Rational drug design of benzothiazole-based derivatives as potent signal transducer and activator of transcription 3 (STAT3) signaling pathway inhibitors

European Journal of Medicinal Chemistry 216 (2021) 1 13333

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European Journal of Medicinal Chemistry

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

Rational drug design of benzothiazole-based derivatives as potent

signal transducer and activator of transcription 3 (STAT3) signaling

pathway inhibitors

,

c

,

Dingding Gao ^{a 1}, Nan Jin ^{b 1}, Yixian Fu ^{b d}, Yueyue Zhu ^a, Yujie Wang ^a, Ting Wang ^b,

Yuehong Chen ^b, Mingming Zhang ^a, Qiang Xiao ^a, Min Huang ^{b d}, Yingxia Li ^a

,

, *

^aSchool of Pharmacy, Fudan University, Shanghai, 201203, China

^bDivision of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai, 201203, China

^cInnovation Research Institute of Traditional Chinese Medicine (IRI), Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China

^dUniversity of Chinese Academy of Sciences, Beijing, 100049, China

a r t i c l e i n f o

a b s t r a c t

Article history:

The cumulative evidence supports STAT3, a transcriptional mediator of oncogenic signaling, as a ther-

apeutic target in cancer. The development of STAT3 inhibitors remain an active area of research as no

inhibitors have yet to be approved for cancer treatment. In a continuing effort to develop more potent

STAT3 inhibitors based on our previously identiﬁed hit compound 16w, a series of benzothiazole de-

rivatives with unique binding mode in SH2 domain of STAT3 were designed, synthesized and biologically

evaluated. Of note, compound B19 demonstrated excellent activity against IL-6/STAT3 signaling pathway

Received 8 December 2020

Received in revised form

19 February 2021

Accepted 20 February 2021

Available online 2 March 2021

with the IC₅₀value as low as 0.067 mM as determined by a luciferase reporter assay. Moreover, multiple

Keywords:

compounds displayed potent antiproliferative activity against MDA-MB-468 and JAK2 mutant HEL cell

lines. Further biochemical study using Western blot assay indicated that B19 blocked the phosphory-

lation of STAT3 at Tyr 705 and Ser 727 and thus suppressed STAT3-mediated gene expression of c-MYC

and MCL-1. Simultaneously, it induced cancer cell G2/M phase arrest and apoptosis both in MDA-MB-468

and HEL cell lines. Finally, molecular docking study along with surface plasmon resonance (SPR) and

ﬂuorescence polarization (FP) assays disclosed the binding mode of B19 in STAT3 SH2 domain. Taken

together, our ﬁnding suggests that B19 is a promising therapeutic STAT3 inhibitor for cancer treatment.

STAT3 signaling pathway

Benzothiazole

Rational design

Antitumor activity

Molecular docking

1. Introduction

various cytokines and growth factors, the Janus kinases (JAKs) are

recruited to the receptor and phosphorylate the tyrosine residues

Signal transducer and activator of transcription 3 (STAT3) is an

intracellular latent transcription factor that belongs to STAT protein

family [1,2]. It contains six functional domains including a dimer-

ization domain at the N-terminus, a coiled-coil domain for protein-

protein interactions, a central DNA-binding domain, a linker

domain that affects DNA-binding stability, a classic Src homology 2

(SH2) domain and a carboxyl transactivation domain [2]. Generally,

STAT3 transmits signals from the cell surface to the nucleus, as well

as directly participates in transcriptional regulation of numerous

gene expression [3,4]. Upon triggered by upstream signals, such as

on the receptor, creating binding sites for proteins possessing SH2

domains, such as STAT3. The latter can bind to the receptor using

SH2 domains and JAKs phosphorylate STAT3 at Tyrosine 705

(Tyr705), which causes STAT3 to dissociate from the receptor. Two

activated STAT3 monomers undergo dimerization through recip-

rocal pTyr-SH2 domain interactions and then translocate into the

nucleus to bind speciﬁc DNA sequence, leading to target gene

transcription [5,6]. Under normal physiological conditions, the

activation of STAT3 is rapid and transient due to the negative

regulation by proteins such as protein inhibitor of activated STAT

(PIAS) and suppressor of cytokine signaling (SOCS) [7e9]. Never-

theless, aberrant activation of STAT3 has a contributing role in

diverse solid and hematological tumors as it is a point of inter-

section of multiple oncogenic signaling pathways [10e14].

* Corresponding author.

E-mail address: liyx417@fudan.edu.cn (Y. Li).

These authors contributed equally to this work.

1

https://doi.org/10.1016/j.ejmech.2021.113333

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

Additionally, STAT3 plays a crucial role in immune cells and is being

regarded as a potent immune checkpoint that mediates tumor-

induced immunosuppression from various perspectives [15e19].

As STAT3 is the main mediator of IL-6 signaling pathway in

Th17 cells, it also plays an important role in pathogenesis of auto-

immune diseases, such as inﬂammatory bowel disease and rheu-

matoid arthritis [20,21]. Some other studies further showed that

different subcellular localization patterns of STAT3 affected auto-

phagy in various ways [22]. Therefore, the discovery of potent

STAT3 inhibitors is of great signiﬁcance for the treatment in many

diseases, especially cancer [23].

So far, a variety of STAT3 inhibitors containing peptide, pepti-

domimetic, small molecule and small-molecule degrader have

been reported (Fig. 1) [24e28], which target the SH2 domain or

DNA-binding domain to inhibit STAT3 transcriptional activity.

However, there are only three small-molecule STAT3 inhibitors

(BBI608, OPB-111077, C188-9) currently under investigation in

clinical trials, perhaps due to the complexity of the biological

functions of STAT3 signaling pathway or the poor physicochemical

properties of STAT3 inhibitors. In detail, BBI608 (Napabucasin), a

direct STAT3 inhibitor that blocks stem cell activity in cancer cells,

is the only STAT3-targeted agent that has been advanced into phase

III trials for the treatment of a variety of cancers [29]. OPB-111077, a

novel inhibitor of STAT3 and mitochondrial oxidative phosphory-

lation, is investigated in phase I trial in unselected advanced can-

cers (NCT01711034) [30]. C188-9, an oral STAT3 inhibitor targeting

SH2 domain, is evaluated in phase I trial in the patients with

advanced cancers (NCT03195699), which is also under develop-

ment for the treatment of ﬁbrosis and inﬂammation [31]. Overall,

the discovery of novel potent STAT3 inhibitors remains to be further

explored and developed.

2. Results and discussion

2.1. Design

Firstly, molecular docking of 16w with STAT3 SH2 domain was

conducted using the X-ray crystal structure of STAT3/DNA complex

(PDB code: 1BG1), and the proposed binding mode was depicted in

Fig. 2A and B. For Fig. 2A, three hydrogen bonds were formed be-

tween compound 16w and the amino acid residues Gln644, Glu638

and Gln635 of STAT3 protein (Fig. 2A). As shown in Fig. 2B and C, a

hydrophobic cleft (S1) enclosed by the Phe716, Trp623, Ile659

residues was located near the ring A and various substituents with

different sizes or electrical properties could be introduced to

explore the inhibitory activities (compounds B1eB14, B17, B18).

The aromatic ring B inserted into a hydrophobic pocket (S2) sur-

rounded by Met648, Tyr640, Ile653, Ile711 residues and hydroxy or

methoxyl was incorporated to form hydrogen-bond or hydrophobic

interaction to improve the activities (compounds B19eB23). We

also investigated the effect of chiral methyl group or the long alkyl

chain (compounds B15, B16, B24eB30) or thioether bond (B31,

B32) on the activity of STAT3 signaling pathway (Fig. 2C).

2.2. Chemistry

To obtain benzothiazole-based derivatives B1eB17, a modiﬁed

literature method was used (Schemes 1 and 2) [32]. Intermediate 2

was prepared from commercially available (S)-1 phenylethan-1-

amine and 2-chloroacetyl chloride using triethylamine in 90%

yield. Then it was condensed with 6-aminobenzothiazole-2-thiol in

reﬂuxing acetone to afford the key intermediate 3 in almost

quantitative yield without affording N-nucleophilic addition

byproduct. Treatment of the substituted 4-hydroxybenzoic acids

(4a-4n) or 7b with 2-bromopentane and potassium hydroxide in

ethanol at 78 ^ꢀC resulted in the formation of 5a-5n or 8c in about

50e80% yield. The intermediates 8a and 8b were prepared by

Mitsunobu reaction among the appropriate substituted alcohols

with 7a in 38.9% and 64.0% yields respectively [33]. Then, com-

pounds 8a-8c were conveniently hydrolyzed to provide the corre-

sponding acid 9a-9c quantitatively. The benzoyl chloride

derivatives (6a-6n, 10a-10c) were prepared from corresponding

benzoic acids (5a-5n, 9a-9c) reacted with SOCl₂. Subsequently,

target compounds B1eB17 were obtained, respectively, by the re-

action between 3 and the corresponding acyl chloride (6a-6n, 10a-

10c) in the presence of potassium carbonate at room temperature

in dichloromethane (36.7e82.7% yields).

In our previous study, molecular docking-based virtual

screening was applied to discover a STAT3 inhibitor with a benzo-

thiazole scaffold targeting the Leu706-Phe 710 binding site of the

STAT3 SH2 domain. Further investigation showed that compound

16w exhibited potent inhibition of IL-6/STAT3 signaling with an

IC₅₀value of 1.6 mM as determined by a luciferase reporter assay

[32]. With the aim of obtaining more potent STAT3 signaling in-

hibitors, rational drug design based on 16w was carried out in this

study. Among those designed compounds, B19 exhibited the most

potent inhibitory activity towards IL-6/STAT3 signaling pathway,

which was 24-fold more potent than hit compound 16w according

to the IC₅₀values (0.067

mM vs 1.6 mM). Moreover, it displayed

potent in vitro antiproliferative activities against MDA-MB-468 cells

and HEL (JAK2 V617F) cells, through inhibiting STAT3 signaling

pathway.

The synthetic route for compounds B18eB26 was shown in

Scheme 3. Formylation of 2-methylbenzene-1, 3-diol (15) with

Fig. 1. Known representative STAT3 inhibitors.

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D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

Fig. 2. (AeB) Docking mode of hit compound 16w with STAT3 SH2 domain (PDB 1BG1), generated by Schrodinger, Maestro suite. Surface representation was created with PyMOL

Viewer. (C) Rational design of target compounds.

Scheme 1. Synthesis of compounds B1eB14. Reagents and conditions: (a) ClCH₂COCl, Et₃N, CH₂Cl₂, 0 ^ꢀC to rt; (b) 6-aminobenzothiazole-2-thiol, K₂CO₃, acetone, reﬂux; (c) 2-

bromopentane, EtOH, KOH, reﬂux; (d) SOCl₂, reﬂux; (e) K₂CO₃, CH₂Cl₂, rt.

Scheme 2. Synthesis of compounds B15eB17. Reagents and conditions: (a) 8a: (S)-pentan-2-ol, triphenylphosphane, DEAD, THF, 0 ^ꢀC to rt; 8b: (R)-pentan-2-ol, triphenylphos-

phane, DEAD, THF, 0 ^ꢀC to rt; 8c: 2-bromopentane, EtOH, KOH, reﬂux; (b) 1 M NaOH (aq.), rt; (c) SOCl₂, reﬂux; (d) 3, K₂CO₃, CH₂Cl₂, rt.

POCl₃eDMF regio-selectively afforded the benzaldehyde derivative

16 in an excellent yield. Sequential O-alkylations were performed in

the DMF to afford 17a-17c in 60.0e75.0% yields [34,35]. After O-

methylation of compounds 16 and 17a-17c, oxidation was carried

out under NaClO₂/NaH₂PO₄$2H₂O buffer solution (pH ¼ 4.5) to

provide 19a-19d in 63.1e83.0% yields. The intermediates 13b-13d

were obtained after demethylation by BBr₃(17% in CH₂Cl₂).

Condensation of the amines (14a-14e) with benzoyl chloride de-

rivatives (20a-20d), which were prepared from the corresponding

carboxylic acid (19a-19e) and SOCl₂, provided the desired com-

pounds B18eB26 in 18.0e70.4% yields (Scheme 3).

After selectively protecting one hydroxyl of the starting material 21

with benzyl (22), the remaining hydroxyl group was methylated

with iodomethane to get compound 23 in almost quantitative yield.

Then compound 26 was obtained by oxidation of the aldehyde

group, removal of the benzyl group, and methylation of carboxyl

group sequentially in a good yield. Subsequent alkylation of 26

under several conditions afforded compounds 27a-27d in

41.9e93.5% yields [36e40], which were hydrolyzed under 1 M

NaOH and CH₃OH solution to afford 28a-28d quantitatively. Finally,

the carboxyl was converted to acyl chloride group (29a-29d) and

condensed with the amino group of 14a to obtain compounds

B27eB30 in 30.0e35.0% yields.

Synthesis of compounds B27eB30 was depicted in Scheme 4.

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D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

Scheme 3. Synthesis of compounds B18eB26. Reagents and conditions: (a) 2-chloroacetyl chloride, K₂CO₃, CH₂Cl₂, 0 ^ꢀC to rt; (b) BBr₃(17% in CH₂Cl₂), CH₂Cl₂, 0 ^ꢀC to rt; (c) 6-

aminobenzothiazole-2-thiol, K₂CO₃, acetone, reﬂux; (d) POCl₃, DMF, ethyl acetate, rt; (e) Bromoalkane, K₂CO₃, KI, DMF, 80 ^ꢀC; (f) CH₃I, K₂CO₃, DMF, 40 ^ꢀC; (g) NaClO₂, NaH₂

-

PO₄$2H₂O, t-BuOH, rt; (h) SOCl₂, CH₂Cl₂, DMF, 0 ^ꢀC to rt; (i) K₂CO₃, CH₂Cl₂, rt.

Scheme 4. Synthesis of compounds B27eB30. Reagents and conditions: (a) Benzyl bromide, KI, K₂CO₃, acetonitrile, 80 ^ꢀC; (b) CH₃I, K₂CO₃, DMF, 40 ^ꢀC; (c)NaClO₂, NaH₂PO₄$2H₂O, t-

BuOH, rt; (d) Pd/C, H₂, rt; (e) SO₂Cl₂, CH₃OH, rt; (f) Cs₂CO₃, anhydrous DMF, 85 ^ꢀC and 1-bromo-2-methoxyethane for 27a, 1-(2-chloroethyl)pyrrolidine hydrochloride and KI for 27b,

2-chloro-N,N-dimethylethan-1-amine hydrochloride and KI for 27c, 4-(2-chloroethyl)morpholine hydrochloride and KI for 27d; (g) 1 M NaOH (aq.), CH₃OH, 50 ^ꢀC, 28a; 6 M HCl

(aq.), 85 ^ꢀC, 28b-28d; (h) SOCl₂, CH₂Cl₂, DMF, rt; (i) 14a, K₂CO₃, CH₂Cl₂, rt.

Scheme 5. Synthesis of compound B31. Reagents and conditions: (a) (tert-butoxycarbonyl)glycine, HATU, HoAt, DIPEA, THF, rt; (b) CF₃COOH, CH₂Cl₂, 0 ^ꢀC; (c) 2-chloro-6-nitrobenzo

[d]thiazole, n-Butanol, Et₃N, reﬂux; (d) Fe powder, NH₄Cl, EtOH/H₂O, reﬂux; (e) 20d, K₂CO₃, CH₂Cl₂, rt.

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D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

The synthetic route of compound B31 was shown in Scheme 5.

Intermediate 31 was prepared by coupling commercially available

compound 30 with N-Boc-glycine using HATU and DIPEA in 92.8%

yield. Then, removing the Boc group and coupling with 2-chloro-6-

nitrobenzo[d]thiazole to get compound 33, which was converted to

B31 by reduction of the nitro group and condensation with 20d

sequentially.

Compound B32 was synthesized as depicted in Scheme 6.

Commercially available 2-chloro-6-aminobenzothiazole was con-

verted to intermediate 36 followed by nucleophilic substitution

reaction with ethyl glycolate to obtain 37 in 64.6% yield. After hy-

drolysis of the ethyl ester group of 37 and then condensation with

intermediate 30, the target compound B32 was afforded.

additional hydrogen-bond or some hydrophobic interaction could

be formed after introducing a hydroxy or methoxy at some posi-

tions of aromatic ring B. As shown in Table 3, compound B19 with a

methoxyl group at the para position exhibited the most potent

activity with an IC₅₀value of 0.067

hydroxy substituted at the same position (B21, IC₅₀¼ 0.20

a methoxyl group at the meta position (B20, IC₅₀¼ 0.27

However, activities of compounds with a hydroxy substituent at the

M) position

m

M, which was better than a

M) and

M).

m

ortho (B23, IC₅₀¼ 0.17

m

M) or meta (B22, IC₅₀¼ 0.15

m

were comparable to that of compound B18. With the goals of

improving solubility or simplify the molecule, we sought to shorten

the long alkyl chain or replaced it with hydrophilic functional

groups (B24eB30). As shown in Table 3, compared with B18, all

resulting compounds showed reduced activities with IC₅₀values

ranging from 0.20 mM to 1.03 mM, which might be due to the hy-

2.3. Biological assays

drophobic interactions between the alkyl chain and the target.

