Design, synthesis, and bioassay of 4-thiazolinone derivatives as influenza neuraminidase inhibitors

European Journal of Medicinal Chemistry 213 (2021) 1 13161

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European Journal of Medicinal Chemistry

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

Design, synthesis, and bioassay of 4-thiazolinone derivatives as

inﬂuenza neuraminidase inhibitors

,

b

,

Mengwu Xiao ^{a 1}, Lvjie Xu ^{c 1}, Ding Lin ^a, Wenwen Lian ^c, Manying Cui ^a, Meng Zhang ^a,

Xiaowei Yan ^a, Shuishi Li ^a, Jun Zhao ^c, Jiao Ye ^{a **}, Ailin Liu ^{c ***}, Aixi Hu ^a

,

, *

^aCollege of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China

^bSchool of Pharmaceutical Science, Hunan University of Chinese Medicine, Changsha, 410208, China

^cInstitute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China

a r t i c l e i n f o

a b s t r a c t

Article history:

A series of 4-thiazolinone derivatives (D1-D58) were designed and synthesized. All of the derivatives

were evaluated in vitro for neuraminidase (NA) inhibitory activities against inﬂuenza virus A (H1N1), and

the inhibitory activities of the ﬁve most potent compounds were further evaluated on NA from two

different inﬂuenza viral subtypes (H3N2 and B), and then their in vitro anti-viral activities were evaluated

using the cytopathic effect (CPE) reduction assay. The results showed that the majority of the target

compounds exhibited moderate to good NA inhibitory activity. Compound D18 presented the most

Received 2 September 2020

Received in revised form

13 December 2020

Accepted 3 January 2021

Available online 12 January 2021

potent inhibitory activity with IC₅₀values of 13.06

selected compounds, D18 and D41 turned out to be the most potent inhibitors against inﬂuenza virus

H3N2 subtype (IC₅₀¼ 15.00 M and IC₅₀¼ 14.97 M, respectively). D25 was the most potent compound

against inﬂuenza B subtype (IC₅₀¼ 16.09 M). In addition, D41 showed low toxicity and greater potency

mM against inﬂuenza H1N1 subtype. Among the

Keywords:

Inﬂuenza

Neuraminidase inhibitors

4-Thiazolinone derivatives

Synthesis

m

than reference compounds Oseltamivir and Amantadine against N1eH275Y variant in cellular assays.

The structure-activity relationship (SAR) analysis showed that introducing 4-CO₂H, 4-OH, 3-OCH₃-4-OH

substituted benzyl methylene can greatly improve the activity of 4-thiazolinones. Further SAR analysis

indicated that 4-thiazolinone and ferulic acid fragments are necessary fragments of target compounds

for inhibiting NA. Molecular docking was performed to study the interaction between compound D41

and the active site of NA. This study may providing important information for new drug development for

anti-inﬂuenza virus including mutant inﬂuenza virus.

Structure-activity relationship

1. Introduction

releasing of the newly formed virions from the infected cells and

circulating to infect surrounding cells. Inhibition of NA can protect

Inﬂuenza viruses remain a major threat to human health due to

its high morbidity and mortality rates. According to statistics from

World Health Organization, seasonal inﬂuenza virus epidemics are

estimated to cause 2e5 million cases of severe illness and up to

250,000e500,000 deaths per year worldwide [1]. These seasonal

ﬂu epidemics cause acute contagious respiratory infections, espe-

cially in children, the elderly and people with chronic diseases.

Inﬂuenza virus neuraminidase (NA) is a sialidase that helps

the host from viral infection and retard its propagation [2]. Because

of the highly conserved structure of the active site of NA, neur-

aminidase has been an attractive target for development of novel

anti-inﬂuenza drugs. Currently, four neuraminidase inhibitors

(NAIs) were approved and are currently in clinical use for inﬂuenza

(Fig. 1A): Relenza™ (zanamivir), Tamiﬂu™ (the phosphate salt of

oseltamivir) are approved worldwide, wheras Inavir™ (laninamivir

octanoate) and Rapivab™ (peramivir) are regionally approved for

* Corresponding author.

** Corresponding author.

*** Corresponding author.

E-mail addresses: yejiao@hnu.edu.cn (J. Ye), liuailin@imm.ac.cn (A. Liu), axhu@hnu.edu.cn (A. Hu).

These two authors contributed equally to this work.

1

https://doi.org/10.1016/j.ejmech.2021.113161

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

Fig. 1. (A) Approved NAIs; (B) Novel NAIs containing amino/guanidino and carboxyl/carboxylate groups.

human use [3]. However, the drug resistance caused by the high

mutability of inﬂuenza virus has become increasingly severe [4,5].

In addition, these classic NA inhibitors contain multiple chiral

centers in their structure, which is difﬁcult to synthesize; and most

of them are polar and have low oral bioavailability. Therefore, it is of

great signiﬁcance to explore new types of NA inhibitors with simple

structure and good pharmacokinetic properties.

were further evaluated with a neuraminidase (NA) activity assay

using two different inﬂuenza viral strains (H3N2 and B), and a

cytopathic effect (CPE) reduction assay. Molecular docking was

further performed to study the interactions between the most

potent compound and the active site of NA.

2. Results and discussion

The catalytic cavity of neuraminidase contains a basic region

composed of three guanidine groups and an acidic region

composed of three carboxyl groups [6]. Acidic groups and basic

groups are the two important active pharmacophores of NA in-

hibitors. In recent years, some novel inﬂuenza NA inhibitors con-

taining amino/guanidino and carboxyl/carboxylate groups have

been discovered (Fig. 1B) [7e14].

2.1. Chemistry

The synthetic route for 4-thiazolinone derivatives (D1-D58) is

outlined in Scheme 1. Using thiazole-2-amine with different sub-

stituents as raw materials, the intermediates C1eC18 were ob-

tained in two steps, including chloroacetylation and cyclic

rearrangement reactions in sequence, and then the intermediates

are condensed with different benzaldehydes to obtain target

compounds D1-D5, D7-D31, D33-D39, and D41-D58. The nitro-

substituted compound D5, D31 and D39 was reducted into its

corresponding amino-substituted compounds D6, D32 and D40,

respectively.

In our preliminary work, inspired by the studies on guanidino-

benzoic acid type NA inhibitors [14e1 7], some amino/guanidino-

thiazole carboxylic acid derivatives (S6eS9, more details in

supporting information) were synthesized with lower NA inhibi-

tion rate, but after being modiﬁed into 4-thiazolinone derivatives,

they showed a signiﬁcant improvement in activity (Fig. 2). These

active compounds have a different scaffold from conventional NAIs

in chemical space and shape, which indicated that they represent a

novel class of NA inhibitors. 4-thiazolinone, also known as thiazo-

lidin-4-one, have a wide range of biological activities [18e21], such

as anti-inﬂammatory [22e24], anti-cancer [25e28], anti-fungal

[29,30], anti-bacterial [31], antiviral [32,33] and so on. Rosiglita-

zone and pioglitazone, both marketed drugs containing 4-

thiazolinone fragments, are used to treat type 2 diabetes [34,35].

In this study, 18 4-thiazolinone derivatives (C1eC18), 58 5-benzyl-

4-thiazolinone derivatives (D1-D58), two 4-thiazolinone analogues

(S1eS2) and three 4-thiazolinone ring-opening analogues (S3eS5)

were designed, synthesized, and evaluated for their H1N1 NA

inhibitory activity. In addition, the ﬁve most potent compounds

To enrich the structure-activity relationship studies, two 4-

thiazolinone analogues (S1eS2) and three 4-thiazolinone ring-

opening analogues (S3eS5) were synthesized as illustrated in

Scheme 2. The compound S1 and S2 was synthesized with the

method similar to that of compounds D. Compounds S3eS4 were

synthesized by reﬂuxing ethyl 2-amino-4-methylthiazole-5-

carboxylate A1 and cinnamoyl isothiocyanates G3-G4. Compound

S4 was hydrolyzed to target compound S5. The intermidieate cin-

namoyl isothiocyanates F3eF4 were obtained by reacting

substituted cinnamic acid and thionyl chloride, which was further

treated with ammonium thiocyanate to get G3-G4. The synthesized

compounds were characterized by elemental analysis, ¹H NMR, ¹³

NMR and mass spectrum.

C

2

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

Fig. 2. Design of target compounds.

Scheme 1. Synthetic route of 5-benzylidene-4-thiazolinones.

2.2. Biological activity

2.2.1. Structure-activity relationship of mother nucleus

Based on the literature study and the principle of bioisosterism,

NA inhibitory fragments were introduced at the thiazole ring to

systematically explore the structure-activity relationship of NA

inhibitory activity of the mother nucleus. The results (Table 1) show

that the intermediates C1 to C18 exhibited moderate NA inhibitory

activity. Among them, C4 displayed the best NA inhibitory activity

All the target compounds (C1eC18, D1-D58, and S1eS5) were

tested for NA inhibitory activity using human inﬂuenza virus A/PR/

8/34 of subtype H1N1. Moreover, the most potent ﬁve compounds

D15, D18, D25, D41, and D44 were selected for further NA inhibition

studies on multiple strains of inﬂuenza viruses. Finally, their

toxicity and anti-inﬂuenza viral effect on drug-resistant inﬂuenza

virus-infected Madin-Darby canine kidney (MDCK) cells were

evaluated.

with IC₅₀value of 33.60

mM, followed by C1, C6, C10, C11 and C16

with moderate activity (IC₅₀less than 60

mM). Comparing S1 with

C1, the activity was signiﬁcantly reduced after the thiazole ring was

replaced with an oxazole ring. Comparing S2 with C1, the activity

3

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

Scheme 2. Synthesis route of 4-thiazolinone analogues.

Table 1

H1N1 NA inhibitory activities of C1 e C18 and S1 e S2.

No.

X

Y

R¹

R²

R³

Inhibition Rates^a/%

IC₅₀/mM

S1

S2

C1

C2

C3

C4

C5

C6

O

S

Se

S

CO₂Et

CO₂H

Me

CF₃

i-Pr

n-Pr

Me

CH₂CO₂Et

t-Bu

H

34.77 4.45

17.92 1.31

78.40 1.01

63.94 3.53

37.05 15.78

68.02 7.42

36.99 10.21

73.88 5.97

74.44 1.43

19.56 5.76

52.12 8.15

52.77 6.71

79.02 3.04

26.41 7.62

43.82 5.98

37.95 9.22

53.39 2.74

77.79 1.38

36.81 4.99

18.75 9.03

NT

58.84 5.75

97.01 13.76

NT

33.60 8.89

NT

53.41 15.66

66.05 27.73

NT

65.05 15.43

55.36 25.79

48.68 4.15

NT

CO₂Et

CO₂Me

CO₂t-Bu

CO₂Et

COCH₃

H

C7

C8

C9

H

Me

n-Bu

Ph

H

C10

C11

C12

C13

C14

C15

C16

C17

C18

Me

H

Br

NO₂

NT

104.38 53.4

41.62 4.46

NT

imidazole-1-yl

1,2,4-triazol-1-yl

H

NT

a

*

b

NA inhibition rates at the concentration of 40

NT: Not tested.

mg/mL.

was also signiﬁcantly reduced after the thiazolidin-4-one ring was

replaced with 1,3-selenazolidin-4-one ring. Comparing C4 with C5

and C6, The 4-position substituent of thiazole has the largest in-

ﬂuence on the activity of the mother nucleus. The relative contri-

bution of the substituents at 4-position to the activity in the mother

when the 4-position of the thiazole ring is tert-butyl, the activity

order of the 5-position substituted group is: nitro

(41.62 mM) > bromine (104.38 mM) > hydrogen, imidazole-1-yl and

1,2,4-triazol-1-yl. Comparing C1 with C8, C9, and C10, 5-position

substitution has no obvious effect on activity. Comparing C1 with

C6 and C7, the alkyl substitution of formate group also have little

effect on the activity. In conclusion, the structure-activity rela-

tionship is summarized in Fig. 3.

nucleus was isopropyl (33.60

Comparing C1 with C3, in terms of the 4-position substituents of

thiazole, methyl (58.84 M) is more active than triﬂuoromethyl.

mM) > methyl (53.41 mM) > n-propyl.

m

Comparing C3 with C4, C8, C9, and C13, the activity contribution

order of the 5-position group on thiazole is acetyl

2.2.2. Structure-activity relationship of side-chains

(48.68

(58.84

(97.01

m

M)

M) and tert-butyl formate (66.05

M). By comparing compounds C14 e C18, it can be seen that

>

methyl formate (53.41

m

M)

>

ethyl formate

In order to further improve the activity of these compounds and

explore the structure-activity relationship, through the aldol

condensation reaction, different substituted benzylidene groups

mM) carboxyl

>

m

4

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

Fig. 3. Structure-activity relationship of the mother nucleus.

were introduced at the 5-position of 4-thiazolinone. This side-

chain expansion may enhance the interaction with NA 430 cavity,

a big hydrophobic cavity adjacent to NA catalytic cavity in both

group-1 and group-2 NAs [36,37]. Herein, C1, C4, C11eC13, and C16

were selected as the main intermediates for lower cost, better ac-

tivity or easy synthesis. The activity results in Table 2 show that:

D25 was the most potent compound against

B

strains

(IC₅₀¼ 19.31 M and IC₅₀¼ 16.09 M, respectively). Therefore, D18

m

possesses the best NA activity against the three inﬂuenza viruses

overall. In order to better understand the relationship between the

structure and the activity, then the logP values of Zanamivir and

selected compounds were calculated by the XLOGP3 [40] (Table 5).

The result demonstrated that D41 (XLOGP3 ¼ 3.46) showed a better

lipid solubility than Zanamivir (XLOGP3 ¼ ꢀ3.19).

(1) The product after introduction of benzylmethylene is

generally more active than its corresponding intermediate.

