Synthesis and structure-activity relationship of new chalcone linked 5-phenyl-3-isoxazolecarboxylic acid methyl esters potentially active against drug resistant Mycobacterium tuberculosis

European Journal of Medicinal Chemistry 222 (2021) 1 13580

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Synthesis and structure-activity relationship of new chalcone linked

5-phenyl-3-isoxazolecarboxylic acid methyl esters potentially active

against drug resistant Mycobacterium tuberculosis

Santosh Kumar Sahoo ^a, Bandela Rani ^a, Nikhil Baliram Gaikwad ^a,

,

Mohammad Naiyaz Ahmad ^{b c}, Grace Kaul ^{b c}, Manjulika Shukla ^b, Srinivas Nanduri ^a,

Arunava Dasgupta ^b^**, Sidharth Chopra ^{b ***}, Venkata Madhavi Yaddanapudi ^a

,

c

,

c

,

, *

^aDepartment of Pharmaceutical Technology and Process Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar,

Hyderabad, 500037, Telangana, India

^bDivision of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Sector 10, Janakipuram Extension, Lucknow, 226031, Uttar Pradesh, India

^cAcSIR: Academy of Scientiﬁc and Innovative Research (AcSIR), Ghaziabad, 201002, India

a r t i c l e i n f o

a b s t r a c t

Article history:

In search of novel therapeutic agents active against emerging drug-resistant Mycobacterium tuberculosis

and to counter the long treatment protocol of existing drugs, herein we present synthesis and biological

evaluation of a new series of 5-phenyl-3-isoxazolecarboxylic acid methyl ester-chalcone hybrids. Among

35 synthesized compounds, 32 analogues displayed potent in-vitro activity against Mycobacterium

Received 12 April 2021

Received in revised form

13 May 2021

Accepted 24 May 2021

Available online 2 June 2021

tuberculosis H37Rv with MIC 0.12e16

to be non-cytotoxic (CC₅₀> 20 g/mL & SI > 10). Most potent compounds with MIC 0.12

7 ab) exhibited selectivity index (SI) in excess of 320. Further studies on activity against drug-resistant

Mycobacterium tuberculosis revealed 7j as the most potent compound with MIC 0.03e0.5 g/mL. Time-

m

g/mL. Cell viability test against Vero cells indicated 29 compounds

m

g/mL (7 b, 7j,

Keywords:

Isoxazole

m

kill kinetic study suggested compound 7j displaying concentration-dependent bactericidal killing ac-

tivity with relatively comparable potency to that of current ﬁrst-line anti-TB drugs. Taken together, 7j

presents a novel hit with potential to be translated into a potent antimycobacterial.

Chalcone

Hybrid

Anti-tubercular

Drug-resistant

Time kill kinetics

1. Introduction

protocol, lack of patient adherence, an alarming increase in the

emergence of resistant strains, the prevalence of latent TB, the

Tuberculosis (TB), an air-borne contagious lung infection caused

mainly by Mycobacterium tuberculosis (Mtb), remains a major

threat to public health and socio-economic status. Frequency of

new drug approval for TB remains low despite TB being one of the

top 10 causes of death worldwide and the foremost cause of death

from a single infectious agent (surpassing HIV/AIDS) [1]. Although

preventable and curable, the lethality of TB lies in its long treatment

complexity of the disease process, and side effect of existing drugs

[2]. All these very reasons constituted major obstacle in the way of

achieving WHO's 2020 milestone of 35% reduction in TB casualty

between 2015 and 2020. In addition, there is an upsurge in drug-

resistant TB with nearly half

a million people developing

rifampicin-resistant TB (RR-TB) in 2019 among 78% of the

multidrug-resistant TB (MDR-TB) population [1]. In this context, it

is of paramount importance to develop new chemical entities (NCE)

exhibiting potent anti-TB activity besides displaying non-toxicity

with improved selectivity.

* Corresponding author.

Though anti-TB drug research has grown in leaps and bounds,

only bedaquiline, delamanid and recently pretomanid [3] have

been approved over the past decade. The anti-TB drug discovery

was mostly based on some of the most privileged structural classes

which include di-aryl ethers, di-arylquinoline, indole carbox-

amides, azaindoles, oxazolidinone, 1,3-benzothiazin-4-ones,

*** Corresponding author. Division of Microbiology, CSIR-Central Drug Research

Institute, Sitapur Road, Sector 10, Janakipuram Extension, Lucknow, 226031, Uttar

Pradesh, India.

** Corresponding author. Division of Microbiology, CSIR-Central Drug Research

Institute, Sitapur Road, Sector 10, Janakipuram Extension, Lucknow, 226031, Uttar

Pradesh, India.

E-mail addresses: a.dasgupta@cdri.res.in (A. Dasgupta), skchopra007@gmail.

com (S. Chopra), yssmadhavi@gmail.com (V.M. Yaddanapudi).

https://doi.org/10.1016/j.ejmech.2021.113580

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

imidazo [1,2-a]pyridines, ﬂuoroquinolines, thiazoles, nitrofuran,

nitroimidazole and their derivatives [4,5]. As isoxazole esters bear

bio-isosteric resemblance (replacement of isoxazole ester with

nitrofuran gives almost similar activity against Mtb) to privileged

anti-TB scaffolds like cycloserine, nitro-furan and nitro-imidazole

(Fig. 1); they have attracted considerable attention as a promising

moiety against Mtb since last decade [6].

tubercular property. Among them, Nitrofuran IX [20,21], Naphthyl

X [22], indolyl XI [23], aryloxy azolyl XII [24], Quinolinyl XIII [25]

chalcones were found to exhibit prominent activity towards Mtb.

As far as mechanism is concerned, naphthyl-chalcones were iden-

tiﬁed as inhibitors of Mtb protein tyrosine phosphatases (PTPs).

Promising inhibitors bearing isoxazole and chalcone moiety active

against Mtb are depicted in Fig. 2.

In addition, nitrofuran, nitroimidazole and other nitro-

containing anti-TB pharmacophores although highly potent have

often been associated with mutagenicity and genotoxicity [7]. This

paves way for alternative scaffolds like isoxazole esters to be

explored. One decade back, 3-isoxazolecarboxylic acid alkyl ester

moiety was developed by combining phenotypic screening and

rational drug design that possessed proven potency against Mtb [8].

In past, various derivatization and combination approaches have

been employed on isoxazole esters to ﬁnd potent anti-TB leads [9].

Benzyloxy I, benzamido II [8], styryl III [10] and meﬂoquine IV [1 1]

linked derivatives of 5-phenyl-3-isoxazolecarboxylic acid ethyl

esters were reported to be highly potent, selective anti-TB com-

pounds active against both replicating and nonreplicating bacteria.

The molecular target of isoxazole ester derivatives remain un-

clear despite several hypothesis proposed by previous literatures

wherein Lilienkampf and his group proposed that cell wall syn-

thetic pathway was not the target for these compounds since these

were active on both replicating and non-replicating TB [9]. Further,

isoxazole esters were having anti-TB activity while corresponding

acids were completely inactive towards Mtb at neutral pH while

exhibiting surprisingly enhanced potency at acidic pH (phenome-

non associated with Pyrazinoic acid, active form of prodrug Pyr-

azinamide which has ambiguous MOA) [12]. In addition, their study

indicated that the introduction of planar moiety/linker to isoxazole

esters enhances potency suggesting increased permeability and

intracellular action as probable mechanism. In this regard, 4,5-

1.1. Rationale

Despite being a highly promising scaffold possessing potent

anti-TB efﬁcacy, isoxazole-based compounds have not made up the

anti-TB drug discovery ladder and clinical pipeline. Lilienkampf

et al. [8], studied different derivatives of 5-phenyl-3-

isoxazolecarboxylic acid ethyl esters as highly potent, selective

anti-TB agents. Among explored compounds, I and II exhibited

potency of 0.6 and 1.4 mM respectively against susceptible Mtb. 3,

methyl ester analogue of II was synthesized and found to display

similar anti-TB activity.

Herein, we report modiﬁcation of II to design (Fig. 3) and syn-

thesize a series of new 5-phenyl-3-isoxazolecarboxylic acid methyl

ester and chalcone hybrids based on encouraging anti-TB efﬁcacy of

isoxazole ester, chalcone moiety and explore their structure-

activity relationship (SAR). Moreover, the rationale was

compelled by both isoxazole acid derivatives and chalcone motifs

sharing same mechanism as potent inhibitor of Mtb protein tyro-

sine phosphatases (MtbPtps) [13,22,26]. In addition, this molecular

hybridization approach was conceptualized with the aim of

investigating whether the resultant hybrids of these two scaffolds,

isoxazole ester and chalcone, could yield

compound.

a potent anti-TB

2. Result and discussion

diarylisoxazole-3-carboxylic acid

V [13] was investigated as

MptpB inhibitor acting as an anti-virulence agent targeting the

pathogen survival mechanism. In contrast to the above-mentioned

classically designed isoxazole derivatives, hybrid analogue i.e., 2-

methylbenzothiazole linked isoxazole-3-carboxamide derivative

VI [14] was reported to be highly active against susceptible Mtb. Not

only isoxazole alkyl ester derivatives but isoxazole incorporated

curcuminoids VII [15] were proven to have inhibitory action against

Mtb. Similarly, 2-aminothiazole based isoxazole-3-carboxamide

VIII was found to have high inhibitory activity towards suscepti-

ble Mtb and was proposed to evade the action of bacterial efﬂux

pump [16].

Chalcones act as Michael acceptors and confer higher Clog P

which is related to permeability and bioavailability [1 7]. These at-

tributes contribute to their wide utility in medicinal chemistry as

anti-microbial and anti-cancer agents [18,19]. Several chalcone

bearing scaffolds and hybrids have been reported to exhibit anti-

2.1. Chemistry

All newly designed compounds were synthesized as described

in Scheme 1. In a 3-step synthesis, 3-Nitroacetophenone 1 was

converted to its corresponding 2, 4-diketo methyl ester interme-

diate in a base-assisted enolate generation by treating with

Dimethyl oxalate. Formed intermediate was readily cyclized to

substituted isoxazole methyl ester upon reﬂuxing with Hydroxyl-

amine hydrochloride in methanol which was reduced with Sodium

dithionite to give the amine fragment, 5-(3-Aminophenyl)iso-

xazole-3-methyl ester 2, as key intermediate. 2 was coupled with 3-

(Triﬂuoromethyl)benzoyl chloride to yield 3 (Scheme 1A).

Meanwhile, a series of chalcone acids 6a-6ai were synthesized

as reported in the literature [27e33] by aldolic condensation of 4-

Formyl benzoic acid 5 and different substituted acetophenones

4a-4ai in presence of Potassium hydroxide as base in methanol. ¹H

Fig. 1. Bio-isosterically similar analogues of isoxazole alkyl ester.

2

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

Fig. 2. Chemical structure of literature cited promising anti-TB agents having isoxazole and chalcone moiety.

NMR revealed that all the chalcone acids synthesized were

conﬁgured trans (E) except 2-Methyl substituted chalcone acid (6u)

that happened to be cis (Z) that complemented with the previous

report [31]. Synthesized chalcone acids 6a-6ai were treated with

methyl 5-(3-aminophenyl)isoxazole-3-carboxylate 2 in presence of

DMAP as base and EDCI to form target hybrid products as isoxazole

methyl esterechalcone derivatives 7a-7ai in moderate to good yield

(Scheme 1B).

Further 8, ethyl ester analogue of 2, was obtained from 3-

Nitroacetophenone by a similar procedure as that of Scheme 1A

wherein Dimethyl oxalate was replaced by Diethyl oxalate in the

1st step. 8, when treated with 6 b (4-Chlorosubstituted chalcone

acid) in presence of EDCI, gave 7aj (Scheme 2A). 10 was obtained

from 4-Nitroacetopnenone by employing a similar procedure as

Scheme 1A, which on treatment with 6 b led to the formation of 7ak

(Scheme 2B). 3-Nitroaniline, upon diazotization followed by sub-

stitution with Sodium azide, yielded 3-Nitrophenyl azide that was

cyclized with Ethyl acetoacetate in presence of base Triethylamine

to 5-Methyl-1-(3-nitrophenyl) ethyl ester. The above product on

reduction with H₂ePd/C led to the synthesis of 12 which upon

treatment with 6 b afforded 7 al (Scheme 2C).

