.94. Compound (5b): yellow solid; mp 116-118 °C; 1H NMR (500 MHz,
chloroform-d, δH ppm): 3.21 (dd, 1H, pyrazoline CH2), 3.36 (dd, 1H, pyrazoline
CH2), 3.88 (s, 3H, OCH3), 4.83 (t, 1H, pyrazoline CH), 5.30 (s, 2H, OCH2), 7.08 (d,
1H, ArH), 7.25– .27 (m, 3H, ArH), 7.30 (d, 1H, ArH), 7.33–7.35 (m, 2H, ArH), 7.85
(d, 1H, ArH). 13C NMR (125 MHz, chloroform-d, δC ppm):41.7, 56.0, 58.3, 61.5,
107.3, 113.2, 118.7, 125.4, 126.7, 128.0, 128.6, 132.5, 133.4, 140.1, 148.7, 150.5,
151.6, 155.7. HRMS Anal. calcd. for C18H17ClN6O2 (M+H)+: 385.118. Found:
385.1174. Anal. calcd. for C18H17ClN6O2: C, 56.17; H, 4.45; N, 21.88. Found: C, 56.
03; H, 4.70; N, 21.78. Compound (5c): yellow solid; mp 188-190 °C; 1H NMR (500
MHz, chloroform-d, δH ppm): 3.21 (dd, 1H, pyrazoline CH2), 3.36 (dd, 1H, pyr-
azoline CH2), 3.88 (s, 3H, OCH3), 4.83 (t, 1H, pyrazoline CH), 5.30 (s, 2H, OCH2), 7.
00-7.03 (m, 2H, ArH), 7.07 (d, 1H, ArH), 7.25–7.28 (m, 1H, ArH), 7.30-7.32 (m, 2H,
ArH), 7.86 (d, 1H, ArH). 13C NMR (125 MHz, chloroform-d, δC ppm): 41.6, 56.0, 58.
6, 61.5, 107.3, 113.2, 115.5, 115.7, 118.7, 126.7, 127.9, 127.9, 138.3, 138.3, 148.7,
150.5, 151.6, 155.7, 161.3, 163.3. HRMS Anal. calcd. for C18H17FN6O2 (M+H)+:
369.1475. Found: 369.1476. Anal. calcd. for C18H17FN6O2: C, 58.69; H, 4.65; N,
22.81. Found: C, 59.15; H, 4.74; N, 24.34. Compound (5d): yellow solid; mp 160-162
°C; 1H NMR (500 MHz, chloroform-d, δH ppm): 3.23 (dd, 1H, pyrazoline CH2), 3.34
(dd, 1H, pyrazoline CH2), 3.82 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 4.87 (t, 1H, pyrazoline CH), 5.30 (s, 2H, OCH2), 6.88 (d, 1H, ArH), 6.91–6.
94 (m, 1H, ArH), 7.07 (d, 1H, ArH), 7.25–7.30 (m, 2H, ArH), 7.90 (d, 1H, ArH). 13C
NMR (125 MHz, chloroform-d, δC ppm): 41.6, 55.9, 56.0, 58.3, 61.5, 107.3, 110.3,
112.4, 113.2, 118.7, 120.3, 122.7, 126.7, 131.4, 148.3, 148.7, 149.4, 150.5, 151.6,
155.7. HRMS Anal. calcd. for C20H22N6O4 (M+H)+: 411.1781. Found: 411.1775.
Anal. calcd. for C20H22N6O4: C, 58.53; H, 5.40; N, 20.48. Found: C, 58.32; H, 5.62;
N, 20.45. Compound (5e): yellow solid; mp 210-212 °C; 1H NMR (500 MHz,
chloroform-d, δH ppm): 3.23 (dd, 1H, pyrazoline CH2), 3.33 (dd, 1H, pyrazoline
CH2), 3.88 (s, 3H, OCH3), 4.86 (t, 1H, pyrazoline CH), 5.30 (s, 2H, OCH2), 6.70
(ddd, 1H, ArH), 6.77 (td, 1H, ArH), 6.99 (ddt, 1H, ArH), 7.07 (d, 1H, ArH), 7.12 (t,
1H, ArH), 7.26 (dd, 1H, ArH), 7.30 (d, 1H, ArH), 7.91 (d, 1H, ArH), 8.25 (s, 1H, OH).