Unfortunately, replacing the S atom of thioether with NH (B31) or O

(B32) brought a signiﬁcant drop of activities.

2.3.1. Inhibitory activity on STAT3 transcription

Compounds B1eB18 were ﬁrstly subjected to test the IL6/STAT3

signaling pathway activities using a STAT3 luciferase reporter sys-

tem and their IC₅₀values were summarized in Table 1 (compounds

LLL12 [26] and 16w were used as positive controls). For the sub-

stituent groups at R¹position, compound B1 with a methyl group

2.3.2. In vitro cell growth inhibitory activity

Compounds that displayed better inhibitory activity on STAT3

transcription were ﬁrstly evaluated for their antiproliferative ac-

tivities against human erythroleukemia (HEL) cells bearing JAK2

V617F mutation that results in constitutive activation of STAT3.

JAK1/2 inhibitors INC18424 and AZD1480 were used as positive

controls. As shown in Table 4, compared to the hit compound 16w,

all of these selected compounds exhibited potent activities toward

HEL (JAK2 V617F) cells except for B1 and B28. Additionally, those

compounds were tested their antiproliferative activities against

STAT3 over-expressed human breast cancer cell line MDA-MB-468

using MTT assay. The IC₅₀values were listed in Table 4 and all the

compounds showed growth inhibitory effect on the MDA-MB-

468 cells with IC₅₀values ranging from sub-micromole to micro-

showed better inhibitory activity (IC₅₀¼ 0.95

pound 16w (IC₅₀¼ 1.60 M). Incorporation of big steric sub-

stituents, such as ethyl (B7) and n-propyl (B8), in this position was

detrimental to the activity (IC₅₀> 10 M). Also, the presence of

electron-withdrawing groups such as ﬂuorine (B3), cyano (B6) and

triﬂuoromethyl (B9) decreased the activity (IC₅₀> 10 M). Com-

pounds B2 and B5 with methoxyl and bromine at this position

displayed similar activities to 16w with IC₅₀values of 1.42 M and

2.15

M, respectively. For the substituent groups at R²position,

mM) than hit com-

m

there exists a similar trend as R¹group. Substituent groups methyl

(B10), methoxy (B11) and chlorine (B13) maintained the activities,

while electron withdrawing group ﬂuorine (B12) led to a signiﬁcant

loss of potency. Since there was a chiral methyl group at the long

alkyl chain, we then investigated its effect on the activity. As shown

in Table 2, the R conﬁguration showed a better luciferase activity

than S conﬁguration (B15 vs B16). Furthermore, the introduction of

a methyl (B17) or a methoxyl group (B18) at R²position resulted in

mole (0.25

the best STAT3-induced luciferase activity (IC₅₀¼ 0.067

displayed the most potent activity against MDA-MB-468 cells

M). In contrast, B18 and B19 showed no anti-

mMe6.57

mM). In particular, compound B19 exhibiting

mM) also

(IC₅₀¼ 0.25

m

proliferative activities against MDA-MB-453 and MCF-7 cell lines,

which were generally reported with low expression levels of STAT3

[42], with IC₅₀values greater than 20

mM (Table 5). These data

a

2-fold or 6-fold increased activity respectively (B17,

together suggested that a subset of compounds with potent anti-

proliferative activity were worth further evaluation.

IC₅₀¼ 0.46 M; B18 IC₅₀¼ 0.16 M). Presumably the formation of

m

an intramolecular hydrogen bond between OCH₃and the adjacent

NH of amide in compound B18 made the amide and the benzene

ring in the same plane，and thus restricted the degrees of freedom.

For this reason, B18 was probably maintained in a preferred

conformation. Therefore, the methyl at R¹position could ﬁt into the

desired pocket more appropriately and increased the binding af-

ﬁnity with the target [41].

2.3.3. Western blot analysis

Based on the results of inhibitory activities on STAT3 tran-

scription and cell proliferation, we next proceeded to evaluate the

inhibitory effect of compounds B18 and B19 on STAT3 signaling

pathway in HEL (JAK2 V617F) and MDA-MB-468 cells by Western

blot analysis. STAT3 phosphorylation at Tyr705 residue can lead to

To further improve the potency of B18, it was envisioned that an

Scheme 6. Synthesis of compound 39. Reagents and conditions: (a) 20d, K₂CO₃, CH₂Cl₂, r. t.; (b) Ethyl-2-hydroxyacetate, NaH, THF, 70 ^ꢀC; (c) 1 M NaOH (aq.), CH₃OH/THF, rt; (d) 11b,

HATU, HoAt, DIPEA, THF, rt.

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European Journal of Medicinal Chemistry 216 (2021) 113333

Table 1

Inhibitory activity on STAT3 transcription as indicated by luciferase reporter gene assay.

Compd.

R¹

R²

IL-6/STAT3 pathway^a

IC₅₀SD (

M)^b

Compd.

R¹

R²

IL-6/STAT3 pathway^a

m

IC₅₀SD (m

M)^b

B1

B2

B3

B4

B5

B6

B7

B8

CH₃

OCH₃

F

Cl

Br

H

0.95 0.22

1.42 0.37

>10

2.15 0.61

>10

B9

B10

B11

B12

B13

B14

16w

LLL12

CF₃

H

CH₃

OCH₃

F

Cl

F

>10

2.72 0.72

2.20 0.27

>10

1.34 0.21

>10

CN

C₂H₅

F

H

>10

H

1.60 0.12

0.098 0.01

CH₂CH₂CH₃

a

STAT3-dependent luciferase reporter gene assay in HepG2 cell.

The data are the mean SD from at least three independent experiments.

b

Table 3

Table 2

Inhibitory activity on STAT3 transcription as indicated by luciferase reporter gene

assay.

Inhibitory activity on STAT3 transcription as indicated by luciferase reporter gene

assay.

Compd.

B18

R³

R⁴

H

X

S

IL-6/STAT3 pathway^a

IC₅₀SD (

M)^b

m

Compd.

B1

R²

H

R³

IL-6/STAT3 pathway^a

IC₅₀SD (

M)^b

0.16 0.03

0.067 0.00

0.27 0.00

0.20 0.11

0.15 0.01

0.17 0.01

m

0.95 0.22

B19

4-OCH₃

3-OCH₃

4-OH

3-OH

2-OH

B15

H

0.90 0.13

B20

B16

H

1.20 0.05

B21

B22

B17

B18

CH₃

0.46 0.01

0.16 0.03

0.098 0.01

B23

OCH₃

B24

B25

4-OCH₃

S

0.20 0.02

0.22 0.00

LLL12

a

STAT3-dependent luciferase reporter gene assay in HepG2 cell.

The data are the mean SD from at least three independent experiments.

b

B26

B27

OCH₃

4-OCH₃

S

0.12 0.03

0.26 0.10

its homodimerization, translocation into nucleus, DNA-binding and

downstream transcriptional activities. There is an increasing un-

derstanding about phosphorylation at Ser727 residue, which is

associated with the transcription, mitochondrial import and other

biological functions of STAT3 [43]. Therefore, the STAT3 phos-

phorylation level at Tyr705 and Ser727 was evaluated. As shown in

Fig. 3, these two compounds could inhibit STAT3 phosphorylation

at Tyr705 and Ser727 in both cell lines without inﬂuence on the

total expression of STAT3 protein after 24 h incubation. Aberrant

STAT3 signaling has been shown to promote tumor progression

through directly or indirectly upregulating the expression of

downstream target genes, such as cell-cycle regulator c-MYC and an

anti-apoptotic gene MCL-1 [44,45]. Compounds B18 and B19

signiﬁcantly reduced the protein level of c-Myc and MCL-1. Overall,

B28

B29

B30

4-OCH₃

S

1.03 0.10

0.52 0.07

0.24 0.01

B31

B32

LLL12

OCH₃

4-OCH₃

NH

O

1.49 0.15

8.86 0.16

0.098 0.01

a

STAT3-dependent luciferase reporter gene assay in HepG2 cell.

The data are the mean SD from at least three independent experiments.

b

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D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

Table 4

investigated the inhibitory effects of compounds B18 and B19 on

eleven receptor tyrosine kinases. Encouragingly, all of our com-

pounds had no effect against these kinases, which clearly man-

ifested that the inhibition of p-STAT3 was not owing to the

inhibition of upstream kinases.

Antiproliferative activity of the representative compounds against HEL (JAK2 V617F)

and MDA-MB-468 cells.

Compd.

HEL (JAK2 V617F)^a

MDA-MB-468^a

IC₅₀SD (

M)^b

IC₅₀SD (

m

M)^b

m

16w

B1

B17

B18

B19

B20

B21

B22

B23

B24

B25

B26

B27

B28

B29

B30

>10

2.21 0.20

0.73 0.02

0.81 0.16

1.04 0.27

0.25 0.05

0.50 0.04

0.70 0.03

2.24 0.18

0.59 0.19

2.02 0.15

1.81 0.09

2.05 0.23

1.16 0.16

3.36 0.32

6.57 0.64

1.77 0.55

0.73 0.03

1.92 0.08

>20

2.3.5. The selectivity against related IFN-

signaling pathways

g

/STAT1 and TNF-

a/NF-kB

0.93 0.16

2.41 0.73

1.11 0.17

0.87 0.19

0.68 0.25

0.62 0.20

0.97 0.22

2.05 1.05

1.29 0.08

1.34 0.06

1.44 0.03

10.4 1.41

4.98 0.61

3.38 1.22

1.46 0.33

1.59 0.35

1.40 0.20

0.73 0.07

To our knowledge, IFN-g/STAT1 and TNF-

a

/NF- B signaling

k

pathways are two closely related intracellular signaling pathways

with IL-6/STAT3 signaling pathway. The former is the structural

homologous signaling pathway, and the latter is the functional

homologous signaling pathway. Therefore, the selectivity of com-

pounds B18 and B19 against these two signaling pathway was

investigated using a STAT1 luciferase reporter gene assay in HepG2

cells and a NF-

shown in Table 7, compounds B18 and B19 did not display any of

TNF- -induced NF- B activations with a concentration as high as

100 M. However, both compounds showed little selectivity for

STAT3 signaling over IFN- /STAT1 signaling pathway, probably

kB luciferase reporter gene assay in HEK-293 cells. As

a

k

LLL12

Stattic

INCB18424

AZD1480

m

g

owing to the high structural homology of the SH2 domain among

the STAT proteins. Thus, improving STAT isoform selectivity re-

mains a goal for future compound optimization.

5.87 0.87

a

The inhibitory effects of these compounds on the proliferation of cancer cell

lines were determined by the MTT assay.

b

The data are the mean SD from at least three independent experiments.

2.3.6. Apoptosis induction and G2/M cell-cycle arrest in MDA-MB-

468 and HEL cells

Table 5

As compounds B18 and B19 displayed potent antiproliferative

potency toward both MDA-MB-468 and HEL (JAK2 V617F) cell lines,

we then investigated the effect of them on the induction of cells

apoptosis by ﬂow cytometry. As shown in Fig. 4A, compounds B18

and B19 induced apoptosis of the test cells at the concentrations of

Antiproliferative activity of B18 and B19 against MDA-MB-453 and MCF-7 cells.

Compd.

MDA-MB-453^a

MCF-7^a

IC₅₀SD (

m

M)^b

IC₅₀SD (m

M)^b

B18

B19

>20

1.25 mM, 2.5 mM and 5.0 mM, which demonstrated that B18 and B19

Stattic

4.40 0.16

1.70 0.10

could effectively inhibit cancer cell growth through apoptosis in-

duction. Also, the effect of B18 and B19 on cell cycle progression of

MDA-MB-468 and HEL (JAK2 V617F) cells was determined using

ﬂow cytometry and the results were shown in Fig. 4B. After treat-

ment with compounds B18 and B19 for 48 h, the proportion of cells

in G2/M phase was increased accompanying with a decrease of cells

in G1 phase in a dose-dependent behavior.

a

The inhibitory effects of these compounds on the proliferation of cancer cell

lines were determined by the MTT assay.

b

The data are the mean SD from at least two independent experiments.

the results indicated these compounds efﬁciently inhibited STAT3

signaling pathway.

2.3.7. SPR assay

2.3.4. In vitro kinases activity test

To determine the binding afﬁnity and kinetic parameters of our

representative compounds toward STAT3 SH2 domain, surface

plasmon resonance (SPR) assay was employed using a Biacore T200

optical biosensor. Firstly, the reported STAT3 SH2 domain binders

5-FAM-GpYLPQTV-NH2 derived peptide (Sub) [46] and Stattic [47]

To further evaluate the selectivity of compounds B18 and B19

against the upstream kinases which could activate STAT3 signaling

pathway, enzyme-linked immunosorbent assay (ELISA) was

employed to test a panel of kinases. As shown in Table 6, we

Fig. 3. Western blot analysis of the inhibition of STAT3 phosphorylation at Tyr705 and Ser727, the downstream target proteins (c-Myc, MCL-1) by compounds B18 and B19 in (A)

HEL (JAK2 V617F) and (B) MDA-MB-468 cells.

7

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

Table 6

Inhibitory activities against a panel of representative kinases.

Compd.

Inhibition rate at 1.0

m

M (%)^a

ErbB2

JAK1

JAK2

JAK3

VEGFR-1

PDGFR-

b

EGFR

ErbB4

Abl

Eph-A2

IGF1R

B18

B19

ꢁ2.2

ꢁ0.6

ꢁ4.7

0.0

/

0.0

/

0.0

/

98.9

/

5.2

9.6

/

93.4

/

0.0

/

88.6

/

4.7

4.1

/

100

/

1.1

0.0

/

100

/

0.0

/

3.3

4.0

ꢁ6.8

INCB018424

Su11248

BIBW2992

Dasatinib

AEW541

80.4

91.7

78.5

/

86.4

76.1

/

100

a

The inhibition rates are the mean of three independent experiments.

Table 7

Inhibitory activity against related IFN-

site of our compounds B18 and B19, FP-based competition binding

assay was conducted according to previously reported method

[47,48]. Firstly, this derived peptide was applied as the ﬂuorescent

probe with the K_Dvalue of 186.4 nM in our experiment, which was

almost equivalent to the reported value (K_D¼ 150 nM) by Berg and

coworker [46]. As shown in Table 8, Stattic could bind to STAT3

g

/STAT1 and TNF-

a

/NF-

k

B signaling pathways.

Compd.

IL-6/STAT3^a

IFN-

g

/STAT1^b

TNF- /NF-

a

k

B^c

IC₅₀SD (

M)^d

m

IC₅₀SD (

M)^d

m

IC₅₀SD (

M)^d

m

B18

B19

BBI608

0.16 0.03

0.067 0.00

0.84 0.07

0.35 0.01

0.32 0.02

0.88 0.08

>100

3.55 0.21

protein in

a concentration-dependent manner, indicating its

a

binding position at pTyr705 site in SH2 domain. However, com-

pounds B18 and B19 did not show any competition with the probe

even at a concentration of 100 mM, which indicates their binding

site was different from Stattic and the probe (Sub).

STAT3-dependent luciferase reporter gene assay in HepG2 cell.

STAT1-dependent luciferase reporter gene assay in HepG2 cell.

b

c

NF-

kB -dependent luciferase reporter gene assay in HEK-293 cell.

d

The data are the mean SD from at least three independent experiments.

2.3.9. Molecular docking study

were used as positive controls. As shown in Fig. 5, the equilibrium

constant K_Dvalues of compounds Sub and Stattic were 28.81 nM

and 1528 nM respectively. For our representative compounds B18

and B19, the response unit (RU) values were proportional to com-

pound concentrations within the selected ranges and the K_Dvalues

were 62.61 nM (B18) and 53.04 nM (B19) respectively, which

indicated a strong binding afﬁnity to STAT3 SH2 domain.

As B19 exhibited the most potent inhibitory activity in luciferase

reporter assay among all designed compounds, a docking model of

B19 bound to SH2 domain was generated on the basis of the crystal

structure of the STAT3b homodimer (PDB code 1BG1) by Schro-

dinger, Maestro suite. As illustrated in Fig. 6B, B19 located at the

Leu706-Phe 710 (Phe710-lys709-thr708-lys707-leu706) binding

site in STAT3 SH2 domain rather than the pY705 site and acted as a

partial peptidomimetic of phosphotyrosine peptide (pYLKTKF). The

left amide NH and the right amide C]O formed two hydrogen

bonds with Gln644 and Glu638 respectively. In addition, the oxy-

gen atom close to the long alkyl chain formed one hydrogen bond

with the amide group of Gln635 (Fig. 6C). The 4-methoxy-S-methyl

2.3.8. Fluorescence polarization (FP) assay

It has been reported that the binding site of 5-FAM-GpYLPQTV-

NH₂derived peptide (Sub) and Sttatic was pTyr705 site in SH2

domain of STAT3 [46,47]. To exclude the directly binding at pTyr705

Fig. 4. Induction of apoptosis and cell-cycle arrest by compounds B18 and B19 in MDA-MB-468 and HEL cells. (A) Effects of compounds B18 and B19 on the induction of apoptosis.