4-[(4-Chlorobenzyl)oxy]benzoic acid 14 was synthesized by al-

kali hydrolysis of ester formed by the reaction between Methyl 4-

hydroxybenzoate with 4-Chlorobenzyl bromide using Potassium

carbonate as base at 85 ^ꢀC. 14 on treatment with 2 formed 7am

(Scheme 3A). 4-(3-(4-Chlorobenzoyl)oxiran-2-yl)benzoic acid 15

was synthesized from 4-Chlorophenacyl bromide and 4-

Formylbenzoic acid 5 using potassium hydroxide as base at rt

3

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

Fig. 3. The rationale of design of new chalcone tethered 5-phenyl-3-isoxazolecarboxylic acid methyl esters; Chemical structures of previously reported potent anti-TB compounds (I,

its linker variant II) and synthetic MtbPtpB inhibitor (X); methyl ester analogue of II synthesized in this research work as lead (3); designed compounds through scaffold hy-

bridization (7a-7ai).

which upon coupling with 2 using EDCI resulted in 7an (Scheme

3B). 4-Chlorocinnamic acid 17 was synthesized following re-

ported method [34] from 4-Chlorobenzaldehyde and Malonic acid

in Pyridine as solvent and catalytic Piperidine under reﬂux condi-

tion. 17 upon treatment with 2 led to the formation of 7ao as

mentioned in Scheme 3C.

compounds, most of the compounds exhibited good to excellent

activity against Mtb H37Rv having MICs in the range of 0.12e32 g/

mL. Screening against bacterial pathogen panel demonstrated no

inhibition by all the synthesized compounds showing their selec-

tivity towards Mtb.

m

A detailed perusal of the anti-TB screening results indicated that

compounds 7 b, 7f, 7j, 7 ab were most potent (having MIC of

0.12

mg/mL). These four compounds have substituents chloro-,

2.2. Antimycobacterial activity

isobutyl-, propoxy- and ﬂuoro-respectively at the 4th (C-4’) posi-

tion of phenyl group (R₁) with 7 ab being disubstituted. Inciden-

tally, MIC of 0.25e1.0 mg/mL was shown by majority of compounds

having halogen or alkyl substitution speciﬁcally at 4th or 2nd po-

sition as compared to 3rd position. Monosubstituted compounds

exhibited better activity than disubstituted compounds. Among

disubstituted compounds 7aa (2,5-diﬂuorophenyl), 7 ab (2,4-

The antimycobacterial activity of synthesized compounds was

evaluated by broth microdilution method with resazurin as an in-

dicator of the metabolic activity of mycobacteria. The concentration

of newly synthesized compounds tested in 64e0.03 mg/mL range.

Isoniazid, Rifampicin, Streptomycin and Ethambutol were used as

reference compounds. The preliminary antimycobacterial

screening indicated that the synthesized compounds were active

against Mycobacterium tuberculosis H37Rv while showing no inhi-

bition of non-tuberculous mycobacteria (NTM) viz. M. abscessus

ATCC 19977, M. fortuitum ATCC 6841 and M. chelonae ATCC 35752

(complete antimycobacterial screening results of compound 7a-7ai

was depicted in Supplementary Material Table S1). For ease of

understanding; compounds were categorized as potent (MIC

diﬂuorophenyl),

7y

(2,4-dichlorophenyl)

and

7z

(2,4-

dimethoxyphenyl) were found to be highly potent. Surprisingly,

compounds bearing OH group 7l and 7w were found to signiﬁ-

cantly reduce activity. A similar result was obtained when aryl

group (R₁) was replaced by heterocyclic groups 7 ag (thiophen-2-

yl) and 7 ah (3-pyridyl) showing poor inhibition of Mtb H37Rv.

Generalized observation from these ﬁndings indicated that

nonpolar groups like halogen and alkyl exhibited potent inhibitory

capacity while polar substituents like hydrogen bond donors or

heterocyclic groups were not tolerated. On the contrary, com-

pounds bearing nitro group (7g, 7q, 7ad, 7ae) exhibited good to

moderate potency despite being a polar substituent which may be

assigned to its electronic interactions (many of privileged anti-TB

scaffolds contain nitro group e.g., nitrofuran, nitro-thiophene,

nitro-imidazole, etc.) [7].

ꢁ0.5

mL), moderately active (MIC ¼ 4

and > 64 g/mL indicates weak and inactive respectively in

m

g/mL), highly active (MIC ¼ 0.5e1.0

mg/mL), good (MIC 2 mg/

g/mL); while MIC of >4

m

following discussion. Anti-TB activity of all the compounds was

listed in Table 1. To determine their selectivity, these compounds

were also tested against panel of bacteria (screening results dis-

played in Supplementary Material Table S2).

As can be observed from Table 1, among 35 evaluated

4

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

Scheme 1. AeB. Synthesis of 3-(triﬂuoromethyl)phenyl and chalcone linked 5-phenyl-3-isoxazolecarboxylic acid methyl esters. Reagents and Conditions: (i) a. Dimethyl oxalate, t-

BuONa, THF, 0 ^ꢀC to rt., 2 h; b. NH₂OH.HCl, MeOH, Reﬂux, 6 h; c. Na₂S₂O₄, MeOH:H₂O (1:1), 65 ^ꢀC, 1 h, 38% (3 steps); (ii) 3-Triﬂuoromethylbenzoyl chloride, DIPEA, DCM, 0 ^ꢀC-rt, 4 h,

85%; (iii) KOH, MeOH, rt, 15e18 h, 40e89%; (iv) EDCI, DMAP, DMF, 12e15 h, rt, 23e69%.

Scheme 2. AeC. Synthesis of 4-Chlorochalcone acid (6 b) linked phenyl isoxazole and triazole esters. Reagents and Conditions: (i) a. Diethyloxalate, t-BuONa, THF, 0 ^ꢀC to rt., 2 h; b.

NH₂OH.HCl, EtOH, Reﬂux, 6 h; c. Na₂S₂O₄, EtOH:H₂O (1:1), 65 ^ꢀC, 1 h, 47% (3 steps); (ii) EDCI, DMAP, DMF, 12e15 h, rt; (iii) a. Dimethyloxalate, t-BuONa, THF, 0 ^ꢀC to rt., 2 h; b.

NH₂OH.HCl, MeOH, Reﬂux, 6 h; c. Na₂S₂O₄, MeOH:H₂O (1:1), 65 ^ꢀC, 1 h, 32% (3 steps); (iv) a. NaNO₂, NaN₃, Conc. HCl:H₂O 1:1, 0 ^ꢀC-rt, 2h; b. Ethyl acetoacetate, TEA, DMF, rt,

Overnight; c. H₂, Pd/C, Dry EtOAc, rt, 2 h, 51% (3 steps).

In general, compounds with non-polar electron-withdrawing

groups (EWG) i.e., halogens were most potent followed by nonpolar

electron-donating groups (EDG) like alkyl followed by methoxy

substitution. Among halogen groups, chloro-is more active than

ﬂuoro-followed by bromo-. Electron donating -OMe substituted

compound (7h, 7p, 7v) demonstrated moderate to good inhibition

of Mtb which drastically improved when replaced by ethoxy and

propoxy i.e., by introducing more non-polarity by increasing alkyl

appendages. Compound having fused ring 7af and tri-substitution

7ai were well tolerated for anti-TB activity. These broad SAR ob-

servations are summarized in Fig. 4.

Lipophilicity and membrane permeability in mycobacteria have

5

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

Scheme 3. AeC. Synthesis of 5-phenyl-3-isoxazolecarboxylic acid methyl ester (2) linked substituted acids. Reagents and Conditions: (i) a. 4-Chlorobenzyl bromide, K₂CO₃, DMF,

80 ^ꢀC, 5 h; b. NaOH, MeOH: THF: H₂O, 3:2:1, reﬂux, 24 h, 63% (2 steps); (ii) EDCI, DMAP, DMF, 12 h, rt; (iii) 4-Chlorophenacyl bromide, KOH, MeOH, rt, overnight, 59%; (iv) Malonic

acid, cat. Piperidine, Pyridine, 100 ^ꢀC, 11 h, 80%.

always been correlated owing to the unique structure of its highly

lipophilic cell wall consisting of mycolic

2.4. Structural variation of the most potent analogue and

veriﬁcation of rationale of design

arabinogalactanepeptidoglycan complex [35]. To understand the

impact of lipophilicity of synthesized compounds on anti-TB po-

tency, Clog P of each was enumerated (Table 1). Clog P provides an

estimation of the lipophilicity of a compound. A generalized eluci-

dation of the plot (Fig. 5) between Clog P and anti-TB MICs indicated

that majority, if not all, of compounds exhibiting potent anti-TB

inhibitory activity, possess higher Clog P values. Most potent com-

pounds 7 b, 7f, 7j, and 7 ab have Clog P values of about 5 or more.

Conversely, weakly active or inactive compounds 7l, 7w, 7 ag, and

7ah were found to have Clog P values of 3.5e4.5. The bulk of the

synthesized compounds possessed Clog P values between 4 and 6.

The observed direct proportional relationship between anti-TB MIC

and Clog P is in agreement with the latest report on analysis of Clog P

with respect to anti-TB potency [36].

Intermediate chalcone acid (6 b, 6f & 6j) and core isoxazole-

methyl ester 2 were subjected to anti-TB testing to investigate

their involvement in the activity of designed molecules. The results

obtained was displayed in Table 2.

Intermediate 2 was found to be inactive while the correspond-

ing chalcone acids exhibited weak to moderate activity. The chal-

cone acids were further subjected to cell viability test against Vero

cells and were found to be highly toxic. Modiﬁcations were per-

formed on the most potent compound 7b in order to seek further

potent molecules showing more selectivity. For this purpose, 7aj

(ethyl ester analogue of 7b) and 7ak (regioisomer of 7b; having

different position of amido-linkage i.e., para to isoxazole ring rather

than meta) was synthesized. Isoxazole ester of 7b was replaced by

triazole ethyl ester moiety to obtain compound 7 al which was

synthesized with an aim to analyze the importance of isoxazole

ester in imparting anti-TB property. To establish difference in po-

tency and selectivity of the newly designed and synthesized hy-

brids 7a-7ai from that of the previously reported molecule (compd.

II, Fig. 2); a similar compound, 3, having isoxazole-methyl ester

moiety was synthesized. Biological evaluation of all these com-

pounds was carried out for their activity against Mtb and Vero cells.

Fig. 6 shows the variations made and their outcome. Altering

methyl ester of 7b to ethyl ester resulted in a decrease in activity

with no signiﬁcant change in SI. Changing the linkage from C-3 to

C-4 (7ak) resulted in a two-fold increase in potency than 7b making

the molecule much more selective for Mtb. Interestingly, complete

loss of anti-TB activity was observed when isoxazole methyl ester

was replaced by triazole ester which proves the essential role

played by the former in displaying anti-TB property. The most

potent analogue 7b was found to be 4 times more potent and se-

lective than reference compound 3 that indicates the role of chal-

cones in the enhancement of anti-TB activity of core isoxazole-

methyl ester moiety. Notably, the increment of potency of 7ak

against Mtb was 10 folds than reference 3.

2.3. Cytotoxicity assay against Vero cells

All the synthesized compounds were tested for their toxicity

against Vero cells using the MTT assay. CC₅₀is deﬁned as the lowest

concentration of compound leading to a 50% reduction in cell

viability. Doxorubicin was used as positive control and each

experiment was repeated in triplicate. Selectivity index (SI) is the

ratio between the concentration of compound that is toxic to

mammalian Vero cells to the concentration that can kill the

mycobacteria. The results indicated that majority of compounds

were non-toxic to Vero cells (CC₅₀ꢂ 20

mg/mL) along with a

selectivity index of >10, which infers that these compounds

selectively inhibit Mtb than normal cells. The results are tabulated

in Table 1. However, compounds 7c and 7h were found to inhibit

Vero cells at conc. ꢁ20

MIC (4 g/mL) which contributed to these compounds having a low

therapeutic index. On the contrary, compound 7f showed excellent

selectivity index >84 despite of having CC₅₀> 10 g/mL (border-

line) owing to its excellent potency (0.12 g/mL). Considering anti-

TB MIC of 0.12 g/mL and a selectivity index in excess of 320;

mg/mL along with having moderate anti-TB

m

On the other hand, to further evaluate the role of chalcones; an

attempt was made to replace chalcone with similar analogues. a, b-

unsaturated carbonyl fragment of chalcone of 7b was altered with

oxy-methylene linker; by an epoxy-carbonyl group; and chalcone

m

compound 7 b, 7i, 7j, and 7 ab were found to be highly potent

amongst all synthesized compounds; most potent being 7 b with a

MIC of 0.12 mg/mL against Mtb H37Rv and selectivity index of >416.

6

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

Table 1

MIC of Chalcone tethered 5-phenyl-3-isoxazolecarboxylic acid methyl esters against Mtb H37Rv, their SI against Vero cells along with Clog P values.

Sl. No.

Compd. code

Structure R₁

Mtb H37Rv MIC

m

g/mL

CC₅₀

mg/mL

SI^a

Clog P^b

1

2

3

4

5

6

7

8

7a

7 b

7c

7d

7e

7f

7g

7h

7i

7j

7k

7l

7 m

7n

7^ꢀ

7p

7q

7r

7s

Phenyl

4-Chlorophenyl

4-Fluorophenyl

4-Bromophenyl

4-Methylphenyl

4-Isobutylphenyl

4-Nitrophenyl

4-Methoxyphenyl

4-Ethoxyphenyl

4-Propoxyphenyl

4-Morpholinophenyl

4-Hydroxyphenyl

3-Fluorophenyl

3-Chlorophenyl

3-Bromophenyl

3-Methoxyphenyl

3-Nitrophenyl

3-Triﬂuoromethylphenyl

2-Chlorophenyl

1

0.12

4

>50

>20

>80

>10

>20

>10

>40

>20

ND

>50

>416

>5.0

>20

>160

>84

>10

>2.5

>160

>320

>40

ND

>20

>10

>25

>50

>20

>100

>40

>100

>40

ND^c

>50

>80

>200

>40

>333

>25

>20

>40

>20

ND^c

>50

e

4.60

5.39

4.82

5.54

5.10

6.56

4.50

4.82

5.35

5.88

4.37

4.48

4.82

5.39

5.54

4.82

4.50

5.62

5.39

5.54

5.10

4.82

4.48

4.50

6.01

4.50

4.92

5.32

4.93

4.44

4.81

4.38

3.50

4.11

ꢃ0.69

2.95

ꢃ7.06

ꢃ0.06

1

0.5

0.12

2

4

9

0.25

0.12

0.5

>64

2

0.25

4

2

1

0.25

1

0.25

1

16

1

0.5

0.25

1

0.12

2

4

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

>40

>2.5^d

>100

>40

>20

>25

>40

>25

>40

ND^c

>50

>40

>50

>40

>50

>80

>40

ND^c

>50

e

7t

7u

7v

7w

7x

2-Bromophenyl

2-Methylphenyl

2-Methoxyphenyl

2-Hydroxyphenyl

3,4-Dimethoxyphenyl

2,4-Dichlorophenyl

2,4-Dimethoxyphenyl

2,5-Diﬂuorophenyl

2,4-Diﬂuorophenyl

3-Fluoro-4-Methylphenyl

4-Methyl-3-Nitrophenyl

4-Methoxy-3-Nitrophenyl

3,4-(Methylenedioxy)phenyl

Thiophen-2-yl

7y

7z

7aa

7 ab

7ac

7ad

7ae

7af

7 ag

7ah

7ai

Std.