13C NMR (125 MHz, chloroform-d, δC ppm): 41.6, 56.0, 58.5, 61.5, 107.3, 113.2,
113.4, 116.0, 118.7, 119.1, 126.7, 130.1, 144.4, 148.7, 150.5, 151.6, 155.7, 158.0.
HRMS Anal. calcd. for C18H18N6O3 (M+H)+: 367.1519. Found: 367.1517. Anal.
calcd. for C18H18N6O3: C, 59.01; H, 4.95; N, 22.94. Found: C, 59.12; H, 4.87; N, 23.
07. Compound (5f): yellow solid; mp 170-172 °C; 1H NMR (500 MHz, chloroform-d,
δH ppm): 3.21 (dd, 1H, pyrazoline CH2), 3.36 (dd, 1H, pyrazoline CH2), 3.78 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 4.84 (t, 1H, pyrazoline CH), 5.30 (s, 2H, OCH2), 6.77-6.
83 (m, 2H, ArH), 7.07 (d, 1H, ArH), 7.24-7.34 (m, 4H, ArH), 7.86 (d, 1H, ArH). 13C
NMR (125 MHz, chloroform-d, δC ppm): 41.6, 55.3, 56.0, 58.6, 61.5, 107.3, 110.3,
113.2, 113.8, 118.7, 126.7, 126.9, 129.3, 136.5, 148.7, 150.5, 151.6, 155.7, 159.1.
HRMS Anal. calcd. for C19H20N6O3 (M+H)+: 381.1675. Found: 381.1670. Anal.
calcd. for C19H20N6O3: C, 59.99; H, 5.30; N, 22.09. Found: C, 60.12; H, 5.39; N, 21.
87. Compound (5g): yellow solid; mp 148-150 °C; 1H NMR (500 MHz, chloroform-d,
δH ppm): 2.34 (s, 3H, CH3), 3.21 (dd, 1H, pyrazoline CH2), 3.36 (dd, 1H, pyrazoline
CH2), 3.88 (s, 3H, OCH3), 4.83 (t, 1H, pyrazoline CH), 5.30 (s, 2H, OCH2), 7.06-7.08
(m, 2H, ArH), 7.20–7.21 (m, 2H, ArH), 7.23–7.26 (m, 1H, ArH), 7.86 (d, 2H, ArH).
13C NMR (125 MHz, chloroform-d, δC ppm): 21.1, 41.7, 56.0, 58.8, 61.5, 107.3, 13.
2, 118.7, 122.7, 126.1, 126.7, 129.4, 133.0, 138.1, 139.7, 148.7, 150.5, 151.6, 155.
7. HRMS Anal. calcd. for C19H20N6O2 (M+H)+: 365.1726. Found: 365.1721.
Anal. calcd. for C19H20N6O2: C, 62.62; H, 5.53; N, 23.06. Found: C, 62.43; H, 5.78;
N, 23.05. Compound (5h): yellow solid; mp 128-130 °C; 1H NMR (500 MHz,
chloroform-d, δH ppm): 3.21 (dd, 1H, pyrazoline CH2), 3.36 (dd, 1H, pyrazoline
CH2), 3.88 (s, 3H, OCH3), 4.84 (t, 1H, pyrazoline CH), 5.30 (s, 2H, OCH2), 6.68- 6.
72 (m, 2H, ArH), 6.96 (s, 1H, ArH), 7.07 (d, 1H, ArH), 7.20 – 7.21 (m, 2H, ArH), 7.
25-7.30 (m, 1H, ArH), 7.86 (d, 1H, ArH). 13C NMR (125 MHz, chloroform-d, δC
ppm): 41.6, 56.0, 58.7, 61.5, 107.3, 113.2, 115.6, 118.7, 122.2, 126.7, 127.7, 133.6,
135.6, 148.7, 150.5, 151.6, 155.7, 157.7. HRMS Anal. calcd. for C18H18N6O3 (M
+H)+: 367.1519. Found: 367.1513. Anal. calcd. for C18H18N6O3: C, 59.01; H, 4.
