Design, synthesis, in vitro and in silico evaluation of new 3-phenyl-4,5-dihydroisoxazole-5-carboxamides active against drug-resistant mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.molstruc.2020.129545

A new series of 3-phenyl-4,5-dihydroisoxazole-5-carboxamides were designed, synthesized, and evaluated for their potency against Mtb H37Rv. Designed molecules were synthesized by one-pot cycloaddition reaction in good to excellent yields. Anti-Tubercular evaluation of all synthesized derivatives identified 6k to be highly potent (MIC 1 μg/mL) against Mtb and drug-resistant strains. All potent derivatives were found to be non-toxic when tested against Vero cells. Also, in silico studies were employed to explore the binding patterns of designed compounds to target Mycobacterial membrane protein Large-3. All derivatives exhibited excellent binding patterns with the receptor. The excellent in silico Absorption, Distribution, Metabolism, and Excretion properties and druggability parameters positions these molecules as promising lead candidates for the future development of new drugs to treat drug-resistant Tuberculosis.

Full text of DOI:10.1016/j.molstruc.2020.129545

Journal Pre-proof
Design, Synthesis, in vitro and in silico evaluation of new
3-phenyl-4,5-dihydroisoxazole-5-carboxamides active against
drug-resistant Mycobacterium tuberculosis
Nikhil Baliram Gaikwad , Pathan Afroz ,
Mohammad Naiyaz Ahmad , Grace Kaul , Manjulika Shukla ,
Srinivas Nanduri , Arunava Dasgupta , Sidharth Chopra ,
Venkata Madhavi Yaddanapudi
PII:
S0022-2860(20)31860-3
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.129545
MOLSTR 129545
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
30 September 2020
26 October 2020
29 October 2020
Please cite this article as: Nikhil Baliram Gaikwad , Pathan Afroz , Mohammad Naiyaz Ahmad ,
Grace Kaul , Manjulika Shukla , Srinivas Nanduri , Arunava Dasgupta , Sidharth Chopra ,
Venkata Madhavi Yaddanapudi , Design, Synthesis, in vitro and in silico evaluation of new 3-phenyl-
4,5-dihydroisoxazole-5-carboxamides active against drug-resistant Mycobacterium tuberculosis,
Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.129545
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Highlights:
•
3-phenyl-4,5-dihydroisoxazole-5-carboxylic acid derivatives were synthesized by click
reaction in good yields.
•
•
•
All the compounds were evaluated for their anti-microbial activity
Compound 6f, 6h, 6k, and 10a were found to be potent against drug-resistant Mtb.
6k is also effective against INH and STR -resistant Mtb with MIC 4ꢀμg/mL and 2 μg/mL
respectively
•
All active derivatives were found non-toxic against Vero cells and exhibited excellent
SI.

Design, Synthesis, in vitro and in silico evaluation of new 3-phenyl-4,5-
dihydroisoxazole-5-carboxamides active against drug-resistant Mycobacterium
tuberculosis.
a
Nikhil Baliram Gaikwad a, Pathan Afroz , Mohammad Naiyaz Ahmad b,c, Grace Kaul b,c, Manjulika
a
Shuklab, Srinivas Nanduri , Arunava Dasgupta*b,c, Sidharth Chopra*b,c and Venkata Madhavi
,
Yaddanapudi *a
a
Department of Pharmaceutical Technology and Process Chemistry, National Institute of
Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad 500037, Telangana, India.
b
Division of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10,
Janakipuram Extension, Lucknow, 226031, Uttar Pradesh, India.
c AcSIR: Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
*Corresponding author: Department of Pharmaceutical Technology and Process Chemistry,
National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad
500037, Telangana, India.
E-mail address: yssmadhavi@gmail.com (Venkata Madhavi Yaddanapudi),
skchopra007@gmail.com (Sidharth Chopra), a.dasgupta@cdri.res.in (Arunava Dasgupta)
Keywords: Anti-TB, Isoxazoline, Mycobacterium tuberculosis, Oximes, Antimycobacterial activity, 3-
phenyl-4,5-dihydroisoxazole-5-carboxamide, In silico ADME studies, Lipinski’s rule of five.
Abstract
A new series of 3-phenyl-4,5-dihydroisoxazole-5-carboxamides were designed, synthesized, and
evaluated for their potency against Mtb H37Rv. Designed molecules were synthesized by one-pot
cycloaddition reaction in good to excellent yields. Anti-Tubercular evaluation of all synthesized
derivatives identified 6k to be highly potent (MIC 1 μg/mL) against Mtb and drug-resistant
strains. All potent derivatives were found to be non-toxic when tested against Vero cells. Also, in
silico studies were employed to explore the binding patterns of designed compounds to target
Mycobacterial membrane protein Large-3. All derivatives exhibited excellent binding patterns
with the receptor. The excellent in silico Absorption, Distribution, Metabolism, and Excretion
properties and druggability parameters positions these molecules as promising lead candidates
for the future development of new drugs to treat drug-resistant Tuberculosis.

1. Introduction
Tuberculosis (TB) is a communicable disease caused due to Mycobacterium tuberculosis (Mtb)
and is one of the top 10 deadly diseases worldwide. In 2018, TB tolled more lives than HIV,
though HIV patients data also involves TB patients, with 10 million infections and 1.5 million
deaths.[1] Many amongst those who developed TB (0.55 million patients) were found to be
resistant to the Rifampicin (RIF), which is a front-line drug utilized against TB along with Isoniazid
(INH). Recently, WHO announced “The End TB strategy” to eradicate TB from the world by
2035.[2, 3] One of the goals in this strategy is to develop new chemical entities with anti-TB
potential as drugs in order to tackle drug-resistance which is a major hindrance to the eradication
of TB. With TB acquiring resistance against the current drug regimen, there is an urgent unmet
need for the development of new drugs.
In line with our efforts towards the development of new anti-TB agents, the Isoxazoline scaffold
was explored based on potential anti-TB molecules reported in the literature. Cycloserine, a
second-line anti-TB drug, inhibits cell wall biosynthesis in Mtb and is a cyclic derivative of D-
alanine existing in isoxazolidinone-isoxazoline tautomeric structures (Fig. 1).[4, 5] Lee et al.
reported compounds based on nitrofuranylisoxazoline scaffold with potent inhibitory activity
against Mtb and the lead molecule was “Lee-878” (Fig. 1). Lee-878's activity was predominantly
due to nitrofuran scaffold and Isoxazoline existed as a bioisosteric replacement for amide
linkage.[6] This paved the way for more research on various isoxazole and isoxazoline derivatives
as anti-TB agents. Thereafter, isoxazoline ester derivative A without a nitrofuran ring was
reported by Tangallapally et al.[6, 7] which also exhibited good anti-TB activity. A study by Reddy
et al.[8] suggested isoxazoline and heterocycle hybrids were found to possess good anti-microbial
activity. Mandawad et al. in 2014 reported novel thiophene substituted isoxazoline derivative D
as Anti-TB agent.[9] In 2006, Viswajanani et al from Ranbaxy Ltd. reported isoxazoline amide B
(Fig. 1) derived from L-phenyl alanine indicated as cell adhesion inhibitor acting as an anti-
inflammatory agent [10] and indicated for bronchial asthma. A further search of reported
literature directed us to BM212 [11] a pyrrole derivative that is active against Mtb and acts as a
MmpL3 inhibitor. Based on this compound, many pyrrole and pyrazole based MmpL3 inhibitors
were designed previously by many research groups using high-throughput screening (HTS) and
scaffold hopping methods.[12, 13] One of the hits was Rimonabant, an anti-obesity drug, and its
analogues have been repurposed for Mtb owing to its similarity to BM212.[14, 15] The lipophilic
nature of Rimonabant enabled it to cross the blood-brain barrier. This property was primarily

suggested to be effective in cerebral TB, later the drug was withdrawn from the market due to
the adverse side effects (e.g. depressive disorder). Thus, there is opportunity for researchers to
reduce the side effects of the Rimonabant without losing its anti-TB potential. So, Rimonabant
was found to be a promising lead for further development. Based on the reported literature on
the Isoxazoline scaffold, a shape-based strategy was adapted incorporating important structural
features of BM212, Rimonabant, and isoxazoline scaffold that led us to 3-phenyl-4,5-
dihydroisoxazole-5-carboxamide derivatives. The designed molecules were found to have good
structural alignment with BM212 as well as Rimonabant (Fig. 2) making it pharmacophorically
more similar in terms of binding interactions with receptor protein, primarily indicating that
designed molecules are plausible MmpL3 inhibitors. This was substantiated through our in silico
studies. 3,5-aryl-substituted isoxazolines, as well as 5-phenyl-4,5-dihydroisoxazole-3-carboxylic
acid derivatives, were reported but, to date not many efforts were put towards the development
of anti-tubercular agents based on the 3-phenyl-4,5-dihydroisoxazoline-5-carboxamide scaffold,
this inspired us to work on this scaffold. Also, interestingly the Kozikowski group has worked
extensively on the indole-2-carboxamide (Fig. 1) derivatives with alicyclic amines as a MmpL3
inhibitors. The reported amides with the aliphatic amines were found to be ∼4-300 times more
potent than the aromatic amide derivatives. Subsequently, the SAR studies suggested that a)
lipophilic and bulky substituents at amide nitrogen were vital for the potent anti-TB activity. b)
the polar group on amidic nitrogen led to loss of activity. Also the designed molecules were
potently active on the MDR and XDR TB strains.[16-18] Based on the aforementioned literature
the isoxazoline caboxamide series with diverse aliphatic amines were designed and synthesized.
The preliminary structure-activity relationships (SAR) of 3-phenyl-4,5-dihydroisoxazole-5-
carboxamide derivatives as anti-TB agents and in silico assessment based on studies involving
molecular docking, Absorption, Distribution, Metabolism, and Excretion studies, and druggability
based on compliance to Lipinski’s rule of five have been presented in the present work.
Figure 1: Previous reports on isoxazoline scaffold and amide conjugate with anti-TB activity.