1

2

32

64

1

0.03

0.5

1

Pyridin-3-yl

3,4,5-Trimethoxyphenyl

Isoniazid

Rifampicin

Streptomycin

e

Ethambutol

e

a

Selectivity index ¼ CC₅₀/MIC.

b

c

Calculated with Chemdraw Professional 20.0, PerkinElmer Informatics Inc.

Not determined.

d

Solubility issue in DMSO at higher concentration.

acid was replaced by cinnamic acid to synthesize 7am, 7an, 7ao

respectively (Fig. 6). The resulted compounds exhibited diminished

anti-TB activity, further emphasizing the fact that introduction of

chalcone to 5-phenyl-3-isoxazolecarboxylic acid methyl esters

resulted in improved potency against Mtb. These ﬁndings from

structural modiﬁcation were summarized in SAR Fig. 4. All com-

pounds obtained by modiﬁcation of 7 b i.e., 7aj-7ao along with

compound 3 were also screened against non-tuberculous myco-

bacteria and panel of bacterial pathogen to determine their selec-

tivity and results obtained were presented in Supplementary

Material Tables S1 and S2 respectively. The results indicated all

above-mentioned compounds were inactive towards non-

tuberculous mycobacteria and bacterial pathogen panel.

H37Rv were further taken to study their activity against drug-

resistant strains. The results obtained are tabulated in Table 3.

Most of the compounds tested exhibited good inhibitory activ-

ity. The most potent compound 7b was inactive against INH and

ETB resistant strains while showing considerable activity against

STR and RIF resistant strains (MIC 2 mg/mL). Surprisingly, its posi-

tional variable analogue 7ak exhibited potent activity against all

strains except STR resistant strain, where its MIC was found to be on

the higher side (8 mg/mL). 7aj, ethyl ester analogue of 7b showed

weak potency. Compound 3 showed inhibition of resistant strains

although at higher concentration. Noteworthy from these results

are compounds 7i and 7j exhibiting potency of MIC <1

mg/mL

against all DR strains. Compound 7k bearing 4-morpholino moiety

also exhibited potent activity. A similar feature among these three

analogues (7i: R₁¼ 4-Ethoxyphenyl, 7j: R₁¼ 4-Propoxyphenyl, and

7k: R₁¼ 4-Morpholinophenyl) is the presence of alkyl fragment

indicating their likely involvement in the inhibition of DR strains.

Owing to the potent inhibitory activity against DR strains and non-

2.5. Determination of MIC against drug-resistant strains of Mtb

(DR-Mtb)

Compounds showing MIC lesser than 0.5

mg/mL against Mtb

7

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

Fig. 4. An overview of structure-activity relationship (SAR) of chalcone tethered 5-phenyl-3-isoxazolecarboxylic acid methyl esters: Highlighted colors of rectangular boxes indicate

the identical color of the part of structure 7a-7ao being referred.

3. Time kill kinetics study

The time-kill kinetics assay provides important information on

the mycobacterial killing dynamics of anti-TB drugs [37]. Com-

pound 7i and 7j were tested to ﬁnd out the rate at which they

decrease Mtb. The timeekill kinetics of INH along with test com-

pounds 7i and 7j are shown in Fig. 7-A and 7-B respectively. As

expected, INH demonstrated concentration-dependent killing ac-

tivity, rapidly reducing Mtb Log₁₀CFU/mL in 6 days and completely

eliminating Mtb load within 3 days in 10x MIC. In comparison, 7i at

10x MIC completely eliminated the bacilli in 6 days while 7j

exhibited a slightly higher killing rate with both 1x and 10x MIC

eliminating bacilli in 7 and 6 days respectively. Taken together, 7j

was found to be the most potent compound of the series with the

best time-kill kinetics.

4. Conclusion

Fig. 5. Correlation between Clog P and MIC against Mtb H37Rv of Chalcone tethered 5-

phenyl-3-isoxazolecarboxylic acid methyl esters.

In this study, a new class of Chalcone tethered 5-phenyl-3-

isoxazolecarboxylic acid methyl ester was conveniently synthe-

sized and evaluated as highly potent anti-TB chemotypes with

several compounds exhibiting low micromolar activity comparable

toxic attributes; both 7i and 7j were forwarded for time-kill kinetic

studies.

to current ﬁrst-line anti-TB drugs. SAR studies demonstrated,

Table 2

MIC of intermediate amine 2 & chalcone acids against Mtb H37Rv and their SI against Vero cells.

Sl. No.

1

Compound code

2

Structure

MIC

64

mg/mL

CC₅₀

ND^b

m

g/mL

SI^a

ND^b

2

3

4

6b

6f

6j

4

<10

32

a

Selectivity index.

Not determined.

b

8

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

Fig. 6. Structural modiﬁcation of most potent compound 7b to obtain ethyl ester analogue 7aj; para amidosubstituted analogue 7ak; triazole ethyl ester variant 7 al; 4-

Chlorobenzyloxy derivative 7am; 4-Chlorobenoyl-oxiranyl analogue 7an; 4-Chlorocinnamic acid derivative 7ao and their effect on MIC against Mtb H37Rv and CC₅₀against

Vero cells.

Table 3

MIC of chalcone tethered 5-phenyl-3-isoxazolecarboxylic acid methyl esters against drug-resistant strain of Mtb.

Sl. No.

Compound code

H37Rv ATCC 27294

INH-res^aATCC 35822

ETB-res^aATCC 35837

STR-res^aATCC 35820

RIF-res^aATCC 35838

1

2

3

4

5

6

7

8

7 b

7e

7f

7i

7j

7k

7n

7s

7u

7y

7z

7 ab

7aj

7ak

3

INH

RIF

STR

ETB

0.12

0.5

0.12

0.25

0.12

0.5

0.25

0.5

0.25

0.12

0.5

>64

0.5

>64

0.5

0.12

0.5

1

>64

0.06

0.12

0.25

0.12

>64

0.25

0.12

4

2

0.25

4

1

0.5

2

>64

16

0.03

0.12

0.5

0.25

64

0.25

0.5

1

2

0.5

2

1

2

64

8

0.03

>64

1

9

1

>64

2

10

11

12

13

14

15

16

17

18

19

1

32

0.5

4

>64

0.03

1

0.06

0.5

0.03

0.06

2

0.25

8

0.03

2

0.03

64

1

4

32

1

a

INH-res: isoniazid-resistant; ETB-res: ethambutol-resistant; STR-res: Streptomycin-resistant; RIF-res: rifampicin-resistance.

halogen and alkyl substitution at all positions on chalcone moiety

in both mono and disubstituted analogue were most potent. The

substitution trend among mono- and disubstituted analogues

suggested the decreasing potency pattern as para > ortho > meta.

An increase in alkyl appendages among para alkoxy analogues led

to enhanced potency and the identiﬁcation of 7j that proved to

inhibit DR strains in micromolar concentrations. Lipophilicity

analysis of this library of compounds revealed that nonpolar

substituents having a higher Clog P value that can likely enhance

Mtb cell permeability were found to show good anti-TB activity. In-

depth SAR observation indicated that isoxazole methyl ester im-

parts anti-TB property while chalcone moiety enhances potency

and selectivity. The most potent compounds 7i and 7j exhibited

MICs of 0.12

strain in a conc. range of 0.03e1

towards Vero cells, compound 7j was discovered to be bactericidal

mg/mL each against Mtb H37Rv while inhibiting DR

mg/mL. Besides being nontoxic

9

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

Fig. 7. (A) Time kill kinetic graph of compound 7i; (B) Time kill kinetic graph of compound 7j.

in nature, completely lowering Mtb load in 6 days. To sum up, we

have successfully potentiated anti-TB activity of the resultant

hybrid formed by scaffold hybridization of isoxazole ester and

chalcone moiety. Further physicochemical optimization and

structural modiﬁcation of potent compounds from this series is

ongoing to accomplish a promising anti-TB agent.

dithionite was gradually added until TLC shows complete con-

sumption of starting material. The reaction mixture was extracted

with ethyl acetate after the removal of residual solvent. The com-

bined organic layer was washed with brine, dried over Na₂SO₄,

evaporated to yield the corresponding amine intermediate which

was puriﬁed by recrystallization from chloroform and hexane.

Yellow solid, Yield 38% (3 steps); mp: 134e136 ^ꢀC; ¹H NMR

(500 MHz, DMSO‑d₆)

d

7.28 (s, 1H), 7.19 (t, J ¼ 7.8 Hz, 1H), 7.10 (t,

5. Materials & methods

J ¼ 1.9 Hz, 1H), 7.09e7.06 (m, 1H), 6.72 (dd, J ¼ 8.0, 2.3, 1.0 Hz, 1H),

5.40 (s, 2H), 3.93 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

All the 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-coated silica gel 60-

F₂₅₄(0.5 mm) aluminium plates under UV light. ¹H and ¹³C NMR

spectra were obtained on Bruker Advance 500 MHz spectrometer

using tetramethylsilane (TMS) as the internal standard and chem-

ical shifts are reported in ppm. Chemical shifts are referenced to

d

172.50, 160.40, 156.98, 149.86, 130.33, 126.98, 116.92, 113.93,

110.68, 100.46, 53.25 ppm; HRMS-QTOF (ESI): m/z calcd. for

[MþH]^þC₁₁H₁₁N₂O₃249.0770; found 249.0773.

5.1.1. Synthesis of methyl 5-(3-(3-(triﬂuoromethyl)benzamido)

phenyl)isoxazole-3-carboxylate (3)

TMS (

d

0.00 for ¹H NMR and ¹³C NMR), CDCl₃(

d

7.26 for ¹H NMR and

A mixture of intermediate aniline 2 (0.1 g, 0.46 mmol) and

DIPEA (0.16 mL, 0.92 mmol, 2 equiv.) in 10 mL DCM was cooled to

0 ^ꢀC. 3-(Triﬂuoromethyl)benzoyl chloride (0.1 g, 0.46 mmol) diluted

with 5 mL DCM was added dropwise followed by stirring at rt for

4 h. Upon completion, DCM (20 mL) was added and the mixture

was washed with saturated NaHCO₃solution, and brine (20 mL).

The organic layer was dried with Na₂SO₄, ﬁltered, and evaporated.

The crude product was puriﬁed by recrystallization from CH₂Cl₂

and hexane to Yield 85% (white crystals). mp: 163e165 ^ꢀC; ¹H NMR

72.6 ¹³C NMR) or DMSO/d₆

(

d

2.50 for ¹H NMR and 39.5 for ¹³C

NMR). Spin multiplicities are reported as s (singlet), brs (broad

singlet), d (doublet), dd (double doublet), t (triplet) and m (multi-

plet). Coupling constant (J) values are reported in hertz (Hz). HRMS

were determined with Agilent QTOF mass spectrometer 6540 se-

ries instrument and were performed in the ESI techniques at 70 eV.

Column chromatography was performed using silica gel 60e120 or

100e200 mesh. Melting point was taken using Stuart® SMP30

apparatus.

(500 MHz, DMSO‑d₆)

d

10.71 (s, 1H), 8.41 (t, J ¼ 1.7 Hz, 1H), 8.35 (s,

1H), 8.31 (d, J ¼ 7.9 Hz, 1H), 8.01 (d, J ¼ 7.8 Hz, 1H), 7.97 (dd, J ¼ 8.2,

1.1 Hz, 1H), 7.83 (t, J ¼ 7.8 Hz, 1H), 7.76 (d, J ¼ 7.8 Hz, 1H), 7.59 (t,

J ¼ 8.0 Hz, 1H), 7.50 (s, 1H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz,

5.1. Synthesis of Methyl 5-(3-aminophenyl)isoxazole-3-carboxylate

(2)

DMSO‑d₆)

d 171.40, 164.74, 160.26, 157.21, 140.14, 135.87, 132.39,

130.37, 130.29, 129.71 (q, J_CF¼ 32.1 Hz),128.85 (dd, J_CF¼ 7.0, 3.5 Hz),

126.84, 124.76 (q, J_CF¼ 3.8 Hz), 124.44 (q, J_CF¼ 272.5 Hz), 123.28,

122.26, 117.67, 101.40, 53.34 ppm; HRMS-QTOF (ESI): m/z calcd. for

[MþNa]^þC₁₉H₁₃F₃N₂NaO₄413.0725; found 413.0727.