95; N, 22.94. Found: C, 58.83; H, 5.26; N, 22.81. General procedure for the synthesis
of substituted 5-((4-(4,5-dihydro-5-phenylisoxazol-3-yl)-2-methoxyphenoxy)me-
thyl)-1H-tetrazole 6a-h: Conventional method: To a solution of substituted (E)-1-(4-
((1H-tetrazol-5-yl)methoxy)-3-methoxyphenyl)-3-phenylprop-2-en-1-one (0.01 mol)
4a-h in 10 mL of ethanol, (0.05 mol) of hydroxylamine hydrochloride was added
dropwise. Reaction mixture was heated under reflux for 4-5 h and progress of the
reaction monitored by TLC. After completion of reaction, resulting solution was
cooled and poured into crushed ice. Solid pyrazolines 6a-h were filtered and re-
crystallized from ethanol. Sonochemical method: To a solution substituted (E)-1-(4-
((1H-tetrazol-5-yl)methoxy)-3-methoxyphenyl)-3-phenylprop-2-en-1-one (0.01 mol)
4a-h in 10 mL of ethanol, (0.05 mol) of hydroxylamine hydrochloride was added
dropwise. Reaction mixture was suspended at centre of the ultrasonic bath and
d6, δH ppm): 2.60 (s, 3H, COCH3), 3.89 (s, 3H, OCH3), 5.62 (s, 2H, OCH2), 7.27 (d,
1H, ArH), 7.54 (s, 1H, ArH), 7.68 (d, 1H, ArH). 13C NMR (125 MHz, DMSO-d6, δC
ppm): 26.9, 56.1, 60.2, 111.2, 113.4, 113.5, 123.2, 131.6, 149.4, 151.4, 197.0.
HRMS Anal. calcd. for C11H12N4O3 (M+H)+: 249.0988. Found: 249.0982. Anal.
calcd. for C11H12N4O3: C, 53.22; H, 4.87; N, 22.57. Found: C, 53.19; H, 4.81; N,
22.62.
43. General procedure for the synthesis of substituted (E)-1-(4-((1H-tetrazol-5-yl)
methoxy)-3-methoxyphenyl)-3-phenylprop-2-en-1-one 4a-h: Conventional method:
To a solution of KOH (0.03 mol) in ethanol (15 mL) was added 1-(4-((1H-tetrazol-5-
yl)methoxy)-3-methoxyphenyl)ethanone 3 (0.01 mol) and substituted benzaldehyde
(0.01 mol) at 0-5°C. The reaction mixture was stirred 3-4 h at room temperature.
Then, reaction mixture was poured over crushed ice and neutralize carefully with 2N
HCl and precipitate so obtained was filtered, washed with water and dried. The crude
product was crystallized with ethanol to afford desired compound. Sonochemical
method: To a solution of KOH (0.03 mol) in ethanol (15 mL) was added 1-(4-((1H-
tetrazol-5-yl)methoxy)-3-methoxyphenyl)ethanone 3 (0.01 mol) and substituted
benzaldehyde (0.01 mol), and resulting mixture subjected to ultrasonic irradiation
for 20-25 min. After completion, resulting mixture was poured into ice-cold water
and then neutralized with 2N HCl. Solid obtained was filtered off, dried and purified
by recrystallization from ethanol. Compound (4a): white solid; mp 166-168°C; 1H
NMR (500 MHz, chloroform-d, δH ppm): 3.88 (s, 3H, OCH3), 5.30 (s, 2H, OCH2), 7.
13 (d, 1H, olefinic proton), 7.36–7.38 (m, 3H, ArH), 7.55–7.59 (m, 4H, ArH), 7.67 (d,
1H, olefinic proton), 7.95 (d, 1H, ArH). 13C NMR (125 MHz, chloroform-d, δC ppm):
56.0, 61.5, 112.6, 114.0, 122.9, 123.7, 127.2, 128.8, 129.1, 130.2, 131.4, 132.4,
135.1, 144.3, 150.2, 152.8, 155.7, 190.0. HRMS Anal. calcd. for C18H16N4O3 (M
+H)+: 337.1301. Found: 337.1292. Anal. calcd. for C18H16N4O3: C, 64.28; H, 4.