Figure 2: Superimposition of Isoxazoline hybrid derivative 6k with BM212 and Rimonabant.
2. Results and discussion
2.1. Chemistry
All newly designed Isoxazoline derivatives were synthesized as outlined in Scheme 1. The synthesis
of final derivatives involved a sequential one-pot click reaction. The first intermediate aldoxime was
synthesized from commercially available substituted benzaldehydes and hydroxylamine sulphate
with sodium hydroxide solution in water using a reported procedure [19, 20] with slight
modifications which generated corresponding aldoximes 2 in good yield and purity. Thus, obtained
aldoximes were further converted to key intermediate substituted hydroxybenzimidoyl chloride by
chlorination using NCS in acetonitrile to get (E/Z)-N-hydroxybenzimidoyl chloride 3.[21] At last, the
key intermediate substituted(E/Z)-N-hydroxybenzimidoyl chlorides 3 were coupled with different

acrylamides using click reaction. The procedure followed a sequential addition of the acrolyl chloride
to the amine and triethylamine solution in DCM leading to form acrylamide as intermediate to which
the substituted (E/Z)-N-hydroxybenzimidoyl chloride 3 along with one equivalent of triethylamine
was added which finally generated the isoxazoline derivatives (6a-6k, 7a-7e, 8a-8e, 9a-9d, 10a-10e,
11a-11e, and 12a-12d) in good yield and purity.[22, 23] The one pot procedure was followed for
synthesizing the entire series of molecules. All the newly synthesized compounds were characterized
by 1H NMR, 13C NMR and HRMS (ESI) spectroscopic techniques.
Scheme 1: Synthetic route to isoxazoline amide compounds.
The analytical data was found in accordance with the reported structures. As a representative
1
compound, the characterization of one of the potent derivative 6k is discussed here. The H NMR
has shown characteristic doublet of doublet peak of the proton present on tertiary carbon
possessing carboxyl group and broad singlet for the amidic proton at 5.12 ppm and 6.65 ppm
respectively which ensured the formation of amide –NH- bond. Methylene Proton of isoxazole C-4 –
CH2- appeared as a doublet of a doublet at 3.65 ppm and also protons of the cyclohexyl group were
observed as multiplets at 1.18-1.21 ppm (2H), 1.32-1.38 ppm (2H), 1.59-1.60 ppm (2H), 1.68-1.75
ppm (2H), 1.82-1.85 (1H), 1.93-1.95ppm (1H) belonging to –CH2- and the proton of the –CH- group of
cyclohexyl ring was observed as a multiplet at 3.74 ppm. Remaining all Protons on phenyl ring
attached to isoxazole at C-3 appeared as doublets at 7.40 ppm and 7.60 ppm. Likewise, 13C NMR
interpretation of compound 6k designates the existence of carbonyl carbon of the amide group at

169.5 ppm. The methylene (–CH2-) carbon of the cyclohexyl ring at the para position to amine was
observed at 25.4 ppm, the meta position methylene carbon at 24.7 ppm, and the ortho position
methylene carbons were observed at 32.9ppm. The -CH- at the ipso position of the cyclohexyl was
present at 48.2ppm. Coming to the isoxazoline ring carbons, the C-3, C-4, and C-5 appeared
correspondingly at 156.2 ppm, 39.4 ppm, and 79.1 ppm. Lastly, the phenyl ring carbons of the ipso,
ortho, meta, and para position of the phenyl ring appeared at 126.9 ppm, 128.2 ppm, 129.2 ppm,
and 136.84 ppm respectively. All the other isoxazoline derivatives were characterized similarly by 1H,
13C NMR, and HRMS and were found in accordance with the depicted structures as presented in the
experimental section. The HRMS (ESI) data of each compound represented as an [M+H]+ or [M+Na]+
peaks were found in compliance with their precise molecular formula.
2.2. In vitro anti-tubercular activity
All molecules (6a-6k, 7a-7e, 8a-8e, 9a-9e, 10a-10e, 11a-11e, and 12a-12e) were screened for their
inhibitory potential against Mtb H37Rv using Microplate Alamar Blue Assay (MABA) assay with INH
and RIF as positive controls. The MIC values of these derivatives along with their CLogP values are
reported in Table 1. To begin with, the cyclohexylamine based amide derivatives 6a-6k (cyclohexyl as
R1 group) were synthesized and screened for their antitubercular potential on M. tuberculosis H37Rv
varying substituents on phenyl ring and their positions. Significantly, all synthesized compounds 6a-
6k showed anti-TB activity (MIC 1-64ꢀμg/mL). Compound 6k with 4-chloro as R substitution exhibited
the most potent activity in this series with (MIC 1ꢀμg/mL, CLogP 4.376). Other halogenated
derivatives also showed promising activity i.e. 6b (3-Cl)-MIC: 16ꢀμg/mL, 6e (4-Br)-MIC: 16ꢀμg/mL, 6f
(4-F)-MIC: 4ꢀμg/mL. Also, 6h with 4-nitro as R showed good activity with MIC of 4ꢀμg/mL, CLogP of
4.61. From the results it was clear that the para substitution was favored for potent antitubercular
activity, as the derivatives with meta or ortho substitutions were found to be less potent, i.e 6a (2-
Nitro: MIC 64 μg/mL), 6j (3-Nitro: MIC 64 μg/mL), and 6h (4-Nitro: MIC 4μg/mL), thus this shift in
nitro group position from para to meta or ortho lead to 16 folds decrease in the activity. Similarly,
the 6k (4-Cl: MIC 1 μg/mL) and 6b (3-Cl: MIC 16 μg/mL) derivatives suggested that a shift in chloro
group position from para to meta lead to the 64 folds decline in potency.
With these results, the effect of ring size on activity was explored taking the 4-Fluoro phenyl group
as constant. 8a-8e with cycloheptyl group as R1 substituent and 11a-11e with cyclopropyl group as
R1 substituent were synthesized and compared with 6a-6n (cyclohexyl as R1 substituent). The results
clearly illustrated the preference for cyclohexyl over cycloheptyl and cyclopropyl ring as R1
substituent as there was no improvement in activity observed in cycloheptyl and cyclopropyl ring,
although activity was retained by these modifications. In cyclopropyl series, the 4-benzyloxybenzyl

group as R was introduced which was the only compound to be moderately active in this series.
Further, to know the effect of acyclic groups, open-chain aliphatic amines were also explored like
octyl amine (7a-7e), pentylamine (9a-9e), and butylamine (10a-10e). Among all octyl amine
derivatives in the 7a-7e subseries, compound 7d was only moderately active, and the remaining
others were found to be weakly active which can be attributed to the higher hydrophobicity as well
as a linear chain of octyl amine. Pentylamine and octylamine series showed similar results, but the
butylamine series showed better results than the other two due to reduced hydrophobicity, which
can be attributed to reduced chain size. This was exemplified by 10a, a butylamine derivative that
was the only derivative in the category of open chain aliphatic amines to have excellent anti-TB
potential with MIC 8ꢀμg/mL and CLogP 3.362. (Fig. 3) Apart from cyclic and acyclic aliphatic amines,
few derivatives with heterocyclic amines (6-methoxy-2-aminbenzothiazole) were synthesized but
these amide derivatives did not show any prominent anti-TB activity, in contrast, a moderate
antimicrobial activity (MIC 16 μg/mL) was observed on S. aureus. Also, all derivatives were tested on
non-tuberculous mycobacteria (NTM) and were found to be inactive. Other substituents such as 4-
Me (6c) and 4- Cyano (6d) also showed good activity compared to unsubstituted derivative 6i. Thus,
a total of 40 isoxazoline carboxamide derivatives with varying R and R1 groups were synthesized. The
synthesized compounds were tested for their anti-TB potential. Based on the results, it was
observed that an increase in the hydrophobicity led to the loss of activity or reduction in potency,
but, apart from hydrophobicity, the nature of the aliphatic group and position of substituent on
phenyl ring also influenced activity.
Table1: In vitro evaluation of anti-TB and anti-microbial activity.
MIC (µg/mL)
Mtb INH-
res ATCC
35822
Mtb ETB-
res ATCC
35837
Mtb STR-
res ATCC
35820
Mtb RIF-
res ATCC
35838
S.
aureus
ATCC 29213
Entry
R
R1
Mtb H37Rv
ATCC 27294
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
7a
7b
7c
7d
2-NO2
3-Cl
4-Me
4-CN
4-Br
4-F
3-F
4-NO2
Ph
3-NO2
4-Cl
4-OH
3-Cl
4-Br
3-F
Ph
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Octyl
64
16
16
16
16
4
64
4
32
64
1
16
32
>64
>64
16
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
NT
NT
NT
NT
NT
32
NT
32
NT
NT
4
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
>64
NT
64
NT
NT
>64
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
32
NT
32
NT
NT
2
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
>64
NT
64
NT
NT
>64
NT
NT
NT
NT
NT
Octyl
Octyl
Octyl

7e
8a
8b
8c
8d
8e
9a
9b
9c
4-F
3-F
4-F
4-Me
4-CN
4-OMe
3-NO2
4-F
4-Cl
2-NO2
4-F
3-F
3-NO2
4-Br
3-Cl
3-NO2
4-F
4-Br
4-Cl
4-
Octyl
Heptyl
Heptyl
Heptyl
Heptyl
Heptyl
Pentyl
Pentyl
Pentyl
Pentyl
Butyl
Butyl
Butyl
Butyl
Butyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
>64
16
32
32
16
32
>64
32
>64
>64
8
32
>64
16
64
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
8
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
>64
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
1
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
>64
NT
NT
NT
NT
NT
NT
NT
NT
9d
10a
10b
10c
10d
10e
11a
11b
11c
11d
64
32
11e
12a
12b
12c
12d
cyclopropyl
16
64
32
64
64
>64
16
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
(OBn)Ph
6-OMe-2-
NH2BTZ*
6-OMe-2-
NH2BTZ*
6-OMe-2-
NH2BTZ*
6-OMe-2-
NH2BTZ*
Ph
3-F
3-Cl
4-F
16
>64
>64
INH
RIF
LEVO
ETB
STR
-
-
-
-
-
-
-
-
-
-
0.03
0.03
NT
0.5
1
NT
NT
0.125
NT
>64
0.03
0.5
2
64
0.03
0.03
0.12
0.5
0.03
>64
0.12
2
0.06
0.06
>64
0.5
NT
1
>64
0.25
INH = Isoniazid, RIF = Rifampicin, LEVO = Levofloxacin, ETB = Ethambutol, STR = Streptomycin, *BTZ = Benzothiazole.
Figure 3: Correlation between MIC and CLogP values of isoxazoline series.