The value of △IC₅₀(IC₅₀product-IC₅₀intermediates) was

used to quantify the change in activity before and after

2.2.5. Cytopathic effect reduction assay

Based on the NA inhibitory activity assay of the compounds, the

active compounds were selected for cellular level activity against

inﬂuenza virus. The anti-inﬂuenza activity was evaluated by the

cytopathic effect of Madin-Darby canine kidney (MDCK) cells under

viral infection. The selected drug-resistant virus, inﬂuenza virus A/

Hebei Xinhua/SWL1106/2017H1N1pdm09, is NA H275Y variants

[41] well known for its severe resistance to Oseltamivir and Per-

amivir. Replacement of the histidine residue at the 275-position of

wild-type H1N1 virus by tyrosine will push the neighboring Glu-

276 away, and cause a large dislocation of the side chain (i.e. pen-

toxy in Oseltamivir and pentyl in Peramivir), thus decreasing the

hydrophobic interactions in the NA active site [42]. As shown in

Table 6, this virus exhibited highly reduced inhibition by Oselta-

mivir and shows normal inhibition by Zanamivir, which is consis-

tent with the literature report [41]. Among ﬁve selected potent

compounds, we are lucky to ﬁnd that the inhibition activity of D41

modiﬁcation. As shown in Table 2, 40/58 of

zero. For example, the activity of intermediate C12 is low

(IC₅₀> 100 M), and all the products D31-D37 are less than

50 M;

(2) The most potent compounds was D15 and D18 with IC₅₀

values of 16.33 and 13.06 M, respectively. And compounds

D16, D25, D41 and D44 also displayed good activity with IC₅₀

values less than 20 M;

DIC₅₀was below

m

(3) As shown in Table 3, by comparing the values of △IC₅₀before

and after the modiﬁcation of each series of compounds, we

can ﬁnd that the NA inhibitory activity of the substituted 4-

thiazolinones, which introduced 4-CO₂H, 4-OH or 3-OCH₃-

4-OH substituted benzyl methylene, was greatly improved.

2.2.3. Structure-activity relationship of the 4-thiazolinone ring

(EC₅₀¼ 30.24

Oseltamivir (EC₅₀> 243.7

and close to Zanamivir (EC₅₀¼ 5.09

lower toxicity (TC₅₀> 278.2

candidate compounds. These results indicate that D41 could be a

promising potential as candidates for further investigation,

contributing to the development of more potent NA inhibitors to

against mutant inﬂuenza viruses.

m

M) is better than the existing anti-inﬂuenza drugs

M) and Amantadine (EC₅₀> 532.7 M),

M). Besides, D41 also has

M, TC₀¼ 41.73 M) than other

As shown in Table 4, the NA inhibitory activity values of the

m

target compounds D1 (64.21

than those of the corresponding 4-thiazolinone ring-opening ana-

logues S3 and S5 (44.00 M), therefore 4-thiazolinone is an

mM) and D18 (13.06

mM) are better

m

essential NA inhibitory active fragment of such compounds.

Moreover, by comparing the activities of S5, S6 and ferulic acid, it

found that the ferulic acid fragment is an important NA inhibitory

activity fragment. Inspired by this ﬁnding, we discovered some

novel ferulic acid derivatives as NA inhibitors recently [39].

2.3. Molecular docking

2.2.4. Inhibitory activity against neuraminidase of inﬂuenza H3N2

and B virus strains

For the purpose of better understanding on the potency of 4-

thiazolinone derivatives and guiding further SAR studies, D41, the

compound with low toxicity, good anti-inﬂuenza activity in both

enzymatic assay and cellular assay, was docked into the active sites

of NA (H1N1, PDB entry: 3TI57) using AutoDock Vina. Meanwhile,

this compound differs from conventional active NA inhibitors in

regard to shape and size, which makes the exploration of the

binding mode of this novel class of inhibitors fascinating for further

modeling studies. PyMOL and Ligplotþ [43] were used for docking

and graphic display, respectively.

As shown in Fig. 4, the predicted binding mode of D41 to the

structure of H1N1 neuraminidase indicates a binding interface

largely non-overlapping with that of Zanamivir. It is obvious that

D41 targets both the catalytic cavity (SA cavity) and the 430 Cavity

(a large cavity adjacent to the SA cavity). This suggested new

The ﬁve most potent compounds D15, D18, D25, D41, and D44

were selected as representatives for the further NA inhibition

studies on A/Minfang/151/2000 (H3N2) and B/Sichuan/01/96 (B)

strains of inﬂuenza virus. As the result shown in Table 5, In general,

the inhibitory activities of these compounds against inﬂuenza vi-

ruses H1N1 and H3N2 have little difference. For inﬂuenza B viruses,

D15, D18, and D25 (with carboxylic acid ester group substitution at

the 5- position of the thiazole ring) show better activity than D41

and D44 (with acyl substitution at the 5-position of the thiazole

ring). Among the selected compounds, D18 exhibited the highest

potency against NA from A/PR/8/34 (H1N1) (IC₅₀¼ 13.06

and D41 turned out to be the most potent inhibitors against H3N2

strains (IC₅₀¼ 15.00 M and IC₅₀¼ 14.97 M, respectively). D18 and

mM). D18

m

5

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

Table 2

H1N1 NA inhibitory activity of target compounds D1-D58.

6

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

opportunities to design highly effective NA inhibitors targeting

both the 430 Cavity and the known active sites [36,37,44e47]. D41

interacts with the NA active site by hydrogen bond interaction with

Arg292 and Arg371, and hydrophobic interaction with Glu276,

Glu277 and Tyr406. These are the key amino acid residues for

Zanamivir to inhibit NA. It is obvious that in the SA cavity, the

interaction between the thiazole moiety of D41 and the key active

site of NA is much weaker than that of Zanamivir. This is consistent

Table 3

Comparison of inhibitory activity between compound D and its corresponding intermediates C.

7

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

Table 4

NA inhibitory activity of D1, D18, S3, S5, and S6.

No

Structure

Inhibition Rates^a/%

72.4 6.10

IC₅₀

/mM

D1

64.21 6.64

S3

23.56 2.10

NT

D18

83.12 5.08

13.06 1.41

S5

80.73 1.07

44.00 3.37

Ferulic acid

e

140 [38]

S6

ꢀ1.32 5.51

NT

a

NA inhibition rates at the concentration of 40 mg/mL; NT: Not tested.

with the conclusion drawn in Table 4 that the thiazole may not

contribute much to the NA inhibitory activity. Therefore, we spec-

ulate that the activity of the target compound is related to the

binding mode of the 5-benzylidenethiazolinone fragment on the

cavity outside the active cavity. Notably, adjacent to the active site,

the hydrophobic region formed by Ile149, Arg430, Pro431, and

Thr439 accommodates the phenyl group with hydrophobic inter-

action. Besides, additional hydrogen bonds interaction with Arg371

signiﬁcantly made benzene moiety more stable in 430 Cavity.

Interestingly, the 4-thiazolinone group acting as a bridge con-

necting the active site and the 430 Cavity.

analysis of the parent framework was conducted, and the following

main conclusions were drawn: i. compound C4 has the best NA

inhibitory activity (IC₅₀¼ 33.60

mM) in C series; ii. When thiazole

ring is replaced by oxazole ring, the activity is signiﬁcantly reduced;

iii. After S is replaced by Se on 4-thiazolinone, the activity is

signiﬁcantly reduced; iv. The 4-position substituent of thiazole has

the largest inﬂuence on the activity of the mother nucleus.

Generally, 5-benzyl-4-thiazolinone derivatives are more active

than its corresponding thiazolinone fragment. Introducing 4-CO₂H,

4-OH, 3-OCH₃-4-OH substituted benzyl methylene to 5-position of

the 4-thiazolinone ring brings signiﬁcant activity improvement.

The compound with the best H1N1 NA inhibitory activity in D se-

ries is D18 (IC₅₀¼ 13.06

mM).

3. Conclusions

In the anti-NAs (H3N2 and B) assay, among the selected ﬁve

most potent compounds, D18 and D41 turned out to be the most

Based on our previous studies, 18 4-thiazolinone derivatives

(C1eC18) and 58 5-benzyl-4-thiazolinone derivatives (D1-D58)

were designed, synthesized, and evaluated for their inﬂuenza H1N1

NA inhibitory activity. A systematic structure-activity relationship

potent inhibitor against H3N2 strains (IC₅₀¼ 15.00

IC₅₀¼ 14.97 M, respectively). D25 was the most potent compound

against B strains (IC₅₀¼ 16.09 M). The cell-based assays indicate

mM and

m

8

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

Table 5

The IC₅₀(mM) values of selected compounds against three NAs and their logP values.

that the activity of D41 (EC₅₀¼ 30.24

anti-inﬂuenza drugs Oseltamivir (EC₅₀> 243.7

dine (EC₅₀> 532.7

M), and close to Zanamivir (EC₅₀¼ 5.09

Besides, D41 also has lower toxicity (TC₅₀> 278.2

M, TC₀¼ 41.73

M) than other candidate compounds. Further structure-activity

relationship analysis and docking studies indicated that 4-

thiazolinone and ferulic acid fragments are necessary NA inhibi-

tory active fragments of such compounds. The 4-thiazolinone

group can be used as a linker to design SA Cavity and 430 Cavity

dual-site NA inhibitors. The results of this work may contribute to

the development of more potent NA inhibitors to against mutant

inﬂuenza virus.

m

M) is better than the existing

M) and Amanta-

M).

m

compound

H1N1^a

H3N2^b

B^c

XLOGP3^d

m

D15

D18

D25

D41

D44

Zanamivir

16.33 1.82

13.06 1.41

19.21 2.88

19.09 3.70

18.28 3.44

1.10 ꢁ 10^ꢀ4

15.45 1.00

15.00 0.88

15.49 1.24

14.97 0.22

24.44 1.95

1.31 ꢁ 10^ꢀ3

19.64 0.75

19.31 0.67

16.09 3.57

36.23 2.78

76.47 0.82

5.13 ꢁ 10^ꢀ4

4.00

3.97

4.39

3.46

3.43

ꢀ3.19

m

a

A/PR/8/34 (H1N1).

A/Minfang/151/2000 (H3N2).

B/Sichuan/01/96 (B).

b

c

d

The values were obtained using the XLOGP3 with the default settings.

Table 6

Inhibitory effects of compounds on the cytopathic effect induced by Oseltamivir and

amantadine-resistant inﬂuenza virus A/Hebei Xinhua/SWL1106/2017H1N1pdm on

MDCK cells

4. Experiment section

4.1. Chemistry

Compound

TC₅₀

(

m

M) ^a

TC₀

(

m

M) ^b

EC₅₀

(m

M) ^c

SI ^d

Reagents were commercial grade and were used as supplied.

Reactions were monitored by analytical thin-layer chromatography

(TLC) on 0.25-mm F254 silica gel precoated sheets, and spots were

visualized with ultraviolet (UV) light. Melting point (mp) was

measured on an X-4 electrothermal digital melting point apparatus

and uncorrected. In addition, The NMR spectra were recorded on a

Bruker NMR AVANCE 400 (400 MHz) or a Bruker AVANCE III HD

600 NMR spectrometer (600 MHz equipped with an UltraCOOL

D15

D18

D25

D41

>256.8

>238.4

29.10 2.48

>278.2

43.29 3.29

>243.7

>532.7

20.54

19.07

19.83

41.73

10.27

>243.7

>532.7

>300.9

>20.54

>19.07

>19.83

30.24 2.00

>10.27

>243.7

>532.7

5.09 0.69

ND

<1.25

>9.85

<3.90

ND

D44

Oseltamivir

Amantadine

Zanamivir

ND

>68.49

>300.9

a

TC₅₀: the average concentration of 50% cytotoxic compound;

TC₀: Maximum non-toxic concentration;

EC₅₀: the average concentration of 50% inhibition rate;

SI: Selection index, TC₅₀/EC₅₀; ND: not determined.

b

c

probe) with TMS as internal standard with chemical shifts (d)

expressed in ppm. Signals are reported as m (multiplet), s (singlet),

d (doublet), t (triplet), q (quartet), bs (broad singlet), bm (broad

d

Fig. 4. Binding mode of Zanamivir (A, B) and D41 (C, D).

9

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

multiplet), br (broad). Coupling constants (J) are given in hertz.

Mass spectra were recorded on a Finnigan LCQ-advantage mass

spectrometer. Elemental analyses were performed on a CHNeO-

Rapid instrument.

acid tert-butyl ester (C7). Yellow solid. m.p. 195e198 ^ꢂC, yield

84.2%. ¹H NMR (400 MHz, CDCl₃)

d

: 1.56 (s, 9H, 3 ꢁ CH₃), 2.63 (s, 3H,

CH₃), 3.91 (s, 2H, SCH 2); Anal. Calcd. for C₁₂H₁₅N₃O₃S₂: C, 45.99; H,

4.82; N, 13.41; found C, 45.95; H, 4.84; N, 13.40.

2-(5-Methyl 4-thiazolinone-2-imino)-4-methyl Thiazole-5-

carboxylic acid ethyl ester (C8). White solid C8, m.p. 183e185 ^ꢂC,

total yield of two steps is 74.3% (based on A8). ¹H NMR (400 MHz,

4.1.1. General procedure for synthesis of 4-thiazolinone

intermediates (C1 e C18)

Aminothiazole (A1-A18, 50 mmol) and 100 mmol of potassium

carbonate were dissolved in dichloromethane (100 mL), chlor-

oacetyl chloride (8.0 mL) was added dropwise under ice bath, and

reacted at room temperature monitored by TLC. When the reaction

is completed, the reaction solution is poured into water, and

washed with sodium bicarbonate solution, saturated saline and

water successively, dry the organic phase with anhydrous sodium

sulfate, remove the solvent under reduced pressure, and recrys-

tallize with ethanol to obtain raw product B1eB18. Then inter-

mediate (30 mmol) and KSCN (45 mmol) were dissolved in 100 mL

of ethanol, and reﬂux for 6 h. The reaction mixture were cooled

down, ﬁltered, and washed with ethanol and water, the crude

product was recrystallized from ethanol/water to afford the pure 4-

thiazolinone intermediate C1eC18.