The cells were treated with increasing concentrations of the test compounds for 48 h and analyzed by annexin V/propidium iodide double staining. The data are representative of

three independent experiments. ****, p < 0.0001, ***, p < 0.001, **, p < 0.01, *, p < 0.05. (B) Cell cycle analysis of compound B18 and B19 by ﬂow cytometry. The cells were treated

with increasing concentrations of test compounds for 48 h.

8

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

Fig. 5. SPR analysis of representative compounds with STAT3 SH2 domain. For Sub, compound concentrations were varied from 0.98 to 62.5 nM; for Stattic, compound con-

centrations were varied from 0.039 to 2.5 mM; For compound B18 and B19, a concentration range of 0.0485e6.25 mM was used. Time was measured in seconds.

Table 8

the downstream gene expression (c-MYC and MCL-1). In addition,

compound B19 was highly selective for the IL-6/STAT3 signaling

FP-based competition binding assay of B18, B19 and Stattic.

pathway over TNF-a/NF-kB signaling pathway and some upstream

Compds.

Binding rate (%)

50

kinases. Mechanistically, we elucidated the predicted binding mode

of B19 at the Leu706-Phe 710 binding site of STAT3 SH2 domain

using SPR, FP and molecular docking studies. All of those results

indicated that targeting the Leu706-Phe710 binding site in STAT3

SH2 could prevent either STAT3 recruitment to the receptor and

phosphorylation at Tyr705 by JAKs, or the formation of STAT3

dimerization, subsequently inhibiting the translocation of STAT3

dimers to nucleus to induce transcription of downstream genes.

However, B19 exhibited poor oral exposures with low area under

the concentration time curve (AUC_last) values (3 mg/kg, 2.50 h ng/

mL) which may be caused by the poor solubility, the oxidative

metabolism of S atom, the hydrolysis of two amide bond or the

cleavage of carbon-sulfur bond. Accordingly, several structural

optimizations to improve metabolism stability should be con-

ducted in the future work, including bioisosteric replacement of the

amide bond, incorporation of methyl, ﬂuoro, gem-dimethyl or gem-

diﬂuoro at the methylene position to improve the oxidative

metabolism, and introduction of heteroatoms to reduce the

lipophilicity.

100

m

M

m

M

20 mM

B18

B19

Stattic

<10

95.0

<10

71.1

<10

62.3

a

The binding rates are the mean of three independent experiments.

benzylamine group occupied the hydrophobic pocket surrounded

by Met648、Tyr640、Ile653、Ile711 residues (S2) in a manner

similar to Phe710 of the phosphotyrosine peptide. The methyl on

the right benzene ring ﬁtted perfectly the expectant hydrophobic

cleft described before and enhanced the binding afﬁnity via hy-

drophobic interactions with Phe716, Ile659 and Trp623. Intrigu-

ingly, the O atom of the methoxyl adjacent to this methyl group

could form an intramolecular hydrogen bond with the NH of amide,

which was conductive to the insertion of methyl into the described

hydrophobic cleft and made a great improvement on activity

(Fig. 6D).

3. Conclusions

4. Experimental section

In this work, we reported a series of potent STAT3 inhibitors by

optimizing the previously identiﬁed benzothiazole derivatives.

Among the tested compounds, we found that compound B19

exhibited the most potent activities against STAT3 transcription

and inhibited cancer cell growth, such as MDA-MB-468 and HEL

(JAK2 V617F) cells, at low micromole concentration. Further

biochemical study demonstrated that B19 could dose-dependently

inhibited STAT3 phosphorylation at Tyr705 and Ser727, as well as

4.1. Chemistry

All the reagents and solvents were purchased from Adamas-

beta®, Energy Chemical or Bide pharmatech and were used

without further puriﬁcation unless otherwise noted. Anhydrous

solvents were purchased from Adamas-beta®. ¹H NMR and ¹³C

NMR spectra were recorded on a 400 MHz Varian spectrometer or a

9

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

Fig. 6. Molecular docking study of B19 in STAT3 SH2 domain (PDB code: 1BG1). (A) Chemical structure of compound B19. (B) Superimposed pose of B19 (yellow) and pY705-Phe 710

peptide (green) bound in the surface of binding site. (C) Predicted hydrogen bonds interaction of B19 (yellow) within STAT3 SH2 domain. (D) B19 (yellow) bound in the surface of

binding site. The ﬁgures were generated using Pymol.

600 MHz Bruker spectrometer at 303 K and referenced to TMS. ¹⁹

NMR spectrum was recorded on a 600 MHz Bruker spectrometer at

303 K. Chemical shifts are reported in parts per million (ppm, ).

F

4.1.1.2. 3-Methoxy-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d] thiazol-6-yl)-4-(pentan-2-yloxy)benzamide (B2).

d

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 3-methoxy-4-(pentan-2-yloxy)benzoyl chloride (6b,

0.24 mmol, 61.6 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (73.1 mg, 64.9% yield). ¹H NMR (400 MHz,

Proton coupling patterns are described as follows: s, singlet; d,

doublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of

triplets; brs, broad singlet. Mass spectra data were obtained on

Agilent Technologies 1260 inﬁnity LC/MS instrument (ESI-MS) and

High-resolution mass spectra (HRMS) data were given by AB

5600 þ Q TOF. Analytical and preparative TLCs were performed on

silica gel HSGF/UV 254. The chromatograms were conducted on

silica gel (200e300 mesh) and visualized under UV light at 254 and

365 nm.

DMSO‑d₆)

d

10.25 (s, 1H), 8.76 (d, J ¼ 7.9 Hz, 1H), 8.44 (d, J ¼ 1.5 Hz,

1H), 7.76 (d, J ¼ 8.8 Hz, 1H), 7.71 (dd, J ¼ 8.9, 1.7 Hz, 1H), 7.56 (dd,

J ¼ 8.4, 1.6 Hz, 1H), 7.51 (d, J ¼ 1.7 Hz, 1H), 7.33e7.22 (m, 4H), 7.17 (t,

J ¼ 6.9 Hz, 1H), 7.07 (d, J ¼ 8.5 Hz,1H), 4.94e4.84 (m, 1H), 4.56e4.46

(m, 1H), 4.16 (d, J ¼ 14.9 Hz, 1H), 4.12 (d, J ¼ 14.9 Hz, 1H), 3.81 (s,

3H),1.69e1.59 (m,1H),1.56e1.46 (m,1H),1.43e1.29 (m, 5H),1.21 (d,

J ¼ 6.0 Hz, 3H), 0.87 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)

4.1.1. General procedures for the synthesis of B1eB14

The synthesis of the products B1eB14 from 4-hydrobenzoic acid

derivatives and (S) methyl benzylamine was previously reported in

ref. 32.

d

165.60, 165.20, 164.62, 150.22, 149.24, 148.91, 144.26, 136.20,

135.29, 128.33, 126.79, 126.70, 126.02, 121.16, 120.89, 120.04, 113.57,

112.82, 111.61, 73.76, 55.74, 48.58, 38.04, 36.77, 22.54, 19.59, 18.22,

14.00. ESI-HRMS [MþH]^þcalcd for C₃₀H₃₄N₃O₄S₂: 564.1985, found:

564.1983.

4.1.1.1. 3-Methyl-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)Benzo[d]thiazol-6-yl)-4-(pentan-2-yloxy)benzamide

(B1).

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 3-methyl-4-(pentan-2-yloxy)benzoyl chloride (6a,

0.24 mmol, 57.8 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (90.2 mg, 82.5% yield). ¹H NMR (400 MHz,

4.1.1.3. 3-Fluoro-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d]

thiazol-6-yl)-4-(pentan-2-yloxy)benzamide

(B3).

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 3-ﬂuoro-4-(pentan-2-yloxy)benzoyl chloride (6c,

0.24 mmol, 58.7 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (91.2 mg, 82.7% yield). ¹H NMR (400 MHz,

DMSO‑d₆)

d

10.24 (s, 1H), 8.78 (d, J ¼ 7.82 Hz, 1H), 8.47 (d,

J ¼ 1.57 Hz, 1H), 7.80e7.70 (m, 4H), 7.31e7.24 (m, 4H), 7.19 (t,

J ¼ 7.04 Hz, 1H), 7.06 (d, J ¼ 8.22 Hz, 1H), 4.95e4.85 (m, 1H),

4.60e4.52 (m, 1H), 4.17 (d, J ¼ 14.9 Hz, 1H), 4.12 (d, J ¼ 14.9 Hz, 1H),

2.18 (s, 3H), 1.70e1.62 (m, 1H), 1.60e1.52 (m, 1H), 1.48e1.33 (m, 5H),

1.25 (d, J ¼ 5.87 Hz, 3H), 0.89 (t, J ¼ 7.43 Hz, 3H). ¹³C NMR (100 MHz,

DMSO‑d₆)

d

10.34 (s, 1H), 8.79 (d, J ¼ 7.8 Hz, 1H), 8.48 (s, 1H),

7.87e7.69 (m, 4H), 7.36e7.15 (m, 6H), 4.95e4.85 (m, 1H), 4.68e4.58

(m, 1H), 4.18 (d, J ¼ 15.0 Hz, 1H), 4.12 (d, J ¼ 15.0 Hz, 1H), 1.72e1.61

(m, 1H), 1.59e1.50 (m, 1H), 1.47e1.34 (m, 5H), 1.27 (d, J ¼ 6.0 Hz,

DMSO‑d₆)

d 165.13, 164.92, 164.02, 158.32, 148.32, 143.77, 135.86,

134.81, 129.78, 127.84, 126.88, 126.31, 125.88, 125.52, 125.43, 120.40,

119.36, 112.06, 111.28, 72.71, 48.11, 37.63, 36.27, 22.05, 19.18, 17.70,

15.86, 13.56. ESI-HRMS [MþH]^þcalcd for C₃₀H₃₄N₃O₃S₂: 548.2036,

found: 548.2039.

3H), 0.89 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)

d 165.93,

165.14, 164.39, 153.31, 150.89, 149.41, 149.01, 144.64, 136.40, 135.71,

128.69, 127.16, 126.41, 125.37, 121.31, 120.31, 116.17, 116.01, 113.14,

75.28, 48.95, 38.27, 37.20, 22.88, 19.89, 18.49, 14.32.¹⁹F NMR

10

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

(565 MHz, CDCl₃)

d

ꢁ131.83. ESI-HRMS [MþH]^þcalcd for

0.24 mmol, 61.2 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (56.7 mg, 50.5% yield). ¹H NMR (400 MHz,

C

₂₉H₃₁FN₃O₃S₂: 552.1785, found: 552.1781.

4.1.1.4. 3-Chloro-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d]thiazol-6-yl)-4-(pentan-2-yloxy)benzamide (B4).

DMSO‑d₆)

d

10.24 (s, 1H), 8.79 (d, J ¼ 7.9 Hz, 1H), 8.47 (d, J ¼ 1.7 Hz,

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 3-chloro-4-(pentan-2-yloxy)benzoyl chloride (6d,

0.24 mmol, 62.7 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (68.8 mg, 60.7% yield). ¹H NMR (400 MHz,

1H), 7.83e7.70 (m, 4H), 7.33e7.23 (m, 4H), 7.19 (t, J ¼ 7.0 Hz, 1H),

7.07 (d, J ¼ 8.8 Hz, 1H), 4.95e4.85 (m, 1H), 4.64e4.54 (m, 1H), 4.18

(d, J ¼ 14.8, 1H), 4.13 (d, J ¼ 14.8, 1H), 2.59 (q, J ¼ 7.5 Hz, 2H),

1.72e1.62 (m, 1H), 1.61e1.51 (m, 1H), 1.50e1.34 (m, 5H), 1.24 (d,

J ¼ 6.0 Hz, 3H), 1.15 (t, J ¼ 7.5 Hz, 3H), 0.89 (t, J ¼ 7.3 Hz, 3H). ¹³

C

NMR (100 MHz, DMSO‑d₆)

d 165.46, 165.29, 164.38, 158.20, 148.71,

DMSO‑d₆)

d

10.38 (s, 1H), 8.79 (d, J ¼ 7.7 Hz, 1H), 8.47 (s, 1H), 8.06 (s,

144.15, 136.23, 135.17, 132.11, 128.73, 128.20, 127.22, 126.66, 125.90,

120.75, 119.78, 112.51, 111.63, 72.66, 48.45, 38.00, 36.65, 23.09,

22.40, 19.39, 18.07, 14.11, 13.91. ESI-HRMS [MþH]^þcalcd for

1H), 7.92 (d, J ¼ 8.6 Hz, 1H), 7.81e7.68 (m, 2H), 7.36e7.16 (m, 5H),

4.95e4.85 (m, 1H), 4.71e4.61 (m, 1H), 4.23e4.09 (m, 2H), 1.74e1.63

(m, 1H),1.63e1.54 (m, 1H),1.51e1.39 (m, 2H),1.36 (d, J ¼ 7.0 Hz, 3H),

1.28 (d, J ¼ 6.0 Hz, 3H), 0.89 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (100 MHz,

C

₃₁H₃₆N₃O₃S₂: 562.2193, found: 562.2193.

DMSO‑d₆)

d

165.45, 164.68, 163.81, 155.84, 148.89, 144.15, 135.87,

4.1.1.8. N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)thio)benzo

[d]thiazol-6-yl)-4-(pentan-2-yloxy)-3-propylbenzamide (B8).

135.19, 129.44, 128.37, 128.20, 127.13, 126.66, 125.90, 121.85, 120.80,

119.80, 114.22, 112.66, 74.74, 48.45, 37.75, 36.65, 22.41, 19.36, 17.94,

13.84. ESI-HRMS [M þ H]^þcalcd for C₂₉H₃₁ClN₃O₃S₂: 568.1490,

found: 568.1487.

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 4-(pentan-2-yloxy)-3-propylbenzoyl chloride (6h,

0.24 mmol, 64.5 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (50.0 mg, 43.5% yield). ¹H NMR (400 MHz,

4.1.1.5. 3-Bromo-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d]thiazol

-6-yl)-4-(pentan-2-yloxy)benzamide

(B5).

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 3-bromo-4-(pentan-2-yloxy)benzoyl chloride (6e,

0.24 mmol, 73.3 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (74.3 mg, 60.6% yield). ¹H NMR (400 MHz,

DMSO‑d₆)

d

10.23 (s, 1H), 8.79 (d, J ¼ 7.9 Hz, 1H), 8.47 (d, J ¼ 1.8 Hz,

1H), 7.83e7.70 (m, 4H), 7.33e7.23 (m, 4H), 7.19 (t, J ¼ 7.0 Hz, 1H),

7.07 (d, J ¼ 8.9 Hz, 1H), 4.95e4.85 (m, 1H), 4.63e4.54 (m, 1H), 4.18

(d, J ¼ 14.9, 1H), 4.13 (d, J ¼ 14.9, 1H), 2.55 (t, J ¼ 7.6 Hz, 2H),

1.73e1.38 (m, 6H), 1.36 (d, J ¼ 7.0 Hz, 3H), 1.24 (d, J ¼ 6.0 Hz, 3H),

0.89 (t, J ¼ 7.3 Hz, 6H). ¹³C NMR (100 MHz, DMSO‑d₆)

d 165.45,

DMSO‑d₆)

d

10.38 (s, 1H), 8.79 (d, J ¼ 7.9 Hz, 1H), 8.46 (d, J ¼ 1.7 Hz,

165.25, 164.36, 158.32, 148.72, 144.14, 136.25, 135.17, 130.54, 129.53,

128.20, 127.26, 126.66, 125.91, 125.77, 120.75, 119.79, 112.52, 111.67,

72.62, 48.45, 38.00, 36.69, 31.88, 22.49, 22.39, 19.34, 18.02, 13.92,

13.88. ESI-HRMS [MþH]^þcalcd for C₃₂H₃₈N₃O₃S₂: 576.2349, found:

576.2350.

1H), 8.21 (d, J ¼ 2.1 Hz, 1H), 7.96 (dd, J ¼ 8.7, 2.0 Hz, 1H), 7.80e7.67

(m, 2H), 7.40e7.10 (m, 6H), 4.95e4.85 (m, 1H), 4.70e4.61 (m, 1H),

4.17 (d, J ¼ 14.9 Hz, 1H), 4.12 (d, J ¼ 14.9 Hz, 1H), 1.73e1.63 (m, 1H),

1.62e1.53 (m, 1H), 1.50e1.33 (m, 5H), 1.28 (d, J ¼ 6.0 Hz, 3H), 0.88 (t,

J ¼ 7.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)

d 165.65, 164.84,

163.89,156.89,149.04,144.29,136.01,135.34,132.60,129.20,128.36,

127.69, 126.83, 126.04, 120.96, 119.98, 114.13, 112.84, 111.72, 74.97,

48.64, 37.94, 36.79, 22.56, 19.52, 18.10, 14.02. ESI-HRMS [MþH]^þ

calcd for C₂₉H₃₁BrN₃O₃S₂: 614.0967, found: 614.0963.

4.1.1.9. N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)thio)benzo

[d]thiazol-6-yl)-4- (pentan-2-yloxy)-3-(triﬂuoromethyl)benzamide

(B9). The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 4-(pentan-2-yloxy)-3-(triﬂuoromethyl) benzoyl

chloride (6i, 0.24 mmol, 70.7 mg). The residue was puriﬁed by ﬂash

column chromatography on silica gel (200e300 mesh) using pe-

troleum ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the

desired product as white solid (56.3 mg, 46.8% yield). ¹H NMR

4.1.1.6. 3-Cyano-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d]thiazol

-6-yl)-4-(pentan-2-yloxy)benzamide

(B6).