2.3. Structure-activity relationship(SAR)
The substituents present on the phenyl ring played an important role in displaying a diverse range of
activity, also the position of these substitutions depicted variation in anti-TB potential. Electron
withdrawing groups (NO2, CN, Cl, and F) at para position of the acid group led to the superior anti-TB
potential. Compounds with an electron-donating group at the para position were weakly active, only
compound with methyl as R substituent (6c) was moderately active. In contrast, the electron-
withdrawing group at meta positions led to the loss of anti-mycobacterial potential. Similar results
were seen with the ortho position substitutions. Only para-substituted compounds were found to
have good to moderate activity. It is apparent from the structure-activity relationship (SAR) analysis
that cyclohexyl as R1 substituent and Strong electron-withdrawing group at a para position such as
NO2, CN, Cl, and F as R substituents are important for the anti-mycobacterial activity. It is significant
to note that the NO2 position on the phenyl ring is crucial as 6h (4-NO2) is more potent than 6a (2-
NO2) and 6j (3-NO2). Also, the NO2 group acts as a hydrogen bond acceptor which plays a crucial role
in binding with the receptor. Thus, similar electronic effects and interactions are seen with cyano
groups and halogens acting as hydrogen bond acceptors. These groups possibly make a more
promising impact on ligand binding which eventually leads to good activity. SAR interpretations are
illustrated in Fig. 4.
Figure 4: SAR inferences on isoxazoline carboxamide derivatives

2.4. Cytotoxicity against Vero cells
Cytotoxicity assay was performed against Vero cells (ATCC CCL-81) by using MTT assay to study
the influence of 6f, 6h, 6k, and 10a on mammalian cells. The results were depicted in CC50 which
is the lowest concentration of compound leading to a 50% reduction in cell viability. All
experiments were performed in triplicate and used Doxorubicin as a reference standard. Results
revealed that compounds 6f, 6h, 6k, and 10a were found nontoxic to Vero cells with a CC50 of
more than 100 µg/mL and have shown encouraging Selectivity Index (SI). The results achieved
are tabulated in Table 2.
Table 2: Cytotoxicity and Selectivity index (SI) of compounds against Vero cells
Entry
Mtb H37Rv MIC
(µg/mL)
CC50 (µg/mL)
SI (CC50/MIC)
2.5.
6f
6h
4.0
4.0
1.0
8.0
>100
>100
>100
>100
>25
>25
6k
>100
>12.5
10a
Activity against drug-resistant TB strains
To study the spectrum of activity of most potent derivatives (6f, 6h, 6k, and 10a), these were further
examined against single drug-resistant strains of Mtb which revealed that all the screened molecules

were active on INH resistant Mtb strain. Compound 6f and 6h showed inhibitory activity with MIC
32µg/mL, compound 6k showed MIC of 4 µg/mL, and compound 10a MIC 8 µg/mL. Against
Ethambutol (ETB) and RIF resistant strains, only compound 6h was found active with MIC 64 µg/mL.
The compounds also showed similar activity on streptomycin (STR) resistant strain except for
compound 10a which exhibited potent activity with MIC of 1 µg/mL. Compound 6k also displayed
excellent activity at MIC of 2 µg/mL. These studies put forward that all tested derivatives were active
on at least two resistant strains and compound 6h found to be active on all single resistant strains
which show its ability to act against drug-resistance strains. The screening results on resistant strains
are presented in Table 1.
2.6. Docking study with Mycobacterial membrane protein Large 3 (MmpL3) of Mtb H37Rv.
The designing of molecules was carried out by using structural alignment and superimposition
module of Schrödinger suite on BM212 and Rimonabant i.e. a reported MmpL3 inhibitor. The
visualization of anti-TB activity inspired us to calculate binding energy studies as well as a binding
pattern of designed molecules on the mycobacterial membrane protein Large (MmpL) protein. The
MmpL transporters are vital for the structure as well as the pathogenesis of Mtb by ferrying
trehalose monomycolates to the mycobacterial cell wall.[15] All synthesized 40 molecules were
docked into the active site of protein as shown in Fig. 5. The binding energies predicted by the
software were listed in Table 3. Each one of these compounds possessed distinctive substitutions at
the 2nd, 3rd, and 4th position of phenyl ring attached on isoxazoline to ensure the effectiveness of a
functional group on binding interaction. Docking experiments carried out by using Schrödinger suites
(2019-4) (Maestro 12.2) was carried out using Glide XP (extra precision) docking. MmpL3 protein
with PDB ID: 6ajj was chosen for molecular docking studies; the protein structure was identified at
2.79 Å and has also been reported previously for docking experiments.[15, 18, 24] The docking
results obtained are presented in Table 3. All the molecules exhibit good binding energy as well as XP
glide scores. The isoxazole ring oxygen had hydrogen bond interaction with ALA682 of the binding
site. (Fig. 6) Also, phenyl ring of isoxazole was having π-π stacking interaction with TYR646 except for
compound 10a which was having interaction with PHE649 instead of TYR646, (Fig. 5d) which
suggested binding in an inverted position concerning other derivatives which allowed amidic -NH- of
10a to make hydrogen bonding with ASH645 similar to the co-crystal ligand. The open aliphatic chain
which can rotate freely makes the molecule more flexible compared to other cyclic ring compounds
and makes it possible to bind in an inverted position. This could be the reason behind the anomalous
activity of the 10a derivative compared to the other aliphatic amide derivatives. Compound 6k had
excellent binding interactions with neighboring amino acids, a p-Cl group made halogen bond
interactions with SER643, GLU647, and VAL648 while the chlorophenyl ring forms π-π stacking

interactions with TYR646.(Fig. 6c) The oxygen of the isoxazole ring had hydrogen bonding with
ASH256, along with a cyclohexyl ring oriented towards the hydrophobic pocket.(Fig. 5b) These
prominent interactions with binding pocket explain the higher anti-TB activity of this compound over
others. All other derivatives were found to have hydrophobic interactions with LEU642, ILE253,
TYR646, VAL648, PHE649, ILE679, ALA682, ILE261, PHE260, TYR257, and VAL290, and polar
hydrophilic interactions with THR644, SER293, THR289, and SER286. The above molecular docking
results insinuated that a plausible mechanism of anti-TB activity of the designed molecules is by
inhibition of MmpL3.
Figure 5: Ligand interactions between most active molecules and receptor protein. a) Compound
6f (Purple) in the binding pocket of MmpL3 protein, exhibited π-π stacking interactions with
TYR646. b) Compound 6k (Sky-blue) in the binding pocket of MmpL3 protein. c) Compound 6h
(yellow) in the binding pocket of MmpL3 protein, exhibited π-π stacking interactions with TYR646.
d) Compound 10a (pink) displayed hydrogen bonding with ASH645. e) Compound 6f, 6h, 6k, and
10a, and co-crystal ligand in the binding pocket depicted a similar binding pattern of all active
derivative except 10a which bound inverted relative to others. f) Co-crystal ligand and compound
6k superimposition show a similar binding pattern.

Figure 6: 2D Ligand interactions between most active molecules, co-crystal, and receptor protein
(6AJJ). a) 2D Ligand interaction diagram representing compound 6f in the binding pocket. b)
Interactions diagram for the compound 6h exhibited π-π stacking interactions with TYR646
shown by the green line. c) Representation of compound 6k associated with multiple interactions
as π-π stacking interactions with TYR646, hydrogen bonding with ALA682, and a yellow line
representing the Halogen bond interactions. d) 2D representation of Co-crystal ligand observed
to have hydrogen bond interactions with ASH645 denoted by the pink line.

Table 3: Anti-tubercular and In-silico study of the synthesized compounds on Mycobacterial
membrane protein Large 3 (MmpL3-6AJJ).
H- bond
MIC (μg/
XP G score
(kcal/mol)
ΔG binding
energy
Entry
ml)
Ligand
Amino acid
6b
6c
6d
6e
16
16
16
16
-10.239
-10.050
-10.531
-10.369
-63.101
-59.822
-71.375
-65.854
-C=O-NH-
-C=O-NH-
-C=O-NH-
-C=O-NH-
-C=O-NH-
-O-N=(ring)
-C=O-NH-
-NO2
-O-N=(ring)
-C=O-NH-
-O-N=(ring)
-C=O-NH-
-C=O-NH-
-C=O-NH-
-C=O-NH-
-C=O-NH-
-C=O-NH-
-C=O-NH-
TYR257
TYR257
TYR257
TYR257
TYR257
ASH256
TYR257
VAL548
ALA682
TYR257
ALA682
TYR257
TYR257
TYR257
ASH645
ASH645
ASH645
TYR257
6f
6h
6k
4
4
1
-10.266
-10.173
-10.413
-54.592
-75.823
-68.141
7a
7d
8a
10a
10d
11e
12b
32
16
16
8
-11.420
-10.402
-11.287
-9.761
-74.777
-76.168
-70.871
-59.56
16
16
32
-10.005
-11.129
-10.908
-63.283
-77.065
-82.547
Co-crystal
ligand
-
-13.204
-88.053
-C=O-NH-
ASH645
2.7 Prediction of in silico absorption, distribution, metabolism, and excretion properties
ADME (absorption, distribution, metabolism, and excretion) parameters of molecules serve as
stalwart points to establish their drug likeliness topographies. Thus, the ADME parameters of
designed isoxazoline amides were determined by using the Qikprop module of the Schrödinger suite.
Many approved drugs are found to follow the predefined set of parameters such as partition
coefficient of n-octanol /water, polar surface area (PSA), aqua solubility (QPlogS), central nervous
system activity (CNS), a number of likely metabolic reactions (metab), Predicted skin permeability
(QPlogKp), predicted IC50 for the blockage of HERG K+ channels (QPloghERG), Caco-2ꢀcell
permeability (QPPCaco), human serum albumin binding (QPlogKhsa), blood/brain partition co-
efficient (QPlogBB), human oral absorption, and percent human oral absorption.[25] The projected
drug likeliness properties of all derivatives were found to be within the mentioned standard
parameters. QPPCaco is a descriptor to predict Caco-2 cell permeability of molecule in nm/sec (>500
is great absorption, <25 is poor absorption), it is a human absorption process by the non-active
transport mechanism across the gut-blood barrier.[26] The predicted QPPCaco value for isoxazoline

derivatives illustrate that designed compounds will certainly get absorbed through the gut layer. For
the target molecule to become CNS active, it must have a CNS value more than or equal to 2 if its –2
(inactive) to +2 (active) scale.[26, 27] The predicted value for the CNS was in the range of -1 to -2
(table with observed values provided in supporting information). Thus, designed molecules will
certainly be inactive on CNS. The Log BB values help to determine the distribution of the compound
in the brain. If the compound is having log BBꢀmore thanꢀ0.3, it is predicted to cross the blood-brain
barrier and if it’s less than -1.0 those compounds will be weakly distributed to the brain. All
molecules exhibited LogBB in a negative value, thus CNS and logBB suggest that the designed
molecules will not affect CNS. Furthermore, the QPlog HERG value denotes the predicted IC50 value
for the blockage of HERG K+ channels. Designed compounds demonstrated good predicted
pharmacological properties like HERG K+ channel (HERG K+), QPlogKhsa which predicts binding to
human serum albumin, QPPMDCK predicts MDCK cell permeability, MDCK cells are considered to be
a good mimic for the blood-brain barrier, and QP logKp predicts the skin permeability of the
molecule. Also, the percent absorption values of the majority of the compound were found to be in
the range of 71-100%. None of the designed derivatives violated the rule of three as well as the rule
of five. The values of predicted descriptors are presented in Table 4. The observed values suggest
that all active molecules of all series exhibit a good ADME profile, which suggests that these
molecules can be taken for further biological evaluation in order to develop potential anti-TB agents.