DMSO‑d₆)

d

: 1.36 (t, J ¼ 7.1 Hz, 3H, CH₃), 1.73 (d, J ¼ 7.3 Hz, 3H,

CHCH₃), 2.67 (s, 3H, CH₃), 4.16 (q, J ¼ 7.3 Hz, 1H, CH), 4.32 (q,

J ¼ 7.1 Hz, 2H, OCH₂); Anal. Calcd. for C₁₁H₁₃N₃O₃S₂: C, 44.13; H,

4.38; N, 14.04; found C, 44.11; H, 4.40; N, 14.01.

2-(5-Butyl-4-thiazolinone-2-imino)-4-methyl

Thiazole-5-

carboxylic acid ethyl ester (C9). Pale yellow solid, m.p.

145e146 ^ꢂC, total yield of two steps is 66.7% (based on raw material

A9). ¹H NMR (400 MHz, CDCl₃)

d

: 0.94 (t, J ¼ 7.1 Hz, 3H, CH₃), 1.36 (t,

J ¼ 7.1 Hz, 3H, OCH₂CH₃), 1.38e1.43 (m, 2H, CH₂), 1.43e1.60 (m, 2H,

CH₂), 2.18e2.22 (m, 2H, CH₂), 2.67 (s, 3H, CH₃), 4.14 (dd, J ¼ 9.0,

4.1 Hz, 1H, CH), 4.32 (q, J ¼ 7.1 Hz, 2H, OCH₂), 11.88 (s, 1H, NH); ¹³

C

NMR (150 MHz, DMSO‑d₆) d: 14.17, 14.62, 17.85, 22.21, 28.55, 31.76,

50.22, 61.30, 117.21, 158.12, 162.04, 164.79, 170.97, 176.80; Anal.

Calcd. for C₁₄H₁₉N₃O₃S₂: C, 49.25; H, 5.61; N, 12.31; found C, 49.21;

H, 5.64; N, 12.29.

2-(5-Phenyl-4-thiazolinone-2-imino)-4-methyl Thiazole-5-

carboxylic acid ethyl ester (C10). Yellow solid, m.p. 198e199 ^ꢂC,

total yield of two steps is 57.6% (based on raw material A10). ¹H

2-(4-Thiazolinone-2-imino)-4-methylthiazole-5-carboxylic

acid ethyl ester (C1). Yellow solid. m.p. 203e204 ^ꢂC, yield 81.9%. ¹H

NMR (400 MHz, DMSO‑d₆)

CH₃), 4.03 (s, 2H, SCH 2), 4.25 (q, J ¼ 7.2 Hz, 2H, OCH₂), 12.28 (s, 1H,

NH); ¹³C NMR (150 MHz, DMSO‑d₆)

: 14.62, 17.84, 35.68, 61.29,

117.06, 158.03, 162.06, 166.69, 171.08, 174.58; Anal. Calcd. for

₁₀H₁₁N₃O₃S₂: C, 42.09; H, 3.89; N, 14.73; found C, 42.05; H, 3.40; N,

14.71.

2-(4-Thiazolinone-2-imino)-4-methylthiazole-5-carboxylic

acid (C2). White solid, m.p. 250e251 ^ꢂC, yield 47.4%. ¹H NMR

d: 1.27 (t, J ¼ 7.2 Hz, 3H, CH₃), 2.58 (s, 3H,

d

NMR (400 MHz, DMSO‑d₆)

d: 1.41 (t, J ¼ 7.0 Hz, 3H, CH₃), 2.56 (s, 3H,

CH₃), 4.48 (q, J ¼ 7.0 Hz, 2H, OCH₂), 5.62 (s, 1H, CH), 7.14e7.17 (m,

1H, C₆H₅4-H), 7.34 (d, J ¼ 7.6 Hz, 2H, C₆H₅3,5-H), 7.58 (d, J ¼ 7.6 Hz,

2H, C₆H₅2,6-H), 12.53 (s, 1H, NH).

C

2-(4-Thiazolinone-2-imino)-4-methyl-5-acetylthiazole (C11).

Yellow solid, m.p. 216e218 ^ꢂC, yield 81.3%. ¹H NMR (400 MHz,

(400 MHz, DMSO‑d₆)

d

: 2.56 (s, 3H, CH₃), 4.02 (s, 2H, SCH 2), 12.23

DMSO‑d₆) d: 2.48 (s, 3H, CH₃), 2.60 (s, 3H, CH₃), 4.03 (s, 2H, CH₂),

12.29 (s, 1H, NH); Anal. Calcd. for C₉H₉N₃O₂S₂: C, 42.34; H, 3.55; N,

16.46; found C, 42.30; H, 3.58; N, 16.39.

(s, 1H, NH); ¹³C NMR (100 MHz, DMSO‑d₆) 1: 17.69, 35.62, 118.66,

157.29,163.60,166.17,170.62,174.63; Anal. Calcd. for C₈H₇N₃O₃S₂: C,

37.35; H, 2.74; N, 16.33; found C, 37.34; H, 2.77; N, 16.29.

2-(4-Thiazolinone-2-imino) Thiazole-4-ethyl acetate (C12).

2-(4-Thiazolinone-2-imino)-4-triﬂuoromethylthiazole-5-

carboxylic acid ethyl ester (C3). Yellow solid. m.p. 228e229 ^ꢂC,

total yield of two steps is 26.4% (based on raw material A3). ¹H NMR

Yellow solid, m.p.162e165 ^ꢂC, yield 81.4%; ¹H NMR (400 MHz,

CDCl₃)

d

:1.29 (t, J ¼ 7.1 Hz, 3H, CH₃), 3.74 (s, 2H, COCH₂), 3.89 (s, 2H,

SCH 2), 4.21 (q, J ¼ 7.1 Hz, 2H, OCH₂), 6.91 (s, 1H, thiazole 5-H); ¹³

C

(400 MHz, DMSO‑d₆)

4.32 (q, J ¼ 7.1 Hz, 2H, OCH₂), 12.48 (s, 1H, NH); Anal. Calcd. for

₁₀H₈F₃N₃O₃S₂: C, 35.40; H, 2.38; N, 12.38; found C, 35.32; H, 2.47;

N, 12.34.

d

: 1.29 (t, J ¼ 7.1 Hz, 3H, CH₃), 4.08 (s, 2H, CH₂),

NMR (150 MHz, DMSO‑d₆) d: 14.59, 35.34, 37.45, 60.85, 113.67,

146.24, 163.28, 169.18,170.26,174.56; Anal. Calcd. for C₁₀H₁₁N₃O₃S₂:

C, 42.09; H, 3.89; N, 14.73; found C, 42.07; H, 3.89; N, 14.70.

2-(4-Thiazolinone-2-imino)-5-methyl Thiazole-4-ethyl ace-

tate (C13). Yellow solid C13, m.p. 170e172 ^ꢂC, yield 46.1%; ¹H NMR

C

2-(4-Thiazolinone-2-imino)-4-isopropyl

Thiazole-5-

carboxylic acid methyl ester (C4). Yellow solid, m.p. 205e207 ^ꢂC,

(400 MHz, CDCl₃)

3.68 (s, 2H, CH₂), 3.89 (s, 2H, SCH 2), 4.19 (q, J ¼ 7.1 Hz, 2H, OCH₂);

¹³C NMR (150 MHz, DMSO‑d₆)

: 14.51, 15.44, 31.72, 35.32, 61.18,

119.82, 146.79, 162.19, 166.81, 170.51, 174.47; Anal. Calcd. for

₁₁H₁₃N₃O₃S₂: C, 44.13; H, 4.38; N, 14.04; found C, 44.10; H, 4.39; N,

14.02.

2-(4-Thiazolinone-2-imino)-4-tert-butylthiazole

White solid, m.p. 200e202 ^ꢂC, ¹H NMR (CDCl₃, 400 MHz)

d: 1.28 (t, J ¼ 7.1 Hz, 3H, CH₃), 2.30 (s, 3H, CH₃),

total yield of two steps is 64.3% (based on raw material A4). ¹H NMR

(400 MHz, DMSO‑d₆)

OCH₃), 3.86 (hept, J ¼ 6.8 Hz, 1H, CH), 4.02 (s, 2H, SCH 2), 12.26 (s,

1H, NH); ¹³C NMR (150 MHz, DMSO‑d₆)

: 22.35, 28.90, 35.70, 52.52,

115.22, 162.25, 167.19, 167.66, 171.70, 174.76; Anal. Calcd. for

₁₁H₁₃N₃O₃S₂: C, 44.13; H, 4.38; N, 14.04; found C, 44.10; H, 4.41; N,

14.02.

2-(4-Thiazolinone-2-imino)-4-propyl Thiazole-5-carboxylic

acid methyl ester (C5). Yellow solid, m.p. 211e212 ^ꢂC, yield

82.4%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.23 (d, J ¼ 6.8 Hz, 6H, 2 ꢁ CH₃), 3.78 (s, 3H,

d

C

(C14).

d

: 1.33 (s,

9H, 3 ꢁ CH₃), 3.88 (s, 2H, CH₂), 6.64 (s, 1H, thiazole-H), 12.24 (s, 1H,

NH); ¹³C NMR (150 MHz, DMSO‑d₆)

: 29.96, 34.94, 35.29, 107.81,

162.94, 168.68, 174.62; EI-MS (m/z): 255.3 [M]^þ; Anal. Calcd. for

₁₀H₁₃N₃OS₂: C, 47.04; H, 5.13; N, 16.46; found C, 47.01; H, 5.16; N,

16.42.

2-(4-Thiazolinone-2-imino)-4-tert-butyl-5-bromothiazole

(C15). Red solid, m.p. 202e203 ^ꢂC, yield 83%. ¹H NMR (CDCl₃,

400 MHz)

d

: 0.92 (t, J ¼ 7.4 Hz, 3H, CH₃),

1.66e1.78 (m, 2H, CH₂), 2.99 (t, J ¼ 7.4 Hz, 2H, Thiazole-CH₂), 3.78 (s,

C

3H, OCH₃), 4.02 (s, 2H, SCH 2), 12.26 (s, 1H, NH) Anal. Calcd. for

C

₁₁H₁₃N₃O₃S₂: C, 44.13; H, 4.38; N, 14.04; found C, 44.09; H, 4.42; N,

14.01.

2-(4-Thiazolinone-2-imino)-4-methyl Thiazole-5-carboxylic

acid methyl ester (C6). Yellow solid, m.p. 215e218 ^ꢂC, yield

84.6%. ¹H NMR (400 MHz, DMSO‑d₆)

: 2.58 (s, 3H, CH₃), 3.78 (s, 3H,

d

: 1.47 (s, 9H, 3 ꢁ CH₃), 3.88 (s, 2H, CH₂), 11.92 (s,1H, NH);

Anal. Calcd. for C₁₀H₁₂BrN₃OS₂: C, 35.93; H, 3.62; N, 12.57; found C,

35.86; H, 3.69; N, 12.52.

d

CH₃), 4.03 (s, 2H, CH₂), 12.28 (s, 1H, NH); Anal. Calcd. for

C₉H₉N₃O₃S₂: C, 39.84; H, 3.34; N, 15.49; found C, 39.80; H, 3.36; N,

15.47.

2-(4-Thiazolinone-2-imino)-4-tert-butyl-5-nitrothiazole

(C16). Yellow solid, m.p. 240e242 ^ꢂC, total yield of two steps is

54.3% (based on raw material A16). ¹H NMR (400 MHz, DMSO‑d₆)

d:

2-(4-Thiazolinone-2-imino)-4-methyl Thiazole-5-carboxylic

1.46 (s, 9H, 3 ꢁ CH₃), 4.08 (s, 2H, CH₂), 12.54 (s, 1H, NH); ¹³C NMR

10

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

(150 MHz, DMSO‑d₆)

d

: 28.62, 36.19, 37.30, 139.44, 164.19, 169.02,

(t, J ¼ 6.8 Hz, 3H, CH₃), 2.62 (s, 3H, CH₃), 4.24 (q, 2H, J ¼ 6.8 Hz,

OCH₂), 7.80e7.82 (m, 1H, C₆H₄5-H), 7.82 (s, 1H, ¼CH), 8.04 (d,

J ¼ 8.0 Hz, 1H, C₆H₄6-H), 8.27 (d, J ¼ 8.0 Hz, 1H, C₆H₄4-H), 8.46 (s,

1H, C₆H₄2-H), 13.00 (s, 1H, NH); Anal. Calcd. for C₁₇H₁₄N₄O₅S₂: C,

48.80; H, 3.37; N, 13.39; found C, 48.80; H, 3.41; N, 13.36.

170.34, 174.82; EI-MS (m/z): 300.0 [M]^þ; Anal. Calcd. for

C

₁₀H₁₂N₄O₃S₂: C, 39.99; H, 4.03; N, 18.65; found C, 39.96; H, 4.05; N,

18.61.

2-((4-(tert-butyl)-5-(1H-imidazole-1-yl)thiazol-2-yl)imino)

thiazolidin-4-one (C17). Yellow solid, m.p. 241e243 ^ꢂC, yield 86%.

¹H NMR (400 MHz, CDCl₃)

2-[5-(3-Hydroxybenzylidene)-4-thiazolinone-2-imino]-4-

methyl Thiazole-5-carboxylic acid ethyl ester (D7). Yellow solid,

d

: 1.20 (s, 9H, 3 ꢁ CH₃), 3.90 (s, 2H, CH₂),

7.09 (s, 1H, imidazole 2-H), 7.19 (s, 1H, imidazole 4-H), 7.70 (s, 1H,

imidazole 5-H), 12.08 (s, 1H, NH); Anal. Calcd. for C₁₃H₁₅N₅OS₂: C,

48.58; H, 4.70; N, 21.79; found C, 48.56; H, 4.72; N, 21.79.

2-((4-(tert-butyl)-5-(1H-1,2,4-triazol-1-yl)thiazol-2-yl)

m.p. 266e268 ^ꢂC, yield 70.2%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.28

(s, 3H, CH₃), 2.66 (s, 3H, CH₃), 4.25 (s, 2H, OCH₂), 6.91 (d, J ¼ 7.8 Hz,

1H, C₆H₄3-H), 7.04 (s, 1H, C₆H₄2-H), 7.09 (d, J ¼ 7.8 Hz, 1H, C₆H₄6-

H), 7.36 (t, J ¼ 7.8 Hz, 1H, C₆H₄5-H), 7.65 (s, 1H, ¼CH), 9.89 (s, 1H,

imino)thiazolidin-4-one (C18). Light yellow solid, m.p.