To an ice-cold solution of Dimethyl oxalate (1.5 equiv.) in 15 mL

of dry THF was added Sodium tertiary butoxide (2.0 g, 21.2 mmol,

3.5 equiv.) under nitrogen atmosphere. Ten minutes later, a solution

of 3-Nitroacetophenone 1 (1.0 g, 6.0 mmol, 1 equiv.) in anhydrous

THF (10 mL) was added dropwise to the mixture, and the resulting

mixture was allowed to stir at room temperature for 1 h. After

completion of the reaction (monitored by TLC; R_f0.2 in EA: Hex

1:3), the reaction mixture was added to crushed ice and quenched

with conc. HCl solution until acidic (pH 2e3); extracted with ethyl

acetate and the combined organic layer was dried over Na₂SO₄,

ﬁltered, concentrated under reduced pressure to give di-keto ester

intermediate as light-yellow crystals; which was taken to the next

step without further puriﬁcation. The crude product was dissolved

in methanol and 1.2 equiv. of hydroxylamine hydrochloride was

added. The solution was reﬂuxed for 6 h. After cooling, the pre-

cipitate formed was ﬁltered and oven-dried to provide the target

isoxazole ester compounds in good yield. A suspension of obtained

product in MeOH/H₂O 1:1 was heated to 65 ^ꢀC and Sodium

5.2. General procedure A for the synthesis of chalcone acids (6a-

6ai)

Intermediates 6a-6ai were prepared according to modiﬁed

literature procedure [27e33]. Brieﬂy, to

a mixture of 4-

formylbenzoic acid (0.5 g, 3.3 mmol, 1 equiv.), and corresponding

substituted acetophenone 4a-4ai (3.3 mmol) in 15 mL of MeOH;

0.56 g of KOH pellet (10.0 mmol, 3 equiv.) was added and the

resulting reaction mixture was stirred for 15e18 h at room temp.

After completion, the mixture was added to 50 mL of cold distilled

water and pH of the solution was adjusted to 4.5 with aqueous HCl

solution (2 M), upon which a precipitate was formed. The precipi-

tate was stirred for 15 min and then subsequently collected by

10

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

suction ﬁltration, dried over CaCl₂and recrystallized from a

mixture of DCM and MeOH to form 6a-6ai in 40e89% yield. In-

termediates 6a, 6b, 6c, 6d, 6e, 6g, 6h, 6l, 6p, 6w, 6x, 6z, 6ae, 6 ag and

6ai were reported in literature cited above. Characterized data for

newly synthesized chalcone acids were presented in supporting

information.

5.3.4. Methyl-(E)-5-(3-(4-(3-(4-bromophenyl)-3-oxoprop-1-en-1-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7d)

Beige solid, Yield 35%, mp: 238e240 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.56 (s, 1H), 8.44 (s, 1H), 8.23 (d, J ¼ 8.5 Hz, 2H),

8.10e8.07 (m, 4H), 8.08 (d, J ¼ 15.5 Hz, 1H), 7.98 (d, J ¼ 8.1 Hz, 1H),

7.84 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 7.8 Hz, 1H), 7.68 (d, J ¼ 8.5 Hz,

2H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.46 (s, 1H), 3.95 (s, 3H) ppm; ¹³C NMR

(125 MHz, DMSO‑d₆) d 188.77, 171.48, 165.53, 160.27, 157.20, 143.65,

5.3. General procedure B for the synthesis of compounds (7a-ai)

140.38, 138.20, 136.84, 136.36, 132.37, 131.12, 130.31, 129.40, 128.73,

128.00, 126.81, 123.98, 123.19, 122.04, 117.59, 101.34, 53.34; ppm;

HRMS-QTOF (ESI): m/z calcd. for [M þ Hþ2]^þC₂₇H₂₀BrN₂O₅

533.0556; found 533.0598.

Equimolar quantities of compound 6a-ai and 2 was stirred in

adequate amount of dry DMF. To this 2.5 equiv. of DMAP was added

followed by the addition of 2.5 equiv. of EDCI. The resulting reaction

mixture was stirred at rt under inert atmosphere. for 12e15 h. After

completion of reaction (monitored by TLC); the reaction mixture

was poured onto crushed ice and then extracted with ethyl acetate.

The organic layer was washed with 10% aq. HCl solution followed

by brine and dried over Na₂SO₄. Evaporation of the organic layer

resulted in the formation of crude product which was puriﬁed by

silica gel column chromatography or recrystallized from methanol

to give compound 7a-ai in 23e69% yield. All the newly synthesized

compounds were characterized by ¹H NMR, ¹³C NMR and HRMS

(ESI). ¹H NMR signals was also determined for some representative

compounds (supporting information). ¹H NMR spectra conﬁrmed

5.3.5. Methyl (E)-5-(3-(4-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)

benzamido)phenyl)isoxazole-3-carboxylate (7e)

Light yellow solid, Yield 39%, mp: 233e235 ^ꢀC; ¹H NMR

(500 MHz, DMSO‑d₆)

d

10.54 (s, 1H), 8.44 (s, 1H), 8.11 (d, J ¼ 8.2 Hz,

2H), 8.09e8.05 (m, 4H), 8.07 (d, J ¼ 15.5 Hz, 1H), 7.98 (d, J ¼ 8.0 Hz,

1H), 7.80 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 7.8 Hz, 1H), 7.58 (t,

J ¼ 8.0 Hz, 1H), 7.46 (s, 1H), 7.41 (d, J ¼ 8.0 Hz, 2H), 3.95 (s, 3H), 2.43

(s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 189.05, 171.49, 165.56,

160.27, 157.20, 144.28, 142.78, 140.40, 138.39, 136.19, 135.39, 130.30,

129.88, 129.25, 128.71, 126.83, 124.39, 123.20, 122.02, 117.61, 101.33,

53.33, 21.68 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþNa]^þ

C₂₈H₂₂N₂NaO₅489.1426; found 489.1427.

that all ﬁnal compounds were conﬁgured E (J_H

7u having Z conﬁguration with coupling constant J_H

_bz 16 Hz) except

a

ꢃH

¼ 7.3 Hz.

a

ꢃHb

5.3.6. Methyl-(E)-5-(3-(4-(3-(4-isobutylphenyl)-3-oxoprop-1-en-

1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7f)

5.3.1. Methyl-(E)-5-(3-(4-(3-oxo-3-phenylprop-1-en-1-yl)

benzamido)phenyl)isoxazole-3-carboxylate (7a)

Off-white solid, Yield 51%, mp: 207e209 ^ꢀC; ¹H NMR (500 MHz,

White solid, Yield 58%, mp: 219e221 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.56 (s, 1H), 8.44 (t, J ¼ 1.8 Hz, 1H), 8.13 (d, J ¼ 8.3 Hz,

DMSO‑d₆)

d

10.55 (s, 1H), 8.44 (s, 1H), 8.20 (d, J ¼ 7.2 Hz, 2H),

2H), 8.11e8.08 (m, 4H), 8.09 (d, J ¼ 15.6 Hz, 1H), 7.98 (dd, J ¼ 8.2,

1.1 Hz, 1H), 7.81 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 8.2 Hz, 1H), 7.57 (t,

J ¼ 8.0 Hz, 1H), 7.48 (s, 1H), 7.39 (d, J ¼ 8.2 Hz, 2H), 3.95 (s, 3H), 2.57

(d, J ¼ 7.2 Hz, 2H), 1.96e1.87 (m, 1H), 0.90 (d, J ¼ 6.6 Hz, 6H) ppm;

8.11e8.07 (m, 4H), 8.09 (d, J ¼ 15.7 Hz, 1H), 7.98 (dd, J ¼ 8.2, 1.1 Hz,

1H), 7.83 (d, J ¼ 15.7 Hz, 1H), 7.74 (d, J ¼ 7.9 Hz, 1H), 7.71 (t,

J ¼ 7.4 Hz,1H), 7.63e7.60 (m, 2H), 7.58 (t, J ¼ 8.0 Hz,1H), 7.46 (s, 1H),

3.95 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 189.66, 171.49,

¹³C NMR (125 MHz, DMSO‑d₆)

d 189.11, 171.48, 165.56, 160.28,

165.55, 160.27, 157.19, 143.16, 140.39, 138.31, 137.88, 136.27, 133.79,

130.30, 129.31, 129.09, 128.73, 126.82, 124.36, 123.19, 122.03, 117.60,

101.33, 53.33 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þ

C₂₇H₂₁N₂O₅453.1450; found 453.1452.

157.20, 147.78, 142.77, 140.40, 138.40, 136.18, 135.69, 130.32, 129.28,

129.13, 128.72, 126.82, 124.41, 123.18, 122.04, 117.56, 101.36, 53.35,

44.95, 30.01, 22.60 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþNa]^þ

C₃₁H₂₈N₂NaO₅531.1896; found 531.1899.

5.3.2. Methyl-(E)-5-(3-(4-(3-(4-chlorophenyl)-3-oxoprop-1-en-1-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7b)

5.3.7. Methyl-(E)-5-(3-(4-(3-(4-nitrophenyl)-3-oxoprop-1-en-1-yl)

benzamido)phenyl)isoxazole-3-carboxylate (7g)

White solid, Yield 53%, mp: 244e246 ^ꢀC; ¹H NMR (500 MHz,

Brown solid, Yield 36%, mp: 137e139 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.56 (s, 1H), 8.44 (s, 1H), 8.23 (d, J ¼ 8.5 Hz, 2H),

DMSO‑d₆)

d

10.57 (s, 1H), 8.44 (s, 1H), 8.41 (s, 4H), 8.12 (d, J ¼ 7.5 Hz,

8.11e8.07 (m, 4H), 8.09 (d, J ¼ 15.7 Hz), 1H 7.98 (d, J ¼ 7.5 Hz, 1H),

7.84 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 7.7 Hz, 1H), 7.68 (d, J ¼ 8.5 Hz,

2H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.48 (s, 1H), 3.95 (s, 3H) ppm; ¹³C NMR

2H), 8.11 (d, J ¼ 15.8 Hz, 1H), 8.10 (d, J ¼ 8.6 Hz, 2H), 7.98 (d,

J ¼ 8.1 Hz, 1H), 7.88 (d, J ¼ 15.7 Hz, 1H), 7.75 (d, J ¼ 7.7 Hz, 1H), 7.58

(t, J ¼ 8.0 Hz, 1H), 7.48 (s, 1H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz,

(125 MHz, DMSO‑d₆) d 188.53, 171.48, 165.50, 160.27, 157.17, 143.60,

DMSO‑d₆)

d 188.88, 171.47, 165.50, 160.27, 157.20, 150.41, 144.63,

140.39, 138.78, 138.19, 136.51, 136.34, 131.00, 130.27, 129.39 (d,

J ¼ 3.4 Hz), 128.72, 126.80, 123.98, 123.18, 122.00, 117.60, 101.31,

53.32 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þC₂₇H₂₀ClN₂O₅

487.1061; found 487.1062.

142.61, 140.36, 138.00, 136.60, 130.47, 130.33, 129.56, 128.75, 126.82,

124.37, 124.10, 123.19, 122.07, 117.59, 101.36, 53.35 ppm; HRMS-

QTOF (ESI): m/z calcd. for [MþNa]^þC₂₇H₁₉N₃NaO₇520.1121;

found 520.1121.

5.3.3. Methyl-(E)-5-(3-(4-(3-(4-ﬂuorophenyl)-3-oxoprop-1-en-1-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7c)

5.3.8. Methyl-(E)-5-(3-(4-(3-(4-methoxyphenyl)-3-oxoprop-1-en-

1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7h)

Off-white solid, Yield 64%, mp: 241e243 ^ꢀC; ¹H NMR (500 MHz,

White solid, Yield 47%, mp: 231e233 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.56 (s, 1H), 8.44 (t, J ¼ 1.7 Hz, 1H), 8.33e8.29 (m, 2H),

DMSO‑d₆)

d

10.54 (s, 1H), 8.44 (s, 1H), 8.21 (d, J ¼ 8.9 Hz, 2H),

8.13e8.08 (m, 5H), 7.98 (dd, J ¼ 8.2, 1.1 Hz, 1H), 7.83 (d, J ¼ 15.6 Hz,

8.11e8.06 (m, 4H), 8.09 (d, J ¼ 15.6 Hz, 1H), 7.98 (dd, J ¼ 8.2, 1.1 Hz,

1H), 7.78 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 8.2 Hz, 1H), 7.58 (t,

J ¼ 8.0 Hz, 1H), 7.47 (s, 1H), 7.12 (d, J ¼ 8.9 Hz, 2H), 3.95 (s, 3H), 3.90

1H), 7.75 (d, J ¼ 8.0 Hz, 1H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.48 (s, 1H), 7.44

(m, 2H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 188.15,

171.47, 165.65 (d, J_CF¼ 252.2 Hz), 165.55, 160.27, 157.21, 143.32,

(s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 187.77, 171.49, 165.57,

140.38, 138.26, 136.30, 134.55 (d, J_CF2.7 Hz), 132.15 (d,

¼

163.86, 160.27, 157.19, 142.29, 140.41, 138.49, 136.07, 131.53, 130.78,

130.29, 129.19, 128.71, 126.81, 124.36, 123.18, 122.01, 117.59, 114.55,

101.33, 56.07, 53.33 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þ

C₂₈H₂₃N₂O₆483.1556; found 483.1565.

J_CF¼ 9.4 Hz), 130.33, 129.38, 128.72, 126.82, 124.10, 123.18, 122.06,

117.56, 116.36 (d, J_CF¼ 21.7 Hz), 101.37, 53.35 ppm; HRMS-QTOF

(ESI): m/z calcd. for [MþH]^þC₂₇H₂₀FN₂O₅471.1356; found 471.1355.

11

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

5.3.9. Methyl-(E)-5-(3-(4-(3-(4-ethoxyphenyl)-3-oxoprop-1-en-1-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7i)

found 493.1185.