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 3-cyano-4-(pentan-2-yloxy)benzoyl chloride (6f,

0.24 mmol, 60.4 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (96.2 mg, 86.2% yield). ¹H NMR (400 MHz,

(400 MHz, CDCl₃)

d

8.40 (d, J ¼ 1.8 Hz, 1H), 8.24 (s, 1H), 8.13e8.06

(m, 2H), 7.87 (d, J ¼ 7.7 Hz, 1H), 7.65 (d, J ¼ 8.7 Hz, 1H), 7.44 (dd,

J ¼ 8.7, 2.0 Hz, 1H), 7.24e7.17 (m, 4H), 7.07 (d, J ¼ 8.7 Hz, 1H),

5.11e5.01 (m, 1H), 4.64e4.54 (m, 1H), 3.96 (d, J ¼ 14.9 Hz, 1H), 3.91

(d, J ¼ 14.9 Hz, 1H), 1.86e1.76 (m, 1H), 1.70e1.60 (m, 2H), 1.56e1.47

(m, 1H), 1.44 (d, J ¼ 6.9 Hz, 3H), 1.37 (d, J ¼ 6.1 Hz, 3H), 0.96 (t,

DMSO‑d₆)

d

10.46 (s, 1H), 8.80 (d, J ¼ 7.9 Hz, 1H), 8.51 (d, J ¼ 2.0 Hz,

1H), 8.39 (d, J ¼ 2.3 Hz, 1H), 8.24 (dd, J ¼ 9.0, 2.3 Hz, 1H), 7.82 (d,

J ¼ 8.8 Hz, 1H), 7.74 (dd, J ¼ 8.9, 2.0 Hz, 1H), 7.46 (d, J ¼ 9.2 Hz, 1H),

7.36e7.27 (m, 4H), 7.25e7.19 (m, 1H), 4.98e4.88 (m, 1H), 4.84e4.74

(m, 1H), 4.21 (d, J ¼ 14.9 Hz, 1H), 4.17 (d, J ¼ 14.9 Hz, 1H), 1.78e1.68

(m,1H),1.67e1.58 (m,1H),1.51e1.36 (m, 5H),1.34 (d, J ¼ 6.0 Hz, 3H),

J ¼ 7.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)

d 165.93, 165.23,

164.36, 158.81, 149.48, 144.64, 136.33, 135.71, 134.40, 128.69, 127.16,

126.41, 126.35, 121.31, 120.41, 114.59, 113.32, 74.96, 48.95, 38.25,

37.20, 22.88, 19.61, 18.24, 14.28.¹⁹F NMR (565 MHz, CDCl₃)

d

ꢁ62.68.

ESI-HRMS [MþH]^þcalcd for C₃₀H₃₁F₃N₃O₃S₂: 602.1753, found:

0.93 (t, J ¼ 7.4 Hz, 3H). ¹³C NMR (150 MHz, DMSO‑d₆)

d

165.31,

602.1751.

164.66, 163.14, 161.71, 148.87, 144.00, 135.61, 135.11, 134.67, 133.26,

128.06, 126.73, 126.53, 125.78, 120.73, 119.63, 115.79, 113.64, 112.52,

100.88, 75.29, 48.33, 37.45, 36.57, 22.23, 19.09, 17.72, 13.66. ESI-

HRMS [MþH]^þcalcd for C₃₀H₃₁N₄O₃S₂: 559.1832, found: 559.1833.

4.1.1.10. 2-Methyl-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d] thiazol-6-yl)-4-(pentan-2-yloxy)benzamide (B10).

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 2-methyl-4-(pentan-2-yloxy)benzoyl chloride (6j,

0.24 mmol, 57.8 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (44.5 mg, 40.7% yield). ¹H NMR (400 MHz,

4.1.1.7. 3-Ethyl-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d]thiazol

-6-yl)-4-(pentan-2-yloxy)benzamide

(B7).

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 3-ethyl-4-(pentan-2-yloxy)benzoyl chloride (6g,

11

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

DMSO‑d₆)

d

10.36 (s, 1H), 8.79 (d, J ¼ 7.9 Hz, 1H), 8.48 (d, J ¼ 1.6 Hz,

J ¼ 2.1 Hz, 1H), 6.99 (dd, J ¼ 8.6, 2.2 Hz, 1H), 4.95e4.84 (m, 1H),

4.61e4.52 (m, 1H), 4.17 (d, J ¼ 14.9 Hz, 1H), 4.12 (d, J ¼ 15.0 Hz, 1H),

1.67e1.59 (m, 1H), 1.56e1.48 (m, 1H), 1.45e1.29 (m, 5H), 1.23 (d,

J ¼ 6.0 Hz, 3H), 0.88 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)

1H), 7.75 (d, J ¼ 8.8 Hz, 1H), 7.64 (dd, J ¼ 8.9, 1.8 Hz, 1H), 7.42 (d,

J ¼ 9.1 Hz, 1H), 7.35e7.15 (m, 5H), 6.81 (d, J ¼ 6.8 Hz, 2H), 4.94e4.84

(m, 1H), 4.54e4.46 (m, 1H), 4.16 (d, J ¼ 14.9 Hz, 1H), 4.11 (d,

J ¼ 14.9 Hz, 1H), 2.36 (s, 3H), 1.67e1.58 (m, 1H), 1.56e1.48 (m, 1H),

d

170.70, 170.13, 169.91, 164.57, 154.05, 150.97, 149.32, 141.04,

1.46e1.32 (m, 5H), 1.21 (d, J ¼ 6.0 Hz, 3H), 0.87 (t, J ¼ 7.3 Hz, 3H). ¹³

C

140.56, 136.51, 135.57, 133.81, 133.41, 131.89, 131.09, 129.02, 126.19,

121.51, 119.34, 78.74, 53.68, 42.93, 41.80, 27.60, 24.50, 23.24, 19.04.

ESI-HRMS [MþNa]^þcalcd for C₂₉H₃₀ClN₃O₃S₂Na: 590.1309, found:

590.1314.

NMR (100 MHz, DMSO‑d₆)

d 167.93, 165.78, 164.42, 159.08, 148.88,

144.35, 138.34, 136.52, 135.51, 129.58, 128.96, 128.45, 126.93, 126.12,

121.11,119.30,117.83,112.20,111.93, 72.89, 48.72, 38.13, 36.81, 22.62,

20.00, 19.71, 18.34, 14.10. ESI-HRMS [MþH]^þcalcd for

C

₃₀H₃₄N₃O₃S₂: 548.2036, found: 548.2034.

4.1.1.14. 2,3-Diﬂuoro-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)

ethyl)thio)benzo[d]

thiazol-6-yl)-4-(pentan-2-yloxy)benzamide

4.1.1.11. 2-Methoxy-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)

ethyl)thio)benzo[d] thiazol-6-yl)-4-(pentan-2-yloxy)benzamide

(B11). The title compound was obtained starting from

(0.20 mmol, 68.7 mg) and 2-methoxy-4-(pentan-2-yloxy)benzoyl

chloride (6k, 0.24 mmol, 61.6 mg). The residue was puriﬁed by ﬂash

column chromatography on silica gel (200e300 mesh) using pe-

troleum ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the

desired product as white solid (45.6 mg, 40.5% yield). ¹H NMR

(B14). The title compound was obtained starting from

3

(0.20 mmol, 68.7 mg) and 2,3-diﬂuoro-4-(pentan-2-yloxy)benzoyl

chloride (6n, 0.24 mmol, 63.0 mg). The residue was puriﬁed by ﬂash

column chromatography on silica gel (200e300 mesh) using pe-

troleum ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the

desired product as white solid (93.7 mg, 82.3% yield). ¹H NMR

3

(400 MHz, DMSO‑d₆)

d

10.55 (s, 1H), 8.79 (d, J ¼ 7.9 Hz, 1H), 8.45 (d,

J ¼ 1.8 Hz, 1H), 7.78 (d, J ¼ 8.8 Hz, 1H), 7.63 (dd, J ¼ 8.8, 1.7 Hz, 1H),

7.49e7.43 (m, 1H), 7.33e7.24 (m, 4H), 7.22e7.14 (m, 2H), 4.95e4.86

(m, 1H), 4.69e4.61 (m, 1H), 4.18 (d, J ¼ 14.9 Hz, 1H), 4.13 (d,

J ¼ 14.9 Hz, 1H), 1.73e1.64 (m, 1H), 1.61e1.53 (m, 1H), 1.47e1.33 (m,

5H), 1.28 (d, J ¼ 6.0 Hz, 3H), 0.89 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR

(400 MHz, CDCl₃)

d

9.91 (s, 1H), 8.55 (d, J ¼ 2.0 Hz, 1H), 8.24 (d,

J ¼ 8.8 Hz, 1H), 7.97 (d, J ¼ 7.7 Hz, 1H), 7.66 (d, J ¼ 8.7 Hz, 1H), 7.38

(dd, J ¼ 8.7, 2.1 Hz, 1H), 7.25e7.16 (m, 5H), 6.64 (dd, J ¼ 8.9, 2.2 Hz,

1H), 6.53 (d, J ¼ 2.1 Hz, 1H), 5.11e5.02 (m, 1H), 4.54e4.44 (m, 1H),

4.05 (s, 3H), 3.96 (d, J ¼ 14.9 Hz, 1H), 3.89 (d, J ¼ 14.9 Hz, 1H),

1.79e1.75 (m, 1H), 1.63e1.57 (m, 1H), 1.54e1.41 (m, 5H), 1.34 (d,

J ¼ 6.1 Hz, 3H), 0.96 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)

(100 MHz, DMSO‑d₆) d 165.92,165.38,162.07,149.52,144.64,136.07,

135.86, 128.69, 127.16, 126.41, 124.85, 121.48, 119.70, 118.33, 118.22,

112.61, 111.57, 76.09, 48.95, 38.17, 37.19, 22.88, 19.88, 18.45, 14.29.¹⁹

F

d

167.39, 165.08, 163.32, 162.78, 158.66, 148.60, 142.88, 136.53,

NMR (565 MHz, CDCl₃)

d

ꢁ137.42 (d, J ¼ 21.4 Hz), ꢁ157.76 (d,

135.86, 134.16, 128.57, 127.22, 125.92, 121.05, 119.37, 113.71, 112.43,

106.96, 100.21, 73.95, 56.23, 49.39, 38.45, 36.32, 22.00, 19.66, 18.71,

14.02. ESI-HRMS [MþH]^þcalcd for C₃₀H₃₄N₃O₄S₂: 564.1985, found:

564.1989.

J ¼ 21.4 Hz). ESI-HRMS [MþH]^þcalcd for C₂₉H₃₀F₂N₃O₃S₂: 570.1691,

found: 570.1696.

4.1.2. Synthesis of B15

4.1.2.1. Methyl

(R)-3-methyl-4-(pentan-2-yloxy)benzoate

(8a).

4.1.1.12. 2-Fluoro-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d] thiazol -6-yl)-4-(pentan-2-yloxy)benzamide (B12).

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 2-ﬂuoro-4-(pentan-2-yloxy)benzoyl chloride (6l,

0.24 mmol, 58.7 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (52.7 mg, 47.8% yield). ¹H NMR (400 MHz,

DEAD (0.695 mL, 4.41 mmol) was added dropwise to a stirred so-

lution of P(Ph)₃(1.16 g, 4.41 mmol) in anhydrous THF at 0 ^ꢀC. When

the reaction mixture became colourless, methyl 4-hydroxy-3-

methylbenzoate (489 mg, 2.94 mmol) was added at 0 ^ꢀC and stir-

red for 10 min. Subsequently, (S)-pentan-2-ol (0.32 mL, 2.94 mmol)

was added at 0 ^ꢀC and stirred for another 10 min. After the reaction

mixture was stirred at room temperature for about 20 h, the solvent

was evaporated under reduced pressure. The residue was dissolved

in ethyl acetate (100 mL) and was washed with distilled water

(100 mL) for three times. The organic phases were combined, dried

over Na₂SO₄, and evaporated under reduced pressure. The crude

compound was puriﬁed by silica gel column chromatography (PE/

EA ¼ 10/1, v/v) to give 8a (270 mg, 38.9% yield). ¹H NMR (400 MHz,

DMSO‑d₆)

d

10.39 (s, 1H), 8.79 (d, J ¼ 7.9 Hz, 1H), 8.46 (d, J ¼ 1.9 Hz,

1H), 7.76 (d, J ¼ 8.8 Hz, 1H), 7.66e7.58 (m, 2H), 7.34e7.23 (m,

4H),7.22e7.16 (m, 1H), 6.94 (dd, J ¼ 12.7, 2.0 Hz, 1H), 6.87 (dd,

J ¼ 8.6, 2.1 Hz, 1H), 4.95e4.84 (m, 1H), 4.63e4.53 (m, 1H), 4.18 (d,

J ¼ 14.9 Hz, 1H), 4.13 (d, J ¼ 14.9 Hz, 1H), 1.67e1.58 (m, 1H),

1.57e1.49 (m, 1H), 1.46e1.30 (m, 5H), 1.24 (d, J ¼ 6.0 Hz, 3H), 0.88 (t,

DMSO‑d₆)

d

7.75 (d, J ¼ 8.6 Hz, 1H), 7.72 (s, 1H), 7.01 (d, J ¼ 8.6 Hz,

J ¼ 7.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)

d

165.96, 165.15,

1H), 4.59e4.48 (m, 1H), 3.77 (s, 3H), 2.12 (s, 3H), 1.72e1.59 (m, 1H),

1.57e1.49 (m, 1H), 1.45e1.30 (m, 2H), 1.22 (d, J ¼ 6.0 Hz, 3H), 0.86 (t,

J ¼ 7.3 Hz, 3H). ESI-MS: calcd for [MþH]^þm/z 237.1, found: 237.1.

163.08, 162.12, 161.72, 159.64, 149.37, 144.63, 136.33, 135.82, 131.70,

128.70, 127.17, 126.40, 121.42, 119.71, 116.72, 112.34, 103.46, 74.17,

48.96, 38.22, 37.17, 22.87, 19.80, 18.52, 14.32.¹⁹F NMR (565 MHz,

CDCl₃)

d

ꢁ110.46. ESI-HRMS [MþH]^þcalcd for C₂₉H₃₁FN₃O₃S₂:

4.1.2.2. (R)-3-methyl-4-(pentan-2-yloxy)benzoic acid (9a). To a so-

lution of 8a (160 mg, 0.677 mmol) in methanol (5 mL) was added

1 M NaOH (5 mL, 5.00 mmol). The resulting mixture was stirred at

50 ^ꢀC for 12 h. Upon cooling the methanol was removed under

vacuum, and the residue was acidiﬁed to pH ¼ 2 or below with HCl

(1 M). Then the solution was extracted with ethyl acetate

(20 mL ꢂ 3) and the combined organic solvents were dried with

sodium sulfate and concentrated in vacuo. The crude compound

was puriﬁed by silica gel column chromatography (DCM/

CH₃OH ¼ 15/1, v/v) to give 9a (130 mg, 86.4% yield). ¹H NMR

552.1785, found: 552.1790.

4.1.1.13. 2-Chloro-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d] thiazol-6-yl)-4-(pentan-2-yloxy)benzamide (B13).

The title compound was obtained starting from 3 (0.20 mmol,

68.7 mg) and 2-chloro-4-(pentan-2-yloxy)benzoyl chloride (6m,

0.24 mmol, 62.7 mg). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (59.5 mg, 52.5% yield). ¹H NMR (400 MHz,

(400 MHz, CDCl₃)

d

7.92 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.89 (d, J ¼ 2.1 Hz,

DMSO‑d₆)

d

10.59 (s, 1H), 8.79 (d, J ¼ 7.9 Hz, 1H), 8.47 (d, J ¼ 1.5 Hz,

1H), 6.83 (d, J ¼ 8.6 Hz, 1H), 4.53e4.43 (m, 1H), 2.22 (s, 3H),

1.82e1.72 (m, 1H), 1.64e1.56 (m, 1H), 1.52e1.38 (m, 2H), 1.32 (d,

J ¼ 6.1 Hz, 3H), 0.94 (t, J ¼ 7.3 Hz, 3H). ESI-MS: calcd for [M ꢁ H]^-m/z

1H), 7.76 (d, J ¼ 8.8 Hz, 1H), 7.62 (dd, J ¼ 8.8, 1.7 Hz, 1H), 7.51 (d,

J ¼ 8.5 Hz, 1H), 7.32e7.22 (m, 4H), 7.19 (t, J ¼ 6.9 Hz, 1H), 7.09 (d,

12

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

221.1, found: 221.1.

afford the desired product as white solid (67.8 mg, 62.0% yield). ¹H

NMR (400 MHz, DMSO‑d₆)

d

10.25 (s, 1H), 8.80 (d, J ¼ 8.0 Hz, 1H),

4.1.2.3. (R)-3-methyl-4-(pentan-2-yloxy)benzoyl chloride (10a).