OH),12.85 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO‑d₆)

d: 14.57, 18.00,

182e185 ^ꢂC, yield 86%. ¹H NMR (400 MHz, CDCl₃)

d

: 1.19 (s, 9H,

3 ꢁ CH₃), 3.90 (s, 2H, CH₂), 8.11 (s, 1H, triazole 3-H), 8.28 (s, 1H,

triazole 5-H), 12.01 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO‑d₆)

61.33, 116.80, 117.78, 118.19, 121.86, 124.43, 130.76, 133.33, 134.83,

158.32, 159.09, 161.94, 167.29, 170.15; Anal. Calcd. for C₁₇H₁₅N₃O₄S₂:

C, 52.43; H, 3.88; N, 10.79; found C, 52.41; H, 3.92; N, 10.76.

2-[5-(3-Methoxybenzylidene)-4-thiazolinone-2-imino]-4-

methyl Thiazole-5-carboxylic acid ethyl ester (D8). Yellow solid,

d

:

29.64, 35.60, 36.23, 120.70, 149.18, 152.80, 157.77, 165.57, 166.16,

174.69; Anal. Calcd. for C₁₂H₁₄N₆OS₂: C, 44.71; H, 4.38; N, 26.07;

found C, 44.67; H, 4.39; N, 26.06.

m.p. 222e223 ^ꢂC, yield 86.4%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.28

(t, J ¼ 7.0 Hz, 3H, CH₃), 2.62 (s, 3H, CH₃), 3.83 (s, 3H, OCH₃), 4.25 (q,

J ¼ 7.0 Hz, 2H, OCH₂), 7.07 (d, J ¼ 7.6 Hz,1H, C₆H₄4-H), 7.21e7.23 (m,

2H, C₆H₄2,6-H), 7.48 (t, J ¼ 7.6 Hz, 1H, C₆H₄5-H), 7.71 (s, 1H, ¼CH),

12.88 (s, 1H, CONH); Anal. Calcd. for C₁₈H₁₇N₃O₄S₂: C, 53.58; H,

4.25; N, 10.41; found C, 53.55; H, 4.29; N, 10.38.

4.1.2. General procedure for synthesis of 5-benzylidene-4-

thiazolinone derivatives (D)

To a solution of intermediate C (1 mmol) and benzaldehydes

(2 mmol) in 15 mL of acetic acid, anhydrous sodium acetate

(6.0 mmol) was added. The mixture was reﬂuxed for 10 h and then

cooled at room temperature. The precipitate was ﬁltered, washed

with ethanol, saturated brine and water in sequence to obtain pure

product D. For the mixture without precipitate, it was poured into

water, ﬁltered, washed with water, and then the ﬁlter residue was

recrystallized with ethanol to obtain the pure product D.

2-(5-Benzylidene-4-thiazolinone-2-imino)-4-methyl Thia-

zole-5-carboxylic acid ethyl ester (D1). Yellow solid, m.p.

2-[5-(3-Fluorobenzylidene)-4-thiazolinone-2-imino]-4-

methyl Thiazole-5-carboxylic acid ethyl ester (D9). Yellow solid,

m.p. 240e242 ^ꢂC, yield 83.7%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.27

(t, J ¼ 6.6 Hz, 3H, CH₃), 2.61 (s, 3H, CH₃), 4.23 (q, J ¼ 6.6 Hz, 2H,

OCH₂), 7.33 (t, J ¼ 7.6 Hz, 1H, C₆H₄4-H), 7.44 (s, 1H, C₆H₄2-H), 7.46

(s, 1H, C₆H₄6-H), 7.58e7.63 (m, 1H, C₆H₄5-H), 7.70 (s, 1H, ¼CH),

12.92 (s, 1H, NH); Anal. Calcd. for C₁₇H₁₄FN₃O₃S₂: C, 52.16; H, 3.61;

N, 10.74; found C, 52.10; H, 3.56; N, 10.70.

215e216 ^ꢂC, yield 75.1%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.28 (t,

2-[5-(3-Chlorobenzylidene)-4-thiazolinone-2-imino]-4-

methyl Thiazole-5-carboxylic acid ethyl ester (D10). Yellow solid,

J ¼ 7.0 Hz, 3H, CH₃), 2.65 (s, 3H, CH₃), 4.25 (q, J ¼ 7.0 Hz, 2H, CH₂),

7.47e7.54 (m,1H, C₆H₅4-H), 7.57 (d, J ¼ 7.2 Hz, 2H, C₆H₅3,5-H), 7.65

(d, J ¼ 7.2 Hz, 2H, C₆H₅2,6-H), 7.75 (s, 1H, ¼CH), 12.90 (s, 1H, NH);

Anal. Calcd. for C₁₇H₁₅N₃O₃S₂: C, 54.68; H, 4.05; N, 11.25; found C,

54.63; H, 4.08; N, 11.22.

m.p. 218e219 ^ꢂC, yield 82.4%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.28

(t, J ¼ 6.8 Hz, 3H, CH₃), 2.62 (s, 3H, CH₃), 4.24 (q, J ¼ 6.8 Hz, 2H,

OCH₂), 7.46e7.64 (m, 4H, C₆H₄2,4,5,6-H), 7.70 (s, 1H, ¼CH), 12.95 (s,

1H, NH); Anal. Calcd. for C₁₇H₁₄ClN₃O₃S₂: C, 50.06; H, 3.46; N,10.30;

found C, 50.01; H, 3.50; N, 10.28.

2-[5-(2-Carboxybenzylidene)-4-thiazolinone-2-imino]-4-

methyl Thiazole-5-carboxylic acid ethyl ester (D2). Yellow solid,

2-[5-(4-Carboxybenzylidene)-4-thiazolinone-2-imino]-4-

methyl Thiazole-5-carboxylate (D11). Yellow solid, m.p.

m.p. 246e247 ^ꢂC, yield 88.4%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.29

(t, J ¼ 7.1 Hz, 3H, CH₃), 2.54 (s, 3H, CH₃), 4.23 (q, J ¼ 7.1 Hz, 2H,

OCH₂), 7.52e7.55 (m, 1H, C₆H₄4-H), 7.69 (d, J ¼ 7.6 Hz, 1H, C₆H₄6-

H), 7.71e7.75 (m, 1H, C₆H₄5-H), 7.95 (d, J ¼ 7.6 Hz, 1H, C₆H₄3-H),

8.23 (s, 1H, ¼CH), 13.16 (s, 1H, NH); Anal. Calcd. for C₁₈H₁₅N₃O₅S₂: C,

51.79; H, 3.62; N, 10.07; found C, 51.74; H, 3.62; N, 10.04.

320e321 ^ꢂC, yield 87.1%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.29 (t,

J ¼ 7.1 Hz, 3H, CH₃), 2.62 (s, 3H, CH₃), 4.26 (d, J ¼ 7.1 Hz, 2H, OCH₂),

7.73 (d, J ¼ 8.0 Hz, 2H, C₆H₄2,6-H), 7.75 (s, 1H, ¼CH), 8.07 (d,

J ¼ 8.0 Hz, 2H, C₆H₄3,5-H); Anal. Calcd. for C₁₈H₁₅N₃O₅S₂: C, 51.79;

H, 3.62; N, 10.07; found C, 51.73; H, 3.67; N, 10.04.

2-[5-(2-Hydroxybenzylidene)-4-thiazolinone-2-imino]-4-

methyl thiazole-5-carboxylic acid ethyl ester (D3). Yellow solid,

2-[5-(4-Methoxycarbonylbenzylidene)-4-thiazolinone-2-

imino]-4-methyl Thiazole-5-carboxylic acid ethyl ester (D12).

Yellow solid, m.p. 222e224 ^ꢂC, yield 78.2%. ¹H NMR (400 MHz,

m.p. 252e254 ^ꢂC, yield 69.3%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.28

(t, J ¼ 7.1 Hz, 3H, CH₃), 2.62 (s, 3H, CH₃), 4.25 (q, J ¼ 7.1 Hz, 2H, CH₂),

7.02 (d, J ¼ 7.6 Hz, 1H, C₆H₄3-H), 7.34 (t, J ¼ 7.6 Hz, 1H, C₆H₄5-H),

7.59 (t, J ¼ 7.6 Hz, 1H, C₆H₄4-H), 7.75 (d, J ¼ 7.6 Hz, 1H, C₆H₄6-H),

8.08 (s, 1H, ¼CH), 10.63 (s, 1H, OH), 12.81 (s, 1H, NH); Anal. Calcd.

For C₁₇H₁₅N₃O₄S₂: C, 52.43; H, 3.88; N, 10.79; found C, 52.40; H,

3.92; N, 10.75.

DMSO‑d₆)

d

: 1.29 (t, J ¼ 6.8 Hz, 3H, CH₃), 2.66 (s, 3H, CH₃), 3.88 (s,

3H, OCH₃), 4.26 (q, J ¼ 6.8 Hz, 2H, OCH₂), 7.77 (d, J ¼ 7.2 Hz, 2H, C₆H₄

2,6-H), 7.78 (s, 1H, ¼CH), 8.09 (d, J ¼ 7.2 Hz, 2H, C₆H₄3,5-H), 13.01

(s, 1H, NH); Anal. Calcd. for C₁₉H₁₇N₃O₅S₂: C, 52.89; H, 3.97; N, 9.74;

found C, 52.84; H, 4.02; N, 9.71.

2-[5-(4-Acetylaminobenzylidene)-4-thiazolinone-2-imino]-

4-methyl Thiazole-5-carboxylic acid ethyl ester (D13). Yellow

2-[5-(3-Methylbenzylidene)-4-thiazolinone-2-imino]-4-

methyl Thiazole-5-carboxylic acid ethyl ester (D4). Yellow solid,

solid, yield 85.7%, m.p. 288e289 ^ꢂC. ¹H NMR (400 MHz, DMSO‑d₆)

d:

m.p. 246e247 ^ꢂC, yield 88.2%. ¹H NMR (400 MHz, DMSO‑d₆)

d

: 1.28

1.29 (t, 8 ¼ 6.9 Hz, 3H, CH₃), 2.09 (s, 3H, CH₃), 2.67 (s, 3H, CH₃), 4.26

(d, J ¼ 6.9 Hz, 2H, OCH₂), 7.60 (d, 0 ¼ 7.2 Hz, 2H, C₆H₄3,5-H), 7.68 (s,

1H, ¼CH), 7.77 (d, J ¼ 7.2 Hz, 2H, C₆H₄2,6-H), 10.29 (s, 1H, NHAc),

12.82 (s, 1H, NH); Anal. Calcd. for C₁₉H₁₈N₄O₄S₂: C, 53.01; H, 4.21; N,

13.01; found C, 52.98; H, 4.29; N, 12.96.

(t, J ¼ 7.0 Hz, 3H, CH₃), 2.38 (s, 3H, PhCH₃), 2.65 (s, 3H, CH₃), 4.26 (q,

J ¼ 7.0 Hz, 2H, OCH₂), 7.32 (s, 1H, C₆H₄2-H), 7.39e7.49 (m, 3H, C₆H₄

4,5,6-H), 7.70 (s, 1H, ¼CH), 12.88 (s, 1H, NH); Anal. Calcd. for

C

₁₈H₁₅N₃O₅S₂: C, 55.80; H, 4.42; N,10.84; found C, 55.79; H, 4.45; N,

10.81.

2-[5-(3-Nitrobenzylidene)-4-thiazolinone-2-imino]-4-

methyl Thiazole-5-carboxylic acid ethyl ester (D5). Yellow solid,

m.p. 279e280 ^ꢂC, yield 89.0%. ¹H NMR (400 MHz, DMSO‑d₆)

: 1.28

2-[5-(4-Nitrobenzylidene)-4-thiazolinone-2-imino]-4-

methyl Thiazole-5-carboxylic acid ethyl ester (D14). Yellow solid,

m.p. 279e280 ^ꢂC, yield 87.8%. ¹H NMR (400 MHz, DMSO‑d₆)

d

: 1.29

d

(t, J ¼ 6.8 Hz, 3H, CH₃), 2.67 (s, 3H, CH₃), 4.27 (q, 2H, J ¼ 6.8 Hz,

11

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

OCH₂), 7.86 (s, 1H, ¼CH), 7.91 (d, J ¼ 8.0 Hz, 2H, C₆H₄2,6-H), 8.40 (d,

J ¼ 8.0 Hz, 2H, C₆H₄3,5-H), 13.09 (s, 1H, NH); Anal. Calcd. for

DMSO‑d₆)

d

: 1.29 (t, J ¼ 7.1 Hz, 3H, CH₃), 2.63 (s, 3H, CH₃), 4.27 (q,

J ¼ 7.1 Hz, 2H, OCH₂), 7.93 (s, 1H, ¼CH), 8.28 (s, 1H, C₆H₂6-H), 8.60

C

₁₇H₁₄N₄O₅S₂: C, 48.80; H, 3.37; N, 13.39; found C, 48.83; H, 3.43; N,

(d, J ¼ 2.9 Hz, 1H, C₆H₂5-H), 12.74 (s, 1H, NH); Anal. Calcd. for

13.35.

2-[5-(4-Hydroxybenzylidene)-4-thiazolinone-2-imino]-4-

methyl Thiazole-5-carboxylic acid ethyl ester (D15). Red solid,

m.p. 291e293 ^ꢂC, yield 67.2%. ¹H NMR (400 MHz, DMSO‑d₆)

: 1.28

C₁₇H₁₃N₅O₈S₂: C, 42.59; H, 2.73; N, 14.61; found C, 42.51; H, 2.83; N,

14.59.