Off-white solid, Yield 49%, mp: 213e215 ^ꢀC; ¹H NMR (500 MHz,

5.3.14. Methyl-(E)-5-(3-(4-(3-(3-chlorophenyl)-3-oxoprop-1-en-1-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7n)

DMSO‑d₆)

d

10.56 (s, 1H), 8.44 (t, J ¼ 1.8 Hz, 1H), 8.20 (d, J ¼ 8.9 Hz,

2H), 8.12e8.07 (m, 5H), 7.98 (dd, J ¼ 8.2, 1.1 Hz, 1H), 7.78 (d,

J ¼ 15.6 Hz, 1H), 7.75 (d, J ¼ 7.7 Hz, 1H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.48

(s, 1H), 7.10 (d, J ¼ 8.9 Hz, 2H), 4.17 (q, J ¼ 7.0 Hz, 2H), 3.95 (s, 3H),

White solid, Yield 55%, mp: 224e226 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.57 (s, 1H), 8.44 (s, 1H), 8.25 (s, 1H), 8.16 (d, J ¼ 7.7 Hz,

1H), 8.13e8.08 (m, 5H), 7.98 (d, J ¼ 7.9 Hz, 1H), 7.86 (d, J ¼ 15.6 Hz,

1H), 7.78 (d, J ¼ 8.0 Hz, 1H), 7.75 (d, J ¼ 7.6 Hz, 1H), 7.65 (t, J ¼ 7.8 Hz,

1H), 7.58 (t, J ¼ 7.9 Hz, 1H), 7.48 (s, 1H), 3.95 (s, 3H) ppm; ¹³C NMR

1.38 (t, J ¼ 7.0 Hz, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 187.72,

171.49, 165.57, 163.18, 160.27, 157.20, 142.24, 140.40, 138.50, 136.06,

131.54, 130.62, 130.30, 129.19, 128.70, 126.82, 124.35, 123.18, 122.02,

117.58, 114.91, 101.34, 64.10, 53.34, 14.96 ppm; HRMS-QTOF (ESI):

m/z calcd. for [MþNa]^þC₂₉H₂₄N₂NaO₆519.1532; found 519.1541.

(125 MHz, DMSO‑d₆) d 188.51, 171.52, 165.58, 160.30, 157.22, 143.94,

140.39, 139.77, 138.20, 136.49, 134.39, 133.49, 131.29, 130.32, 129.50,

128.70, 127.72, 126.86, 124.07, 123.27, 122.07, 117.73, 101.33,

53.33 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þC₂₇H₂₀ClN₂O₅

487.1061; found 487.1017.

5.3.10. Methyl-(E)-5-(3-(4-(3-oxo-3-(4-propoxyphenyl)prop-1-en-

1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7j)

Off-white solid, Yield 56%, mp: 221e223 ^ꢀC; ¹H NMR (500 MHz,

5.3.15. Methyl-(E)-5-(3-(4-(3-(3-bromophenyl)-3-oxoprop-1-en-1-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7^ꢀ)

DMSO‑d₆)

d

10.56 (s, 1H), 8.44 (s, 1H), 8.20 (d, J ¼ 8.7 Hz, 2H),

8.12e8.06 (m, 5H), 7.98 (d, J ¼ 8.1 Hz, 1H), 7.78 (d, J ¼ 15.6 Hz, 1H),

7.75 (d, J ¼ 7.7 Hz, 1H), 7.58 (t, J ¼ 7.9 Hz, 1H), 7.48 (s, 1H), 7.11 (d,

J ¼ 8.7 Hz, 2H), 4.07 (t, J ¼ 6.5 Hz, 2H), 3.95 (s, 3H), 1.83e1.74 (m,

2H), 1.01 (t, J ¼ 7.4 Hz, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

Brown solid, Yield 61%, mp: 224e226 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.58 (s, 1H), 8.44 (s, 1H), 8.38 (s, 1H), 8.20 (d, J ¼ 7.8 Hz,

1H), 8.14e8.07 (m, 5H), 7.97 (d, J ¼ 8.1 Hz, 1H), 7.91 (dd, J ¼ 7.9,

1.0 Hz, 1H), 7.85 (d, J ¼ 15.6 Hz, 1H), 7.75 (d, J ¼ 7.7 Hz, 1H), 7.57 (t,

J ¼ 7.9 Hz, 2H), 7.48 (s, 1H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz,

d

187.75, 171.48, 165.60, 163.35, 160.28, 157.20, 142.26, 140.39,

138.50, 136.06, 131.55, 130.61, 130.33, 129.20, 128.70, 126.82, 124.36,

123.20, 122.03, 117.59, 114.95, 101.34, 69.87, 53.35, 22.39,

10.79 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþNa]^þ

C₃₀H₂₆N₂NaO₆533.1689; found 533.1690.

DMSO‑d₆) d 188.37, 171.48, 165.58, 160.28, 157.23, 143.97, 140.37,

139.87, 138.17, 136.45, 131.59, 131.57, 130.36, 129.55, 128.71, 128.19,

128.11, 126.83, 123.90, 123.23, 122.86, 122.10, 117.60, 101.38,

53.37 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþNa]^þ

C₂₇H₁₉BrN₂NaO₅553.0375; found 553.0370.

5.3.11. Methyl-(E)-5-(3-(4-(3-oxo-3-(4-morpholinophenyl)prop-1-

en-1 yl)benzamido)phenyl)isoxazole-3-carboxylate (7k)

5.3.16. Methyl-(E)-5-(3-(4-(3-(3-methoxyphenyl)-3-oxoprop-1-en-

1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7p)

Yellow solid, Yield 59%, mp: 225e227 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.55 (s, 1H), 8.44 (s, 1H), 8.12 (d, J ¼ 9.0 Hz, 2H),

White solid, Yield 56%, mp: 186e188 ^ꢀC; ¹H NMR (500 MHz,

8.11e8.04 (m, 5H), 7.98 (d, J ¼ 8.1 Hz, 1H), 7.74 (d, J ¼ 15.5 Hz, 1H),

7.74 (d, J ¼ 7.7 Hz, 1H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.48 (s, 1H), 7.06 (d,

J ¼ 9.0 Hz, 2H), 3.95 (s, 3H), 3.80e3.73 (m, 4H), 3.39e3.35 (m, 4H)

DMSO‑d₆) d 10.55 (s, 1H), 8.44 (s, 1H), 8.11e8.05 (m, 4H), 8.07 (d,

J ¼ 15.7 Hz, 1H), 7.98 (d, J ¼ 8.1 Hz, 1H), 7.82 (d, 15.5 Hz, 1H), 7.82 (s,

1H), 7.74 (d, J ¼ 7.7 Hz, 1H), 7.66 (s, 1H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.53

(t, J ¼ 7.9 Hz, 1H), 7.47 (s, 1H), 7.28 (dd, J ¼ 8.1, 2.2 Hz, 1H), 3.95 (s,

ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 186.88, 171.49, 165.61, 160.28,

157.21, 154.67, 141.43, 140.42, 138.71, 135.87, 131.18, 130.33, 129.11,

128.70, 127.79, 126.81, 124.54, 123.18, 122.03, 117.54, 113.52, 101.38,

66.33, 53.36, 47.15 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþNa]^þ

C₃₁H₂₇N₃NaO₆560.1798; found 560.1797.

3H), 3.88 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 189.40,

171.49, 165.56, 160.28, 160.09, 157.20, 143.24, 140.39, 139.33, 138.30,

136.28, 130.46, 130.31, 129.37, 128.71, 126.83, 124.41, 123.20, 122.04,

121.65, 119.81, 117.61, 113.63, 101.34, 55.89, 53.33 ppm; HRMS-QTOF

(ESI): m/z calcd. for [MþH]^þC₂₈H₂₃N₂O₆505.1376; found 505.1377.

5.3.12. Methyl(E)-5-(3-(4-(3-(4-hydroxyphenyl)-3-oxoprop-1-en-

1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7l)

5.3.17. Methyl-(E)-5-(3-(4-(3-(3-nitrophenyl)-3-oxoprop-1-en-1-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7q)

Yellow solid, Yield 38%, mp: 280e282 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.55 (s, 1H), 10.47 (s, 1H), 8.44 (s, 1H), 8.12 (d,

Brown solid, Yield 23%, mp: 242e244 ^ꢀC; ¹H NMR (500 MHz,

J ¼ 8.1 Hz, 2H), 8.10e8.03 (m, 5H), 7.98 (d, J ¼ 7.9 Hz, 1H), 7.76 (d,

J ¼ 15.9 Hz,1H), 7.75 (m,1H), 7.58 (t, J ¼ 7.7 Hz,1H), 7.48 (s,1H), 6.93

(d, J ¼ 8.0 Hz, 2H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

DMSO‑d₆)

d

10.59 (s,1H), 8.88 (s,1H), 8.65 (d, J ¼ 7.7 Hz,1H), 8.53 (d,

J ¼ 8.1 Hz, 1H), 8.44 (s, 1H), 8.18 (d, J ¼ 15.6 Hz, 1H), 8.14 (d,

J ¼ 8.2 Hz, 2H), 8.10 (d, J ¼ 8.3 Hz, 2H), 7.97 (d, J ¼ 8.2 Hz, 1H), 7.92

(d,15.7 Hz,1H), 7.91 (s,1H), 7.75 (d, J ¼ 7.7 Hz,1H), 7.58 (t, J ¼ 8.0 Hz,

1H), 7.48 (s, 1H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d

187.54, 171.50, 165.60, 162.85, 160.28, 157.22, 141.88, 140.42,

138.60, 136.00, 131.79, 130.32, 129.49, 129.12, 128.71, 126.84, 124.52,

123.21, 122.04, 117.62, 115.93, 101.35, 53.34 ppm; HRMS-QTOF (ESI):

m/z calcd. for [MþH]^þC₂₇H₂₁N₂O₆469.1400; found 469.1359.

d

188.11,171.47, 165.57,160.28, 157.22,148.74,144.64,140.35, 139.06,

138.05, 136.60, 135.29, 131.15, 130.36, 129.64, 128.74, 127.99, 126.83,

123.77, 123.43, 123.23, 122.10, 117.61, 101.38, 53.37 ppm; HRMS-

QTOF (ESI): m/z calcd. for [MþNa]^þC₂₇H₁₉N₃NaO₇520.1121;

found 520.1113.

5.3.13. Methyl-(E)-5-(3-(4-(3-(3-ﬂuorophenyl)-3-oxoprop-1-en-1-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7 m)

Off-white solid, Yield 69%, mp: 232e234 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.57 (s, 1H), 8.44 (s, 1H), 8.14e8.08 (m, 4H), 8.09 (d,

5.3.18. Methyl-(E)-5-(3-(4-(3-oxo-3-(3-(triﬂuoromethyl)phenyl)

prop-1-en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7r)

Off-white solid, Yield 44%, mp: 157e159 ^ꢀC; ¹H NMR (500 MHz,

J ¼ 15.4 Hz, 1H), 8.06 (d, J ¼ 7.8 Hz, 1H), 8.02 (dd, J ¼ 9.6, 2.0 Hz, 1H),

7.98 (d, J ¼ 8.1 Hz, 1H), 7.85 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 7.7 Hz,

1H), 7.67 (td, J ¼ 7.9, 6.1 Hz, 1H), 7.60e7.54 (m, 2H), 7.47 (s, 1H), 3.95

DMSO‑d₆)

d

10.59 (s, 1H), 8.52 (d, J ¼ 7.9 Hz,1H), 8.49 (s, 1H), 8.44 (s,

(s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d

188.48, 171.49, 165.56,

1H), 8.19 (d, J ¼ 15.6 Hz, 1H), 8.14 (d, J ¼ 8.4 Hz, 2H), 8.11e8.07 (m,

3H), 7.97 (d, J ¼ 8.1 Hz, 1H), 7.90 (d, J ¼ 15.6 Hz, 1H), 7.86 (d,

J ¼ 7.8 Hz, 1H), 7.75 (d, J ¼ 7.7 Hz, 1H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.49 (s,

162.87 (d, J_CF¼ 243.6 Hz), 160.28, 157.22, 143.87, 140.39, 140.10 (d,

J_CF¼ 6.8 Hz), 138.18, 136.43, 131.52 (d, J_CF¼ 7.9 Hz), 130.33, 129.49,

128.73, 126.83, 125.30, 123.99, 123.22, 122.07, 120.72 (d,

J_CF¼ 22.1 Hz), 117.62, 115.64 (d, J_CF¼ 22.8 Hz), 101.36, 53.35 ppm;

HRMS-QTOF (ESI): m/z calcd. for [MþH]^þC₂₇H₂₀FN₂O₅493.1176;

1H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 188.49,

171.47, 165.55, 160.27, 157.20, 144.24, 140.36, 138.55, 138.11, 136.49,

133.08, 130.65, 130.33, 130.17 (q, J_CF31.9 Hz), 130.10 (q,

¼

12

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

J_CF¼ 3.4 Hz), 129.58, 128.71, 126.81, 125.47 (q, J_CF¼ 3.4 Hz), 124.39

(q, J_CF¼ 272.8 Hz), 123.77, 123.20, 122.06, 117.57, 101.36, 53.35 ppm;

HRMS-QTOF (ESI): m/z calcd. for [MþH]^þC₂₈H₂₀F₃N₂O₅521.1324;

found 521.1327.

J ¼ 7.9 Hz, 1H), 7.04 (d, J ¼ 8.1 Hz, 1H), 3.95 (s, 3H) ppm; ¹³C NMR

(125 MHz, DMSO‑d₆) d 193.96, 171.47, 165.53, 162.25, 160.27, 157.21,

143.78, 140.36, 138.06, 136.96, 136.51, 131.47, 130.34, 129.56, 128.76,

126.82, 124.24, 123.18, 122.07, 121.35, 119.73, 118.23, 117.56, 101.38,

53.36 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þC₂₇H₂₁N₂O₆

469.1400; found 469.1404.