Table 4: In-silico ADME properties of compounds of series 6, 7, 8, 9, 10, 11, and 12.
%
Huma
Huma
n Oral
absorp
tion
Rule
Of
three
QPlog
S
QPlog
HERG
QPP
Caco
QP
logBB
QPP
MDCK
QP
logKp
QP log
Khsa
n Oral
absorp
tion
Rule
Of five
ENTRY
CNS
Metab
6a
6b
-1
0
-3.028
-4.272
-4.105
-4.331
-4.398
-3.913
-3.898
-3.516
-3.556
-3.506
-4.303
-3.391
-5.281
-5.321
-4.903
-4.464
-4.901
-4.293
-4.334
-4.542
-4.933
-4.175
-3.503
-3.765
-4.134
-3.433
-3.346
-3.339
-3.145
-3.831
-3.707
-2.913
-2.913
-3.391
-3.285
-4.907
-4.75
-3.37
-3.787
-3.794
-3.995
-3.823
-3.747
-3.742
-3.806
-3.865
-3.802
-3.8
338.65
1184.9
1180.9
248.1
-0.837
-0.119
-0.3
263.52
2509.1
1015.7
185.25
2701.4
1832.2
1833.5
101.97
1016.5
102.64
2501.0
278.89
1676.3
2013.9
1229.5
759.80
1334.8
1569.6
1585.2
875.3
-3.095
-2.163
-2.194
-3.349
-2.166
-2.134
-2.132
-3.904
-1.997
-3.899
-2.167
-3.059
-1.893
-1.811
-1.859
-1.639
-1.797
-2.245
-2.239
-2.302
-3.455
-2.208
-3.859
-2.155
-2.189
-3.49
3
2
3
2
2
2
2
3
2
3
2
3
2
2
2
2
2
2
2
3
2
3
3
2
2
3
2
2
3
2
2
2
2
2
2
4
3
4
-0.289
-0.063
-0.021
-0.346
-0.039
-0.133
-0.134
-0.226
-0.174
-0.227
-0.062
-0.304
0.184
0.185
0.109
0.046
0.089
-0.01
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
81.62
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6c
0
96.82
78.47
100
6d
-2
0
-1.101
-0.109
-0.172
-0.17
6e
1187.2
1180.7
1177.9
141.23
1189.6
141.55
1190.3
362.33
746.29
874.90
747.72
874.46
875.37
1051.1
1061.8
1060.2
220.48
1058.0
104.48
821.27
819.45
151.53
820.74
819.40
97.54
6f
0
96.40
96.37
74.12
95.09
74.14
100
6g
0
6h
-2
0
-1.339
-0.278
-1.335
-0.122
-0.886
-0.782
-0.701
-0.83
6i
6j
-2
0
6k
6l
-1
-1
-1
-1
-1
-1
0
-3.754
-4.674
-4.585
-4.647
-4.668
-4.552
-3.777
-3.822
-3.876
-4.057
-3.844
-4.111
-4.08
81.713
100
7a
7b
100
7c
100
7d
-0.868
-0.777
-0.238
-0.239
-0.372
-1.175
-0.429
-1.741
-0.566
-0.517
-1.543
-0.477
-0.477
-1.67
100
7e
100
8a
96.97
100
8b
0
-0.01
8c
0
0.104
-0.224
-0.048
-0.339
-0.244
-0.172
-0.341
-0.357
-0.357
-0.447
-0.263
-0.286
-0.471
-0.471
-0.382
-0.401
0.06
100
8d
-2
0
160.2
79.04
96.16
71.79
93.20
94.69
75.44
90.99
90.97
68.95
92.94
92.45
87.083
87.083
89.089
88.583
100
8e
873.1
9a
-2
0
74.08
9b
1220.4
1664.9
110.61
1219.0
1217.8
67.59
9c
0
-4.118
-4.112
-3.878
-3.876
-3.944
-3.95
9d
-2
0
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
12a
12b
-2.253
-2.252
-4.024
-2.289
-2.286
-2.489
-2.489
-2.52
0
-2
0
821.21
819.61
722.64
722.64
731.58
722.88
735.73
1084.3
1081.1
-0.42
1787.6
1662.3
1152.2
1152.1
1700.6
1571.8
644.82
1004.5
1806.4
0
-3.917
-3.774
-3.774
-3.837
-3.822
-5.545
-6.516
-6.287
-0.429
-0.366
-0.366
-0.305
-0.319
-0.79
0
0
0
0
-2.522
-1.528
-1.807
-2.042
-1
0
-0.471
-0.453
-0.02
100
0
-5.319
0.019
100
*Calculations were done by using the Qikprop module of Schrödinger Suite 2019-02

2.7. Lipinski's rule-of-five
Additionally, to evaluate the drug-likeness of these synthesized derivatives, these were checked for
compliance with Lipinski's rule-of-five(RO5)/ Pfizer's rule of five i.e. Molecular weight (MW) ≤ 500D,
partition coefficient (logP) values ≤5, Number of hydrogen bond donors ≤ 5, Number of hydrogen
bond acceptors ≤10, No of rotatable bonds ≤10. [28, 29] (Details of these findings along with
descriptors are depicted in Table 5). The results illustrate that all the derivatives of this series exhibit
excellent pharmacokinetic properties and might be useful for the future development of novel lead
molecules. Descriptor values for the most potent derivatives are described in Table 5

Table 5: Descriptors of Lipinski’s rule-of-five and calculated values.
No. of
Entry
Mol_MW
HB Donor
HB Acceptor
Rotatable
bonds
QPlogPo/w
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
317.344
306.791
286.373
297.356
351.242
290.337
290.337
317.344
272.346
317.344
306.791
288.346
336.861
381.312
320.406
302.416
320.406
304.363
304.363
300.4
311.383
316.399
305.137
278.143
294.113
305.137
264.299
264.299
291.306
325.204
280.753
248.256
248.256
309.162
264.711
336.39
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6.2
5.2
5.2
6.7
5.2
5.2
5.2
6.2
5.2
6.2
5.2
5.95
5.2
5.2
5.2
5.2
5.2
5.2
5.2
5.2
6.7
5.95
6.2
5.2
5.2
6.2
5.2
5.2
6.2
5.2
5.2
5.2
5.2
5.2
5.2
5.95
6.7
7.45
7.45
7.45
3
2
2
2
2
3
2
2
2
2
3
3
8
8
8
8
8
2
2
2
3
3
6
5
5
6
4
4
5
4
4
2
1.605
2.727
2.543
1.48
2.805
2.472
2.471
1.485
2.238
1.485
2.729
1.531
3.817
3.871
3.557
3.302
3.531
2.723
2.726
2.8
1.733
2.576
1.487
2.407
2.664
1.617
2.031
2.029
1.094
2.362
2.284
1.532
1.532
1.858
1.787
3.213
2.786
3.104
4.161
3.104
9d
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
12a
12b
12c
12d
2
2
2
5
2
3
3
3
323.369
371.385
387.0444
371.385
Std. range
<500
0-5
5-10
0-15
-2.5-6.5
*Calculations were done by using the Qikprop module of Schrödinger Suite 2019-02

3. Conclusions
In conclusion, a series of new 3-phenyl-4,5-dihydroisoxazole-5-carboxamide derivatives (6a-6m),
(7a-7e), (8a-8e), (9a-9d), (10a-10e), (11a-11e), (12a-12d) were designed and synthesized in very
good yields and evaluated for anti-TB potential. The synthesized derivatives were characterized
by NMR and HRMS. All derivatives were evaluated for their in vitro anti-TB activity against Mtb
H37Rv. Amongst all, compound 6f (MIC: 4ꢀµg/mL), 6k (MIC: ꢀ1µg/mL), 6h (MIC: 4ꢀµg/mL), and 10a
(MIC: 8ꢀµg/mL) were most active derivatives. Also, these molecules were active against drug-
resistant strains of Mtb. Cytotoxicity assessment on Vero cells (ATCC CCL-81) by using MTT assay
demonstrated that all the derivatives tested were notably non-toxic along with excellent SI (6k
SI-100). To substantiate the design of these molecules, activity studies, and to identify the binding
pattern of these derivatives, molecular modelling studies were carried out on the MmpL3
protein,(PDB id: 6ajj), which illustrated excellent binding energy together with binding
interactions with the receptor-binding pocket. Docking results demonstrated the synergy in the
binding energy with the activity of reported molecules. This insinuates MmpL3 as a plausible
target for these molecules. Further, these molecules were evaluated for in silico ADME
properties. Results stated that all observed descriptor values for designed molecules were in
agreement with standard values. In addition, physicochemical parameters of all derivatives were
following Lipinski’s rule-of-five, thus exhibiting excellent druggability. As there is an urgent unmet
need for newer anti-TB agents active against drug-resistant TB, an attempt has been made to
overcome the shortcomings of Rimonabant by incorporating the isoxazoline carboxamide scaffold
and studying the effect of lipophilicity through ADME studies of the synthesized derivatives.
These results primarily indicate that this scaffold has the potential for further optimization and
improvement. Further efforts in this direction are in progress.
4. Experimental section
4.1. Materials and methods
All the required chemicals, reagents, and starting materials were procured from commercial
providers and were used as such. The monitoring of reactions was performed by TLC-MERCK pre-
1
coated silica gel 60-F254 (0.5 mm) aluminium plates under UV light. H and 13C NMR spectra were
obtained on Bruker Avance 500 MHz spectrometer operating at 500 MHz for proton (1H) and 125
MHz for carbon (13C) spectrometry using tetramethylsilane (TMS) as the internal standard and
1
chemical shifts are reported in ppm. Chemical shifts are in reference to TMS (δ 0.00 for H NMR and
1
1
13C NMR), DMSO-D6 appeared at (δ 2.50 for H NMR and 39.7 for 13C NMR) or CDCl3 (δ 7.26 for H