79; N, 16.66. Found: C, 64.26; H, 4.83; N, 16.71. Compound (4b): white solid; mp
132-134 °C; 1H NMR (500 MHz, chloroform-d, δH ppm): 3.88 (s, 3H, OCH3), 5.30 (s,
2H, OCH2), 7.13 (d, 1H, olefinic proton), 7.42-7.44 (m, 2H, ArH), 7.54-7.57 (m, 4H,
ArH), 7.67 (d, 1H, olefinic proton), 7.77 (dd, 1H, ArH). 13C NMR (125 MHz,
chloroform-d, δC ppm): 56.0, 61.5, 112.6, 114.0, 123.3, 123.7, 126.3, 129.4, 129.9,
132.4, 134.0, 134.8, 140.3, 144.0, 150.2, 152.8, 155.7, 190.0. HRMS Anal. Calcd.
For C18H15ClN4O3 (M+H)+: 371.0911. Found: 371.091. Anal. calcd. for
C18H15ClN4O3: C, 58.31; H, 4.08; N, 15.11. Found: C, 58.28; H, 4.12; N, 15.04.
Compound (4c): white solid; mp 152-154 °C; 1H NMR (500 MHz, chloroform-d, δH
ppm): 3.89 (s, 3H, OCH3), 5.05 (s, 2H, OCH2), 7.02 (d, 1H, olefinic proton), 7.20
(dd, 2H, ArH), 7.52 (d, 1H, ArH), 7.56-7.58 (m, 2H, ArH), 7.62 (d, 1H ArH), 7.67 (dd,
1H, ArH), 7.77 (d, 1H, olefinic proton). 13C NMR (125 MHz, chloroform-d, δC ppm):
55.9, 61.7, 112.7, 113.9, 115.8, 122.7, 123.9, 130.4, 130.5, 131.7, 132.4, 144.2,
150.2, 152.7, 156.3, 162.5, 164.5, 190.0. HRMS Anal. Calcd. For C18H15FN4O3 (M
+H)+: 355.1206. Found: 355.1202. Anal. calcd. for C18H15FN4O3: C, 61.01; H, 4.
27; N, 15.81. Found: C, 60.92; H, 4.36; N, 15.98. Compound (4d): white solid; mp
176-178 °C; 1H NMR (500 MHz, chloroform-d, δH ppm): 3.84 (s, 3H, OCH3), 3.85 (s,
3H, OCH3), 3.88 (s, 3H, OCH3), 5.30 (s, 2H, OCH2), 6.88 (d, 1H, ArH), 7.13 (d, 1H,
olefinic proton), 7.21 (d, 1H, olefinic proton), 7.24 (dd, 1H, ArH), 7.52–7.59 (m, 2H,
ArH), 7.68 (dd, 1H, ArH), 7.77–7.81(m, 1H, ArH). 13C NMR (125 MHz, chloroform-
d, δC ppm): 55.9, 55.9, 56.0, 61.5, 111.1, 112.5, 112.6, 114.0, 122.4, 123.1, 123.7,
129.8, 132.4, 144.0, 150.2, 150.5, 151.0, 152.8, 155.7, 189.9. HRMS Anal. Calcd.
For C20H20N4O5 (M+H)+: 397.1512. Found: 397.1501. Anal. calcd. for
C20H20N4O5: C, 60.60; H, 5.09; N, 14.13. Found: C, 60.43; H, 5.31; N, 14.18.
Compound (4e): white solid; mp 190-192 °C; 1H NMR (500 MHz, chloroform-d, δH
ppm): 3.88 (s, 3H, OCH3), 5.30 (s, 2H, OCH2), 6.84 (d, 1H, ArH), 7.09 (d, 1H, ArH),
7.13 (d, 1H, olefinic proton), 7.20-7.26 (m, 1H, ArH), 7.30 (d, 1H, ArH), 7.52-7.59
(m, 2H, ArH), 7.67 (d, 1H, olefinic proton), 7.72–7.79 (m, 1H, ArH), 8.40 (s, 1H,
OH). 13C NMR (125 MHz, chloroform-d, δC ppm): 56.0, 61.5, 112.6, 114.0, 116.0,
117.8, 122.3, 122.8, 123.7, 130.3, 132.4, 137.5, 143.2, 150.2, 152.8, 155.7, 158.5,
189.9. ESI-MS Anal. calcd. for C18H16N4O4 (M+H)+: 353.125. Found: 353.
12Anal. calcd. for C18H16N4O4: C, 61.36; H, 4.58; N, 15.90. Found: C, 61.31; H, 4.