2-[5-(3-Methoxy-4-hydroxybenzylidene)-4-thiazolinone-2-

d

imino]-4-methyl Thiazole-5-carboxylic acid (D22). Off-white

(t, J ¼ 7.0 Hz, 3H, CH₃), 2.66 (s, 3H, CH₃), 4.25 (q, J ¼ 7.0 Hz, 2H,

OCH₂), 6.95 (d, J ¼ 8.2 Hz, 2H, C₆H₄3,5-H), 7.52 (d, J ¼ 8.2 Hz, 2H,

C₆H₄2,6-H), 7.66 (s, 1H, ¼CH), 10.36 (s, 1H, OH), 12.75 (s, 1H, NH);

solid, m.p. 245e247 ^ꢂC, yield 35.7%. ¹H NMR (400 MHz, DMSO‑d₆)

d

: 13.16 (s, 1H, CO₂H), 12.72 (s, 1H, NH), 10.01 (s, 1H, OH), 7.68 (s,

1H, ¼CH), 7.26 (s, 1H, C₆H₃2-H), 7.16 (d, J ¼ 8.0 Hz, 1H, C₆H₃5-H),

6.96 (d, J ¼ 8.0 Hz, 1H, C₆H₃6-H), 3.87 (s, 3H, OCH₃), 2.62 (s, 3H,

CH₃); Anal. Calcd. for C₁₆H₁₃N₃O₅S₂: C, 49.10; H, 3.35; N, 10.74;

found C, 49.04; H, 3.41; N, 10.70.

2-[5-(3-Methoxy-4-hydroxybenzylidene)-4-thiazolinone-2-

imino]-4-methyl Thiazole-5-carboxylic acid methyl ester (D23).

Yellow solid, m.p. 262e263 ^ꢂC, yield 26.3%. ¹H NMR (400 MHz,

¹³C NMR (150 MHz, DMSO‑d₆)

d: 14.58, 18.02, 61.29, 116.79, 117.45,

120.11, 124.61, 133.07, 133.67, 158.30, 159.31, 160.42, 161.98, 167.48,

170.29; Anal. Calcd. for C₁₇H₁₅N₃O₄S₂: C, 52.43; H, 3.88; N, 10.79;

found C, 52.40; H, 3.90; N, 10.78.

2-[5-(2-Hydroxy-3-methoxybenzylidene)-4-thiazolinone-2-

imino]-4-methyl Thiazole-5-carboxylic acid ethyl ester (D16).

Yellow solid, m.p. 242e244 ^ꢂC, yield 75.6%. ¹H NMR (400 MHz,

DMSO‑d₆) d: 3.73 (s, 3H, OCH₃), 3.84 (s, 3H, CO₂CH₃), 6.91 (d,

DMSO‑d₆)

d

: 1.28 (t, J ¼ 6.6 Hz, 3H, CH₃), 2.64 (s, 2H, CH₃), 3.85 (s,

J ¼ 8.0 Hz, 1H, C₆H₃5-H), 7.07 (d, J ¼ 8.0 Hz, 1H, C₆H₃6-H), 7.13 (s,

3H, OCH₃), 4.25 (q, J ¼ 6.6 Hz, 2H, OCH₂), 6.97 (t, J ¼ 7.6 Hz, 1H, C₆H₃

5-H), 7.06 (d, J ¼ 7.6 Hz,1H, C₆H₃4-H), 7.10 (d, J ¼ 7.6 Hz,1H, C₆H₃6-

H), 8.02 (s, 1H, ¼CH), 9.75 (s, 1H, OH), 12.80 (s, 1H, NH); Anal. Calcd.

for C₁₈H₁₇N₃O₅S₂: C, 51.54; H, 4.09; N, 10.02; found C, 51.45; H, 4.15;

N, 9.98.

2-[5-(Benzo[d][1,3]dioxol-5-ylmethylene)-4-thiazolinone-2-

imino]-4-methyl Thiazole-5-carboxylic acid ethyl ester (D17).

Yellow solid, m.p. 255e256 ^ꢂC, yield 73.9%. ¹H NMR (400 MHz,

1H, C₆H₃2-H), 7.55 (s, 1H, ¼CH), 10.06 (s, 1H, NH); Anal. Calcd. for

C

₁₇H₁₅N₃O₅S₂: C, 50.36; H, 3.73; N, 10.36; found C, 50.34; H, 3.76; N,

10.32.

2-[5-(3-Methoxy-4-hydroxybenzylidene)-4-thiazolinone-2-

imino]-4-methyl Thiazole-5-carboxylic acid tert-butyl ester

(D24). Yellow solid. m.p. 263e265 ^ꢂC, yield 35.7%. ¹H NMR

(400 MHz, DMSO‑d₆) d: 2.51 (s, 9H, C(CH₃)₃), 2.60 (s, 3H, CH₃), 3.87

(s, 3H, OCH₃), 6.96 (d, J ¼ 8.0 Hz, 1H, C₆H₃5-H), 7.15 (d, J ¼ 8.0 Hz,

1H, C₆H₃6-H), 7.25 (s, 1H, C₆H₃2-H), 7.66 (s, 1H, ¼CH), 10.02 (s, 1H,

NH); Anal. Calcd. for C₂₀H₂₁N₃O₅S₂: C, 53.68; H, 4.73; N, 9.39; found

C, 53.63; H, 4.77; N, 9.37.

DMSO‑d₆)

d: 1.30 (t, J ¼ 7.0 Hz, 3H, CH₃), 2.65 (s, 3H, CH₃), 4.27 (q,

J ¼ 7.0 Hz, 2H, OCH₂), 6.16 (s, 2H, OCH₂O), 7.14 (d, 8 ¼ 8.0 Hz,1H,1,3-

Benzodioxole 6-H), 7.17 (s, 1H, 1,3-Benzodioxole 2-H), 7.22 (d,

J ¼ 8.0 Hz, 1H, 1,3-Benzodioxole 7-H), 7.67 (s, 1H, ¼CH), 12.82 (s, 1H,

2-[5-(4-Hydroxybenzylidene)-4-thiazolinone-2-imino]-4-

isopropyl Thiazole-5-carboxylate (D25). Yellow solid, m.p.

NH); ¹³C NMR (100 MHz, DMSO‑d₆)

d: 14.63, 18.03, 61.40, 102.54,

109.69, 117.67, 122.01, 126.76, 127.84, 133.36, 148.60, 149.81, 158.30,

159.10, 162.01, 167.39, 170.26; Anal. Calcd. for C₁₈H₁₅N₃O₅S₂: C,

51.79; H, 3.62; N, 10.07; found C, 51.73; H, 3.68; N, 10.05.

2-[5-(3-Methoxy-4-hydroxybenzylidene)-4-thiazolinone-2-

imino]-4-methyl Thiazole-5-carboxylic acid ethyl ester (D18).

Yellow solid. m.p. 237e239 ^ꢂC, yield 77.6%. ¹H NMR (400 MHz,

250e252 ^ꢂC, yield 77.1%. ¹H NMR (400 MHz, DMSO‑d₆) ¹H NMR

d:

(400 MHz, DMSO‑d₆)

d

: 1.31 (d, J ¼ 6.6 Hz, 6H, 2 ꢁ CH₃), 3.80 (s, 3H,

OCH₃), 3.84e3.91 (m, 1H, CH), 6.92 (d, J ¼ 8.4 Hz, 2H, C₆H₄3,5-H),

7.53 (d, J ¼ 8.4 Hz, 2H, C₆H₄2,6-H), 7.67 (s, 1H, ¼CH), 10.37 (s, 1H,

OH), 12.78 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO‑d₆)

d: 22.29,

29.03, 52.45, 115.64, 116.58, 120.74, 124.85, 132.94, 133.59, 160.10,

160.33, 162.13, 167.60, 170.91, 172.49; Anal. Calcd. for C₁₈H₁₇N₃O₄S₂:

C, 53.58; H, 4.25; N, 10.41; found C, 53.55; H, 4.29; N, 10.38.

2-[5-(2-Hydroxy-3-methoxybenzylidene)-4-thiazolinone-2-

imino]-4-isopropyl Thiazole-5-carboxylic acid methyl ester

(D26). Red solid, m.p. 247e249 ^ꢂC, yield 66.8%. ¹H NMR (400 MHz,

DMSO‑d₆)

d

: 1.28 (t, J ¼ 6.9 Hz, 3H, CH₃), 2.64 (s, 2H, CH₃), 3.85 (s,

3H, OCH₃), 4.26 (q, J ¼ 6.9 Hz, 2H, OCH₂), 6.98 (d, J ¼ 7.8 Hz,1H, C₆H₃

5-H), 7.05e7.11 (m, 2H, C₆H₃2,6-H), 8.03 (s, 1H, ¼CH), 9.75 (s, 1H,

OH), 12.81 (s, 1H, NH); ¹³C NMR (150 MHz, DMSO‑d₆)

d: 14.60,17.86,

39.96, 55.77, 61.35, 114.09, 116.62, 117.46, 120.41, 125.10, 125.57,

133.99, 148.40, 149.97, 158.11, 159.66, 162.00, 167.48, 170.40; Anal.

Calcd. for C₁₈H₁₇N₃O₅S₂: C, 51.54; H, 4.09; N, 10.02; found C, 51.44;

H, 4.12; N, 4.02.

DMSO‑d₆)

d

: 1.28 (d, J ¼ 6.6 Hz, 6H, 2 ꢁ CH₃), 3.80 (s, 3H, OCH₃), 3.85

(s, 3H, CO₂CH₃), 3.87e3.93 (m, 1H, CH), 6.93 (t, 1H, C₆H₃5-H),

7.03e7.14 (m, 2H, C₆H₃4,6-H), 8.02 (s, 1H, ¼CH), 9.77 (s, 1H, OH),

12.83 (s,1H, NH); Anal. Calcd. for C₁₉H₁₉N₃O₅S₂: C, 52.64; H, 4.42; N,

9.69; found C, 52.63; H, 4.45; N, 9.65.

2-[5-(2,4-Dihydroxybenzylidene)-4-thiazolinone-2-imino]-

4-methyl Thiazole-5-carboxylic acid ethyl ester (D19). Red solid,

m.p. > 300 ^ꢂC, yield 51.7%. ¹H NMR (400 MHz, DMSO‑d₆)

d

: 1.28 (s,

2-[5-(3,4-Dihydroxybenzylidene)-4-thiazolinone-2-imino]-

4-isopropyl Thiazole-5-carboxylic acid methyl ester (D27). Yel-

low solid, m.p. 254e257 ^ꢂC, yield 19.8%. ¹H NMR (400 MHz,

3H, CH₃), 2.62 (s, 3H, CH₃), 4.24 (s, 2H, OCH₂), 6.43 (s, 1H, C₆H₃3-H),

7.10 (s, 1H, C₆H₃5-H), 7.63e7.68 (m, 1H, C₆H₃6-H), 8.43 (s,

1H, ¼CH), 10.36 (s, 1H, OH),11.06 (s, 1H, OH), 12.57 (s, 1H, NH); Anal.

Calcd. for C₁₇H₁₅N₃O₅S₂: C, 50.36; H, 3.73; N, 10.36; found C, 50.27;

H, 3.81; N, 10.31.

2-[5-(3,4-Dihydroxybenzylidene)-4-thiazolinone-2-imino]-

4-methyl Thiazole-5-carboxylic acid ethyl ester (D20). Red-

brown solid, m.p. > 300 ^ꢂC, yield 39.4%. ¹H NMR (400 MHz,

DMSO‑d₆)

d

: 1.31 (d, J ¼ 6.5 Hz, 6H, 2 ꢁ CH₃), 3.80 (s, 3H, OCH₃),

3.82e3.94 (m, 1H, CH), 6.89 (d, J ¼ 8.0 Hz, 1H, C₆H₃6-H), 7.03 (d,

J ¼ 8.0 Hz, 1H, C₆H₃5-H), 7.08 (s, 1H, C₆H₃2-H), 7.59 (s, 1H, ¼CH),

9.31 (s, 1H, OH), 10.07 (s, 1H, OH), 12.76 (s, 1H, NH); Anal. Calcd. for

C

₁₈H₁₇N₃O₅S₂: C, 51.54; H, 4.09; N, 10.02; found C, 51.50; H, 4.10; N,

9.99.

2-[5-(3-Methoxy-4-hydroxybenzylidene)-4-thiazolinone-2-

DMSO‑d₆)

d: 1.29 (t, J ¼ 6.6 Hz, 3H, CH₃), 2.68 (s, 3H, CH₃), 4.26 (q,

J ¼ 6.6 Hz, 2H, OCH₂), 6.90 (d, J ¼ 7.4 Hz, 1H, C₆H₃5-H), 7.03 (d,

J ¼ 7.9 Hz, 1H, C₆H₃6-H), 7.07 (s, 1H, C₆H₃2-H), 7.58 (s, 1H, ¼CH),

9.57 (s, 1H, OH), 9.95 (s, 1H, OH), 12.77 (s, 1H, NH); Anal. Calcd. for

imino]-4-isopropyl Thiazole-5-carboxylic acid methyl ester

(D28). Red solid, m.p. 242e245 ^ꢂC, yield 32.8%. ¹H NMR (400 MHz,

DMSO‑d₆)

d

: 1.28 (d, J ¼ 6.6 Hz, 6H, 2 ꢁ CH₃), 3.80 (s, 3H, OCH₃),

C

₁₇H₁₅N₃O₅S₂: C, 50.36; H, 3.73; N, 10.36; found C, 50.31; H, 3.76; N,

10.32.

2-[5-(2-Hydroxy-3,5-dinitrobenzylidene)-4-thiazolinone-2-

3.89e3.87 (s, 3H, CO₂CH₃), 3.89e3.93 (m, 1 H, CH), 6.92 (d,

J ¼ 8.0 Hz, 1H, C₆H₃5-H), 7.18e7.21 (m, 2H, C₆H₃2,6-H), 7.68 (s,

1H, ¼CH), 10.07 (s, 1H, OH), 12.80 (s, 1H, NH); Anal. Calcd. for

imino]-4-methyl Thiazole-5-carboxylic acid ethyl ester (D21).