NMR and 77.00 or 77.16 for 13C NMR) or combination of CDCl3 and DMSO-D6. Spin multiplicities are
reported as s (singlet), brs (broad singlet), d (doublet), dd (double doublet), t (triplet), and m
(multiplet). Coupling constant (J) values are reported in hertz (Hz). HRMS were determined with
Agilent QTOF mass spectrometer 6540 series instrument and were performed in the ESI techniques
at 70 eV. Melting points were taken using Stuart® SMP30 apparatus.
5. Synthesis and procedures
5.1. General procedure for the preparation of compounds 2
Compound 2a-l were synthesized according to the procedures described in the literature [19, 20]To
a solution of substituted aldehyde (1 mmol) in ethanol/ methanol (5 mL) the solution of
(NH2OH)2.SO4 (0.6 mmol, 0.6 eq.) and NaOH (1.2 mmol, 1.2 eq.) in water (25ml) at 0oC were added
dropwise. After addition cooling was removed and the reaction mixture was continued to stir at
room temperature for 1-2 h (depending on aldehyde). After the completion of reaction indicated by
the TLC, the reaction mixture was evaporated under reduced pressure to remove organic solvent
which leaves oily liquid at the base of aqueous layer. To which crushed ice was added and resulting
solid was filtered. Crude product was crystallised of from EtOH gave the compound 2
5.2. General procedure for the preparation of compounds 3a-3l
Compound 3a-l were synthesized based on previous synthetic procedures.[21] To a solution of
substituted aldoxime (0.5 mmol, 1eq.) in acetonitrile (10 mL) the n-chlorosuccinimide (NCS)(0.55
mmol, 1.1 eq.) was added slowly at 0oC. The reaction mixture stirred for 15min at 0oC then cooling
was removed and stir at room temperature for 1-2h. Completion of reaction indicated by the TLC;
the reaction mixture was evaporated under reduced pressure. To the liquid residue obtained 20 mL
water was added and the resulting aqueous solution was extracted with Ethyl acetate (20 mL x 3).
Combined organic layer was washed with dil. Hydrochloric acid (0.1N) to remove residual
succinimide, followed by brine wash. The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo to obtain a clear crystalline compound 3a, which was directly used in the next
step without further purification. Substituted chloro-aldoxime 3b-3l were synthesized using same
method.
5.3. General procedure for the preparation of compounds 6, 7, 8, 9, 10, 11, and 12 series
To a cooled (0oC) solution of amine 4 (0.2mmol, 1 eq.), triethylamine (0.21mmol, 1.05eq.) in
dichloromethane (DCM) (10 mL) acrolyl chloride (0.21mmol, 1.05 eq.) was added slowly over 4-5 min
and stirred for 45 min at 0oC. Reaction monitored by TLC, after completion of amine another portion

of triethylamine (0.21mmol, 1.05eq.) was added to the reaction mixture and stirred for another
10min and substituted chloroaldoximes 3a-3l were added slowly in cooling condition. After 15 min
cooling bath was removed and continued to stir at room temperature for overnight. Upon
completion of the reaction DCM was evaporated in vacuo and 25ml water was added. The aqueous
layer was extracted with ethyl acetate (20 mL x 3). The combined organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure to get off white coloured crude solid
product. Crude product obtained was crystallised from ethanol : ACN mixture.
5.3.1. N-cyclohexyl-3-(2-nitrophenyl)-4,5-dihydroisoxazole-5-carboxamide (6a).
1
White solid, Yield: 68%, m.p. 101oC, H NMR (500 MHz, CDCl3) δ 8.10 (d, J = 8.1 Hz, 1H), 7.71 (t, J =
7.5 Hz, 1H), 7.67 – 7.61 (m, 1H), 7.51 (d, J = 7.6 Hz, 1H), 6.67 (s, 1H), 5.18 (dd, J = 11.2, 5.5 Hz, 1H),
3.86 – 3.79 (m, 1H), 3.63 (td, J = 17.3, 11.5 Hz, 2H), 1.96 (t, J = 12.3 Hz, 2H), 1.78 – 1.71 (m, 3H), 1.66
– 1.60 (m, 1H), 1.43 – 1.34 (m, 2H), 1.24 – 1.18 (m, 2H).; 13C NMR (126 MHz, CDCl3) δ 169.29, 156.37,
147.92, 133.59, 131.08, 130.72, 125.03, 124.53, 79.42, 48.28, 42.50, 32.93, 32.88, 25.43, 24.86,
24.82.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C16H19N3O4 318.1454; found,318.1454.
5.3.2. 3-(3-chlorophenyl)-N-cyclohexyl-4,5-dihydroisoxazole-5-carboxamide (6b).
Off White solid; Yield: 86%; m.p. 95 oC,; 1H NMR (500 MHz, CDCl3) δ 7.60 (d, J = 8.7 Hz, 2H), 7.40 (d, J
= 8.7 Hz, 2H), 6.65 (s, 1H), 5.11 (dd, J = 10.7, 6.8 Hz, 1H), 3.79 – 3.70 (m, 1H), 3.65 (dd, J = 8.7, 4.4 Hz,
2H), 1.94 (d, J = 16.1 Hz, 1H), 1.83 (d, J = 16.3 Hz, 1H), 1.77 – 1.66 (m, 2H), 1.66 – 1.58 (m, 2H), 1.35
(tt, J = 16.3, 10.3 Hz, 2H), 1.21 – 1.14 (m, 2H).; 13C NMR (126 MHz, CDCl3) δ 169.55, 156.37, 136.83,
129.18, 128.25, 126.91, 79.16, 48.25, 39.47, 32.93, 32.88, 25.39, 24.78.; HRMS –QTOF MS/MS: m/z
[M+H]+ calcd for C16H20N2O2Cl: 307.1213; found, 307.1211.
5.3.3. N-cyclohexyl-3-(p-tolyl)-4,5-dihydroisoxazole-5-carboxamide (6c).
Off White solid; Yield: 89%; m.p. 130oC; 1H NMR (500 MHz, CDCl3) δ 7.56 (d, J = 8.1 Hz, 2H), 7.22 (d, J
= 8.0 Hz, 2H), 6.69 (s, 1H), 5.09 (dd, J = 9.6, 7.9 Hz, 1H), 3.78 – 3.71 (m, 1H), 3.69 – 3.63 (m, 2H), 2.39
(s, 3H), 1.94 (d, J = 15.1 Hz, 1H), 1.82 (d, J = 15.5 Hz, 1H), 1.75 – 1.66 (m, 2H), 1.39 – 1.30 (m, 2H),
1.25 (s, 1H), 1.22 – 1.12 (m, 3H).; 13C NMR (126 MHz, CDCl3) δ 169.90, 157.18, 141.13, 129.56,
126.98, 125.60, 78.86, 48.17, 39.87, 32.94, 32.88, 25.41, 24.75, 21.46.; HRMS –QTOF MS/MS: m/z
[M+H]+ calcd for C17H23N2O2: 287.1760; found, 287.1760.
5.3.4. 3-(4-cyanophenyl)-N-cyclohexyl-4,5-dihydroisoxazole-5-carboxamide (6d).
White solid; Yield: 92%; m.p. 96oC; 1H NMR (500 MHz, CDCl3) δ 7.75 (dd, J = 31.1, 7.5 Hz, 4H), 6.62 (s,
1H), 5.17 (dd, J = 11.3, 6.1 Hz, 1H), 3.74 (dd, J = 13.1, 7.3 Hz, 1H), 3.67 (dd, J = 19.4, 6.1 Hz, 2H), 1.95

(d, J = 11.7 Hz, 1H), 1.83 (d, J = 12.0 Hz, 1H), 1.76 – 1.67 (m, 2H), 1.62 (d, J = 12.5 Hz, 1H), 1.35 (dd, J =
15.5, 13.0 Hz, 2H), 1.23 – 1.13 (m, 3H).; 13C NMR (126 MHz, CDCl3) δ 169.12, 156.04, 132.69, 132.61,
127.49, 118.10, 114.13, 79.62, 48.34, 38.89, 32.90, 32.85, 25.37, 24.75.; HRMS –QTOF MS/MS: m/z
[M+H]+ calcd for C17H20N3O2: 298.1556; found, 298.1554.
5.3.5. 3-(4-bromophenyl)-N-cyclohexyl-4,5-dihydroisoxazole-5-carboxamide (6e).
White solid; Yield: 85%; m.p. 103oC; 1H NMR (500 MHz, CDCl3) δ 7.55 (q, J = 8.8 Hz, 4H), 6.64 (s, 1H),
5.12 (dd, J = 10.6, 6.9 Hz, 1H), 3.79 – 3.70 (m, 1H), 3.65 (dd, J = 8.7, 3.8 Hz, 2H), 1.94 (d, J = 14.4 Hz,
1H), 1.83 (d, J = 16.0 Hz, 1H), 1.75 – 1.66 (m, 2H), 1.40 – 1.30 (m, 2H), 1.24 (d, J = 8.8 Hz, 1H), 1.22 –
1.12 (m, 3H).; 13C NMR (126 MHz, CDCl3) δ 169.51, 156.47, 150.54, 132.14, 128.44, 127.36, 125.17,
79.18, 76.77, 48.25, 39.39, 32.93, 32.88, 25.39, 24.78.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for
C16H19N2O2Br: 353.0692; found, 353.0692.
5.3.6. N-cyclohexyl-3-(4-fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide (6f).
White solid; Yield: 92%; m.p. 98oC; 1H NMR (500 MHz, CDCl3) δ 7.73 – 7.62 (m, 2H), 7.12 (t, J = 8.7 Hz,
2H), 6.67 (s, 1H), 5.11 (dd, J = 10.6, 6.9 Hz, 1H), 3.78 – 3.71 (m, 1H), 3.69 – 3.60 (m, 2H), 1.94 (d, J =
14.2 Hz, 1H), 1.83 (d, J = 12.6 Hz, 1H), 1.74 – 1.60 (m, 4H), 1.39 – 1.29 (m, 2H), 1.19 (dd, J = 20.7, 8.1
Hz, 2H).; 13C NMR (126 MHz, CDCl3) δ 169.64, 165.14, 163.14, 156.29, 129.10, 129.03, 124.71,
116.17, 116.00, 79.06, 48.23, 39.70, 32.94, 32.89, 25.40, 24.78.; HRMS –QTOF MS/MS: m/z [M+H]+
calcd for C16H19N2O2F: 291.1509; found, 291.1509.
5.3.7. N-cyclohexyl-3-(3-fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide (6g).
White solid; Yield: 78%; m.p. 80oC; 1H NMR (500 MHz, CDCl3) δ 7.41 (dd, J = 7.5, 3.8 Hz, 3H), 7.15 (dd,
J = 9.7, 8.0 Hz, 1H), 6.65 (s, 1H), 5.13 (dd, J = 10.3, 7.2 Hz, 1H), 3.79 – 3.71 (m, 1H), 3.66 (dd, J = 8.8,
2.5 Hz, 2H), 1.94 (d, J = 16.2 Hz, 1H), 1.83 (d, J = 16.1 Hz, 1H), 1.72 (ddd, J = 17.9, 12.3, 6.7 Hz, 3H),
1.64 – 1.58 (m, 1H), 1.39 – 1.31 (m, 2H), 1.18 (ddd, J = 12.7, 10.4, 3.6 Hz, 2H).; 13C NMR (126 MHz,
CDCl3) δ 169.43, 163.72, 161.75, 156.41, 130.53, 122.84, 117.81, 113.87, 79.18, 48.21, 39.41, 32.89,
32.84, 25.36, 24.75.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C16H19N2O2F : 291.1509; found,
291.1507.
5.3.8. N-cyclohexyl-3-(4-nitrophenyl)-4,5-dihydroisoxazole-5-carboxamide (6h).
1
White solid; Yield: 84%; m.p. 110oC; H NMR (500 MHz, CDCl3) δ 7.41 (dd, J = 7.5, 3.8 Hz, 3H), 7.15
(dd, J = 9.7, 8.0 Hz, 1H), 6.65 (s, 1H), 5.13 (dd, J = 10.3, 7.2 Hz, 1H), 3.79 – 3.71 (m, 1H), 3.66 (dd, J =
8.8, 2.5 Hz, 2H), 1.94 (d, J = 16.2 Hz, 1H), 1.83 (d, J = 16.1 Hz, 1H), 1.72 (ddd, J = 17.9, 12.3, 6.7 Hz,
3H), 1.64 – 1.58 (m, 1H), 1.39 – 1.31 (m, 2H), 1.18 (ddd, J = 12.7, 10.4, 3.6 Hz, 2H).; 13C NMR (126