Thionyl chloride (10 mL, 1378 mmol) was added to 9a (111 mg,

0.50 mmol) and stirred for 3 h under reﬂux condition. After cooling

to room temperature, the thionyl chloride was evaporated under

reduced pressure to obtain a yellow oil (10a), which was used

directly for the following synthesis.

8.47 (d, J ¼ 1.7 Hz,1H), 7.84e7.66 (m, 4H), 7.33e7.16 (m, 5H), 7.05 (d,

J ¼ 8.5 Hz, 1H), 4.95e4.86 (m, 1H), 4.61e4.52 (m, 1H), 4.18 (d,

J ¼ 14.9, 1H), 4.13 (d, J ¼ 14.9, 1H), 2.18 (s, 3H), 1.71e1.61 (m, 1H),

1.60e1.52 (m, 1H), 1.46e1.31 (m, 5H), 1.25 (d, J ¼ 6.0 Hz, 3H), 0.88 (t,

J ¼ 7.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)

d 165.49, 165.27,

164.38, 158.69, 148.69, 144.13, 136.23, 135.17, 130.14, 128.20, 127.24,

126.67, 126.24, 125.89, 125.80, 120.76, 119.72, 112.42, 111.64, 73.06,

48.46, 37.98, 36.63, 22.39, 19.53, 18.05, 16.21, 13.90. ESI-HRMS

[MþH]^þcalcd for C₃₀H₃₄N₃O₃S₂: 548.2036, found: 548.2041.

4.1.2.4. 3-Methyl-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d]thiazol-6-yl)-4-(((R)-pentan-2-yl)oxy)benzamide (B15).

Compound B15 was prepared in a similar manner as described for

compounds B1eB14, starting from 3 (68.7 mg, 0.20 mmol) and 10a

(57.8 mg, 0.24 mmol). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to afford the desired

product as white solid (40.2 mg, 36.7% yield). ¹H NMR (400 MHz,

4.1.4. Synthesis of B17

4.1.4.1. 2,3-Dimethyl-4-(pentan-2-yloxy)benzoic

acid

(9c).

Compound 8c was synthesized following the synthetic procedure

of compounds 5a-5n and the crude product was obtained. Subse-

quently, NaOH (1 M) water solution (3 mL, 3.00 mmol) and CH₃OH

(3 mL) were added and stirred for 1 h. Then the methanol was

removed under vacuum, and the residue was acidiﬁed to pH ¼ 2 or

below with HCl (1 M). Then the solution was extracted with ethyl

acetate (20 mL ꢂ 3) and the combined organic solvents were dried

with sodium sulfate and concentrated in vacuo. The crude com-

pound was puriﬁed by silica gel column chromatography (DCM/

CH₃OH ¼ 15/1, v/v) to give 9c (white solid, two steps 51.0% yield). ¹H

DMSO‑d₆)

d

10.24 (s, 1H), 8.79 (d, J ¼ 7.9 Hz, 1H), 8.48 (d, J ¼ 1.8 Hz,

1H), 7.83e7.68 (m, 4H), 7.34e7.16 (m, 5H), 7.06 (d, J ¼ 8.5 Hz, 1H),

4.95e4.86 (m, 1H), 4.61e4.52 (m, 1H), 4.18 (d, J ¼ 14.9, 1H), 4.13 (d,

J ¼ 14.9, 1H), 2.18 (s, 3H), 1.71e1.63 (m, 1H), 1.60e1.52 (m, 1H),

1.49e1.31 (m, 5H), 1.25 (d, J ¼ 6.0 Hz, 3H), 0.89 (t, J ¼ 7.3 Hz, 3H). ¹³

C

NMR (100 MHz, DMSO‑d₆)

d 165.47, 165.25, 164.36, 158.68, 148.68,

144.13, 136.24, 135.17, 130.14, 128.19, 127.23, 126.66, 126.23, 125.89,

125.82, 120.76, 119.70, 112.41, 111.64, 73.06, 48.45, 37.98, 36.64,

22.39, 19.53, 18.05, 16.20, 13.90. ESI-HRMS [MþH]^þcalcd for

NMR (400 MHz, DMSO‑d₆)

d

12.40 (s, 1H), 7.60 (d, J ¼ 8.7 Hz, 1H),

6.83 (d, J ¼ 8.8 Hz, 1H), 4.54e4.41 (m, 1H), 2.40 (s, 3H), 2.07 (s, 3H),

1.70e1.59 (m, 1H), 1.56e1.48 (m, 1H), 1.45e1.27 (m, 2H), 1.20 (d,

J ¼ 6.0 Hz, 3H), 0.86 (t, J ¼ 7.3 Hz, 3H). ESI-MS: calcd for [M ꢁ H]^-m/

z 235.1, found: 235.1.

C

₃₀H₃₄N₃O₃S₂: 548.2036, found: 548.2041.

4.1.3. Synthesis of B16

4.1.3.1. Methyl (S)-3-methyl-4-(pentan-2-yloxy)benzoate

(8b).

Compound 8b was prepared in a similar manner as described for

compound 8a, starting from 7a (489 mg, 2.94 mmol) and (R)-

pentan-2-ol (0.32 mL, 2.94 mmol). The residue was puriﬁed by ﬂash

column chromatography on silica gel (200e300 mesh) using pe-

troleum ether/ethyl acetate eluent (PE/EA ¼ 10/1, v/v) to afford the

desired product as colorless oil (444 mg, 64.0% yield). ¹H NMR

4.1.4.2. 2,3-Dimethyl-4-(pentan-2-yloxy)benzoyl chloride (10c).

Compound 10c was prepared in a similar manner as described for

compound 10a, starting from 9c (118 mg, 0.50 mmol).

4.1.4.3. 2,3-Dimethyl-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)

ethyl)thio)benzo[d]

thiazol-6-yl)-4-(pentan-2-yloxy)benzamide

(400 MHz, CDCl₃)

d

7.85 (d, J ¼ 2.0 Hz, 1H), 7.82 (s, 1H), 6.80 (d,

(B17). Compound B17 was prepared in a similar manner as

described for compounds B1eB14, starting from 3 (68.7 mg,

0.20 mmol) and 10c (61.1 mg, 0.24 mmol). The residue was puriﬁed

by ﬂash column chromatography on silica gel (200e300 mesh)

using petroleum ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to

afford the desired product as white solid (67.3 mg, 60.0% yield). ¹H

J ¼ 8.5 Hz, 1H), 4.52e4.36 (m, 1H), 3.87 (s, 3H), 2.21 (s, 3H),

1.80e1.70 (m, 1H), 1.65e1.53 (m, 1H), 1.52e1.37 (m, 2H), 1.31 (d,

J ¼ 6.1 Hz, 3H), 0.93 (t, J ¼ 7.3 Hz, 3H). ESI-MS: calcd for [MþH]^þm/z

237.1, found: 237.1.

4.1.3.2. (S)-3-methyl-4-(pentan-2-yloxy)benzoic

acid

(9b).

NMR (400 MHz, DMSO‑d₆)

d

10.40 (s, 1H), 8.79 (d, J ¼ 7.6 Hz, 1H),

Compound 9b was prepared in a similar manner as described for

compound 9a, starting from 8b (472 mg, 2.00 mmol). The residue

was puriﬁed by ﬂash column chromatography on silica gel

(200e300 mesh) using dichloromethane/methanol eluent (DCM/

CH₃OH ¼ 15/1, v/v) to afford the desired product as colorless oil

8.50 (s, 1H), 7.75 (d, J ¼ 8.9 Hz, 1H), 7.64 (d, J ¼ 8.1 Hz, 1H), 7.33e7.15

(m, 6H), 6.88 (d, J ¼ 8.5 Hz, 1H), 4.95e4.85 (m, 1H), 4.55e4.45 (m,

1H), 4.18 (d, J ¼ 14.9, 1H), 4.13 (d, J ¼ 14.9, 1H), 2.25 (s, 3H), 2.10 (s,

3H), 1.70e1.61 (m, 1H),1.60e1.51 (m, 1H),1.46e1.33 (m, 5H), 1.22 (d,

J ¼ 5.6 Hz, 3H), 0.88 (t, J ¼ 7.0 Hz, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)

(444 mg,100.0% yield). ¹H NMR (400 MHz, CDCl₃)

d

8.02 (dd, J ¼ 8.6,

d 168.53, 165.45, 164.31, 156.41, 148.65, 144.15, 136.38, 135.40,

2.1 Hz,1H), 7.99 (d, J ¼ 2.1 Hz,1H), 6.93 (d, J ¼ 8.6 Hz,1H), 4.63e4.53

(m, 1H), 2.32 (s, 3H), 1.90e1.80 (m,1H), 1.72e1.64 (m,1H),1.60e1.46

(m, 2H), 1.42 (d, J ¼ 6.1 Hz, 3H), 0.93 (t, J ¼ 7.3 Hz, 3H). ESI-MS: calcd

for [M ꢁ H]^-m/z 221.1, found: 221.1.

135.32, 130.07, 128.21, 126.68, 125.93, 125.64, 120.89, 118.93, 111.53,

109.80, 73.18, 48.45, 38.09, 36.69, 22.40, 19.63, 18.13, 16.66, 13.94,

11.78. ESI-HRMS [MþH]^þcalcd for C₃₁H₃₆N₃O₃S₂: 562.2193, found:

562.2190.

4.1.3.3. (S)-3-methyl-4-(pentan-2-yloxy)benzoic

acid

(10b).

4.1.5. General procedures for the synthesis of B18eB26

Compound 10b was prepared in a similar manner as described for

compound 10a, starting from 9b (111 mg, 0.50 mmol).

4.1.5.1. 2, 4-dihydroxy-3-methylbenzaldehyde (16). DMF (6.2 mL,

80.56 mmol) and POCl₃(8.1 mL, 88.61 mmol) were mixed and

added to a stirred solution of 2-methylbenzene-1,3-diol (15, 5.0 g,

0.806 mmol) in ethyl acetate (100 mL) at room temperature. Then

water (5 mL) was added to the mixture and stirred for about 2 h.

The reaction mixture was extracted with ethyl acetate (20 mL ꢂ 3).

The combined organic phases were dried with sodium sulfate and

concentrated in vacuo. The crude compound was puriﬁed by silica

gel column chromatography (PE/EA ¼ 5/1, v/v) to give 16 (white

4.1.3.4. 3-Methyl-N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)

thio)benzo[d]thiazol

-6-yl)-4-(((R)-pentan-2-yl)oxy)benzamide

(B16). Compound B16 was prepared in a similar manner as

described for compounds B1eB14, starting from 3 (68.7 mg,

0.20 mmol) and 10b (57.8 mg, 0.24 mmol). The residue was puriﬁed

by ﬂash column chromatography on silica gel (200e300 mesh)

using petroleum ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v) to

solid, 79.9% yield). ¹H NMR (400 MHz, DMSO‑d₆)

d 11.56 (s, 1H),

13

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

10.74 (s, 1H), 9.65 (d, J ¼ 3.0 Hz, 1H), 7.38 (dd, J ¼ 8.4, 3.8 Hz, 1H),

6.51 (d, J ¼ 8.5 Hz, 1H), 1.93 (s, 3H). ESI-MS: calcd for [M ꢁ H]^-m/z

151.1, found: 151.1.

2-butene (4 mL, 47.6 mmol) were added NaClO₂(543 mg,

6.0 mmol) and NaH₂PO₄$2H₂O (468 mg, 3.0 mmol) buffer solution

(pH ¼ 4.5, 8 mL) at 0 ^ꢀC. Then the mixture was stirred at room

temperature for about 1 h. The solution was extracted with ethyl

acetate (20 mL ꢂ 3) and the organic layer was dried with sodium

sulfate and concentrated in vacuo. The crude compounds were

puriﬁed by silica gel column chromatography (DCM/CH₃OH ¼ 10/1,

v/v) to give 19a-19d.

4.1.5.2. General procedures for the synthesis of compounds 17a-17c.

To a stirred solution of 16 (152 mg, 1.0 mmol) in DMF (10 mL) were

added bromoalkane (302 mg, 2.0 mmol, 2-bromopentane for 17a;

274 mg, 2.0 mmol, 1-bromo-2-methyl-propan for 17b; 246 mg,

2.0 mmol, 2-bromopropane for 17c)、K₂CO₃(553 mg, 4.0 mmol)

and KI (16.6 mg, 0.1 mmol). The mixture was stirred at 80 ^ꢀC for

about 6 h. After cooling and ﬁltration on Celite, the solvent was

removed under reduced pressure to obtain an oily residue, which

was puriﬁed by ﬂash column chromatography on silica (PE/

EA ¼ 10/1, v/v) to give 17a-17c.

4.1.5.4.1. 2-Methoxy-3-methyl-4-(pentan-2-yloxy) benzoic acid

(19a). White solid, 63.1% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d

12.40 (s, 1H), 7.60 (d, J ¼ 8.8 Hz, 1H), 6.78 (d, J ¼ 8.9 Hz, 1H),

4.55e4.42 (m, 1H), 3.66 (s, 3H), 2.01 (s, 3H), 1.69e1.58 (m, 1H),

1.56e1.48 (m, 1H), 1.44e1.29 (m, 2H), 1.21 (d, J ¼ 6.0 Hz, 3H), 0.86 (t,

J ¼ 7.3 Hz, 3H). ESI-MS: calcd for [M ꢁ H]^-m/z 251.1, found: 251.1.

4.1.5.4.2. 4-Isobutoxy-2-methoxy-3-methylbenzoic acid (19b).

4.1.5.2.1. 2-Hydroxy-3-methyl-4-(pentan-2-yloxy) benzaldehyde

(17a). Reddish oil, 60.0% yield, ¹H NMR (400 MHz, DMSO‑d₆)

White solid, 83.0% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d 12.39 (s,

d

11.40 (s, 1H), 9.76 (s, 1H), 7.56 (d, J ¼ 8.7 Hz, 1H), 6.74 (d, J ¼ 8.8 Hz,

1H), 7.59 (d, J ¼ 8.7 Hz, 1H), 6.76 (d, J ¼ 8.7 Hz, 1H), 3.76 (d,

J ¼ 6.2 Hz, 2H), 3.66 (s, 3H), 2.04 (s, 3H), 2.04e1.95 (m, 1H), 0.96 (d,

J ¼ 6.6 Hz, 6H). ESI-MS: calcd for [M ꢁ H]^-m/z 237.1, found: 237.1.

4.1.5.4.3. 4-Isopropoxy-2-methoxy-3-methylbenzoic acid (19c).

1H), 4.65e4.56 (m, 1H), 1.95 (s, 3H), 1.70e1.59 (m, 1H), 1.58e1.48

(m, 1H), 1.47e1.27 (m, 2H), 1.23 (d, J ¼ 6.0 Hz, 3H), 0.86 (t, J ¼ 7.3 Hz,

3H). ESI-MS: calcd for [M ꢁ H]^-m/z 221.1, found: 221.1.

4.1.5.2.2. 2-Hydroxy-4-isobutoxy-3-methylbenzaldehyde (17b).

White solid, 80.0% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d 12.39 (s,

Colorless oil, 75.0% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d

11.37 (s,

1H), 7.60 (d, J ¼ 8.7 Hz, 1H), 6.81 (d, J ¼ 8.8 Hz, 1H), 4.71e4.58 (m,

1H), 3.67 (s, 3H), 2.02 (s, 3H), 1.27 (d, J ¼ 6.0 Hz, 6H). ESI-MS: calcd

for [M ꢁ H]^-m/z 223.1, found: 223.1.

1H), 9.79 (s, 1H), 7.59 (d, J ¼ 8.6 Hz, 1H), 6.72 (d, J ¼ 8.7 Hz, 1H), 3.86

(d, J ¼ 6.4 Hz, 2H), 2.11e2.02 (m, 1H), 2.00 (s, 3H), 0.98 (d, J ¼ 6.7 Hz,

6H). ESI-MS: calcd for [MþH]^þm/z 209.1, found: 209.1.

4.1.5.4.4. 2,4-Dimethoxy-3-methylbenzoic acid (19d). White

4.1.5.2.3. 4-Isopropoxy-2-methoxy-3-methylbenzaldehyde (17c).

solid, 79.6% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d 12.41 (s, 1H), 7.62

Colorless oil, 60.4% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d

11.39 (s,

(d, J ¼ 8.9 Hz, 1H), 6.79 (d, J ¼ 8.8 Hz, 1H), 3.80 (s, 3H), 3.66 (s, 3H),

1H), 9.78 (s, 1H), 7.57 (d, J ¼ 8.6 Hz, 1H), 6.76 (d, J ¼ 8.8 Hz, 1H),

4.85e4.67 (m,1H),1.96 (s, 3H),1.28 (d, J ¼ 6.0 Hz, 6H). ESI-MS: calcd

for [MþH]^þm/z 195.1, found:195.1.

2.02 (s, 3H). ESI-MS: calcd for [M ꢁ H]^-m/z 195.1, found: 195.1.

4.1.5.5. General procedures for the synthesis of 20a-20d. To a solu-

tion of 19a-19d (0.51 mmol) in 5 mL anhydrous CH₂Cl₂were added

SOCl₂(607 mg, 5.1 mmol) and DMF (1 drop) at 0 ^ꢀC. The mixture

was stirred at room temperature for 15 min. Then the solution was

concentrated in vacuo giving 20a-20d, which were used directly for

the following synthesis.