C₁₈H₁₇N₃O₅S₂: C, 51.54; H, 4.09; N, 10.02; found C, 51.50; H, 4.10; N,

Red solid, m.p. 258e260 ^ꢂC, yield 45.3%. ¹H NMR (400 MHz,

10.00.

12

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

2-[5-(3,5-Dimethoxy-4-hydroxybenzylidene)-4-

m.p.157e159 ^ꢂC, yield 33.6%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.20

thiazolinone-2-imino]-4-isopropyl Thiazole-5- carboxylic acid

(t, J ¼ 7.0 Hz, 3H, CH₃), 3.78 (s, 2H, CH₂), 3.87 (s, 3H, OCH₃), 4.13 (q,

J ¼ 7.0 Hz, 2H, OCH₂), 6.95 (d, J ¼ 8.2 Hz, 1H, C₆H₃5-H), 7.14 (d,

J ¼ 8.2 Hz, 1H, C₆H₃6-H), 7.23 (s, 1H, C₆H₃2-H), 7.25 (s, 1H, thiazole

5-H), 7.63 (s, 1H, ¼CH), 10.02 (s, 1H, OH), 12.54 (s, 1H, NH); Anal.

Calcd. for C₁₈H₁₇N₃O₅S₂: C, 51.54; H, 4.09; N, 10.02; found C, 51.50;

H, 4.15; N, 9.98.

methyl ester (D29). Red solid. m.p. 273e275 ^ꢂC, yield 71.8%. ¹H

NMR (400 MHz, DMSO‑d₆)

(s, 3H, CO₂CH₃), 3.85 (s, 6H, 2 ꢁ OCH₃), 3.87e3.92 (m, 1H, CH), 7.00

(s, 2H, C₆H₂2,6-H), 7.69 (s, 1H, ¼CH), 9.43 (s, 1H, OH), 12.79 (s, 1H,

d

: 1.26 (d, J ¼ 6.7 Hz, 6H, 2 ꢁ CH₃), 3.79

NH); ¹³C NMR (150 MHz, DMSO‑d₆)

d: 22.46, 28.85, 52.61, 56.36,

108.69, 115.75, 120.67, 123.92, 134.51, 139.17, 148.69, 159.83, 162.18,

167.55, 167.73, 171.00; Anal. Calcd. for C₂₀H₂₁N₃O₆S₂: C, 51.82; H,

4.57; N, 9.07; found C, 51.76; H, 4.65; N, 9.02.

2-[2-(3-Methoxy-4-hydroxybenzylidene)-4-thiazolinone-2-

imino]-5-methyl Thiazole-4-ethyl acetate (D38). Yellow solid,

m.p. 138e140 ^ꢂC, yield 41.6%; ¹H NMR (400 MHz, CDCl₃)

d: 1.29 (t,

2-[5-(3-Methoxy-4-hydroxybenzylidene)-4-thiazolinone-2-

imino]-4-propyl Thiazole-5-carboxylic acid methyl ester (D30).

Yellow solid, m.p. 274e276 ^ꢂC, yield 69.9%. ¹H NMR (400 MHz,

J ¼ 6.9 Hz, 3H, CH₃), 2.32 (s, 3H, CH₃), 3.70 (s, 2H, CH₂), 3.97 (s, 3H,

OCH₃), 4.20 (q, J ¼ 6.9 Hz, 2H, OCH₂), 7.01e7.03 (m, 2H, C₆H₃5,6-H),

7.11 (s, 1H, C₆H₃2-H), 7.77 (s, 1H, ¼CH); Anal. Calcd. for

DMSO‑d₆)

d

: 0.96 (t, J ¼ 7.2 Hz, 3H, CH₃), 1.74e1.79 (m, 2H, CH₂),

C₁₉H₁₉N₃O₅S₂: C, 52.64; H, 4.42; N, 9.69; found C, 52.68; H, 4.48; N,

2.99 (t, J ¼ 7.2 Hz, 2H, Thiazole-CH₂), 3.76 (s, 3H, OCH₃), 3.86 (s, 3H,

CO₂CH₃), 6.91 (d, J ¼ 8.1 Hz, 1H, C₆H₃5-H), 7.13 (d, J ¼ 8.1 Hz, 1H,

C₆H₃6-H), 7.16 (s, 1H, C₆H₃2-H), 7.61 (s, 1H, ¼CH), 9.99 (s, 1H, OH),

12.68 (s, 1H, CONH); Anal. Calcd. for C₁₉H₁₉N₃O₅S₂: C, 52.64; H,

4.42; N, 9.69; found C, 52.63; H, 4.47; N, 9.65.

9.64.

2-((5-Acetyl-4-methylthiazol-2-yl)

nitrobenzylidene) thiazolidin-4-one (D39). Yellow solid, m.p.

303e306 ^ꢂC, yield 75%. ¹H NMR (400 MHz, DMSO‑d₆)

: 2.51 (s, 3H,

imino)-5-(3-

d

CH₃), 2.67 (s, 3H, CH₃), 7.88 (s, 1H, ¼CH), 8.08e8.50 (m, 4H, C₆H₄),

13.04 (s,1H, CONH); Anal. Calcd. for C₁₆H₁₂N₄O₄S₂: C, 49.48; H, 3.11;

N, 14.42; found C, 49.43; H, 3.14; N, 14.40.

2-[2-(3-Nitrobenzylidene)-4-thiazolinone-2-imino]

zole-4-ethyl acetate (D31). Yellow solid, m.p.170e172 ^ꢂC, yield

89.2%. ¹H NMR (400 MHz, CDCl₃)

Thia-

d

:1.31 (t, J ¼ 7.1 Hz, 3H, CH₃), 3.79

2-((5-Acetyl-4-methylthiazol-2-yl)imino)-5-(3-

(s, 2H, COCH₂), 4.23 (q, J ¼ 7.1 Hz, 2H, OCH₂), 7.00 (s, 1H, thiazole 5-

H), 7.70 (t, J ¼ 8.0 Hz, 1H, C₆H₄5-H), 7.83e7.87 (m, 2H, ¼CH, C₆H₄6-

H), 8.28 (dd, J ¼ 8.0, 1.3 Hz, 1H, C₆H₄4-H), 8.42 (s, 1H, C₆H₄2-H);

Anal. Calcd. for C₁₇H₁₄N₄O₅S₂: C, 48.80; H, 3.37; N, 13.39; found C,

48.78; H, 3.43; N, 13.38.

hydroxybenzylidene)thiazolidin-4-one(D41). Yellow solid, m.p.

246e249 ^ꢂC, yield 31%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 2.49 (s, 3H,

CH₃), 2.67 (s, 3H, CH₃), 6.90e7.38 (m, 4H, C₆H₄), 7.64 (s, 1H, ¼CH),

9.91 (s, 1H, OH), 12.88 (s, 1H, CONH); ¹³C NMR (150 MHz, DMSO‑d₆)

d

: 19.03, 30.59, 116.74, 118.24, 121.94, 124.40, 129.44, 130.83, 133.41,

2-[2-(4-Carboxybenzylidene)-4-thiazolinone-2-imino] Thia-

134.82, 156.23, 158.32, 159.27, 167.33, 170.07, 190.81; EI-MS (m/z):

359.0 [M]þ; Anal. Calcd. for C₁₆H₁₃N₃O₃S₂: C, 53.47; H, 3.65; N,

11.69; found C, 53.45; H, 3.68; N, 11.67.

zole-4-ethyl acetate (D33). Yellow solid. m.p. 289e290 ^ꢂC, yield

82.4%; ¹H NMR (400 MHz, DMSO‑d₆)

d

: 1.21 (t, J ¼ 7.1 Hz, 3H, CH₃),

3.82 (s, 2H, COCH₂), 4.15 (q, J ¼ 7.1 Hz, 2H, OCH₂), 7.28 (s, 1H,

thiazole 5-H), 7.72e7.79 (m, 3H, C₆H₄2,6-H, ¼CH), 8.06 (d,

J ¼ 8.4 Hz, 2H, C₆H₄3,5-H), 12.78 (s, 1H, CO₂H), 13.23 (s, 1H, CONH);

Anal. Calcd. for C₁₈H₁₅N₃O₅S₂: C, 51.79; H, 3.62; N, 10.07; found C,

51.75; H, 3.71; N, 10.03.

4-((2-((5-Acetyl-4-methylthiazol-2-yl)imino)-4-

oxothiazolidin-5-ylidene)methyl)benzoic acid(D42). Yellow

solid, m.p. 313e315 ^ꢂC, yield 92%. ¹H NMR (400 MHz, DMSO‑d₆)

d:

2.50 (s, 3H, CH₃), 2.67 (s, 3H, CH₃), 7.71e7.77 (m, 3H, ¼CH, C₆H₄2,6-

H), 8.07 (d, J ¼ 5.2 Hz, 2H, C₆H₄3,5-H), 12.99 (s, 1H, CONH), 13.25 (s,

1H, CO₂H); Anal. Calcd. for C₁₇H₁₃N₃O₄S₂: C, 52.70; H, 3.38; N,

10.85; found C, 52.67; H, 3.43; N, 10.82.

2-[2-(4-Hydroxybenzylidene)-4-thiazolinone-2-imino] Thia-

zole-4-ethyl acetate (D34). Yellow solid. m.p. 215e217 ^ꢂC, yield

81.8%. ¹H NMR (400 MHz, DMSO‑d₆)

d

:1.23 (t, J ¼ 7.1 Hz, 3H, CH₃),

2-((5-Acetyl-4-methylthiazol-2-yl)imino)-5-(4-

3.81 (s, 2H, COCH₂), 4.16 (q, J ¼ 7.1 Hz, 2H, OCH₂), 6.96 (d, ¼ 8.4 Hz,

2H, C₆H₄3,5-H), 7.24 (s, 1H, thiazole 5-H), 7.51 (d, J ¼ 8.4 Hz, 2H,

C₆H₄2,6-H), 7.62 (s, 1H, ¼CH),10.51 (s,1H, OH),12.56 (s, 1H, CONH);

Anal. Calcd. for C₁₇H₁₅N₃O₄S₂: C, 52.43; H, 3.88; N, 10.79; found C,

52.40; H, 3.93; N, 10.75.

hydroxybenzylidene)thiazolidin-4-one(D43). Yellow solid, m.p.

302e305 ^ꢂC, yield 60%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 2.50 (s, 3H,

CH₃), 2.68 (s, 3H, CH₃), 6.95 (d, J ¼ 8.0 Hz, 2H, C₆H₄3,5-H), 7.53 (d,

J ¼ 8.0 Hz, 2H, C₆H₄2,6-H), 7.67 (s, 1H, ¼CH), 10.38 (s, 1H, OH), 12.77

(s, 1H, CONH); ¹³C NMR (150 MHz, DMSO‑d₆)

d: 19.07, 30.59, 116.84,

2-[2-(2-Hydroxy-3-methoxybenzylidene)-4-thiazolinone-2-

120.06, 124.62, 129.18, 133.13, 133.80, 156.28, 159.47, 160.45, 167.52,

170.21, 190.79; Anal. Calcd. for C₁₆H₁₃N₃O₃S₂: C, 53.47; H, 3.65; N,

11.69; found C, 53.41; H, 3.70; N, 11.65.

imino] Thiazole-4-ethyl acetate (D35). Tan solid, m.p. 180e182 ^ꢂC,

yield 24.3%. ¹H NMR (400 MHz, DMSO‑d₆)

d

:1.21 (t, J ¼ 7.1 Hz, 3H,

CH₃), 3.78 (s, 2H, CH₂), 3.85 (s, 3H, OCH₃), 4.14 (q, J ¼ 7.1 Hz, 2H,

OCH₂), 6.92 (t, J ¼ 8.0 Hz, 1H, C₆H₃4-H), 7.05 (d, J ¼ 8.0 Hz, 1H, C₆H₃

5-H), 7.10 (d, J ¼ 8.0 Hz,1H, C₆H₃6-H), 7.26 (s,1H, thiazole 5-H), 7.99

(s, 1H, ¼CH), 9.70 (s, 1H, OH), 12.60 (s, 1H, CONH); Anal. Calcd. for

2-((5-Acetyl-4-methylthiazol-2-yl)imino)-5-(3-hydroxy-4-

methoxybenzylidene)thiazolidin-4-one (D44). Yellow solid, m.p.

259e263 ^ꢂC, yield 51%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 2.51 (s, 3H,

CH₃), 2.70 (s, 3H, CH₃), 3.85 (s, 3H, OCH₃), 7.10 (s, 1H, C₆H₃2-H),

7.13e7.16 (m, 2H, C₆H₃5,6-H), 7.62 (s, 1H, ¼CH), 9.58 (s, 1H, OH),

12.80 (s, 1H, CONH); Anal. Calcd. for C₁₇H₁₅N₃O₄S₂: C, 52.43; H,

3.88; N, 10.79; found C, 52.41; H, 3.41; N, 10.76.

C

₁₈H₁₇N₃O₅S₂: C, 51.54; H, 4.09; N, 10.02; found C, 51.51; H, 4.05; N,

10.03.

2-[2-(4-Methoxy-3-hydroxybenzylidene)-4-thiazolinone-2-

imino] Thiazole-4-ethyl acetate (D36). Off-white solid, m.p.

168e170 ^ꢂC, yield 42.7%. ¹H NMR (400 MHz, DMSO‑d₆)

:1.22 (t,

2-((5-Acetyl-4-methylthiazol-2-yl)imino)-5-(4-hydroxy-3-

methoxybenzylidene)thiazolidin-4-one (D45). Yellow solid, m.p.

d

J ¼ 6.8 Hz, 3H, CH₃), 3.82 (s, 2H, CH₂), 3.86 (s, 3H, OCH₃), 4.17 (q,

J ¼ 6.8 Hz, 2H, OCH₂), 7.05e7.15 (m, 3H, C₆H₃2,5,6-H), 7.25 (s, 1H,

thiazole 5-H), 7.58 (s, 1H, ¼CH), 9.45 (s, 1H, OH), 12.58 (s, 1H, NH);

255e260 ^ꢂC, yield 31%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 2.47 (s, 3H,

CH₃), 2.61 (s, 3H, CH₃), 3.86 (s, 3H, OCH₃), 7.01 (d, J ¼ 8.0 Hz, 1H,

C₆H₃5-H), 7.12 (d, J ¼ 8.0 Hz, 1H, C₆H₃6-H), 7.21 (s, 1H, C₆H₃2-H),

7.63 (s, 1H, ¼CH), 10.17 (s, 1H, OH), 12.80 (s, 1H, CONH); Anal. Calcd.

for C₁₇H₁₅N₃O₄S₂: C, 52.43; H, 3.88; N, 10.79; found C, 52.39; H,

3.40; N, 10.77.