MHz, CDCl3) δ 169.43, 163.72, 161.75, 156.41, 130.53, 122.84, 117.81, 113.87, 79.18, 48.21, 39.41,
32.89, 32.84, 25.36, 24.75.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C16H19N3O4: 318.1454; found,
318.1454.
5.3.9. N-cyclohexyl-3-phenyl-4,5-dihydroisoxazole-5-carboxamide (6i).
1
White solid; Yield: 76%; m.p. 102oC; H NMR (500 MHz, CDCl3) δ 7.67 (d, J = 6.3 Hz, 2H), 7.47 – 7.40
(m, 3H), 6.68 (s, 1H), 5.11 (dd, J = 10.1, 7.3 Hz, 1H), 3.79 – 3.71 (m, 1H), 3.70 – 3.65 (m, 2H), 1.94 (d, J
= 16.1 Hz, 1H), 1.83 (d, J = 16.3 Hz, 1H), 1.72 (ddd, J = 15.2, 8.8, 2.7 Hz, 2H), 1.63 – 1.58 (m, 1H), 1.41
– 1.29 (m, 2H), 1.17 (ddd, J = 15.9, 13.5, 3.6 Hz, 3H).; 13C NMR (126 MHz, CDCl3) δ 157.25, 130.78,
128.90, 128.41, 127.07, 79.07, 77.30, 77.04, 76.79, 48.24, 39.90, 32.99, 32.94, 25.41, 24.81.; HRMS –
QTOF MS/MS: m/z [M+H]+ calcd for C16H19N2O2 :273.1603; found, 273.1605.
5.3.10. N-cyclohexyl-3-(3-nitrophenyl)-4,5-dihydroisoxazole-5-carboxamide (6j).
1
White solid; Yield: 73%; m.p. 83oC; H NMR (500 MHz, CDCl3) δ 8.52 (t, J = 1.9 Hz, 1H), 8.30 (d, J =
10.3 Hz, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 6.63 (s, 1H), 5.19 (dd, J = 11.6, 5.9 Hz,
1H), 3.77 (t, J = 6.5 Hz, 1H), 3.75 – 3.64 (m, 2H), 1.96 (d, J = 10.7 Hz, 1H), 1.84 (d, J = 14.0 Hz, 1H),
1.74 – 1.62 (m, 4H), 1.40 – 1.31 (m, 2H), 1.23 – 1.16 (m, 2H).; 13C NMR (126 MHz, CDCl3) δ 169.11,
155.71, 148.54, 132.47, 130.29, 130.01, 125.12, 121.89, 79.59, 48.34, 39.09, 32.91, 32.87, 25.38,
24.76.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C16H19N3O4: 318.1454; found, 318.1454.
5.3.11. 3-(4-chlorophenyl)-N-cyclohexyl-4,5-dihydroisoxazole-5-carboxamide (6k).
White solid; Yield: 90%; m.p. 83oC; 1H NMR (500 MHz, CDCl3) δ 7.60 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.7
Hz, 2H), 6.65 (s, 1H), 5.12 (dd, J = 10.6, 6.8 Hz, 1H), 3.78 – 3.71 (m, 1H), 3.65 (dd, J = 8.7, 4.1 Hz, 2H),
1.97 – 1.91 (m, 1H), 1.86 – 1.80 (m, 1H), 1.76 – 1.67 (m, 2H), 1.59 (d, J = 3.8 Hz, 2H), 1.40 – 1.30 (m,
2H), 1.21 – 1.14 (m, 2H).; 13C NMR (126 MHz, CDCl3) δ 169.12, 156.04, 132.61, 127.49, 118.10,
114.13, 79.62, 48.34, 38.89, 32.90, 32.85, 25.37, 24.75.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for
C16H20N2O2Cl: 307.1213; found, 307.1213.
5.3.12. N-cyclohexyl-3-(4-hydroxyphenyl)-4,5-dihydroisoxazole-5-carboxamide (6l).
1
White solid; Yield: 70%; m.p. 123oC; H NMR (500 MHz, DMSO-D6) δ 9.96 (s, 1H), 7.99 (d, J = 8.1 Hz,
1H), 7.52 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 4.97 (dd, J = 11.3, 7.2 Hz, 1H), 3.61 – 3.55 (m,
2H), 3.50 – 3.44 (m, 1H), 1.70 (d, J = 16.5 Hz, 5H), 1.25 (dd, J = 16.2, 9.9 Hz, 5H).; 13C NMR (126 MHz,
CDCl3) δ 170.00, 159.79, 156.89, 128.60, 126.71, 115.93, 78.52, 48.08, 39.94, 32.82, 32.77, 25.32,
24.73, 24.71.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C16H19N2O3: 289.1552; found, 289.1552.

5.3.13. 3-(3-chlorophenyl)-N-octyl-4,5-dihydroisoxazole-5-carboxamide (7a).
White solid; Yield: 78%; m.p. 56oC; 1H NMR (500 MHz, CDCl3) δ 7.68 (t, J = 1.8 Hz, 1H), 7.53 (d, J = 9.0
Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 7.36 (t, J = 7.9 Hz, 1H), 6.78 (s, 1H), 5.15 (dd, J = 10.9, 6.3 Hz, 1H),
3.66 (dd, J = 8.6, 6.0 Hz, 2H), 3.31 (dt, J = 13.5, 6.7 Hz, 1H), 3.22 (dt, J = 13.3, 6.5 Hz, 1H), 1.54 – 1.48
(m, 2H), 1.29 – 1.22 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H).; 13C NMR (126 MHz, CDCl3) δ 170.40, 156.37,
134.96, 130.75, 130.14, 130.11, 127.00, 125.12, 79.19, 39.40, 39.31, 31.74, 29.35, 29.17, 29.15,
26.81, 22.62, 14.07.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C18H26N2O2Cl: 337.1683; found,
337.1683. m/z [M+Na]+ calcd for C18H26N2O2ClNa: 359.1502; found, 359.1502.
5.3.14. 3-(4-bromophenyl)-N-octyl-4,5-dihydroisoxazole-5-carboxamide (7b).
1
White solid; Yield: 72%; m.p. 106oC; H NMR (500 MHz, CDCl3) δ 7.60 – 7.49 (m, 4H), 6.78 (s, 1H),
5.14 (dd, J = 10.7, 6.4 Hz, 1H), 3.66 (dd, J = 8.6, 5.2 Hz, 2H), 3.32 (td, J = 13.5, 7.0 Hz, 1H), 3.20 (td, J =
13.1, 7.2 Hz, 1H), 1.54 – 1.48 (m, 2H), 1.29 – 1.22 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H).; 13C NMR (126
MHz, CDCl3) δ 170.48, 156.56, 132.15, 128.44, 127.30, 125.21, 79.16, 39.44, 39.34, 31.74, 29.35,
29.17, 29.15, 26.81, 22.62, 14.07.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C18H26N2O2Br:
381.1178; found, 381.1178.
5.3.15. 3-(3-fluorophenyl)-N-octyl-4,5-dihydroisoxazole-5-carboxamide (7c).
1
White solid; Yield: 84%; m.p. 74oC; H NMR (500 MHz, CDCl3) δ 7.41 (ddd, J = 9.2, 4.7, 3.2 Hz, 3H),
7.19 – 7.11 (m, 1H), 6.78 (s, 1H), 5.15 (dd, J = 10.5, 6.7 Hz, 1H), 3.71 – 3.62 (m, 2H), 3.32 (td, J = 13.5,
7.0 Hz, 1H), 3.24 – 3.17 (m, 1H), 1.54 – 1.48 (m, 2H), 1.29 – 1.23 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H).; 13
C
NMR (126 MHz, CDCl3) δ 170.42, 163.75, 161.78, 156.53, 130.57, 122.88, 117.87, 113.90, 79.20,
39.49, 39.34, 31.74, 29.35, 29.17, 29.15, 26.81, 22.61, 14.06.; HRMS –QTOF MS/MS: m/z [M+H]+
calcd for C18H26N2O2F : 321.1978; found, 321.1978. m/z [M+Na]+ calcd for C18H26N2O2F.Na : 343.1798;
found, 343.1798.
5.3.16. N-octyl-3-phenyl-4,5-dihydroisoxazole-5-carboxamide (7d).
1
White solid; Yield: 88%; m.p. 103oC; H NMR (500 MHz, CDCl3) δ 7.60 (d, J = 8.5 Hz, 2H), 7.40 (d, J =
8.4 Hz, 2H), 6.78 (s, 1H), 5.14 (dd, J = 10.8, 6.4 Hz, 1H), 3.70 – 3.61 (m, 2H), 3.32 (dt, J = 13.6, 6.8 Hz,
1H), 3.20 (td, J = 13.1, 7.2 Hz, 1H), 1.55 – 1.49 (m, 2H), 1.34 – 1.25 (m, 5H), 0.87 (t, J = 6.9 Hz, 3H).;
13C NMR (126 MHz, CDCl3) δ 170.72, 157.34, 130.80, 128.88, 128.35, 127.03, 78.95, 39.74, 39.31,
31.75, 29.37, 29.18, 29.15, 26.82, 22.62, 14.08.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for
C18H27N2O2: 303.2073; found, 303.2070
5.3.17. 3-(4-fluorophenyl)-N-octyl-4,5-dihydroisoxazole-5-carboxamide (7e).