62; N, 15.88. Compound (4f): white solid; mp 158-160 °C; 1H NMR (500 MHz,
chloroform-d, δH ppm): 3.82 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 5.30 (s, 2H, OCH2),
6.92–6.94 (m, 2H, ArH), 7.12 (s, 1H, ArH), 7.56–7.59 (m, 4H, ArH), 7.68 (d, 1H,
olefinic proton), 7.78 (d, 1H, ArH). 13C NMR (125 MHz, chloroform-d, δC ppm): 55.
3, 56.0, 61.5, 112.6, 114.0, 114.0 114.3, 120.6, 123.3, 123.7, 128.9, 130.4, 132.4,
144.1, 150.2, 152.8, 155.7, 161.5, 190.0. HRMS Anal. calcd. for C19H18N4O4 (M
+H)+: 367.1406. Found: 367.1407. Anal. calcd. for C19H18N4O4: C, 62.29; H, 4.
95; N, 15.29. Found: C, 62.14; H, 5.24; N, 15.22. Compound (4g): white solid; mp
124-126 °C; 1H NMR (500 MHz, chloroform-d, δH ppm): 2.38 (s, 3H, CH3), 3.88 (s,
3H, OCH3), 5.30 (s, 2H, OCH2), 7.13 (d, 1H, olefinic proton), 7.24-7.29 (m, 2H,
ArH), 7.45-7.51 (m, 2H, ArH), 7.53-7.59 (m, 2H, ArH), 7.67 (d, 1H, olefinic proton),
7.78 (dd, 1H, ArH); 13C NMR (125 MHz, chloroform-d, δC ppm): 21.4, 56.0, 61.5,
112.6, 114.0, 120.3, 123.3, 123.7, 129.6, 129.9, 132.4, 133.1, 138.3, 139.8, 144.2,
150.2, 152.8, 155.7, 190.0. HRMS Anal. Calcd. For C19H18N4O3 (M+H)+: 351.
1457. Found: 351.1451 Anal. calcd. for C19H18N4O3: C, 65.13; H, 5.18; N, 15.99.
Found: C, 65.08; H, 5.22; N, 16.08. Compound (4h): white solid; mp 156-158 °C; 1H
NMR (500 MHz, chloroform-d, δH ppm): 3.88 (s, 3H, OCH3), 5.30 (s, 2H, OCH2), 6.
08 (s, 1H, OH), 6.79–6.81 (m, 2H, ArH), 7.13 (d, 1H, ArH), 7.51–7.57 (m, 4H, ArH),
7.67 (d, 1H, olefinic proton), 7.78 (d, 1H, olefinic proton). 13C NMR (125 MHz,
DMSO-d6, δC ppm): 56.0, 61.5, 112.6, 114.0, 116.8, 120.1, 123.3, 123.7, 127.5, 131.
1, 132.4, 135.6, 144.3, 150.2, 152.8, 155.7, 160.0, 190.0. HRMS Anal. calcd. for
C18H16N4O4 (M+H)+: 353.125. Found: 353.1244. Anal. calcd. for C18H16N4O4:
C, 61.36; H, 4.58; N, 15.90. Found: C, 61.17; H, 4.83; N, 15.84. General procedure for
the synthesis of substituted 5-((4-(4,5-dihydro-5-phenyl-1H-pyrazol-3-yl)-2-methox-
yphenoxy)methyl)-1H-tetrazole 5a-h: Conventional method: To a solution of sub-
stituted (E)-1-(4-((1H-tetrazol-5-yl)methoxy)-3-methoxyphenyl)-3-phenylprop-2-en-
1-one (0.01 mol) 4a-h in 10 mL of ethanol, (0.05 mol) of hydrazine hydrate (99%)
7

V.S. Dofe, et al.
sonicated for 10-15 min. After completion of reaction, separated solid was collected
Bioorganic&MedicinalChemistryLetters30(2020)127592
Anal. calcd. for C18H17N5O4: C, 58.85; H, 4.66; N, 19.06. Found: C, 58.69; H, 4.93;
N, 19.01. Compound (6f): yellow solid; mp 130-132 °C; 1H NMR (500 MHz,
chloroform-d, δH ppm): 3.67–3.74 (m, 2H, isoxazoline CH2), 3.78 (s, 3H, OCH3), 3.