5.3.19. Methyl-(E)-5-(3-(4-(3-(2-chlorophenyl)-3-oxoprop-1-en-1-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7s)

White solid, Yield 67%, mp: 167e169 ^ꢀC; ¹H NMR (500 MHz,

5.3.24. Methyl-(E)-5-(3-(4-(3-(3,4-dimethoxyphenyl)-3-oxoprop-

1-en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7x)

Light yellow solid, Yield 39%, mp: 179e181 ^ꢀC; ¹H NMR

DMSO‑d₆)

d

10.56 (s, 1H), 8.43 (t, J ¼ 1.8 Hz, 1H), 8.06 (d, J ¼ 8.4 Hz,

2H), 7.98 (d, J ¼ 8.5 Hz, 2H), 7.97e7.92 (m, 1H), 7.76e7.71 (m, 1H),

7.65e7.61 (m, 2H), 7.59 (dd, J ¼ 8.0, 1.7 Hz, 1H), 7.57 (t, J ¼ 8.0 Hz,

1H), 7.53 (td, J ¼ 7.3, 1.5 Hz, 1H), 7.52 (d, J ¼ 16.1 Hz, 1H), 7.47 (s, 1H),

7.45 (d, J ¼ 16.2 Hz, 1H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz,

(500 MHz, DMSO‑d₆)

d

10.55 (s, 1H), 8.44 (t, J ¼ 1.8 Hz, 1H), 8.10 (m,

5H), 7.97 (dt, J ¼ 8.5, 2.0 Hz, 2H), 7.79 (d, J ¼ 15.6 Hz, 1H), 7.74 (d,

J ¼ 7.7 Hz, 1H), 7.64 (d, J ¼ 2.0 Hz, 1H), 7.57 (t, J ¼ 8.0 Hz, 1H), 7.47 (s,

1H), 7.14 (d, J ¼ 8.5 Hz, 1H), 3.95 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H)

DMSO‑d₆)

d 193.59, 171.47, 165.43, 160.27, 157.21, 145.21, 140.36,

138.92, 137.72, 136.59, 132.52, 130.65, 130.40, 130.33, 129.89, 129.37,

128.79, 128.37, 127.94, 126.82, 123.17, 122.07, 117.55, 101.37,

53.35 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þC₂₇H₂₀ClN₂O₅

487.1061; found 487.1024.

ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 187.75, 171.48, 165.59, 160.28,

157.20, 153.90, 149.35, 142.24, 140.39, 138.51, 136.06, 130.82, 130.32,

129.24, 128.70, 126.82, 124.27, 124.12, 123.18, 122.04, 117.57, 111.38,

111.22, 101.35, 56.29, 56.10, 53.35 ppm; HRMS-QTOF (ESI): m/z

calcd. for [MþH]^þC₂₉H₂₅N₂O₇513.1662; found 513.1659.

5.3.20. Methyl-(E)-5-(3-(4-(3-(2-bromophenyl)-3-oxoprop-1-en-

1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7t)

Off-white solid, Yield 42%, mp: 159e161 ^ꢀC; ¹H NMR (500 MHz,

5.3.25. Methyl-(E)-5-(3-(4-(3-(2,4-dichlorophenyl)-3-oxoprop-1-

en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7y)

DMSO‑d₆)

d

10.57 (s,1H), 8.43 (s,1H), 8.06 (d, J ¼ 8.4 Hz, 2H), 7.98 (d,

J ¼ 8.3 Hz, 2H), 7.97e7.95 (m, 1H), 7.78 (d, J ¼ 7.9 Hz, 1H), 7.74 (d,

J ¼ 7.7 Hz,1H), 7.60 (m, 1H), 7.57 (t, J ¼ 7.7 Hz, 2H), 7.50 (m, 1H), 7.49

(d, J ¼ 16.1 Hz, 1H), 7.48 (s, 1H), 7.42 (d, J ¼ 16.2 Hz, 1H), 3.94 (s, 3H)

White solid, Yield 62%, mp: 181e183 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.56 (s, 1H), 8.43 (t, J ¼ 1.6 Hz, 1H), 8.06 (d, J ¼ 8.4 Hz,

2H), 7.99 (d, J ¼ 8.4 Hz, 2H), 7.96 (d, J ¼ 9.1 Hz, 1H), 7.82 (d,

J ¼ 1.9 Hz, 1H), 7.74 (d, J ¼ 7.8 Hz, 1H), 7.68 (d, J ¼ 8.2 Hz, 1H), 7.62

(dd, J ¼ 8.2, 1.9 Hz, 1H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.54 (d, J ¼ 16.0 Hz,

1H), 7.47 (s, 1H), 7.43 (d, J ¼ 16.2 Hz, 1H), 3.95 (s, 3H) ppm; ¹³C NMR

ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 194.55, 171.47, 165.43, 160.28,

157.21, 145.44, 140.96, 140.35, 137.71, 136.58, 133.71, 132.50, 130.34,

129.75, 129.38, 128.80, 128.39, 128.22, 126.81, 123.17, 122.08, 119.11,

117.55, 101.38, 53.36 ppm; HRMS-QTOF (ESI): m/z calcd. for

[M þ Hþ2]^þC₂₇H₂₀BrN₂O₅533.0556; found 533.0540.

(125 MHz, DMSO‑d₆) d 192.79, 171.47, 165.42, 160.27, 157.19, 145.82,

140.34, 137.67, 137.64, 136.66, 136.29, 131.79, 131.31, 130.31, 130.22,

129.41, 128.78, 128.17, 128.15, 126.81, 123.16, 122.06, 117.57, 101.34,

53.34 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þ

C₂₇H₁₉Cl₂N₂O₅521.0671; found 521.0674.

5.3.21. Methyl (Z)-5-(3-(4-(3-oxo-3-(o-tolyl)prop-1-en-1-yl)

benzamido)phenyl)isoxazole-3-carboxylate (7u)

White solid, Yield 48%, mp: 185e187 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.53 (s, 1H), 8.43 (m, 1H), 8.07 (d, J ¼ 8.4 Hz, 2H), 7.99

5.3.26. Methyl-(E)-5-(3-(4-(3-(2,4-dimethoxyphenyl)-3-oxoprop-

1-en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7z)

White solid, Yield 28%, mp: 204e206 ^ꢀC; ¹H NMR (500 MHz,

(d, J ¼ 8.5 Hz, 2H), 7.96 (m, 1H), 7.75e7.72 (m, 1H), 7.70 (d, J ¼ 7.1 Hz,

1H), 7.58 (d, J ¼ 8.1 Hz, 1H), 7.57e7.55 (m, 2H), 7.50e7.46 (m, 1H),

7.46 (s, 1H), 7.39 (s, 1H), 7.37 (d, J ¼ 7.5 Hz, 1H), 3.95 (s, 3H), 2.42 (s,

DMSO‑d₆)

d

10.54 (s, 1H), 8.44 (s, 1H), 8.06 (d, J ¼ 8.3 Hz, 2H), 7.96

3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 195.23, 171.48, 165.50,

(dd, J ¼ 8.1,1.2 Hz,1H), 7.92 (d, J ¼ 8.3 Hz, 2H), 7.74 (d, J ¼ 7.7 Hz,1H),

7.69 (d, J ¼ 15.9 Hz, 1H), 7.66 (d, J ¼ 8.6 Hz, 1H), 7.62 (d, J ¼ 15.8 Hz,

1H), 7.57 (t, J ¼ 8.0 Hz, 1H), 7.48 (s, 1H), 6.73 (d, J ¼ 2.2 Hz, 1H), 6.68

160.28, 157.21, 143.80, 140.39, 138.96, 138.08, 137.13, 136.29, 131.80,

131.41, 130.33, 129.23, 129.00, 128.75, 128.40, 126.83, 126.29, 123.17,

122.06, 117.57, 101.37, 53.36, 20.54 ppm; HRMS-QTOF (ESI): m/z

calcd. for [MþH]^þC₂₈H₂₃N₂O₅467.1607; found 467.1573.

(dd, J ¼ 8.6, 2.2 Hz,1H), 3.94 (d, J ¼ 7.2 Hz, 6H), 3.88 (s, 3H) ppm; ¹³

C

NMR (125 MHz, DMSO‑d₆)

d 189.67, 171.49, 165.62, 164.66, 160.89,

160.28, 157.21, 140.43, 140.27, 138.64, 135.95, 132.62, 130.32, 129.40,

128.82, 128.77, 126.82, 123.13, 122.01, 121.70, 117.51, 106.60, 101.36,

99.15, 56.52, 56.13, 53.34 ppm; HRMS-QTOF (ESI): m/z calcd. for

[MþH]^þC₂₉H₂₅N₂O₇513.1662; found 513.1617.

5.3.22. Methyl-(E)-5-(3-(4-(3-(2-methoxyphenyl)-3-oxoprop-1-

en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7v)

White solid, Yield 44%, mp: 199e201 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.54 (s, 1H), 8.44 (s, 1H), 8.06 (d, J ¼ 8.4 Hz, 2H), 7.96

(dd, J ¼ 8.3 Hz, 0.9 Hz, 1H), 7.93 (d, J ¼ 8.4 Hz, 2H), 7.74 (d, J ¼ 7.9 Hz,

1H), 7.61e7.53 (m, 4H), 7.60 (d, J ¼ 15.9 Hz, 1H), 7.46 (s, 1H), 7.23 (d,

J ¼ 8.3 Hz, 1H), 7.10 (t, J ¼ 7.4 Hz, 1H), 3.95 (s, 3H), 3.90 (s, 3H) ppm;

5.3.27. Methyl-(E)-5-(3-(4-(3-(2,5-diﬂuorophenyl)-3-oxoprop-1-

en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7aa)

White solid, Yield 31%, mp: 230e232 ^ꢀC; ¹H NMR (500 MHz,

¹³C NMR (125 MHz, DMSO‑d₆)

d 192.54, 171.49, 165.57, 160.28,

158.36, 157.21, 141.57, 140.40, 138.31, 136.18, 133.75, 130.32, 130.08,

129.20, 129.13, 128.96, 128.81, 126.82, 123.15, 122.03, 121.09, 117.54,

112.91, 101.35, 56.37, 53.34 ppm; HRMS-QTOF (ESI): m/z calcd. for

[MþH]^þC₂₈H₂₃N₂O₆483.1556; found 483.1556.

DMSO‑d₆)

d

10.55 (s, 1H), 8.43 (s, 1H), 8.08 (d, J ¼ 8.3 Hz, 2H), 8.01

(d, J ¼ 8.4 Hz, 2H), 7.97 (d, J ¼ 8.2 Hz, 1H), 7.75 (s, 1H), 7.74 (d,

J ¼ 15.7 Hz, 1H), 7.68 (dd, J ¼ 8.6, 5.4, 3.3 Hz, 1H), 7.61 (dd, J ¼ 16.0,

1.9 Hz, 1H), 7.59e7.53 (m, 2H), 7.49 (dd, J ¼ 9.6, 4.2 Hz, 1H), 7.46 (s,

1H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 188.27,

5.3.23. Methyl-(E)-5-(3-(4-(3-(2-hydroxyphenyl)-3-oxoprop-1-en-

1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7w)

171.48, 165.49, 160.27, 158.49 (dd, J_CF¼ 241.8, 1.4 Hz), 157.20, 156.83

(dd, J_CF¼ 247.9, 1.7 Hz),144.51, 140.36, 137.77, 136.65, 130.32, 129.37,

128.80,128.33 (dd, J_CF¼ 15.9, 6.8 Hz),127.22 (d, J_CF¼ 3.5 Hz),126.83,

123.19, 122.06, 121.32 (dd, J_CF¼ 24.4, 9.3 Hz), 119.12 (dd, J_CF¼ 25.6,

8.4 Hz), 117.60, 117.02 (dd, J_CF¼ 25.3, 3.0 Hz), 101.34, 53.33 ppm;

HRMS-QTOF (ESI): m/z calcd. for [MþNa]^þC₂₇H₁₈F₂N₂NaO₅

511.1081; found 511.1087.

Yellow solid, Yield 46%, mp: 230e232 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆) d 12.41 (s, 1H), 10.58 (s, 1H), 8.44 (t, 1.7 Hz, 1H), 8.29 (dd,

J ¼ 8.0, 1.1 Hz, 1H), 8.18 (d, J ¼ 15.6 Hz, 1H), 8.13e8.08 (m, 4H), 7.98

(dd, J ¼ 8.2, 1.1 Hz, 1H), 7.91 (d, J ¼ 15.6 Hz, 1H), 7.75 (d, J ¼ 7.7 Hz,

1H), 7.62e7.57 (m, 1H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.49 (s, 1H), 7.05 (d,

13

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

5.3.28. Methyl-(E)-5-(3-(4-(3-(2,4-diﬂuorophenyl)-3-oxoprop-1-

en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7 ab)

White solid, Yield 32%, mp: 213e215 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.55 (s, 1H), 8.44 (s, 1H), 8.11e8.05 (m, 4H), 8.08 (d,

J ¼ 15.5 Hz, 1H), 7.98 (d, J ¼ 8.1 Hz, 1H), 7.92 (dd, J ¼ 8.2, 1.6 Hz, 1H),

7.78 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 7.8 Hz, 1H), 7.70 (d, J ¼ 1.5 Hz,

1H), 7.57 (t, J ¼ 8.0 Hz, 1H), 7.47 (d, J ¼ 6.8 Hz, 1H), 7.12 (d, J ¼ 8.2 Hz,

1H), 6.19 (s, 2H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

DMSO‑d₆)

d

10.56 (s, 1H), 8.43 (s, 1H), 8.08 (d, J ¼ 8.3 Hz, 2H), 8.00

(d, J ¼ 8.4 Hz, 2H), 7.96 (d, J ¼ 8.5 Hz, 2H), 7.74 (d, J ¼ 15.8 Hz, 1H),

7.74 (d, J ¼ 7.8 Hz, 1H), 7.63 (dd, J ¼ 15.9, 2.2 Hz, 1H), 7.57 (t,

J ¼ 8.0 Hz, 1H), 7.51e7.45 (m, 2H), 7.30 (td, J ¼ 8.5, 2.3 Hz, 1H), 3.95

d

187.34, 171.48, 165.55, 160.27, 157.19, 152.21, 148.55, 142.55,

140.40, 138.44, 136.10, 132.57, 130.29, 129.25, 128.68, 126.81, 125.79,

124.21, 123.16, 122.01, 117.57, 108.66, 108.44, 102.62, 101.33,

53.33 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þC₂₈H₂₁N₂O₇

497.1349; found 497.1348.