4.1.5.3. General procedures for the synthesis of compounds 18a-18d.

To a solution of 16 or 17a-17c (1.0 mmol) in DMF (5 mL) were added

CH₃I (124 mL, 2.0 mmol for 18a-18c, 248 mL, 4.0 mmol for 18d) and

K₂CO₃(690 mg, 5.0 mmol). The mixture was stirred at 40 ^ꢀC. for

about 6 h. After cooling and ﬁltration on Celite, the mixture was

extracted with ethyl acetate (20 mL ꢂ 3) and the combined organic

solvents were washed with water (20 mL). Then the organic phase

was dried with sodium sulfate and concentrated in vacuo. The crude

compounds were puriﬁed by silica gel column chromatography

(PE/EA ¼ 7/1, v/v) to give 18a-18d.

4.1.5.6. General procedures for the synthesis of 12b-12d.

2-Chloroacetyl chloride (169 mg, 1.5 mmol) was added dropwise to

a mixture of (S)-1-(4-methoxyphenyl)ethan-1-amine derivatives

(1.0 mmol) and potassium carbonate (414 mg, 3.0 mmol) in 5 mL of

dioxane originally at 0 ^ꢀC with stirring. Then the resulting mixture

was warmed to ambient temperature (25 ^ꢀC) and stirred for 6 h.

After removal of dioxane solvents under reduced pressure, water

was added and extracted with ethyl acetate (20 mL ꢂ 3). The

organic layer was combined and dried with sodium sulfate. The

solution was evaporated to give the crude product, which was

puriﬁed by silica gel column chromatography (PE/EA ¼ 15/1, v/v) to

obtain 12b-12d.

4.1.5.3.1. 2-Methoxy-3-methyl-4-(pentan-2-yloxy) benzaldehyde

(18a). Yellow oil, 97.2% yield, ¹H NMR (400 MHz, CDCl₃)

d 10.21 (s,

1H), 7.70 (d, J ¼ 8.7 Hz, 1H), 6.71 (d, J ¼ 8.7 Hz, 1H), 4.54e4.42 (m,

1H), 3.85 (s, 3H), 2.14 (s, 3H), 1.82e1.70 (m, 1H), 1.64e1.55 (m, 1H),

1.54e1.36 (m, 2H), 1.32 (d, J ¼ 6.1 Hz, 3H), 0.94 (t, J ¼ 7.3 Hz, 3H).

ESI-MS: calcd for [MþH]^þm/z 237.3, found: 237.2.

4.1.5.3.2. 4-Isobutoxy-2-methoxy-3-methylbenzaldehyde (18b).

Colorless oil, 93.0% yield, ¹H NMR (400 MHz, CDCl₃)

d

10.21 (s, 1H),

4.1.5.6.1. (S)-2-chloro-N-(1-(4-methoxyphenyl)ethyl)acetamide

7.71 (d, J ¼ 8.7 Hz, 1H), 6.70 (d, J ¼ 8.7 Hz, 1H), 3.85 (s, 3H), 3.79 (d,

J ¼ 6.4 Hz, 2H), 2.17 (s, 3H), 2.17e2.06 (m, 1H), 1.06 (s, 3H), 1.04 (s,

3H). ESI-MS: calcd for [MþH]^þm/z 223.1, found: 223.1.

(12b). White solid, 27.5% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d 8.59

(d, J ¼ 7.5 Hz, 1H), 7.24 (d, J ¼ 7.6 Hz, 2H), 6.89 (d, J ¼ 7.2 Hz, 2H),

4.92e4.82 (m, 1H), 4.06 (s, 2H), 3.73 (d, J ¼ 1.2 Hz, 3H), 1.35 (d,

J ¼ 6.5 Hz, 3H). ESI-MS: calcd for [MþH]^þm/z 228.1, 230.1, found:

228.1, 230.1.

4.1.5.3.3. 4-Isopropoxy-2-methoxy-3-methylbenzaldehyde (18c).

Colorless oil, yield 90.0%, ¹H NMR (400 MHz, DMSO‑d₆)

d 10.08 (s,

1H), 7.60 (d, J ¼ 8.7 Hz, 1H), 6.94 (d, J ¼ 8.8 Hz, 1H), 4.78e4.65 (m,

1H), 3.78 (s, 3H), 2.04 (s, 3H), 1.28 (d, J ¼ 6.0 Hz, 6H). ESI-MS: calcd

for [MþH]^þm/z 209.1, found: 209.1.

4.1.5.6.2. (S)-2-chloro-N-(1-(3-methoxyphenyl)ethyl)acetamide

(12c). White solid, 35.5% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d 8.61

(d, J ¼ 7.9 Hz, 1H), 7.20 (t, J ¼ 8.1 Hz, 1H), 6.85 (d, J ¼ 7.2 Hz, 2H),

6.79e6.75 (m, 1H), 4.89e4.79 (m, 1H), 4.04 (s, 2H), 3.71 (s, 3H), 1.32

(d, J ¼ 7.0 Hz, 3H). ESI-MS: calcd for [MþH]^þm/z 228.1, 230.1, found:

228.1, 230.1.

4.1.5.3.4. 2,4-Dimethoxy-3-methylbenzaldehyde

(18d).

White solid, 85.0% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d

10.05 (s,

1H), 7.60 (d, J ¼ 8.7 Hz, 1H), 6.91 (d, J ¼ 8.7 Hz, 1H), 3.83 (s, 3H), 3.75

(s, 3H), 2.03 (s, 3H). ESI-MS: calcd for [MþH]^þm/z 181.1,

found:181.1.

4.1.5.6.3. (S)-2-chloro-N-(1-(2-methoxyphenyl)ethyl)acetamide

(12d). White solid, 79.7% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d 8.55

(d, J ¼ 7.8 Hz, 1H), 7.25e7.15 (m, 2H), 6.94 (d, J ¼ 8.2 Hz, 1H), 6.88 (t,

J ¼ 7.4 Hz, 1H), 5.20e5.08 (m, 1H), 4.05 (s, 2H), 3.77 (s, 3H), 1.25 (d,

J ¼ 6.9 Hz, 3H). ESI-MS: calcd for [MþH]^þm/z 228.1, 230.1, found:

4.1.5.4. General procedures for the synthesis of compounds 19a-19d.

To a solution of 18a-18d (1.0 mmol) in t-BuOH (8 mL) and 2-methyl-

14

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

228.1, 230.1.

1.48e1.32 (m, 2H),1.44 (d, J ¼ 5.7 Hz, 3H), 1.36 (d, J ¼ 3.0 Hz, 3H),

0.98 (t, J ¼ 6.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)

d 167.20, 165.02,

4.1.5.7. General procedures for the synthesis of 13b-13d. To a solu-

tion of 12b-12d (1.0 mmol) in anhydrous dichloromethane (10 mL)

was added boron tribromide (17% in CH₂Cl₂, 1.25 g, 5.0 mmol) at

0 ^ꢀC. Water (10 mL) was added after the reaction was stirred for 3 h.

Then the mixture was stirred for another 30 min before the

extraction with dichloromethane (10 mL ꢂ 3). The organic layer

was combined and dried with sodium sulfate. The solvent was

evaporated to give the crude product, which was puriﬁed by silica

gel column chromatography (PE/EA ¼ 10/1, v/v) to give 13b-13d.

4.1.5.7.1. (S)-2-chloro-N-(1-(4-hydroxyphenyl)ethyl)acetamide

163.65, 160.72, 158.70, 157.44, 148.76, 136.64, 135.96, 135.05, 130.12,

127.14, 121.24, 120.58, 118.95, 117.66, 113.90, 112.13, 108.79, 74.11,

61.76, 55.25, 48.80, 38.60, 36.36, 21.82, 19.77, 18.71, 14.08, 9.25. ESI-

HRMS [MþH]^þcalcd for C₃₂H₃₈N₃O₅S₂: 608.2247, found: 608.2243.

4.1.5.9.3. 2-Methoxy-N-(2-((2-(((S)-1-(3-methoxyphenyl)ethyl)

amino)-2-oxoethyl)thio) benzo[d]thiazol-6-yl)-3-methyl-4-(pentan-

2-yloxy)benzamide (B20). White solid, 20.0% yield, ¹H NMR

(400 MHz, CDCl₃)

d

10.09 (s, 1H), 8.58 (s, 1H), 8.02 (t, J ¼ 7.9 Hz, 2H),

7.70 (d, J ¼ 8.7 Hz, 1H), 7.39 (d, J ¼ 8.7 Hz, 1H), 7.15 (t, J ¼ 7.9 Hz, 1H),

6.82e6.70 (m, 4H), 5.04 (quint,1H), 4.51 (sext,1H), 3.93 (s, 2H), 3.87

(s, 3H), 3.69 (s, 3H), 2.20 (s, 3H), 1.83e1.71 (m, 1H), 1.67e1.53 (m,

3H),1.42 (d, J ¼ 6.8 Hz, 3H),1.33 (d, J ¼ 6.0 Hz, 3H), 0.95 (t, J ¼ 7.2 Hz,

(13b). White solid, 85.0% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d 9.29

(s, 1H), 8.50 (d, J ¼ 7.7 Hz, 1H), 7.09 (d, J ¼ 8.3 Hz, 2H), 6.68 (d,

J ¼ 8.3 Hz, 2H), 4.80 (quint, 1H), 4.02 (s, 2H), 1.31 (d, J ¼ 6.9 Hz, 3H).

ESI-MS: calcd for [MþH]^þm/z 214.1, 216.1, found:214.1, 216.1.

4.1.5.7.2. (S)-2-chloro-N-(1-(3-hydroxyphenyl)ethyl)acetamide

3H). ¹³C NMR (150 MHz, CDCl₃)

d 166.72, 164.58, 163.02, 160.10,

159.14, 156.82, 148.08,144.03,136.00,135.38, 129.50,129.01, 120.66,

119.95, 118.35, 117.56, 117.08, 111.91, 111.49, 111.20, 108.19, 73.50,

61.12, 54.48, 48.75, 37.98, 35.75, 21.39, 19.14, 18.07, 13.43, 8.60. ESI-

HRMS [MþH]^þcalcd for C₃₂H₃₇N₃O₅S₂: 608.2247, found: 608.2241.

4.1.5.9.4. N-(2-((2-(((S)-1-(4-hydroxyphenyl)ethyl)amino)-2-

(13c). White solid, 85.5% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d 9.34

(s, 1H), 8.57 (d, J ¼ 7.9 Hz, 1H), 7.07 (t, J ¼ 7.8 Hz, 1H), 6.72e6.65 (m,

2H), 6.59 (dd, J ¼ 8.0, 1.5 Hz, 1H), 4.83e4.73 (m, 1H), 4.02 (s, 2H),

1.30 (d, J ¼ 7.0 Hz, 3H). ESI-MS: calcd for [MþH]^þm/z 214.1, 216.1,

found:214.1, 216.1.

oxoethyl)thio)benzo[d]

tan-2-yloxy)benzamide (B21). White solid, 18.0% yield, ¹H NMR

(400 MHz, CDCl₃)

thiazol-6-yl)-2-methoxy-3-methyl-4-(pen-

4.1.5.7.3. (S)-2-chloro-N-(1-(2-hydroxyphenyl)ethyl)acetamide

d

10.05 (s, 1H), 8.14 (d, J ¼ 1.8 Hz 1H), 8.05 (d,

(13d). White solid, 82.8% yield, ¹H NMR (400 MHz, DMSO‑d₆)

d

9.55

J ¼ 8.8 Hz, 1H), 7.60 (d, J ¼ 8.6 Hz, 1H), 7.35 (dd, J ¼ 8.6, 1.8 Hz, 1H),

7.23 (d, J ¼ 7.8 Hz, 1H), 6.86e6.73 (m, 4H), 6.41 (d, J ¼ 8.4 Hz, 2H),

4.96 (quint, 1H), 4.50 (sext, 1H), 3.92 (brd, J ¼ 14.7, 2H), 3.89 (s, 3H),

2.20 (s, 3H),1.84e1.72 (m,1H),1.70e1.56 (m,1H),1.55e1.44 (m, 2H),

1.42 (d, J ¼ 6.8 Hz, 3H),1.34 (d, J ¼ 6.0 Hz, 3H), 0.96 (t, J ¼ 7.3 Hz, 3H).

(s, 1H), 8.50 (d, J ¼ 8.1 Hz, 1H), 7.13 (d, J ¼ 7.6 Hz, 1H), 7.04e6.98 (m,

1H), 6.77e6.69 (m, 2H), 5.14e5.06 (m, 1H), 4.05 (s, 2H), 1.27 (d,

J ¼ 6.9 Hz, 3H). ESI-MS: calcd for [MþH]^þm/z 214.1, 216.1,

found:214.1, 216.1.

¹³C NMR (100 MHz, CDCl₃)

d 166.53, 164.90, 164.87, 160.98, 157.76,

4.1.5.8. General procedures for the synthesis of 14a-14e. The syn-

theses of the compounds 14a-14e starting from 6-

aminobenzothiazole-2-thiol (91.1 mg, 0.50 mmol) and 12b-12c or

13b-13d (0.50 mmol) were previously reported in ref. 32 (syntheses

of 13a-g) and the obtained intermediates were directly used for the

following step.

155.49, 149.78, 136.40, 134.59, 133.75, 130.31, 126.93, 121.67, 121.58,

120.64, 116.99, 115.45, 115.16, 108.73, 74.14, 61.77, 48.70, 38.58,

36.15, 20.71, 19.76, 18.70, 14.07, 9.27. ESI-HRMS [MþH]^þcalcd for

C

₃₁H₃₅N₃O₅S₂: 594.2091, found: 594.2089.

4.1.5.9.5. N-(2-((2-(((S)-1-(3-hydroxyphenyl)ethyl)amino)-2-

oxoethyl)thio)benzo[d]

tan-2-yloxy)benzamide (B22). White solid, 25.0% yield, ¹H NMR

(400 MHz, CDCl₃)

thiazol-6-yl)-2-methoxy-3-methyl-4-(pen-

4.1.5.9. General procedures for the synthesis of B18eB26. The syn-

theses of the products B18eB26 from 20a-20d (0.24 mmol) and 3

(68.7 mg, 0.20 mmol) or 14a-14e (0.20 mmol) were similar to the

previously reported method published in ref. 32. The residues were

puriﬁed by ﬂash column chromatography on silica gel (200e300

mesh) using petroleum ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v)

to afford the desired products.

4.1.5.9.1. 2-Methoxy-3-methyl-N-(2-((2-oxo-2-(((S)-1-

phenylethyl)amino)ethyl)thio) benzo[d]thiazol-6-yl)-4-(pentan-2-

yloxy)benzamide (B18). White solid, 50.7% yield, ¹H NMR

d

10.09 (s, 1H), 8.38 (s, 1H), 8.05 (d, J ¼ 8.7 Hz,1H),

7.76 (d, J ¼ 7.3 Hz,1H), 7.66 (d, J ¼ 8.6 Hz,1H), 7.38 (d, J ¼ 8.6 Hz,1H),

7.02 (t, J ¼ 7.8 Hz, 1H), 6.80 (d, J ¼ 8.9 Hz, 1H), 6.72 (d, J ¼ 7.1 Hz,1H),

6.60 (d, J ¼ 12.7 Hz, 2H), 4.96 (quint, 1H), 4.50 (sext, 1H), 3.92 (brd,

J ¼ 14.7, 2H), 3.88 (s, 3H), 2.21 (s, 3H), 1.86e1.74 (m, 1H), 1.63e1.55

(m, 1H),1.55e1.43 (m, 2H), 1.42 (d, J ¼ 6.8 Hz, 3H),1.35 (d, J ¼ 6.0 Hz,

3H), 0.97 (t, J ¼ 7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)

d 165.10,

162.88, 161.86, 158.57, 155.32, 154.22, 146.74, 142.03, 134.17, 133.05,

127.76, 127.24, 119.13, 118.35, 117.86, 115.52, 115.13, 112.32, 111.00,

106.58, 71.93, 59.57, 47.14, 36.30, 33.97, 19.25, 17.57, 16.51, 11.88,

7.06. ESI-HRMS [M þ H]^þcalcd for C₃₁H₃₅N₃O₅S₂: 594.2091, found:

594.2094.