88 (s, 3H, OCH3), 5.30 (s, 2H, OCH2), 5.79 (dd, 1H, isoxazoline CH), 6.83–6.90 (m,
2H, ArH), 7.01 (d, 1H, ArH), 7.26–7.30 (m, 4H, ArH). 13C NMR (125 MHz,
chloroform-d, δC ppm): 41.1, 55.3, 56.0, 61.5, 76.4, 110.1, 113.4, 113.9, 118.7, 121.
9, 125.6, 127.3, 130.3, 133.6, 149.1, 150.7, 155.1, 155.7, 159.8. HRMS Anal. calcd.
for C19H19N5O4 (M+H)+: 382.1515. Found: 382.1512. Anal. calcd. for
C19H19N5O4: C, 59.84; H, 5.02; N, 18.36. Found: C, 59.55; H, 5.40; N, 18.35.
Compound (6g): yellow solid; mp 152-154 °C; 1H NMR (500 MHz, chloroform-d, δH
ppm): 2.34 (s, 2H, CH3), 3.68–3.74 (m, 2H, isoxazoline CH2), 3.88 (s, 3H, OCH3), 5.
30 (s, 2H, OCH2), 5.79 (dd, 1H, isoxazoline CH), 7.01 (d, 1H, ArH), 7.15 (dq, 2H,
ArH), 7.26–7.30 (m, 5H, ArH). 13C NMR (125 MHz, chloroform-d, δC ppm): 21.1,
41.1, 56.0, 61.5, 76.5, 110.1, 113.4, 121.1, 121.9, 125.6, 126.6, 129.6, 137.6, 137.6,
137.9, 149.1, 150.7, 155.1, 155.7. HRMS Anal. calcd. for C19H19N5O3 (M+H)+:
366.1566. Found: 366.1561. Anal. calcd. for C19H19N5O3: C, 62.46; H, 5.24; N, 19.
17. Found: C, 62.28; H, 5.56; N, 19.08. Compound (6h): yellow solid; mp 102-104 °C;
1H NMR (500 MHz, chloroform-d, δH ppm): 3.67–3.74 (m, 2H, isoxazoline CH2), 3.
88 (s, 3H, OCH3), 5.30 (s, 2H, OCH2), 5.79 (dd, 1H, isoxazoline CH), 6.78–6.80 (m,
2H, ArH), 7.01 (d, 1H, ArH), 7.26–7.28 (m, 3H, ArH), 7.29 (dd, 1H, ArH), 8.17 (s,
1H, OH). 13C NMR (125 MHz, chloroform-d, δC ppm): 41.1, 56.0, 61.5, 76.5, 110.1,
113.4, 115.7, 119.5, 121.9, 125.6, 127.3, 129.0, 132.5, 149.1, 150.7, 155.1, 155.7,
158.2. HRMS Anal. calcd. for C18H17N5O4 (M+H)+: 368.1359. Found: 368.1351.
Anal. calcd. for C18H17N5O4: C, 58.85; H, 4.66; N, 19.06. Found: C, 58.72; H, 4.90;
N, 18.89.
by filtration, washed with water and recrystallized from ethanol. Compound (6a):
yellow solid; mp 140-142 °C; 1H NMR (500 MHz, chloroform-d, δH ppm): 3.67–3.74
(m, 2H, isoxazoline CH2), 3.88 (s, 3H, OCH3), 5.30 (s, 2H, OCH2), 5.79-5.81 (t, 1H,
isoxazoline CH), 7.01 (d, 1H, ArH), 7.26-7.35 (m, 5H, ArH), 7.40–7.41 (m, 2H, ArH).
13C NMR (125 MHz, chloroform-d, δC ppm): 41.1, 56.0, 61.5, 79.8, 110.1, 113.4,
121.9, 123.0, 125.6, 126.5, 128.4, 128.5, 132.8, 141.0, 149.1, 150.7, 155.1, 155.7.
HRMS Anal. calcd. for C18H17N5O3 (M+H)+: 352.141. Found: 352.1402. Anal.
calcd. for C18H17N5O3: C, 61.53; H, 4.88; N, 19.93. Found: C, 61.34; H, 5.12; N, 19.