(s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 187.94, 171.48, 166.23,

165.18 (dd, J_CF¼ 260.3, 7.9 Hz), 161.69 (dd, J_CF¼ 255.6, 12.3 Hz),

160.27, 157.20, 143.81, 140.37, 137.87, 136.52, 133.21 (dd, J_CF¼ 11.3,

3.6 Hz), 130.32, 129.27, 128.81, 127.41, 126.82, 123.96 (dd, J_CF¼ 13.2,

2.9 Hz), 123.16, 122.05, 117.55, 112.83 (dd, J_CF¼ 21.1, 3.8 Hz), 105.56

(t, J_CF¼ 26.8 Hz), 101.35, 53.34 ppm; HRMS-QTOF (ESI): m/z calcd.

for [MþH]^þC₂₇H₁₉F₂N₂O₅489.1262; found 489.1219.

5.3.33. Methyl-(E)-5-(3-(4-(3-oxo-3-(thiophen-2-yl)prop-1-en-1-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7 ag)

Beige solid, Yield 44%, mp: 252e254 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d 10.55 (s, 1H), 8.44 (s, 1H), 8.41e8.39 (m, 1H), 8.11 (dd,

5.3.29. Methyl-(E)-5-(3-(4-(3-(4-ﬂuoro-3-methylphenyl)-3-

oxoprop-1-en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate

(7ac)

J ¼ 4.9, 0.8 Hz, 1H), 8.09 (m, 4H), 8.03 (d, J ¼ 15.6 Hz, 1H), 7.98 (dd,

J ¼ 8.2, 1.0 Hz, 1H), 7.81 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 7.7 Hz, 1H),

7.58 (t, J ¼ 8.0 Hz, 1H), 7.47 (s, 1H), 7.36 (dd, J ¼ 4.8, 3.9 Hz, 1H), 3.95

White solid, Yield 36%, mp: 223e225 ^ꢀC; ¹H NMR (500 MHz,

(s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 182.02, 171.48, 165.56,

DMSO‑d₆)

d

10.54 (s, 1H), 8.43 (s, 1H), 8.19 (d, J ¼ 7.3 Hz, 1H),

160.28, 157.21, 145.82, 142.32, 140.38, 138.16, 136.40, 136.31, 134.55,

130.32,129.45,129.33,128.73,126.83,124.20, 123.20,122.05,117.59,

101.35, 53.34 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þ

C₂₅H₁₉N₂O₅S 459.1015; found 459.1015.

8.10e8.05 (m, 5H), 8.09 (d, J ¼ 15.5 Hz, 1H), 7.98 (d, J ¼ 8.0 Hz, 1H),

7.82 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 7.7 Hz, 1H), 7.58 (t, J ¼ 8.0 Hz,

1H), 7.46 (s, 1H), 7.36 (t, J ¼ 9.0 Hz, 1H), 3.95 (s, 3H), 2.37 (s, 3H)

ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 188.17, 171.48, 165.54, 164.24

(d, J_CF¼ 251.5 Hz), 160.27, 157.20, 143.13, 140.39, 138.30, 136.27,

134.25 (d, J_CF¼ 2.9 Hz), 133.07 (d, J_CF¼ 6.4 Hz), 130.31, 129.44 (d,

J_CF¼ 9.3 Hz), 129.33, 128.70, 126.82, 125.55 (d, J_CF¼ 17.7 Hz), 124.12,

123.18, 122.04, 117.58, 115.93 (d, J_CF¼ 22.8 Hz), 101.34, 53.34, 14.59

(d, J_CF¼ 3.0 Hz) ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þ

C₂₈H₂₂FN₂O₅485.1513; found 485.1470.

5.3.34. Methyl-(E)-5-(3-(4-(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)

benzamido)phenyl)isoxazole-3-carboxylate (7 ah)

Off-white solid, Yield 37%, mp: 199e201 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.56 (s,1H), 9.38 (s,1H), 8.86 (d, J ¼ 3.6 Hz,1H), 8.51 (d,

J ¼ 7.9 Hz, 1H), 8.44 (s, 1H), 8.14e8.08 (m, 5H), 7.98 (d, J ¼ 8.1 Hz,

1H), 7.87 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 7.7 Hz, 1H), 7.65 (dd, J ¼ 7.8,

4.8 Hz, 1H), 7.58 (t, J ¼ 7.9 Hz, 1H), 7.47 (s, 1H), 3.95 (s, 3H) ppm; ¹³

C

5.3.30. Methyl-(E)-5-(3-(4-(3-(4-methyl-3-nitrophenyl)-3-

oxoprop-1-en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate

(7ad)

NMR (125 MHz, DMSO‑d₆) d 188.97, 171.48, 165.55, 160.28, 157.22,

153.96, 150.27, 143.86, 140.37, 138.10, 136.54, 136.49, 133.16, 130.35,

129.53, 128.74, 126.83, 124.48, 124.12, 123.21, 122.08, 117.60, 101.37,

53.36 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þC₂₆H₂₀N₃O₅

454.1403; found 454.1405.

Yellow solid, Yield 33%, mp: 233e235 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.57 (s, 1H), 8.70 (d, J ¼ 1.7 Hz, 1H), 8.46e8.41 (m, 2H),

8.15 (d, J ¼ 15.5 Hz, 1H), 8.12 (d, J ¼ 8.2 Hz, 2H), 8.09 (d, J ¼ 8.5 Hz,

2H), 7.97 (d, J ¼ 8.2 Hz, 1H), 7.89 (d, J ¼ 15.6 Hz, 1H), 7.75 (d,

J ¼ 8.2 Hz, 2H), 7.57 (t, J ¼ 8.0 Hz, 1H), 7.47 (s, 1H), 3.95 (s, 3H), 2.62

5.3.35. Methyl-(E)-5-(3-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl)

prop-1-en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate (7ai)

White solid, Yield 30%, mp: 226e228 ^ꢀC; ¹H NMR (500 MHz,

(s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 187.52, 171.47, 165.50,

160.27, 157.19, 149.87, 144.23, 140.36, 138.23, 138.08, 136.74, 136.48,

133.91, 133.12, 130.30, 129.55, 128.70, 126.81, 124.64, 123.59, 123.18,

122.04, 117.57, 101.34, 53.34, 19.99 ppm; HRMS-QTOF (ESI): m/z

calcd. for [MþNa]^þC₂₈H₂₁N₃NaO₇534.1277; found 534.1280.

DMSO‑d₆)

d

10.57 (s, 1H), 8.44 (t, J ¼ 1.8 Hz, 1H), 8.11 (d, J ¼ 8.7 Hz,

2H), 8.11e8.09 (m, 1H), 8.09 (d, J ¼ 8.2 Hz, 2H), 7.97 (dd, J ¼ 8.2,

1.1 Hz, 1H), 7.83 (d, J ¼ 15.6 Hz, 1H), 7.76e7.73 (m, 1H), 7.58 (t,

J ¼ 8.0 Hz, 1H), 7.48e7.44 (m, 3H), 3.94 (s, 3H), 3.93 (s, 6H), 3.79 (s,

3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 188.36, 171.49, 165.59,

5.3.31. Methyl-(E)-5-(3-(4-(3-(4-methoxy-3-nitrophenyl)-3-

oxoprop-1-en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate

(7ae)

160.28, 157.22, 153.45, 143.02, 142.73, 140.39, 138.39, 136.25, 133.27,

130.33,129.39,128.68,126.84,124.25, 123.22,122.06,117.62,106.88,

101.35, 60.70, 56.78, 53.34 ppm; HRMS-QTOF (ESI): m/z calcd. for

[MþH]^þC₃₀H₂₇N₂O₈543.1767; found 543.1723.

Brown solid, Yield 37%, mp: 214e216 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.55 (s, 1H), 8.71 (d, J ¼ 2.2 Hz, 1H), 8.49 (dd, J ¼ 8.9,

2.2 Hz, 1H), 8.43 (t, J ¼ 1.8 Hz, 1H), 8.14 (d, J ¼ 15.6 Hz, 1H), 8.11 (d,

J ¼ 8.9 Hz, 2H), 8.09 (d, J ¼ 8.7 Hz, 2H), 7.97 (dd, J ¼ 8.2, 1.2 Hz, 1H),

7.86 (d, J ¼ 15.6 Hz, 1H), 7.74 (d, J ¼ 8.0 Hz, 1H), 7.58 (d, J ¼ 8.0 Hz,

1H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.56 (d, J ¼ 9.0 Hz, 1H), 7.46 (s, 1H), 4.06

5.4. Synthesis of 4-Chlorochalcone acid (6 b) linked phenyl

isoxazole and triazole esters (7aj-7al)

5.4.1. Synthesis of ethyl-(E)-5-(3-(4-(3-(4-chlorophenyl)-3-

oxoprop-1-en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate

(7aj)

Compound 7aj was synthesized from 8 (synthesis described in

supporting information) and 6 b by employing general procedure

B; White solid, Yield 72%, mp: 235e237 ^ꢀC; ¹H NMR (500 MHz,

(s, 3H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 186.62,

171.48, 165.56, 160.27, 157.21, 155.75, 143.67, 140.37, 139.92, 138.21,

136.40, 135.01, 130.32, 130.15, 129.47, 128.69, 126.83, 125.89, 123.59,

123.22, 122.05, 117.63, 115.00, 101.34, 57.81, 53.34 ppm; HRMS-

QTOF (ESI): m/z calcd. for [MþNa]^þC₂₈H₂₁N₃NaO₈550.1226;

found 550.1226.

DMSO‑d₆)

d

10.57 (s, 1H), 8.44 (s, 1H), 8.23 (d, J ¼ 8.4 Hz, 2H),

8.14e8.06 (m, 4H), 8.10 (d, J ¼ 15.4 Hz, 1H), 7.98 (d, J ¼ 7.8 Hz, 1H),

7.84 (d, J ¼ 15.6 Hz, 1H), 7.75 (d, J ¼ 7.7 Hz, 1H), 7.68 (d, J ¼ 8.4 Hz,

2H), 7.58 (t, J ¼ 8.0 Hz, 1H), 7.47 (s, 1H), 4.42 (q, J ¼ 7.1 Hz, 2H), 1.36

5.3.32. Methyl-(E)-5-(3-(4-(3-(benzo[d] [1,3]dioxol-5-yl)-3-

oxoprop-1-en-1-yl)benzamido)phenyl)isoxazole-3-carboxylate

(7af)

(t, J ¼ 7.1 Hz, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 188.55,

Brown solid, Yield 29%, mp: 237e239 ^ꢀC; ¹H NMR (500 MHz,

171.44, 165.53, 159.81, 157.40, 143.63, 140.37, 138.80, 138.21, 136.51,

14

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

136.36, 131.04, 130.33, 129.44, 128.72, 126.84, 124.01, 123.16, 122.05,

117.57, 101.35, 62.42, 14.44 ppm; HRMS-QTOF (ESI): m/z calcd. for

[MþNa]^þC₂₈H₂₁ClN₂NaO₅523.1037; found 523.1035.

[MþNa]^þC₂₇H₁₉ClN₂NaO₆525.0829; found 525.0828.

5.5.3. Synthesis of methyl-(E)-5-(3-(3-(4-chlorophenyl)acrylamido)

phenyl)isoxazole-3-carboxylate (7ao)

5.4.2. Synthesis of methyl-(E)-5-(4-(4-(3-(4-chlorophenyl)-3-

oxoprop-1-en-1 yl)benzamido)phenyl)isoxazole-3-carboxylate

(7ak)

2 upon coupling with 4-Chloro cinnamic acid 17 (synthesized by

previously reported procedure [34]) by general procedure B yielded

7ao. White solid, Yield 66%, mp: 187e189 ^ꢀC; ¹H NMR (500 MHz,

Intermediate 10 (synthesis detailed in supporting information)

was treated by general procedure B with 6 b as starting material to

afford 7ak as beige solid, Yield 53%, mp: 257e259 ^ꢀC; ¹H NMR

DMSO‑d₆)

d

10.50 (s, 1H), 8.34 (s, 1H), 7.81 (d, J ¼ 8.1 Hz, 1H), 7.69 (t,

J ¼ 8.2 Hz, 3H), 7.63 (d, J ¼ 15.7 Hz, 1H), 7.54 (t, J ¼ 8.1 Hz, 3H), 7.47

(s, 1H), 6.85 (d, J ¼ 15.7 Hz, 1H), 3.94 (s, 3H) ppm; ¹³C NMR

(500 MHz, DMSO‑d₆)

d

10.62 (s, 1H), 8.23 (dt, J ¼ 8.6, 2.3, 1.9 Hz, 2H),

(125 MHz, DMSO‑d₆) d 171.43, 164.14, 160.26, 157.16, 140.49, 139.76,

8.10e8.08 (m, 3H), 8.06 (d, J ¼ 8.8 Hz, 2H), 8.01 (d, J ¼ 9.0 Hz, 2H),

7.98 (d, J ¼ 9.0 Hz, 2H), 7.83 (d, J ¼ 15.6 Hz, 1H), 7.68 (dt, J ¼ 8.6, 2.3,

1.9 Hz, 2H), 7.43 (s, 1H), 3.94 (s, 3H) ppm; ¹³C NMR (125 MHz,

134.82, 134.02, 130.47, 129.96, 129.55, 126.94, 123.20, 122.05, 121.71,

116.29, 101.36, 53.33 ppm; HRMS-QTOF (ESI): m/z calcd. for

[MþH]^þC₂₀H₁₆ClN₂O₄383.0799; found 383.0827.