(400 MHz, DMSO‑d₆)

d

10.30 (s, 1H), 8.79 (d, J ¼ 7.9 Hz, 1H), 8.49 (s,

1H), 7.75 (d, J ¼ 8.6 Hz, 1H), 7.66 (d, J ¼ 8.7 Hz, 1H), 7.46 (d,

J ¼ 8.6 Hz, 1H), 7.33e7.16 (m, 5H), 6.87 (d, J ¼ 8.7 Hz, 1H), 4.95e4.85

(m,1H), 4.55e4.47 (m,1H), 4.16 (d, J ¼ 14.9,1H), 4.12 (d, J ¼ 14.9,1H)

3.71 (s, 3H), 2.08 (s, 3H), 1.70e1.61 (m, 1H), 1.59e1.50 (m, 1H),

4.1.5.9.6. N-(2-((2-(((S)-1-(2-hydroxyphenyl)ethyl)amino)-2-

oxoethyl)thio)benzo[d]

tan-2-yloxy)benzamide (B23). White solid, 22.0% yield, ¹H NMR

(400 MHz, CDCl₃)

thiazol-6-yl)-2-methoxy-3-methyl-4-(pen-

1.47e1.27 (m, 5H), 1.23 (d, J ¼ 6.0 Hz, 3H), 0.88 (t, J ¼ 7.3 Hz, 3H). ¹³

C

d

10.13 (s, 1H), 8.77 (s, 1H), 8.60 (d, J ¼ 2.0 Hz, 1H),

NMR (100 MHz, DMSO‑d₆)

d

165.52, 164.85, 164.45, 158.76, 156.74,

8.27 (d, J ¼ 8.0 Hz, 1H), 8.05 (d, J ¼ 8.8 Hz, 1H), 7.78 (d, J ¼ 8.7 Hz,

1H), 7.45 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.21e7.14 (m, 2H), 6.93 (d,

J ¼ 7.7 Hz, 1H), 6.85 (td, J ¼ 7.5, 1.0 Hz, 1H), 6.81 (d, J ¼ 9.0 Hz, 1H),

5.26 (quint,1H), 4.50 (sext,1H), 4.00 (d, J ¼ 15.1 Hz,1H), 3.90 (s, 3H),

3.86 (d, J ¼ 15.0 Hz, 1H), 2.23 (s, 3H), 1.86e1.76 (m, 1H), 1.66e1.58

(m, 1H), 1.56e1.42 (m, 2H),1.54 (d, J ¼ 6.8 Hz, 3H), 1.36 (d, J ¼ 6.1 Hz,

148.73, 144.10, 136.03, 135.33, 128.21, 127.79, 126.68, 125.88, 121.27,

120.92, 119.80, 119.16, 111.82, 108.25, 73.41, 61.48, 48.47, 37.99,

36.60, 22.37, 19.55, 18.07, 13.89, 8.99. ESI-HRMS [MþH]^þcalcd for

C

₃₁H₃₆N₃O₄S₂: 578.2142, found: 578.2144.

4.1.5.9.2. 2-Methoxy-N-(2-((2-(((S)-1-(4-methoxyphenyl)ethyl)

amino)-2-oxoethyl)thio) benzo [d]thiazol-6-yl)-3-methyl-4-(pentan-

3H), 0.98 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃)

d 168.83,

2-yloxy)benzamide (B19). White solid, 30.0% yield, ¹H NMR

164.06, 163.09, 160.14, 156.84, 154.36, 148.01, 136.04, 135.43, 129.51,

128.45, 127.82, 125.20, 120.60, 119.97, 119.65, 118.56, 117.54, 117.00,

111.64, 108.19, 73.52, 61.13, 43.21, 37.98, 35.34, 19.14, 18.63, 18.07,

13.43, 8.61. ESI-HRMS [MþH]^þcalcd for C₃₁H₃₅N₃O₅S₂: 594.2091,

found: 594.2093.

(400 MHz, CDCl₃)

d

10.12 (s, 1H), 8.62 (s, 1H), 8.06 (d, J ¼ 8.9 Hz, 1H),

7.85 (d, J ¼ 6.2 Hz,1H), 7.69 (d, J ¼ 7.7 Hz,1H), 7.42 (d, J ¼ 8.1 Hz,1H),

7.15 (d, J ¼ 7.6 Hz, 2H), 6.82 (d, J ¼ 8.7 Hz, 1H), 6.77 (d, J ¼ 7.5 Hz,

2H), 5.04 (quint, 1H), 4.51 (sext, 1H), 3.92 (brd, J ¼ 14.7, 2H), 3.90 (s,

3H), 3.77 (s, 3H), 2.23 (s, 3H), 1.85e1.72 (m, 1H), 1.58e1.48 (m, 1H),

15

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

4.1.5.9.7. (S)-4-isobutoxy-2-methoxy-N-(2-((2-((1-(4-

methoxyphenyl)ethyl)amino)-2- oxoethyl)thio) benzo [d]thiazol-6-

yl)-3-methylbenzamide (B24). White solid, 70.4% yield, ¹H NMR

was extracted with ethyl acetate (100 mL) twice. The combined

organic layers were washed with brine and the organic phase was

dried with sodium sulfate and concentrated in vacuo. Finally, the

crude compound was puriﬁed by silica gel column chromatography

(PE/EA ¼ 10/1, v/v) to give 23 as a white solid (3.3 g, 98.9% yield). ¹H

(400 MHz, DMSO‑d₆)

d

10.31 (s, 1H), 8.69 (d, J ¼ 8.0 Hz, 1H), 8.52 (s,

1H), 7.76 (d, J ¼ 8.8 Hz, 1H), 7.68 (d, J ¼ 8.7 Hz, 1H), 7.50 (d,

J ¼ 8.5 Hz, 1H), 7.22 (d, J ¼ 8.4 Hz, 2H), 6.86 (d, J ¼ 8.8 Hz, 1H), 6.82

(d, J ¼ 8.6 Hz, 2H), 4.87 (quint, 1H), 4.13 (brd, J ¼ 14.8 Hz, 2H), 3.81

(d, J ¼ 6.2 Hz, 2H), 3.74 (s, 3H), 3.70 (s, 3H), 2.14 (s, 3H), 2.10e2.01

(m, 1H), 1.34 (d, J ¼ 6.9 Hz, 3H), 1.00 (d, J ¼ 6.6 Hz, 6H). ¹³C NMR

NMR (400 MHz, DMSO‑d₆)

d

10.08 (s, 1H), 7.61 (d, J ¼ 8.7 Hz, 1H),

7.44 (d, J ¼ 7.4 Hz, 2H), 7.38 (t, J ¼ 7.4 Hz, 2H), 7.31 (t, J ¼ 7.2 Hz, 1H),

7.02 (d, J ¼ 8.7 Hz, 1H), 5.21 (s, 2H), 3.78 (s, 3H), 2.11 (s, 3H). ESI-MS:

calcd for [MþH]^þm/z 257.1, found:257.1.

(150 MHz, CDCl₃)

d 166.51, 164.45, 162.99, 160.86, 158.09, 156.59,

148.13, 136.02, 135.33, 134.44, 129.69, 126.50, 120.63, 119.29, 118.33,

117.41, 113.29, 111.51, 107.10, 74.16, 61.18, 54.61, 48.17, 35.75, 27.73,

21.17, 18.62, 8.41. ESI-HRMS [MþH]^þcalcd for C₃₁H₃₅N₃O₅S₂:

594.2091, found: 594.2091.

4.1.5.9.8. (S)-4-isopropoxy-2-methoxy-N-(2-((2-((1-(4-

methoxyphenyl)ethyl)amino)- 2-oxoethyl)thio) benzo[d]thiazol-6-

yl)-3-methylbenzamide (B25). White solid, 68.5% yield, ¹H NMR

4.1.6.3. 4-(benzyloxy)-2-methoxy-3-methylbenzoic

acid

(24).

To a solution of 23 (3.0 g, 11.7 mmol) in t-BuOH (30 mL) and 2-

methyl-2-butene (10 mL) were added NaClO₂(1.59 g,

17.55 mmol) and NaH₂PO₄$2H₂O (4.2 g, 35.1 mmol) buffer solution

(pH ¼ 4.5, 35 mL) at 0 ^ꢀC. Then the mixture was stirred at room

temperature for about 1 h. The solution was extracted with ethyl

acetate (100 mL ꢂ 3) and the organic layer was dried with sodium

sulfate and concentrated in vacuo. The crude compound was puri-

ﬁed by silica gel column chromatography (DCM/CH₃OH ¼ 10/1, v/v)

(400 MHz, DMSO‑d₆)

d

10.31 (s, 1H), 8.69 (d, J ¼ 7.9 Hz, 1H), 8.52 (s,

1H), 7.75 (d, J ¼ 8.8 Hz, 1H), 7.67 (d, J ¼ 8.8 Hz, 1H), 7.47 (d,

J ¼ 8.4 Hz, 1H), 7.21 (d, J ¼ 8.4 Hz, 2H), 6.88 (d, J ¼ 8.8 Hz, 1H), 6.81

(d, J ¼ 8.4 Hz, 2H), 4.86 (quint, 1H), 4.71e4.58 (m, 1H), 4.14 (brd,

J ¼ 14.8 Hz, 2H), 3.70 (d, J ¼ 12.2 Hz, 6H), 2.09 (s, 3H), 1.33 (d,

J ¼ 6.9 Hz, 3H), 1.28 (d, J ¼ 5.9 Hz, 6H). ¹³C NMR (150 MHz, CDCl₃)

to give 24 (3.18 g, 100% yield). ¹H NMR (400 MHz, DMSO‑d₆)

d 12.04

(s, 1H), 7.60 (d, J ¼ 8.7 Hz, 1H), 7.44 (d, J ¼ 7.4 Hz, 2H), 7.38 (t,

J ¼ 7.5 Hz, 2H), 7.31 (t, J ¼ 7.0 Hz, 1H), 6.88 (d, J ¼ 8.7 Hz, 1H), 5.15 (s,

2H), 3.67 (s, 3H), 2.08 (s, 3H). ESI-MS: calcd for [M ꢁ H]^-m/z 271.3,

found: 271.3.

d

166.53, 164.43, 163.01, 159.86, 158.09, 156.84, 148.12, 136.02,

135.34, 134.44, 129.47, 126.50, 120.63, 120.02, 118.32, 117.18, 113.29,

111.51, 108.35, 69.96, 61.11, 54.61, 48.17, 35.75, 21.49, 21.17, 8.61. ESI-

HRMS [MþH]^þcalcd for C₃₀H₃₃N₃O₅S₂: 580.1934, found: 580.1937.

4.1.5.9.9. (S)-2,4-dimethoxy-N-(2-((2-((1-(4-methoxyphenyl)

ethyl)amino)-2-oxoethyl) thio)benzo[d]thiazol-6-yl)-3-

methylbenzamide (B26). White solid, 65.0% yield, ¹H NMR

4.1.6.4. 4-Hydroxy-2-methoxy-3-methylbenzoic acid (25). A solu-

tion of compound 24 (3.0 g, 11.03 mmol) in methanol (30 mL) was

hydrogenated in the presence of 10% Pd/C (300 mg) and H₂(balloon

pressure) at room temperature for 1 h. The mixture was then

ﬁltered through a Celite pad and the ﬁltrate was concentrated in

vacuo to give 25, which was used directly for the following syn-

thesis. ESI-MS: calcd for [M ꢁ H]^-m/z 181.1, found: 181.2.

(400 MHz, DMSO‑d₆)

d

10.30 (s, 1H), 8.67 (d, J ¼ 7.4 Hz, 1H), 8.49 (s,

1H), 7.73 (d, J ¼ 8.8 Hz,1H), 7.65 (d, J ¼ 7.9 Hz,1H), 7.49 (d, J ¼ 8.4 Hz,

1H), 7.19 (d, J ¼ 8.2 Hz, 2H), 6.86 (d, J ¼ 8.6 Hz, 1H), 6.79 (d,

J ¼ 8.3 Hz, 2H), 4.84 (quint, 1H), 4.12 (brd, J ¼ 14.8 Hz, 2H), 3.81 (s,

4.1.6.5. Methyl

4-hydroxy-2-methoxy-3-methylbenzoate

(26).

3H), 3.70 (s, 3H), 3.67 (s, 3H), 2.08 (s, 3H), 1.31 (d, J ¼ 6.7 Hz, 3H). ¹³

C

SOCl₂(796 L, 10.98 mmol) was slowly added to a solution of 26

m

NMR (150 MHz, CDCl₃)

d

166.52, 164.49, 162.94, 161.28, 158.09,

(2.0 g, 10.98 mmol) in dry methanol (25 mL) and the solution was

stirred at rt for 5 h. After removing the solvent under reduced

pressure, the residue was dissolved in ethyl acetate (50 mL),

washed with water and brine. Then the organic phase was dried

with sodium sulfate and concentrated in vacuo. The crude com-

pound was puriﬁed by silica gel column chromatography (PE/

EA ¼ 6/1, v/v) to give 26 as a white solid（2.1 g，98.0% yield）. ¹H

156.56, 148.15, 136.02, 135.28, 134.43, 129.75, 126.50, 120.64, 119.23,

118.33, 117.74, 113.29, 111.53, 106.31, 61.20, 55.23, 54.62, 48.17,

35.75, 21.17, 8.40. ESI-HRMS [MþH]^þcalcd for C₂₈H₂₉N₃O₅S₂:

552.1621, found: 552.1617.

4.1.6. General procedures for the synthesis of B27eB30

4.1.6.1. 4-(benzyloxy)-2-hydroxy-3-methylbenzaldehyde

(22).

NMR (400 MHz, DMSO‑d₆)

d

10.24 (s, 1H), 7.47 (d, J ¼ 8.6 Hz, 1H),

To a solution of 21 (3.0 g, 19.72 mmol) in acetonitrile were added

benzyl bromide (2.58 mL, 21.69 mmol), KI (327 mg, 1.972 mmol)

and potassium carbonate (6.0 g, 43.38 mmol). The mixture was

stirred at reﬂux for about 6 h and the completion of the reaction

was monitored by TLC and LC-MS. After cooling to room temper-

ature, the solvent was removed under reduced pressure. The res-

idue was dissolved in ethyl acetate (100 mL), and washed with

water and brine. Then the organic phase dried with sodium sulfate

and concentrated in vacuo. The crude compound was puriﬁed by

silica gel column chromatography (PE/EA ¼ 15/1, v/v) to give 22 as a

white solid (4.0 g，88.2% yield). ¹H NMR (400 MHz, DMSO‑d₆)

6.62 (d, J ¼ 8.6 Hz,1H), 3.72 (s, 3H), 3.65 (s, 3H), 2.00 (s, 3H). ESI-MS:

calcd for [MþH]^þm/z 197.1, found:197.1.

4.1.6.6. General procedures for the synthesis of 27a-27d. To a solu-

tion of 26 (196 mg, 1.0 mmol) in anhydrous DMF were added

different alkyl halides (278 mg, 2.0 mmol, 1-bromo-2-

methoxyethane for 27a, 340 mg, 2.0 mmol, 1-(2-chloroethyl)pyr-

rolidine hydrochloride for 27b, 288 mg, 2.0 mmol, 2-chloro-N,N-

dimethylethan-1-amine hydrochloride for 27c, 372 mg, 2.0 mmol,

4-(2-chloroethyl)morpholine hydrochloride for 27d), KI (16.6 mg,

0.1 mmol) and potassium carbonate (414 mg, 3.0 mmol). The

mixture was stirred at 100 ^ꢀC for about 2 h and the completion of

the reaction was monitored by TLC and LC-MS. After cooling to

room temperature, water was added. The mixture was extracted

with ethyl acetate (20 mL) twice. The combined organic layers were

washed with brine and the organic phase was dried with sodium

sulfate and concentrated in vacuo. The crude compound was puri-

ﬁed by silica gel column chromatography (PE/EA ¼ 10/1, v/v) to give

27a-27d.

d

11.35 (s, 1H), 9.78 (s, 1H), 7.59 (d, J ¼ 8.7 Hz, 1H), 7.43 (d, J ¼ 7.5 Hz,

2H), 7.38 (t, J ¼ 7.4 Hz, 2H), 7.31 (t, J ¼ 7.2 Hz, 1H), 6.82 (d, J ¼ 8.7 Hz,

1H), 5.23 (s, 2H), 2.02 (s, 3H). ESI-MS: calcd for [MþH]^þm/z 231.1,

found:231.1.

4.1.6.2. 4-(benzyloxy)-2-methoxy-3-methylbenzaldehyde

(23).

To a solution of 22 (3.0 g, 13.03 mmol) in anhydrous DMF were

added CH₃I (3.25 mL, 52.12 mmol) and potassium carbonate (3.96 g,

28.67 mmol). The mixture was stirred at 40 ^ꢀC for about 3 h and the

completion of the reaction was monitored by TLC and LC-MS. After

cooling to room temperature, water was added and the mixture

4.1.6.6.1. Methyl 2-methoxy-4-(2-methoxyethoxy)-3-

methylbenzoate (27a). Brown oil, 93.5% yield, ¹H NMR (400 MHz,

DMSO‑d₆)

d

7.59 (d, J ¼ 8.8 Hz,1H), 6.81 (d, J ¼ 8.8 Hz,1H), 4.14e4.10

16

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

(m, 2H), 3.75 (s, 3H), 3.68e3.63 (m, 5H), 3.29 (s, 3H), 2.04 (s, 3H).

ESI-MS: calcd for [MþH]^þm/z 255.1, found:255.2.