86. Compound (6b): yellow solid; mp 126-128 °C; 1H NMR (500 MHz, chloroform-d,
δH ppm): 3.68–3.73 (m, 2H, isoxazoline CH2), 3.88 (s, 3H, OCH3), 5.30 (s, 2H,
OCH2), 5.78 (t, 1H, isoxazoline CH), 7.01 (d, 1H, ArH), 7.26 (d, 1H, ArH), 7.32–7.27
(m, 1H, ArH), 7.35–7.29 (m, 1H, ArH), 7.32 (s, 1H, ArH). 13C NMR (125 MHz,
chloroform-d, δC ppm): 41.1, 56.0, 61.5, 76.3, 110.1, 113.4, 121.9, 124.3, 125.6,
127.9, 128.8, 131.4, 134.1, 138.2, 149.1, 150.7, 155.1, 155.7. HRMS Anal. calcd. for
C18H16ClN5O3 (M+H)+: 386.102. Found: 386.1014. Anal. calcd. for
C18H16ClN5O3: C, 56.04; H, 4.18; N, 18.15. Found: C, 55.89; H, 4.43; N, 18.11.
Compound (6c): yellow solid; mp 138-140 °C; 1H NMR (500 MHz, chloroform-d, δH
ppm): 3.75–3.65 (m, 2H, isoxazoline CH2), 3.88 (s, 3H, OCH3), 5.30 (s, 2H, OCH2),
5.79 (dd, 1H, isoxazoline CH), 7.01 (d, 1H, ArH), 7.07–7.10 (m, 1H, ArH), 7.26 (d,
1H, ArH), 7.29 (dd, 1H, ArH), 7.38–7.41 (m, 2H, ArH). 13C NMR (125 MHz,
chloroform-d, δC ppm): 41.1, 56.0, 61.5, 110.1, 113.4, 115.8, 115.9, 121.9, 125.6,
127.7, 127.8, 135.7, 135.7, 149.1, 150.7, 155.1, 155.7, 162.1, 164.2. HRMS Anal.
calcd. for C18H16FN5O3 (M+H)+: 370.1315. Found: 370.1313. Anal. calcd. for
C18H16FN5O3: C, 58.53; H, 4.37; N, 18.96. Found: C, 58.36; H, 4.63; N, 18.92.
Compound (6d): yellow solid; mp 120-122 °C; 1H NMR (500 MHz, chloroform-d, δH
ppm): 3.71 (dd, 2H, isoxazoline CH2), 3.81 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.84
(s, 3H, OCH3), 5.30 (s, 1H, OCH2), 5.79 (t, 1H, isoxazoline CH), 7.00 (d, 1H, ArH), 7.
06–7.10 (m, 3H, ArH), 7.26 (d, 1H, ArH), 7.29 (dd, 1H, ArH). 13C NMR (125 MHz,
chloroform-d, δC ppm): 41.1, 55.9, 56.0, 61.5, 81.5, 110.1, 110.6, 112.6, 113.4, 120.
3, 121.9, 125.6, 128.5, 132.7, 148.9, 149.1, 149.1, 150.7, 155.1, 155.7. HRMS Anal.
calcd. for C20H21N5O5 (M+H)+: 412.1621. Found: 412.1612. Anal. calcd. for
C20H21N5O5: C, 58.39; H, 5.14; N, 17.02. Found: C, 58.26; H, 5.30; N, 16.86.
Compound (6e): yellow solid; mp 168-170 °C; 1H NMR (500 MHz, chloroform-d, δH
ppm): 3.70 (dd, 2H, isoxazoline CH2), 3.88 (s, 3H, OCH3), 5.30 (s, 1H, OCH2), 5.70
(t, 1H, isoxazoline CH), 6.69–6.71 (m, 1H, ArH), 6.83 (dd, 1H, ArH), 7.01 (d, 1H,
ArH), 7.16–7.21 (m, 2H, ArH), 7.26 (d, 1H, ArH), 7.29 (dd, 1H, ArH), 8.46 (s, 1H,
OH). 13C NMR (125 MHz, chloroform-d, δC ppm): 41.1, 56.0, 61.5, 81.8, 110.1, 113.
4, 114.4, 115.8, 118.3, 121.9, 125.6, 130.1, 139.1, 149.1, 150.7, 155.1, 155.7, 157.
5. HRMS Anal. calcd. for C18H17N5O4 (M+H)+: 368.1359. Found: 368.1358.
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