DMSO‑d₆)

d 188.55, 171.49, 165.67, 160.38, 157.09, 143.62, 141.99,

138.80, 138.22, 136.50, 136.42, 131.03, 129.43, 129.40, 128.79, 127.04,

124.00, 121.70, 120.92, 100.36, 53.31 ppm; HRMS-QTOF (ESI): m/z

calcd. for [MþH]^þC₂₇H₂₀ClN₂O₅487.1061; found 487.1061.

5.6. Antibiotic susceptibility testing against mycobacteria

Antimycobacterial susceptibility testing was carried out on

newly synthesized compounds by using broth microdilution assay

[38]. 10 mg/mL stock solutions of test and control compounds were

prepared in DMSO and stored in ꢃ20 ^ꢀC. Mycobacterial cultures

were inoculated in Middlebrook 7H9 enriched (Difco, Becton, NJ,

USA) media supplemented with 10% ADC-Tween-80 (Bovine Serum

Albumin, Dextrose, 0.2% glycerol and 0.05% Tween-80) and OD₆₀₀

of cultures was measured, followed by dilution to achieve ~10⁶CFU/

mL. The newly synthesized compounds were tested from 64 to

5.4.3. Synthesis of ethyl (E)-1-(3-(4-(3-(4-chlorophenyl)-3-

oxoprop-1-en-1-yl)benzamido)phenyl)-5-methyl-1H-1,2,3-triazole-

4-carboxylate (7 al)

Intermediate 12 (synthesis given in supporting information) on

treatment with 6 b by general procedure B formed 7 al as white

solid, Yield 58%, mp: 226e228 ^ꢀC; ¹H NMR (500 MHz, DMSO‑d₆)

d

10.67 (s, 1H), 8.22 (d, J ¼ 8.7 Hz, 2H), 8.13 (t, J ¼ 2.0 Hz,1H), 8.08 (d,

J ¼ 15.7 Hz, 1H), 8.11e8.06 (m, 4H), 8.03e8.00 (m, 1H), 7.83 (d,

J ¼ 15.6 Hz, 1H), 7.69e7.64 (m, 3H), 7.40 (dd, J ¼ 7.9, 2.0, 0.8 Hz, 1H),

0.03

concentration added per well of a 96-well round bottom microtiter

plate. Later, 97.5 L of bacterial suspension was added to each well

mg/mL in two-fold serial diluted fashion with 2.5 mL of each

4.38 (q, J ¼ 7.1 Hz, 2H), 2.57 (s, 3H), 1.36 (t, J ¼ 7.1 Hz, 3H) ppm; ¹³

C

m

NMR (125 MHz, DMSO‑d₆)

d

188.56, 165.70, 161.55, 143.59, 140.69,

containing the test compound along with appropriate controls.

Presto blue (Thermo Fisher, USA) resazurin-based dye was used for

the visualized identiﬁcation of active compounds [39]. MIC of active

compound was determined as lowest concentration of compound

that inhibited visible growth after incubation period. The MIC re-

sults evaluated were obtained from one among the conc. of 0.03,

139.69, 138.80, 138.29, 136.51, 136.41, 136.29, 135.78, 131.03, 130.44,

129.43, 128.75, 124.07, 121.96, 120.81, 117.50, 60.95, 14.66,

10.30 ppm; HRMS-QTOF (ESI): m/z calcd. for [MþH]^þC₂₈H₂₄ClN₄O₄

515.1486; found 515.1478.

5.5. Synthesis of 5-phenyl-3-isoxazolecarboxylic acid methyl ester

(2) linked substituted acids (7am-7ao)

0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 and > 64 mg/mL of test

solution. MIC was detected on basis of visual color change of culture

medium from blue to pink [40]. The lowest conc. that prevents this

color change was recorded as MIC, the determination of which was

performed in triplicate and consistent MIC value obtained was re-

ported as ﬁnal analyzed MIC. i.e., The MIC result reported is the one

obtained twice or thrice out of three times experiment. Average of

the thrice replicate was not taken as MIC, as it would represent

unjustiﬁed concentrations. The MIC plates were incubated at 37 ^ꢀC

for 7 days for Mtb and 48 h for other mycobacterial pathogens.

5.5.1. Synthesis of methyl 5-(3-(4-((4-chlorobenzyl)oxy)

benzamido)phenyl)isoxazole-3-carboxylate (7am)

Compound 7am was synthesized from 2 to 14 (synthetic pro-

cedure presented in supporting information) by general procedure

B. White solid, Yield 73%, mp: 199e201 ^ꢀC; ¹H NMR (500 MHz,

DMSO‑d₆)

d

10.32 (s, 1H), 8.41 (t, J ¼ 1.8 Hz, 1H), 8.00 (d, J ¼ 8.9 Hz,

2H), 7.94 (dd, J ¼ 8.2, 1.9, 0.8 Hz, 1H), 7.70 (dd, J ¼ 8.2, 1.9, 0.8 Hz,

1H), 7.56e7.53 (t, J ¼ 8.0 Hz, 1H), 7.53e7.47 (m, 4H), 7.45 (s, 1H), 7.17

(dt, J ¼ 8.9, 2.0 Hz, 2H), 5.23 (s, 2H), 3.95 (s, 3H) ppm; ¹³C NMR

5.7. Antibiotic susceptibility testing against bacterial pathogen

panel

(125 MHz, DMSO‑d₆) d 171.56, 165.56, 161.42, 160.28, 157.19, 140.68,

136.17, 133.03, 130.25, 130.17, 130.08, 128.97, 127.36, 126.75, 123.02,

121.68, 117.38, 115.01, 101.29, 69.04, 53.35 ppm; HRMS-QTOF (ESI):

m/z calcd. for [MþNa]^þC₂₅H₂₀ClN₂NaO₅485.0880; found

485.0880.

Antibiotic susceptibility testing was carried out on the newly

synthesized compounds by determining the Minimum Inhibitory

Concentration (MIC) with reference to the standard CLSI guidelines

[41]. MIC is deﬁned as the minimum concentration of compound at

which visible bacterial growth is inhibited. Bacterial cultures were

grown in Mueller-Hinton cation supplemented broth (CAMHB).

Optical density (OD₆₀₀) of the cultures was measured, followed by

dilution for ~10⁶CFU/mL. This inoculum was added into a series of

test wells in a microtiter plate that contained various concentra-

5.5.2. Synthesis of methyl-5-(3-(4-(3-(4-chlorobenzoyl)oxiran-2-

yl)benzamido)phenyl)isoxazole-3-carboxylate (7an)

15 (synthesis outlined in supporting information) on treatment

with 2 by general procedure B gave 7an as yellow solid, Yield 26%,

mp: 222e224 ^ꢀC; ¹H NMR (500 MHz, DMSO‑d₆)

d 10.52 (s, 1H), 8.44

(s, 1H), 8.09e8.03 (m, 4H), 7.96 (dd, J ¼ 8.2, 1.1 Hz, 1H), 7.74 (d,

J ¼ 7.8 Hz,1H), 7.67 (d, J ¼ 8.6 Hz, 2H), 7.63 (d, J ¼ 8.3 Hz, 2H), 7.57 (t,

J ¼ 8.0 Hz,1H), 7.48 (s,1H), 4.90 (d, J ¼ 1.9 Hz,1H), 4.29 (d, J ¼ 1.8 Hz,

tions of compound under test ranging from 64 to 0.03 mg/mL.

Controls i.e., cells alone and media alone (without

compound þ cells) and levoﬂoxacin used as a reference standard.

Plates were incubated at 37 ^ꢀC for 16e18 h followed by observations

of MIC values by the absence or presence of visible growth. For each

compound, MIC determinations were performed independently

thrice using duplicate samples each time.

1H), 3.95 (s, 3H) ppm; ¹³C NMR (125 MHz, DMSO‑d₆)

d 192.44,

171.49, 165.75, 160.28, 157.21, 140.42, 139.87, 139.59, 135.39, 134.36,

130.66, 130.32, 129.65, 128.44, 126.94, 126.82, 123.15, 122.01, 117.53,

101.35, 60.35, 58.60, 53.35 ppm; HRMS-QTOF (ESI): m/z calcd. for

15

S.K. Sahoo, B. Rani, N.B. Gaikwad et al.

European Journal of Medicinal Chemistry 222 (2021) 113580

Derivatives of 3-isoxazolecarboxylic acid esters: a potent and selective com-

pound class against replicating and nonreplicating Mycobacterium tubercu-

losis, Curr. Top. Med. Chem. 12 (2012) 729e734.

5.8. Cell cytotoxicity assay

The active newly synthesized compounds were screened for

their cell toxicity against Vero cells using MTT assay [42]. ~10³cells/

well were seeded in 96 well plate and incubated at 37 ^ꢀC with a 5%

CO₂atmosphere. After 24 h, compound was added ranging from

100 to 5 mg/l and incubated for 72 h at 37 ^ꢀC with 5% CO₂atmo-

sphere. After the incubation was over, MTT was added at 5 mg/l in

each well, incubated at 37 ^ꢀC for further 4 h, residual medium was

discarded, 0.1 mL of DMSO was added to solubilise the formazan

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Synthesis, biological evaluation, and Structure ꢃ Activity relationships for 5-

[(E)-2-Arylethenyl]-3-isoxazolecarboxylic acid alkyl ester derivatives as

valuable antitubercular chemotypes, J. Med. Chem. 52 (2009) 6287e6296.

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crystals and OD was taken at 540 nm for the calculation of CC₅₀

CC₅₀is deﬁned as the lowest concentration of compound which

leads to a 50% reduction in cell viability.

.

5.9. Time-kill kinetic study

The presence or absence of bactericidal activity was assessed by

time-kill method. Mtb H37Rv was diluted to ~10⁵CFU/mL, added to

96 well plate along with compounds and appropriate controls at 1x

and 10x MIC, followed by incubation at 37 ^ꢀC for 6 days. For eval-

uating the reduction in CFU, 0.03 mL sample was removed at

various time-points, serially two-fold diluted in 0.27 mL normal

saline and 0.1 mL of the respective dilution was spread on Mid-

dlebrook 7H11 agar plate supplemented with OADC. The plates

were incubated at 37 ^ꢀC for 1 week and colonies were enumerated.

Kill curves were constructed by counting the colonies from plates

and plotting the CFU/mL of surviving bacteria at each time point in

the presence and absence of compound.

Declaration of competing interest

[21] M.N. Gomes, R.C. Braga, E.M. Grzelak, B.J. Neves, E. Muratov, R. Ma, L.L. Klein,

S. Cho, G.R. Oliveira, S.G. Franzblau, C.H. Andrade, QSAR-driven design, syn-

thesis and discovery of potent chalcone derivatives with antitubercular ac-

tivity, Eur. J. Med. Chem. 137 (2017) 126e138.

[22] L.D. Chiaradia, P.G.A. Martins, M.N.S. Cordeiro, R.V.C. Guido, G. Ecco,

A.D. Andricopulo, R.A. Yunes, J. Vernal, R.J. Nunes, H. Terenzi, Synthesis, bio-

logical evaluation, and molecular modeling of chalcone derivatives as potent

inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatases (PtpA

and PtpB), J. Med. Chem. 55 (2012) 390e402.

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Acknowledgements

SKS, BR and NBG are thankful to Department of Pharmaceuti-

cals, Ministry of Chemicals and Fertilizers, Govt. of India, New Delhi

for award of NIPER fellowship. This article bears NIPER, Hyd

communication number NIPER-H/2021/167.

[23] D. Ramesh, A. Joji, B.G. Vijayakumar, A. Sethumadhavan, M. Mani, T. Kannan,

Indole chalcones: design, synthesis, in vitro and in silico evaluation against

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[24] V.K. Marrapu, V. Chaturvedi, S. Singh, S. Singh, S. Sinha, K. Bhandari, Novel

aryloxy azolyl chalcones with potent activity against Mycobacterium tuber-

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P.M.S. Chauhan, Substituted quinolinyl chalcones and quinolinyl pyrimidines

Appendix A. Supplementary data

as

a new class of anti-infective agents, Eur. J. Med. Chem. 44 (2009)

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.1 13580.

2081e2091.

[26] N.J. Beresford, D. Mulhearn, B. Szczepankiewicz, G. Liu, M.E. Johnson,

A. Fordham-Skelton, C. Abad-Zapatero, J.S. Cavet, L. Tabernero, Inhibition of

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survival in macrophages, J. Antimicrob. Chemother. 63 (2009) 928e936.

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17

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Synthesis and structure-activity relationship of new chalcone linked 5-phenyl-3-isoxazolecarboxylic acid methyl esters potentially active against drug resistant Mycobacterium tuberculosis

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