¹³C NMR (150 MHz, DMSO‑d₆)

d: 14.66, 37.44, 56.18, 60.96, 112.71,

114.36, 117.13, 121.62, 123.57, 126.59, 132.75, 146.74, 147.34, 150.35,

156.37, 167.59, 168.55, 170.31; Anal. Calcd. for C₁₈H₁₇N₃O₅S₂: C,

51.54; H, 4.09; N, 10.02; found C, 51.51; H, 4.12; N, 10.01.

2-((5-Acetyl-4-methylthiazol-2-yl)imino)-5-(benzo[d][1,3]

dioxol-5-ylmethylene)thiazolidin-4-one (D46). Tan solid, m.p.

2-[2-(3-Methoxy-4-hydroxybenzylidene)-4-thiazolinone-2-

imino]

Thiazole-4-ethyl

acetate

(D37).

Yellow

solid,

13

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

283e285 ^ꢂC, yield 51%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 2.50 (s, 3H,

2-[5-(4-Nitrobenzylidene)-4-thiazolinone-2-imino]-4-tert-

butyl-5-nitrothiazole (D55). Yellow solid. m.p. 288e290 ^ꢂC, yield

CH₃), 2.66 (s, 3H, CH₃), 6.14 (s, 2H, CH₂), 7.13 (d, J ¼ 7.2 Hz, 1H, C₆H₃

5-H), 7.17 (s, 1H, C₆H₃2-H), 7.22 (d, J ¼ 7.2 Hz, 1H, C₆H₃6-H), 7.68 (s,

1H, ¼CH), 12.83 (s, 1H, CONH); Anal. Calcd. for C₁₇H₁₃N₃O₄S₂: C,

52.70; H, 3.38; N, 10.85; found C, 52.63; H, 3.46; N, 10.82.

2-((5-Acetyl-4-methylthiazol-2-yl)imino)-5-(3,4-

88.0%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.53 (s, 9H, C(CH₃)₃), 7.56 (d,

J ¼ 8.0 Hz, 2H, C₆H₄2,6-H), 7.68 (s, 1H, ¼CH), 7.71 (d, J ¼ 8.0 Hz, 2H,

C₆H₄3,5-H), 12.99 (s, 1H, CONH); Anal. Calcd. for C₁₇H₁₅N₅O₅S₂: C,

47.11; H, 3.49; N, 16.16; found C, 47.09; H, 3.55; N, 16.13.

dihydroxybenzylidene)thiazolidin-4-one (D47). Dark brown

2-[5-(4-Hydroxybenzylidene)-4-thiazolinone-2-imino]-4-

solid, m.p. 275e278 ^ꢂC, yield 56%. ¹H NMR (400 MHz, DMSO‑d₆)

d:

tert-butyl-5-nitrothiazole (D56). Yellow solid. m.p. 275e277 ^ꢂC,

2.50 (s, 3H, CH₃), 2.69 (s, 3H, CH₃), 6.89 (d, J ¼ 7.6 Hz, 1H, C₆H₃5-H),

7.02 (d, J ¼ 7.6 Hz, 1H, C₆H₃6-H), 7.07 (s, 1H, C₆H₃2-H), 7.57 (s,

1H, ¼CH), 9.55 (s, 1H, 3-OH), 9.93 (s, 1H, 4-OH), 12.75 (s, 1H, CONH);

Anal. Calcd. for C₁₆H₁₃N₃O₄S₂: C, 51.19; H, 3.49; N, 11.19; found C,

51.14; H, 3.46; N, 11.17.

yield 72.5%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.53 (s, 9H, C(CH₃)₃),

6.91 (d, J ¼ 8.5 Hz, 2H, C₆H₄3,5-H), 7.53 (d, ¼ 8.5 Hz, 2H, C₆H₄2,6-

H), 7.72 (s, 1H, ¼CH), 10.39 (s, 1H, OH), 13.02 (s, 1H, CONH); Anal.

Calcd. for C₁₇H₁₆N₄O₄S₂: C, 50.48; H, 3.99; N, 13.85; found C, 50.46;

H, 4.05; N, 13.82.

2-[5-(2-Carboxybenzylidene)-4-thiazolinone-2-imino]-4-

2-[5-(2-Hydroxy-3-methoxybenzylidene)-4-thiazolinone-2-

imino]-4-tert-butyl-5-nitro-thiazole (D57). Yellow solid, m.p.

tert-butyl-5-nitrothiazole (D48). Yellow solid, m.p. 278e279 ^ꢂC,

yield 89.3%. ¹H NMR (400 MHz, DMSO‑d₆)

d

: 1.43 (s, 9H, C(CH₃)₃),

274e275 ^ꢂC, yield 74.6%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.51 (s, 9H,

7.61 (t, J ¼ 7.6 Hz,1H, C₆H₄4-H), 7.68e7.73 (m, 2H, C₆H₄5,6-H), 8.03

(d, J ¼ 7.6 Hz, 1H, C₆H₄3-H), 8.40 (s, 1H, ¼CH), 13.17 (s, 1H, CONH);

Anal. Calcd. for C₁₈H₁₆N₄O₅S₂: C, 49.99; H, 3.73; N, 12.96; found C,

49.92; H, 3.77; N, 12.91.

C(CH₃)₃), 3.86 (s, 3H, OCH₃), 6.90 (t, J ¼ 8.0 Hz, 1H, C₆H₃5-H), 7.05

(d, J ¼ 8.0 Hz, 1H, C₆H₃4-H), 7.10 (d, J ¼ 8.0 Hz, 1H, C₆H₃6-H), 8.07

(s, 1H, ¼CH), 9.80 (s, 1H, OH), 13.08 (s, 1H, CONH); Anal. Calcd. for

C

₁₈H₁₈N₄O₅S₂: C, 49.76; H, 4.18; N, 12.90; found C, 49.80; H, 4.22; N,

2-[5-(2-Hydroxybenzylidene)-4-thiazolinone-2-imino]-4-

12.86.

2-[5-(3-Methoxy-4-hydroxybenzylidene)-4-thiazolinone-2-

imino]-4-tert-butyl-5-nitro-thiazole (D58). Yellow solid, m.p.

285e286 ^ꢂC, yield 78.1%. ¹H NMR (400 MHz, DMSO‑d₆)

: 1.50 (s,

9H, C(CH₃)₃), 3.83 (s, 3H, OCH₃), 6.90 (s, 1H, C₆H₃5-H), 7.05e7.23

(m, 2H, C₆H₃2,6-H), 7.67 (s, 1H, ¼CH), 9.98 (s, 1H, OH), 12.94 (s, 1H,

CONH); Anal. Calcd. for C₁₈H₁₈N₄O₅S₂: C, 49.76; H, 4.18; N, 12.90;

found C, 49.74; H, 4.22; N, 12.88.

tert-butyl-5-nitrothiazole (D49). Yellow solid, m.p. 262e264 ^ꢂC,

yield 84.9%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.50 (s, 9H, C(CH₃)₃),

6.91 (t, J ¼ 7.7 Hz, 1H, C₆H₄5-H), 6.99 (d, J ¼ 7.7 Hz, 1H, C₆H₄3-H),

7.33 (t, J ¼ 7.7 Hz, 1H, C₆H₄4-H), 7.42 (d, J ¼ 7.7 Hz, 1H, C₆H₄6-H),

8.03 (s, 1H, ¼CH), 10.58 (s, 1H, CONH); Anal. Calcd. for

d

C

₁₇H₁₆N₄O₄S₂: C, 50.48; H, 3.99; N, 13.85; found C, 50.45; H, 4.03; N,

13.81.

2-[5-(3-Nitrobenzylidene)-4-thiazolinone-2-imino]-4-tert-

butyl-5-nitrothiazole (D50). Yellow solid, m.p. 260e261 ^ꢂC, yield

88.2%. ¹H NMR (DMSO‑d₆, 400 MHz)

: 1.50 (s, 9H, C(CH₃)₃),

4.1.3. General procedure for synthesis of D6, D32 and D40

d

Nitro-substituted raw material D5, D31 and D39 (1 mmol) was

dissolved in a mixed solution of 10 mL of acetic acid, 10 mL of

dichloromethane and 1 mL of water, and add an appropriate

amount of reducing iron powder. The reaction proceeded at room

temperature and monitored by TLC. The reaction solution was

ﬁltered, washed with dichloromethane, water. Then the mixture

was washed with sodium bicarbonate solution. The organic phase

was dried with anhydrous sodium sulfate, ﬁltered and evaporated

to afford pure target compounds D6, D32 and D40, respectively.

2-[5-(3-Aminobenzylidene)-4-thiazolinone-2-imino]-4-

7.67e7.81 (m, 1H, C₆H₄5-H), 7.86 (s, 1H, ¼CH), 8.08 (d, J ¼ 8.0 Hz,

1H, C₆H₄6-H), 8.29 (d, J ¼ 8.0 Hz, 1H, C₆H₄4-H), 8.38 (s, 1H, C₆H₄2-

H), 13.27 (s, 1H, CONH); Anal. Calcd. for C₁₇H₁₅N₅O₅S₂: C, 47.11; H,

3.49; N, 16.16; found C, 47.08; H, 3.50; N, 16.10.

2-[5-(3-Methoxybenzylidene)-4-thiazolinone-2-imino]-4-

tert-butyl-5-nitrothiazole (D51). Yellow solid, m.p. 255e256 ^ꢂC,

yield 84.5%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.50 (s, 9H, C(CH₃)₃),

3.80 (s, 3H, OCH₃), 7.07 (d, J ¼ 7.6 Hz, 1H, C₆H₄4-H), 7.17 (s, 1H, C₆H₄

2-H), 7.21 (d, ¼ 7.6 Hz, 1H, C₆H₄6-H), 7.42 (t, J ¼ 7.6 Hz, 1H, C₆H₄5-

H), 7.74 (s, 1H, ¼CH), 13.13 (s, 1H, CONH); Anal. Calcd. for

methyl Thiazole-5-carboxylic acid ethyl ester (D6). Yellow solid.

m.p. 202e203 ^ꢂC, yield 69.2%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.29

C

₁₈H₁₈N₄O₄S₂: C, 51.66; H, 4.34; N, 13.39; found C, 51.62; H, 4.36; N,

13.34.

2-[5-(3-Fluorobenzylidene)-4-thiazolinone-2-imino]-4-tert-

butyl-5-nitrothiazole (D52). Yellow solid, m.p. 244e245 ^ꢂC, yield

86.4%. ¹H NMR (400 MHz, DMSO‑d₆)

: 1.49 (s, 9H, C(CH₃)₃), 7.32

(td, J ¼ 8.0, 2.2 Hz, 1H, C₆H₄4-H), 7.41 (d, 4 ¼ 8.0 Hz, 1H, C₆H₄2-H),

7.45 (d, J ¼ 8.0 Hz, 1H, C₆H₄6-H), 7.51e7.57 (m, 1H, C₆H₄5-H), 7.74

(s, 1H, ¼CH), 13.18 (s, 1H, CONH)); Anal. Calcd. for C₁₇H₁₅FN₄O₃S₂: C,

50.24; H, 3.72; N, 13.78; found C, 50.16; H, 3.83; N, 13.76.

(t, J ¼ 6.8 Hz, 3H, CH₃), 2.67 (s, 3H, CH₃), 4.26 (q, 2H, J ¼ 6.8 Hz,

OCH₂), 6.70 (d, J ¼ 7.6 Hz, 1H, C₆H₄2-H), 6.79 (s, 1H, C₆H₄4-H), 6.81

(s, 1H, C₆H₄6-H), 7.18e7.22 (m, 1H, C₆H₄5-H), 7.82 (s, 1H, ¼CH);

Anal. Calcd. for C₁₇H₁₄N₄O₅S₂: C, 48.80; H, 3.37; N, 13.39; found C,

48.77; H, 3.39; N, 13.35.

d

2-[2-(3-Aminobenzylidene)-4-thiazolinone-2-imino] Thia-

zole-4-ethyl acetate (D32). Yellow solid, m.p. 144e146 ^ꢂC, yield

51.4%; ¹H NMR (400 MHz, DMSO‑d₆)

d

: 1.21 (t, J ¼ 7.1 Hz, 3H, CH₃),

2-[5-(3-Chlorobenzylidene)-4-thiazolinone-2-imino]-4-tert-

3.82 (s, 2H, COCH₂), 4.15 (q, J ¼ 7.1 Hz, 2H, OCH₂), 5.41 (s, 2H, NH₂),

6.69 (d, J ¼ 7.5 Hz, 1H, C₆H₄4-H), 6.78e6.81 (m, 2H, C₆H₄2,6-H),

7.18 (t, J ¼ 7.5 Hz, 1H, C₆H₄5-H), 7.25 (s, 1H, thiazole 5-H), 7.51 (s,

1H, ¼CH), 12.69 (s, 1H, CONH); Anal. Calcd. for C₁₇H₁₆N₄O₃S₂: C,

52.56; H, 4.15; N, 14.42; found C, 52.56; H, 4.15; N, 14.42.

2-((5-Acetyl-4-methylthiazol-2-yl)imino)-5-(3-

butyl-5-nitrothiazole (D53). Yellow solid, m.p. 253e254 ^ꢂC, yield

85.2%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.50 (s, 9H, C(CH₃)₃),

7.49e7.55 (m, 2H, C₆H₄2,5-H), 7.59 (d, J ¼ 8.4 Hz, 1H, C₆H₄4-H),

7.63 (s, 1H, C₆H₄6-H), 7.72 (s, 1H, ¼CH), 13.18 (s, 1H, CONH); Anal.