1
White solid; Yield: 85%; m.p. 92oC; H NMR (500 MHz, CDCl3) δ 7.46 – 7.35 (m, 3H), 7.19 – 7.11 (m,
1H), 6.79 (s, 1H), 5.15 (dd, J = 10.6, 6.6 Hz, 1H), 3.71 – 3.62 (m, 2H), 3.32 (td, J = 13.4, 7.1 Hz, 1H),
3.21 (td, J = 13.1, 7.2 Hz, 1H), 1.54 – 1.48 (m, 2H), 1.26 (d, J = 9.5 Hz, 10H), 0.86 (t, J = 7.0 Hz, 3H).; 13
C
NMR (126 MHz, CDCl3) δ 170.45, 163.74, 161.78, 156.52, 130.57, 122.88, 117.86, 113.89, 79.21,
39.51, 39.35, 31.73, 29.35, 29.17, 29.14, 26.81, 22.61, 14.06.; HRMS –QTOF MS/MS: m/z [M+H]+
calcd for C18H26N2O2F: 321.1978; found, 321.1978.
5.3.18. N-cycloheptyl-3-(3-fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide (8a).
White solid; Yield: 78%; m.p. 105oC; 1H NMR (500 MHz, CDCl3) δ 8.09 (dd, J = 8.1, 0.9 Hz, 1H), 7.71
(td, J = 7.5, 1.3 Hz, 1H), 7.64 (td, J = 8.0, 1.5 Hz, 1H), 7.51 (dd, J = 7.6, 1.5 Hz, 1H), 6.79 (s, 1H), 5.20
(dd, J = 11.4, 5.4 Hz, 1H), 3.69 – 3.57 (m, 2H), 3.33 (dd, J = 13.4, 7.0 Hz, 2H), 1.58 (dd, J = 14.4, 7.4 Hz,
2H), 1.39 – 1.28 (m, 5H), 0.90 (t, J = 7.0 Hz, 3H).; 13C NMR (126 MHz, CDCl3) δ 172.92., 168.35,
166.41, 160.98, 136.23, 128.13, 122.24, 118.61, 84.70, 55.14, 43.36, 39.34, 39.28, 32.90, 32.89,
29.01, 28.98; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C17H22N2O2F: 305.1667; found, 305.1665.
5.3.19. N-cycloheptyl-3-(4-fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide (8b).
1
White solid; Yield: 89%; m.p. 125oC; H NMR (500 MHz, CDCl3) δ 7.43 – 7.37 (m, 3H), 7.15 (ddd, J =
9.6, 4.2, 2.5 Hz, 1H), 6.73 (s, 1H), 5.13 (dd, J = 10.4, 7.1 Hz, 1H), 3.94 (dt, J = 12.9, 4.4 Hz, 1H), 3.69 –
3.63 (m, 2H), 1.97 – 1.92 (m, 1H), 1.87 – 1.82 (m, 1H), 1.61 (dd, J = 16.9, 10.0 Hz, 4H), 1.54 – 1.47 (m,
4H), 1.46 – 1.38 (m, 2H).; 13C NMR (126 MHz, CDCl3) δ 169.21, 163.76, 156.46, 130.56, 130.53,
122.88, 117.84, 113.90, 79.22, 50.44, 39.44, 34.97, 34.89, 27.92, 27.89, 24.06, 24.00.; HRMS –QTOF
MS/MS: m/z [M+H]+ calcd for C17H22N2O2F: 305.1667; found, 305.1664.
5.3.20. N-cycloheptyl-3-(p-tolyl)-4,5-dihydroisoxazole-5-carboxamide(8c).
1
White solid; Yield: 82%; m.p. 112oC; H NMR (500 MHz, CDCl3) δ 7.55 (d, J = 8.2 Hz, 2H), 7.22 (d, J =
8.0 Hz, 2H), 6.78 (s, 1H), 5.09 (dd, J = 9.6, 7.7 Hz, 1H), 3.93 (ddt, J = 13.0, 8.7, 4.4 Hz, 1H), 3.68 – 3.63
(m, 2H), 2.38 (s, 3H), 1.97 – 1.91 (m, 1H), 1.86 – 1.81 (m, 1H), 1.61 (ddd, J = 10.4, 6.7, 1.8 Hz, 4H),
1.50 (dd, J = 14.6, 6.9 Hz, 4H), 1.45 – 1.37 (m, 2H).; 13C NMR (126 MHz, CDCl3) δ 169.61, 157.19,
141.13, 129.56, 126.97, 125.57, 78.83, 50.36, 39.81, 34.96, 34.89, 27.93, 27.90, 24.07, 24.01, 21.48.;
HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C18H25N2O2: 301.1916; found, 301.1915.
5.3.21. 3-(4-cyanophenyl)-N-cycloheptyl-4,5-dihydroisoxazole-5-carboxamide (8d).
White solid; Yield: 82%; m.p. 130oC; 1H NMR (500 MHz, CDCl3) δ 7.80 – 7.70 (m, 4H), 6.68 (d, J = 7.9
Hz, 1H), 5.17 (dd, J = 11.5, 6.1 Hz, 1H), 3.97 – 3.90 (m, 1H), 3.74 – 3.62 (m, 2H), 1.98 – 1.93 (m, 1H),
1.87 – 1.82 (m, 1H), 1.66 – 1.58 (m, 4H), 1.49 (dd, J = 14.0, 5.2 Hz, 4H), 1.42 (dd, J = 20.2, 9.2 Hz, 2H).;

13C NMR (126 MHz, CDCl3) δ 168.83, 156.06, 132.68, 132.62, 127.50, 118.12, 114.14, 79.63, 50.52,
38.91, 34.97, 34.89, 27.91, 27.87, 24.04, 23.99.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for
C18H22N3O2: 312.1712; found, 312.1710, m/z [M+H]+ calcd for C18H22N3O2: 334.1531; found,
334.1532.
5.3.22. N-cycloheptyl-3-(4-methoxyphenyl)-4,5-dihydroisoxazole-5-carboxamide (8e).
White solid; Yield: 71%; m.p. 82oC; 1H NMR (500 MHz, CDCl3) δ 7.61 (d, J = 8.9 Hz, 2H), 6.93 (d, J =
8.9 Hz, 2H), 6.78 (d, J = 8.1 Hz, 1H), 5.08 (dd, J = 9.7, 7.6 Hz, 1H), 3.92 (dd, J = 8.6, 4.3 Hz, 1H), 3.67 –
3.63 (m, 2H), 1.93 (dd, J = 11.1, 3.9 Hz, 1H), 1.86 – 1.81 (m, 1H), 1.60 (dd, J = 17.2, 10.6 Hz, 4H), 1.50
(dd, J = 16.6, 8.8 Hz, 4H), 1.42 (dd, J = 11.7, 8.5 Hz, 2H).; 13C NMR (126 MHz, CDCl3) δ 169.68, 161.56,
156.78, 128.63, 120.89, 114.28, 78.75, 55.40, 50.36, 39.91, 34.97, 34.90, 27.94, 27.90, 24.07, 24.01.;
HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C18H25N2O3: 317.1865; found, 317.1862.
5.3.23. 3-(3-nitrophenyl)-N-pentyl-4,5-dihydroisoxazole-5-carboxamide (9a).
White solid; Yield: 78%; m.p. 99oC; 1H NMR (500 MHz, CDCl3) δ 8.52 (t, J = 1.9 Hz, 1H), 8.30 (ddd, J =
8.2, 2.2, 1.0 Hz, 1H), 8.02 – 7.99 (m, 1H), 7.64 (t, J = 8.0 Hz, 1H), 6.78 (s, 1H), 5.22 (dd, J = 11.5, 5.7
Hz, 1H), 3.80 – 3.68 (m, 2H), 3.34 (dd, J = 13.5, 6.4 Hz, 1H), 3.22 (dt, J = 13.2, 6.5 Hz, 1H), 1.56 – 1.50
(m, 2H), 1.34 – 1.28 (m, 5H), 0.88 (t, J = 7.0 Hz, 3H).; 13C NMR (126 MHz, CDCl3) δ 170.08, 155.80,
148.51, 132.49, 130.23, 130.03, 125.16, 121.89, 79.58, 39.40, 39.14, 29.04, 28.96, 22.28, 13.94.;
HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C15H19N3O4: 306.1454; found, 306.1450, m/z [M+Na]+
calcd for C15H19N3O4: 328.1273; found, 328.1270.
5.3.24. 3-(4-fluorophenyl)-N-pentyl-4,5-dihydroisoxazole-5-carboxamide (9b).
White solid; Yield: 89%; m.p. 110oC; 1H NMR (500 MHz, CDCl3) δ 7.45 – 7.37 (m, 3H), 7.14 (dt, J = 4.6,
4.0 Hz, 1H), 6.81 (s, 1H), 5.17 (dd, J = 10.5, 6.8 Hz, 1H), 3.71 – 3.63 (m, 2H), 3.37 – 3.30 (m, 1H), 3.21
(td, J = 13.1, 7.2 Hz, 1H), 1.55 – 1.49 (m, 2H), 1.34 – 1.25 (m, 5H), 0.87 (t, J = 7.0 Hz, 3H).; 13C NMR
(126 MHz, CDCl3) δ 170.49, 163.74, 156.51, 130.51, 130.40, 122.86, 117.70, 113.71, 79.20, 39.50,
39.34, 29.04, 28.95, 22.28, 13.93.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C16H19N3O4F:
279.1509; found, 279.1507.
5.3.25. 3-(4-chlorophenyl)-N-pentyl-4,5-dihydroisoxazole-5-carboxamide (9c).
1
White solid; Yield: 93%; m.p. 122oC; H NMR (500 MHz, CDCl3) δ 7.60 (d, J = 8.5 Hz, 2H), 7.40 (d, J =
8.4 Hz, 2H), 6.78 (s, 1H), 5.14 (dd, J = 10.8, 6.4 Hz, 1H), 3.70 – 3.61 (m, 2H), 3.32 (dt, J = 13.6, 6.8 Hz,
1H), 3.20 (td, J = 13.1, 7.2 Hz, 1H), 1.55 – 1.49 (m, 2H), 1.34 – 1.25 (m, 5H), 0.87 (t, J = 6.9 Hz, 3H).;
13C NMR (126 MHz, CDCl3) δ 170.48, 156.46, 136.85, 129.18, 128.25, 126.86, 79.14, 39.49, 39.31,