Calcd. for C₁₇H₁₅ClN₄O₃S₂: C, 48.28; H, 3.58; N, 13.25; found C,

48.29; H, 3.64; N, 13.21.

nitrobenzylidene) thiazolidin-4-one (D40). Yellow solid, m.p.

225e227 ^ꢂC, yield 24%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 2.50 (s, 3H,

2-[5-(4-Acetamidobenzylidene)-4-thiazolinone-2-imino]-4-

tert-butyl-5-nitrothiazole (D54). Yellow solid. m.p. 287e288 ^ꢂC,

CH₃), 2.66 (s, 3H, CH₃), 6.79e7.19 (m, 4H, C₆H₄), 7.55 (s, 1H, ¼CH),

10.18 (s,1H, CONH); Anal. Calcd. for C₁₆H₁₄N₄O₂S₂: C, 53.62; H, 3.94;

N, 15.63; found C, 53.64; H, 3.96; N, 15.58.

yield 82.6%. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.53 (s, 9H, C(CH₃)₃),

2.08 (s, 3H, COCH₃), 7.58 (d, J ¼ 8.0 Hz, 2H, C₆H₄3,5-H), 7.71 (d,

0 ¼ 8.0 Hz, 2H, C₆H₄2,6-H), 7.74 (s, 1H, ¼CH), 10.24 (s, 1H, NH),

13.02 (s,1H, CONH); Anal. Calcd. for C₁₉H₁₉N₅O₄S₂: C, 51.22; H, 4.30;

N, 15.72; found C, 51.20; H, 4.35; N, 15.69.

4.1.4. Procedure for synthesis of 4-thiazolinone analogue S1

Ethyl 2-amino-4-methyloxazole-5-carboxylate (A19, 50 mmol)

14

M. Xiao, L. Xu, D. Lin et al.

European Journal of Medicinal Chemistry 213 (2021) 113161

and potassium carbonate (100 mmol) were dissolved in dichloro-

methane (100 mL), chloroacetyl chloride (8.0 mL) was added

dropwise under ice bath, and reacted at room temperature moni-

tored by TLC. When the reaction is completed, the reaction solution

is poured into water, and washed with sodium bicarbonate solu-

tion, saturated sodium chloride solution and water successively,

dry the organic phase with anhydrous sodium sulfate, remove the

solvent under reduced pressure to get the crude product, and the

residue was puriﬁed by silica-gel column chromatography to give

the intermediate B19. Yield 30.5%, mp 173e174 ^ꢂC; ¹H NMR

J ¼ 15.2 Hz, 1H, CH), 9.87 (s, 1H, OH), 12.13 (s, 1H, NH), 14.33 (s, 1H,

NH); Anal. Calcd. for C₁₈H₁₉N₃O₅S₂: C, 51.29; H, 4.54; N, 9.97; found

C, 51.25; H, 4.60; N, 9.95.

4.2. Determination of NA inhibitory activity

All the target compounds were tested for their NA inhibitory

activity in vitro. NA inhibition assays were performed using NA from

inﬂuenza A/PR/8/34 (H1N1), A/Minfang/151/2000 (H3N2) and B/Si

chuan/01/96 virus. Under the hydrolysis of NA, the NA substrate 2⁰-

(400 MHz, CDCl₃)

d

: 1.39 (t, J ¼ 7.0 Hz, 3H, CH₃), 2.49 (s, 3H, oxazole-

(4-methylumbellifer-yl)-a-D-acetyl neuraminic acid (MUNANA)

CH₃), 4.34 (s, 2H, CH₂Cl), 4.38 (q, J ¼ 7.0 Hz, 2H, OCH₂). Similar to the

method of 4.1.1, the intermediate B19 (10 mmol) and KSCN

(15 mmol) were reﬂuxed for 6 h to obtain yellow solid S1. Yield

generates a ﬂuorescent product that could be quantiﬁed. The in-

tensity of ﬂuorescence can reﬂect the activity of NA sensitively [48].

Inhibition of inﬂuenza virus NA activity was determined by a

standard ﬂuorimetric method [49,50] using MUNANA as substrate,

in 96-well microplates. The reaction mixture containing the com-

pounds and NA enzyme in MES buffer (32.5 mM MES, 4 mM CaCl₂,

pH 6.5) was incubated at 37 ^ꢂC for 60 min. After incubation, the

reaction was terminated by adding NaOH (34 mM). Fluorescence

intensity (M) was quantiﬁed with excitation wavelength at 360 nm

and emission wavelength at 450 nm. Percentage inhibition was

calculated relative to a blank reaction mixture (solvent control)

62.1%, m.p. 213e214 ^ꢂC. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.29 (t,

J ¼ 7.1 Hz, 3H, CH₃), 2.38 (s, 3H, oxazole-CH₃), 4.06 (s, 2H, SCH 2),

4.27 (q, J ¼ 7.1 Hz, 2H, OCH₂), 12.44 (s, 1H, NH); Anal. Calcd. for

C

₁₀H₁₁N₃O₄S: C, 44.60; H, 4.12; N, 15.61; found C, 44.58; H, 4.16; N,

15.57.

4.1.5. Procedure for synthesis of 4-thiazolinone analogue S2

Similar to the method of 4.1.1, the intermediate ethyl 2-(2-

chloroacetamido)-4-methylthiazole-5- carboxylate B1 (5 mmol)

and KSeCN (5 mmol) were reﬂuxed for 6 h to obtain yellow solid S2.

containing virus NA and solvent (Inhibition rate ¼ [1- (M_compound

/

M

_control)] ꢁ 100%). The 50% inhibitory concentration (IC₅₀) was

Yield 78.9%, m.p. 254e255 ^ꢂC. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.28

deﬁned as the concentration of NA inhibitor necessary to reduce NA

activity by 50% relative to a blank reaction mixture. IC₅₀values

displayed represent the mean of three individual determinations

each performed in triplicate assays. Zanamivir (Sigma) was used as

the reference compound.

(t, J ¼ 6.9 Hz, 3H, CH₃), 2.52 (s, 3H, CH₃), 3.94 (s, 2H, CH₂), 4.23 (q,

J ¼ 6.9 Hz, 2H, OCH₂), 12.55 (s, 1H, NH); Anal. Calcd. for

C

₁₀H₁₁N₃O₃SSe: C, 36.15; H, 3.34; N, 12.65; found C, 36.18; H, 3.36;

N, 12.61.

4.1.6. Synthesis of 4-thiazolinone ring-opening analogue S3

4.3. Cytotoxicity test

Acetoferulic acid E3 (2 mmol) was suspended in toluene (2 mL),

thionyl chloride (0.5 mL), 1 drop of N, N-dimethylformamide were

added dropwise in turn. The mixture reacted at 60 ^ꢂC for 5 h. Then

remove the solvent and remaining thionyl chloride to obtain crude

acetylferulyl chloride F3. Potassium thiocyanate (2.5 mmol) was

dissolved in 5 mL acetonitrile, and 5 mL of the acetonitrile solution

of the F3 was added dropwise to it. After the addition was

completed, the reaction was carried out at room temperature for

5 h, and then ﬁltered to obtain acetyl ferulyl isocyanate (G3)

acetonitrile solution. Ethyl 2-amino-4-methyl- thiazole-5-

carboxylate (1.5 mmol, A1) was added to this solution in two

batches, heated to 75 ^ꢂC and reacted for 4 h, cooled to room tem-

perature, sealed and refrigerated, ﬁltered, washed with ethanol,

and dried to obtain target compounds S3. m.p. 237e239 ^ꢂC, yield

Madin-Darby canine kidney (MDCK) cells grown in 96-well

plates until reaching 100% conﬂuence. Cells were treated with the

test samples at concentrations ranging from 0.1 to 100 mg/mL at

37 ^ꢂC and 5% CO₂for 48 h. Blank medium was added to the control

wells. Cell viability was determined using the crystal violet staining

method. The crystal violet staining solution was added to each well

and the plates were incubated for 30 min. The excess staining so-

lution was wash off and ethanol-acetic acid eluent was used to

dissolve the product. After being homogenized, the absorbance of

plates was read at a wavelength of 570 nm using spectrophotom-

eter. The maximal non-toxic concentration (TC₀) and the median

toxic concentration (TC₅₀

compounds.

) values were calculated for all

44.2%; ¹H NMR (400 MHz, DMSO‑d₆)

d

: 1.29 (t, J ¼ 6.8 Hz, 3H, CH₃),

2.56 (s, 3H, thiazole-CH₃), 4.24 (q, J ¼ 6.8 Hz, 2H, OCH₂), 6.99 (d,

J ¼ 15.8 Hz, 1H, ¼CH), 7.46e7.49 (m, 3H, C₆H₅), 7.60e7.68 (m, 2H,

C₆H₅), 7.79 (d, J ¼ 15.8 Hz, 1H, ¼CH), 12.24 (s, 1H, NH), 14.17 (s, 1H,

NH); Anal. Calcd. for C₁₇H₁₇N₃O₃S₂: C, 54.38; H, 4.56; N,11.19; found

C, 36.13; H, 3.38; N, 12.64.

4.4. Cytopathic effect (CPE) reduction assay

The antiviral activities of the test samples were measured using

the CPE reduction assay. MDCK cells were seeded into 96-well

plates and incubated until reaching monolayer. The 6 serial di-

lutions of each test sample were added to the cells 1 h after the

adsorption of 100TCID₅₀A/HebeiXinhua/SWL1106/2017 (Oselta-

mivir & amantadine-resistant H1N1). The TC₀was used as the

highest concentration. The control drugs (Oseltamivir, Amantadine

and Zanamivir), negative control wells and viral wells were set as

comparison on each plate. The plates were incubated at 34 ^ꢂC for

48 h. The CPE was then assessed using the crystal violet staining

method (as above). Resulting data were used to calculate the half

inhibitory concentration (IC₅₀). The experiments were repeated at

least three times.

4.1.7. Synthesis of 4-thiazolinone ring-opening analogue S5

The

raw

material

S4

(ethyl

2-(3-(3-(4-acetoxy-3-

thiazole-5-

methoxyphenyl)acryloyl)

thioureido)-4-methyl-

carboxylate) was synthesized according to the 4.1.6 method. In-

termediate S4 was dissolved in 3 mL tetrahydrofuran, and 2 mL

0.6 mol/L lithium hydroxide solution was added dropwise. The

mixture reacted at room temperature for 5 h, and the pH was

adjusted to 2 to 3 with dilute hydrochloric acid. The precipitate was

ﬁltered and washed with water to obtain a light yellow powder S5.

Yield 75.0%, m.p. 230e231 ^ꢂC. ¹H NMR (400 MHz, DMSO‑d₆)

d: 1.32

(t, J ¼ 6.6 Hz, 3H, CH₃), 2.59 (s, 3H, CH₃), 3.86 (s, 3H, OCH₃), 4.29 (q,

J ¼ 6.6 Hz, 2H, OCH₂), 6.86 (d, J ¼ 15.2 Hz,1H, CH), 6.89 (d, J ¼ 7.2 Hz,

1H, C₆H₃), 7.17 (d, 2 ¼ 7.2 Hz, 1H, C₆H₃), 7.24 (s, 1H, C₆H₃), 7.76 (d,

4.5. Docking procedure

The crystal structure data of H1N1 neuraminidase-Zanamivir

15

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complex (PDB Code: 3TI5) is downloaded from RSCB Protein Data

Bank. The protein was prepared in the standard manner using

AutoDock Tools (version 1.5.6) [51]. And the conformation of the

compounds was optimized using Corina Classic online demo

(https://www.mn-am.com/online_demos/corina_demo_

interactive). Then AutoDock Vina [52] was used for docking D41 to

the X-ray structures. A grid of 19.4 Å ꢁ 17.5 Å ꢁ 18.7 Å was con-

structed by GetBox Plugin (https://github.com/MengwuXiao/

GetBox-PyMOL-Plugin), centered on the active site [29.7,

12.9, ꢀ21.5]. Default settings were applied for molecular docking.

We veriﬁed that the docking procedures are reliable using re-

docking method (more details in supporting information). The re-

sults were visualized using PyMOL (https://pymol.org). The mo-

lecular interactions (hydrogen bonds and hydrophobic interactions

between the target proteins and compounds) were studied using

LigPlot þ version 2.2 [39].

Declaration of competing interest

[14] S. Singh, M.J. Jedrzejas, G.M. Air, M. Luo, W.G. Laver, W.J. Brouillette, Structure-

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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.

Acknowledgments

[16] P. Chand, Y.S. Babu, S. Bantia, N. Chu, L.B. Cole, P.L. Kotian, W.G. Laver,

J.A. Montgomery, V.P. Pathak, S.L. Petty, D.P. Shrout, D.A. Walsh, G.M. Walsh,

Design and synthesis of benzoic acid derivatives as inﬂuenza neuraminidase

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[17] V.R. Atigadda, W.J. Brouillette, F. Duarte, Y.S. Babu, S. Bantia, P. Chand, N. Chu,

J.A. Montgomery, D.A. Walsh, E. Sudbeck, J. Finley, G.M. Air, M. Luo,

G.W. Laver, Hydrophobic benzoic acids as inhibitors of inﬂuenza neuramini-

This work was ﬁnancially supported by the Hunan Provincial

Natural Science Foundation of China (No. 2019JJ40030), the Drug

Innovation Major Project (No. 2018ZX09711001-003-002), National

Natural Science Foundation of China (No. 81673480) and the Hunan

Provincial Innovation Foundation for Postgraduate (No.

CX2016B114). The Institute of the Chinese Academy of Medical

Sciences and Peking Union Medical College provided the biological

activity assays.

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Appendix A. Supplementary data

Supplementary data associated with this article can be found in

the online version, at https://doi.org/10.1016/j.ejmech.2021.1 13161.

These data include MOL ﬁles and InChiKeys of the most important

compounds described in this article.

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17

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Design, synthesis, and bioassay of 4-thiazolinone derivatives as influenza neuraminidase inhibitors

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