29.04, 28.95, 22.28, 13.93.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C16H19N3O4Cl: 295.1213;
found, 295.1210.
5.3.26. 3-(2-nitrophenyl)-N-pentyl-4,5-dihydroisoxazole-5-carboxamide (9d).
1
White solid; Yield: 72%; m.p. 115oC; H NMR (500 MHz, CDCl3) δ 8.09 (dd, J = 8.1, 0.9 Hz, 1H), 7.71
(td, J = 7.5, 1.3 Hz, 1H), 7.64 (td, J = 8.0, 1.5 Hz, 1H), 7.51 (dd, J = 7.6, 1.5 Hz, 1H), 6.79 (s, 1H), 5.20
(dd, J = 11.4, 5.4 Hz, 1H), 3.69 – 3.57 (m, 2H), 3.33 (dd, J = 13.4, 7.0 Hz, 2H), 1.58 (dd, J = 14.4, 7.4 Hz,
2H), 1.39 – 1.28 (m, 5H), 0.90 (t, J = 7.0 Hz, 3H).; 13C NMR (126 MHz, CDCl3) δ 170.20, 156.36, 133.53,
131.08, 130.72, 125.00, 124.45, 79.46, 42.37, 39.38, 29.10, 28.98, 22.33, 13.97.; HRMS –QTOF
MS/MS: m/z [M+H]+ calcd for C15H19N3O4: 306.1454; found, 306.1452.
5.3.27. N-butyl-3-(4-fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide (10a).
1
White solid; Yield: 87%; m.p. 99oC; H NMR (500 MHz, CDCl3) δ 7.44 – 7.38 (m, 3H), 7.17 – 7.12 (m,
1H), 6.76 (s, 1H), 5.15 (dd, J = 10.6, 6.7 Hz, 1H), 3.70 – 3.63 (m, 2H), 3.34 (td, J = 13.5, 7.0 Hz, 1H),
3.22 (dt, J = 13.2, 6.5 Hz, 1H), 1.53 – 1.47 (m, 2H), 1.36 – 1.32 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H).; 13C
NMR (126 MHz, CDCl3) δ 170.45, 163.74, 156.51, 130.53, 130.38, 122.87, 117.90, 113.90, 79.19,
39.49, 39.06, 31.41, 20.00, 14.15, 13.70.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C14H18N2O2F:
265.1352; found, 265.1347.
5.3.28. N-butyl-3-(3-fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide (10b).
1
White solid; Yield: 83%; m.p. 109oC; H NMR (500 MHz, CDCl3) δ 7.44 – 7.37 (m, 3H), 7.15 (ddd, J =
9.4, 6.5, 2.5 Hz, 1H), 6.77 (brs, 1H), 5.15 (dd, J = 10.6, 6.7 Hz, 1H), 3.70 – 3.62 (m, 2H), 3.33 (dt, J =
13.5, 6.7 Hz, 1H), 3.25 – 3.18 (m, 1H), 1.50 (ddd, J = 14.6, 10.0, 4.9 Hz, 2H), 1.34 (dd, J = 15.1, 7.4 Hz,
2H), 0.91 (t, J = 7.3 Hz, 3H).; 13C NMR (126 MHz, CDCl3) δ 170.45, 163.74, 156.53, 130.52, 130.39,
122.87, 117.72, 113.72, 79.19, 39.48, 39.05, 31.41, 20.00, 13.70.; HRMS –QTOF MS/MS: m/z [M+H]+
calcd for C14H18N2O2F : 265.1352; found, 265.1347.
5.3.29. N-butyl-3-(3-nitrophenyl)-4,5-dihydroisoxazole-5-carboxamide (10c).
1
White solid; Yield: 80%; m.p. 63oC; H NMR (500 MHz, CDCl3) δ 8.51 (s, 1H), 8.3 – 8.27 (m, 1H), 8.01
(d, J = 0.5 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 6.75 (s, 1H), 5.22 (dd, J = 11.6, 5.7 Hz, 1H), 3.75 (dq, J =
17.2, 5.7 Hz, 2H), 3.34 (dt, J = 13.6, 6.7 Hz, 1H), 3.23 (dt, J = 13.6, 6.7 Hz, 1H), 1.51 (dd, J = 15.0, 7.6
Hz, 2H), 1.35 (dd, J = 15.0, 7.6 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H).; 13C NMR (126 MHz, CDCl3) δ 170.08,
155.80, 148.51, 132.50, 130.22, 130.04, 125.18, 121.91, 79.57, 39.13, 31.40, 20.01, 13.70.; HRMS –
QTOF MS/MS: m/z [M+H]+ calcd for C14H18N3O4 : 292.1297; found, 292.1295.

5.3.30. 3-(4-bromophenyl)-N-butyl-4,5-dihydroisoxazole-5-carboxamide (10d).
1
White solid; Yield: 86%; m.p. 65oC; H NMR (500 MHz, CDCl3) δ 7.54 (q, J = 8.8 Hz, 4H), 6.77 (s, 1H),
5.14 (dd, J = 10.8, 6.4 Hz, 1H), 3.66 (dd, J = 8.6, 5.7 Hz, 2H), 3.33 (dt, J = 13.5, 6.7 Hz, 1H), 3.25 – 3.18
(m, 1H), 1.53 – 1.47 (m, 2H), 1.36 – 1.32 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H).; 13C NMR (126 MHz, CDCl3)
δ 170.49, 156.56, 132.15, 128.45, 127.28, 125.21, 79.15, 39.42, 39.05, 31.41, 20.00, 13.70.; HRMS –
QTOF MS/MS: m/z [M+H]+ calcd for C14H18N2O2Br: 325.0552; found, 325.0551.
5.3.31. N-butyl-3-(3-chlorophenyl)-4,5-dihydroisoxazole-5-carboxamide (10e).
White solid; Yield: 88%; m.p. 81oC; 1H NMR (500 MHz, CDCl3) δ 7.68 (t, J = 1.8 Hz, 1H), 7.54 – 7.51 (m,
1H), 7.42 (ddd, J = 8.0, 2.0, 1.1 Hz, 1H), 7.36 (t, J = 7.9 Hz, 1H), 6.76 (s, 1H), 5.15 (dd, J = 10.9, 6.3 Hz,
1H), 3.70 – 3.61 (m, 2H), 3.34 (dd, J = 13.5, 6.4 Hz, 1H), 3.21 (dd, J = 13.5, 5.9 Hz, 1H), 1.53 – 1.48 (m,
2H), 1.34 (d, J = 7.6 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H).; 13C NMR (126 MHz, CDCl3) δ 170.43, 156.38,
134.96, 130.78, 130.16, 130.09, 127.02, 125.14, 79.18, 39.40, 39.07, 31.41, 20.00, 13.70.; HRMS –
QTOF MS/MS: m/z [M+Na]+ calcd for C14H18N2O2Cl : 303.0806; found, 303.0807.
5.3.32. N-cyclopropyl-3-(3-nitrophenyl)-4,5-dihydroisoxazole-5-carboxamide (11a).
White solid; Yield: 83%; m.p. 92oC; 1H NMR (500 MHz, CDCl3) δ 8.51 (t, J = 1.7 Hz, 1H), 8.30 (d, J = 9.6
Hz, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 6.83 (s, 1H), 5.20 (dd, J = 11.7, 5.6 Hz, 1H),
3.74 (ddd, J = 29.0, 17.3, 8.7 Hz, 2H), 2.76 (tq, J = 7.4, 3.8 Hz, 1H), 0.85 – 0.78 (m, 2H), 0.62 – 0.52 (m,
2H).; 13C NMR (126 MHz, CDCl3) δ 171.59, 155.76, 148.52, 132.50, 130.16, 130.04, 125.19, 121.91,
79.54, 39.08, 22.45, 6.46, 6.37.; HRMS –QTOF MS/MS: m/z [M+H]+ calcd for C13H13N3O4 : 276.0984;
found, 276.0979.
5.3.33. N-cyclopropyl-3-(4-fluorophenyl)-4,5-dihydroisoxazole-5-carboxamide (11b).
1
White solid; Yield: 78%; m.p. 93oC; H NMR (500 MHz, CDCl3) δ 7.45 – 7.39 (m, 3H), 7.20 – 7.13 (m,
1H), 6.85 (s, 1H), 5.15 (dd, J = 11.2, 6.2 Hz, 1H), 3.73 – 3.63 (m, 2H), 2.76 (tq, J = 7.4, 3.8 Hz, 1H), 0.85
– 0.78 (m, 2H), 0.61 – 0.53 (m, 2H).; 13C NMR (126 MHz, CDCl3) δ 171.98, 163.75, 156.49, 130.60,
130.39, 122.90, 117.92, 113.91, 79.15, 39.42, 22.39, 6.43, 6.35.; HRMS –QTOF MS/MS: m/z [M+H]+
calcd for C13H13N2O2F : 249.1039; found, 249.1034.
5.3.34. 3-(4-bromophenyl)-N-cyclopropyl-4,5-dihydroisoxazole-5-carboxamide (11c).
White solid; Yield: 82%; m.p. 114oC; 1H NMR (500 MHz, CDCl3) δ 7.54 (q, J = 8.6 Hz, 4H), 6.84 (s, 1H),
5.12 (dd, J = 11.1, 5.8 Hz, 1H), 3.74 – 3.58 (m, 2H), 2.75 (dt, J = 10.6, 3.5 Hz, 1H), 0.83 – 0.75 (m, 2H),
0.55 (dt, J = 13.9, 11.6 Hz, 2H).; 13C NMR (126 MHz, CDCl3) δ 172.10, 156.54, 132.16, 128.46, 127.20,