4.1.6.10.2. (S)-2-methoxy-N-(2-((2-((1-(4-methoxyphenyl)ethyl)

amino)-2-oxoethyl)thio) benzo[d]thiazol-6-yl)-3-methyl-4-(2-(pyr-

rolidin-1-yl)ethoxy)benzamide (B28). White solid, 35.0% yield, ¹H

4.1.6.6.2. Methyl

2-methoxy-3-methyl-4-(2-(pyrrolidin-1-yl)

ethoxy)benzoate (27b). Brown oil, 41.9% yield, ¹H NMR (400 MHz,

CDCl₃)

NMR (400 MHz, CDCl₃)

d

9.94 (s, 1H), 8.49 (d, J ¼ 1.7 Hz, 1H), 7.98 (d,

d

7.72 (d, J ¼ 8.7 Hz, 1H), 6.63 (d, J ¼ 8.7 Hz, 1H), 4.15 (t,

J ¼ 8.8 Hz, 1H), 7.72 (d, J ¼ 7.6 Hz, 1H), 7.60 (d, J ¼ 8.7 Hz, 1H), 7.33

(dd, J ¼ 8.7, 1.9 Hz, 1H), 7.06 (d, J ¼ 8.6 Hz, 2H), 6.74 (d, J ¼ 8.9 Hz,

1H), 6.67 (d, J ¼ 8.6 Hz, 2H), 4.99e4.90 (m, 1H), 4.34 (t, J ¼ 5.0 Hz,

2H), 3.85 (brd, J ¼ 14.7, 2H), 3.80 (s, 3H), 3.67 (s, 3H), 3.24 (t,

J ¼ 5.0 Hz, 2H), 3.08e3.02 (m, 4H), 2.16 (s, 3H), 2.00e1.92 (m, 4H),

J ¼ 5.9 Hz, 2H), 3.87 (s, 3H), 3.79 (s, 3H), 2.94 (t, J ¼ 5.9 Hz, 2H),

2.70e2.62 (m, 4H), 2.15 (s, 3H), 1.84e1.76 (m, 4H). ESI-MS: calcd for

[MþH]^þm/z 294.2, found:294.2.

4.1.6.6.3. Methyl 4-(2-(dimethylamino)ethoxy)-2-methoxy-3-

methylbenzoate (27c). Brown oil, 84.3% yield, ¹H NMR (400 MHz,

1.34 (d, J ¼ 6.8 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)

d 167.17, 165.20,

DMSO‑d₆)

d

7.59 (d, J ¼ 8.8 Hz, 1H), 6.81 (d, J ¼ 8.8 Hz, 1H), 4.07 (t,

163.34, 160.21, 158.70, 157.28, 148.83, 136.64, 135.78, 135.05, 130.54,

127.13, 121.27, 119.84, 119.17, 118.98, 113.91, 112.21, 107.82, 65.67,

61.93, 55.26, 54.60, 54.24, 48.81, 36.37, 23.40, 21.82, 9.32. ESI-HRMS

[MþH]^þcalcd for C₃₃H₃₈N₄O₅S₂: 635.2356, found: 635.2358.

J ¼ 5.7 Hz, 2H), 3.75 (s, 3H), 3.66 (s, 3H), 2.63 (t, J ¼ 5.6 Hz, 2H), 2.20

(s, 6H), 2.03 (s, 3H). ESI-MS: calcd for [MþH]^þm/z 268.2,

found:268.1.

4.1.6.6.4. Methyl 2-methoxy-3-methyl-4-(2-morpholinoethoxy)

benzoate (27d). Brown oil, 90.5% yield, ¹H NMR (400 MHz,

4.1.6.10.3. (S)-4-(2-(dimethylamino)ethoxy)-2-methoxy-N-(2-((2-

((1-(4-methoxyphenyl) ethyl)amino)-2-oxoethyl)thio)benzo[d]thia-

zol-6-yl)-3-methylbenzamide (B29). White solid, 32.0% yield, ¹H

DMSO‑d₆)

d

7.60 (d, J ¼ 8.7 Hz, 1H), 6.83 (d, J ¼ 8.8 Hz, 1H), 4.12 (t,

J ¼ 5.7 Hz, 2H), 3.75 (s, 3H), 3.66 (s, 3H), 3.57e3.51 (m, 4H), 2.70 (t,

J ¼ 5.7 Hz, 2H), 2.03 (s, 3H). ESI-MS: calcd for [MþH]^þm/z 310.1,

found:310.2.

NMR (400 MHz, DMSO‑d₆)

d

10.34 (s, 1H), 8.72 (d, J ¼ 8.0 Hz, 1H),

8.52 (s, 1H), 7.78 (d, J ¼ 8.8 Hz, 1H), 7.69 (dd, J ¼ 8.8, 1.5 Hz, 1H), 7.52

(d, J ¼ 8.6 Hz, 1H), 7.23 (d, J ¼ 8.6 Hz, 2H), 6.91 (d, J ¼ 8.7 Hz, 1H),

6.83 (d, J ¼ 8.6 Hz, 2H), 4.92e4.82 (m, 1H), 4.20e4.07 (m, 4H), 3.74

(s, 3H), 3.71 (s, 3H), 2.72 (t, J ¼ 5.5 Hz, 2H), 2.27 (s, 6H), 2.13 (s, 3H),

4.1.6.7. 2-Methoxy-4-(2-methoxyethoxy)-3-methylbenzoic

acid

(28a). Compound 28a was prepared in a similar manner as

described for compound 9c, starting from 27a (139 mg,

0.545 mmol). The residue was puriﬁed by ﬂash column chroma-

tography on silica gel (200e300 mesh) using dichloromethane/

methanol eluent (DCM/CH₃OH ¼ 10/1, v/v) to afford the desired

product as white solid (131 mg, 100.0% yield). ¹H NMR (400 MHz,

1.35 (d, J ¼ 6.9 Hz, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)

d 165.86,

165.29, 164.95, 159.88, 158.50, 157.06, 149.27, 136.53, 135.86, 128.42,

127.58, 122.26, 121.46, 119.68, 114.03, 112.36, 107.71, 67.08, 62.02,

57.96, 55.49, 48.34, 45.96, 37.18, 22.81, 9.40. ESI-HRMS [MþH]^þ

calcd for C₃₁H₃₆N₄O₅S₂: 609.2200, found: 609.2195.

DMSO‑d₆)

d

7.59 (d, J ¼ 8.7 Hz, 1H), 6.79 (d, J ¼ 8.7 Hz, 1H),

4.1.6.10.4. (S)-2-methoxy-N-(2-((2-((1-(4-methoxyphenyl)ethyl)

amino)-2-oxoethyl) thio)benzo[d]thiazol-6-yl)-3-methyl-4-(2-

morpholinoethoxy)benzamide (B30). White solid, 30.0% yield, ¹H

4.15e4.07 (m, 2H), 3.67e3.63 (m, 5H), 3.29 (s, 3H), 2.04 (s, 3H). ESI-

MS: calcd for [M ꢁ H]^-m/z 239.1, found:239.1.

NMR (400 MHz, CDCl₃)

d

9.93 (s, 1H), 8.49 (d, J ¼ 2.0 Hz,1H), 7.99 (d,

4.1.6.8. General procedures for the synthesis of 28b-28d. 6 M HCl

aqueous solution (5 mL) was added to 27b-27d (0.545 mmol) and

the solution was stirred at 80 ^ꢀC for about 6 h. After solvent evap-

oration under reduced pressure, the residue was used for the

following synthesis.

J ¼ 8.8 Hz, 1H), 7.71 (d, J ¼ 7.6 Hz, 1H), 7.60 (d, J ¼ 8.7 Hz, 1H), 7.33

(dd, J ¼ 8.7, 2.1 Hz, 1H), 7.06 (d, J ¼ 8.6 Hz, 2H), 6.75 (d, J ¼ 8.9 Hz,

1H), 6.67 (d, J ¼ 8.7 Hz, 2H), 4.99e4.90 (m, 1H), 4.37 (t, J ¼ 4.5 Hz,

2H), 3.92e3.82 (m, 6H), 3.81 (s, 3H), 3.67 (s, 3H), 3.14 (t, J ¼ 4.5 Hz,

2H), 2.99e2.87 (m, 4H), 2.15 (s, 3H), 1.35 (d, J ¼ 6.9 Hz, 3H). ¹³C NMR

(100 MHz, CDCl₃)

d 167.19, 165.26, 163.29, 158.71, 157.31, 148.86,

4.1.6.9. General procedures for the synthesis of 29a-29d.

Compounds 29a-29d were prepared in a similar manner as

described for compounds 20a-20d, starting from 28a-28d

(0.50 mmol).

136.65, 135.73, 135.04, 130.58, 127.14, 121.29, 119.85, 118.98, 113.91,

112.23, 107.84, 65.24, 61.95, 57.09, 55.26, 53.40, 48.82, 36.36, 21.81,

9.31. ESI-HRMS [MþH]^þcalcd for C₃₃H₃₈N₄O₆S₂: 651.2306, found:

651.2315.

4.1.6.10. General procedures for the synthesis of B27eB30. The

syntheses of the products B27eB30 from 29a-29d (0.24 mmol) and

14a (74.7 mg, 0.20 mmol) were similar to the previously reported

method published in ref. 32. The residues were puriﬁed by ﬂash

column chromatography on silica gel (200e300 mesh) using

dichloromethane/methanol eluent (DCM/CH₃OH ¼ 10/1, v/v) to

afford the desired products.

4.1.7. General procedures for the synthesis of B31

4.1.7.1. Tert-butyl (S)-(2-((1-(4-methoxyphenyl)ethyl)amino)-2-

oxoethyl)carbamate (31). To a solution of (tert-butoxycarbonyl)

glycine (1.0 g, 5.7 mmol) in anhydrous THF (30 mL) were added

HATU (2.17 g, 5.7 mmol) and HOAT (776 mg, 5.7 mmol) at 0 ^ꢀC for

15 min. Then (S)-1-(4-methoxyphenyl)ethan-1-amine (860 mg,

5.7 mmol) and DIPEA (3.0 mL, 17.1 mmol) were added. The mixture

was stirred at room temperature for about 8 h and the completion

of the reaction was monitored by TLC. Then the aqueous layer was

extracted with ethyl acetate (100 mL) twice, and the combined

organic layers were washed with brine, dried over sodium sulfate,

and concentrated under reduced pressure. The residue was puriﬁed

by column chromatography on silica gel (PE/EA ¼ 10/1, v/v) to

afford intermediate 31 (colorless oil, 92.8% yield). ¹H NMR

4.1.6.10.1. (S)-2-methoxy-4-(2-methoxyethoxy)-N-(2-((2-((1-(4-

methoxyphenyl)ethyl) amino)-2-oxoethyl)thio)benzo[d]thiazol-6-yl)-

3-methylbenzamide (B27). White solid, 35.0% yield, ¹H NMR

(400 MHz, DMSO‑d₆)

d

10.33 (s, 1H), 8.69 (d, J ¼ 8.0 Hz, 1H), 8.53 (d,

J ¼ 1.8 Hz, 1H), 7.77 (d, J ¼ 8.8 Hz, 1H), 7.69 (dd, J ¼ 8.9, 2.0 Hz, 1H),

7.51 (d, J ¼ 8.6 Hz, 1H), 7.23 (d, J ¼ 8.6 Hz, 2H), 6.90 (d, J ¼ 8.7 Hz,

1H), 6.83 (d, J ¼ 8.7 Hz, 2H), 4.93e4.83 (m, 1H), 4.20e4.13 (m, 4H),

3.75 (s, 3H), 3.73e3.69 (m, 5H), 3.34 (s, 3H), 2.14 (s, 3H), 1.35 (d,

(600 MHz, CDCl₃)

d

7.22 (d, J ¼ 8.6 Hz, 2H), 6.85 (d, J ¼ 8.7 Hz, 2H),

6.58 (s, 1H), 5.33 (s, 1H), 5.12e5.00 (m, 1H), 3.78 (s, 3H), 3.75 (d,

J ¼ 4.2 Hz, 2H), 1.46 (d, J ¼ 6.9 Hz, 3H), 1.43 (s, 9H). ESI-MS: calcd for

[M þ Na]^þm/z 331.2, found:331.0.

J ¼ 7.0 Hz, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)

d 165.77, 165.27,

164.91, 159.88, 158.53, 157.08, 149.27, 136.60, 136.55, 135.86, 128.34,

127.59, 122.52, 121.44, 119.77, 119.65, 114.05, 112.32, 107.88, 70.86,

68.35, 62.01, 58.85, 55.51, 48.31, 37.23, 22.84, 9.41. ESI-HRMS

[MþH]^þcalcd for C₃₀H₃₃N₃O₆S₂: 596.1884, found: 596.1887.

4.1.7.2. (S)-2-amino-N-(1-(4-methoxyphenyl)ethyl)acetamide (32).

To a solution of 31 (1.0 g, 3.25 mmol) in anhydrous CH₂Cl₂(15 mL)

17

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

was added triﬂuoroacetic acid (1.5 mL) at 0 ^ꢀC and stirred for 3 h.

After removal of the solvents under reduced pressure, saturated

sodium bicarbonate solution and CH₂Cl₂were added. The organic

layer was washed with brine, dried with sodium sulfate and

concentrated in vacuo to give the crude product, which was used

directly for the next step. ESI-MS: calcd for [M þ H]^þm/z 209.1,

found:209.2.

2.2 Hz, 1H), 6.80 (d, J ¼ 8.8 Hz, 2H), 3.90 (s, 3H), 3.86 (s, 3H), 2.21 (s,

3H). ESI-MS: calcd for [MþH]^þm/z 363.0, found:363.1.

4.1.8.2. N-(2-chlorobenzo[d]thiazol-6-yl)-2,4-dimethoxy-3-

methylbenzamide (37). To a solution of 36 (363 mg, 1.0 mmol) in

anhydrous THF were added ethyl 2-hydroxyacetate (257 mg,

1.2 mmol) and NaH (60 mg, 1.5 mmol, 60% dispersion in mineral

oil). The mixture was stirred at reﬂux for about 2 h and the

completion of the reaction was monitored by TLC. After adding

water (2 mL), the mixture was extracted with ethyl acetate

(10 mL ꢂ 3). Then the organic phase was dried with sodium sulfate

and concentrated in vacuo to give the crude product, which was

puriﬁed by silica gel column chromatography (PE/EA ¼ 6/1, v/v) to

obtain compound 37 as colorless oil (278 mg, 64.6% yield). ¹H NMR

4.1.7.3. (S)eN-(1-(4-methoxyphenyl)ethyl)-2-((6-nitrobenzo[d]thia-

zol-2-yl)amino) acetamide (33). To

nitrobenzo [d]thiazole (114 mg, 0.53 mmol) in n-butyl alcohol

L,

a solution of 2-chloro-6-

(5 mL) were added 32 (110 mg, 0.53 mmol) and Et₃N (242

m

1.75 mmol) at 0 ^ꢀC. The mixture was stirred at reﬂux for about 3 h

and the completion of the reaction was monitored by TLC. Reducing

temperature led to a precipitation of compound 33, which was

ﬁltered and dried (yellow solid, 73.5% yield). ¹H NMR (600 MHz,

(400 MHz, CDCl₃)

d

9.98 (s, 1H), 8.44 (d, J ¼ 2.1 Hz, 1H), 8.07 (d,

J ¼ 8.8 Hz, 1H), 7.62 (d, J ¼ 8.6 Hz, 1H), 7.35 (dd, J ¼ 8.7, 2.2 Hz, 1H),

6.81 (d, J ¼ 8.9 Hz, 1H), 5.08 (s, 2H), 4.29 (q, J ¼ 7.1 Hz, 2H), 3.90 (s,

3H), 3.86 (s, 3H), 2.21 (s, 3H), 1.30 (t, J ¼ 7.1 Hz, 3H). ESI-MS: calcd

for [M þ H]^þm/z 431.1, found: 431.1.

DMSO‑d₆)

d

8.97 (t, J ¼ 5.5 Hz, 1H), 8.72 (d, J ¼ 2.3 Hz, 1H), 8.51 (d,

J ¼ 8.0 Hz, 1H), 8.13 (dd, J ¼ 8.9, 2.4 Hz, 1H), 7.47 (d, J ¼ 8.9 Hz, 1H),

7.26 (d, J ¼ 8.6 Hz, 2H), 6.87 (d, J ¼ 8.6 Hz, 2H), 4.96e4.88 (m, 1H),

4.14 (d, J ¼ 5.1 Hz, 2H), 3.72 (s, 3H), 1.36 (d, J ¼ 6.9 Hz, 3H). ESI-MS:

calcd for [M þ H]^þm/z 387.1, found:387.2.

4.1.8.3. 2-((6-(2,4-dimethoxy-3-methylbenzamido)benzo[d]thiazol-

2-yl)oxy)acetic acid (38). The compound 37 (270 mg, 0.63 mmol)

was stirred in NaOH (1 M) water solution (5 mL) and THF (5 mL) for

5 min. Then the THF was removed under vacuum, and the residue

was acidiﬁed to pH ¼ 2 or below with HCl (1 M). Then the solution

was extracted with ethyl acetate (20 mL ꢂ 3) and the combined

organic solvents were dried with sodium sulfate and concentrated

in vacuo. The crude product was used directly for next step. ESI-MS:

calcd for [M ꢁ H]^-m/z 401.1, found:401.1.

4.1.7.4. (S)-2-((6-aminobenzo[d]thiazol-2-yl)amino)-N-(1-(4-

methoxyphenyl)ethyl) acetamide (34). To a solution of 33 (150 mg,

0.39 mmol) in CH₃CH₂OH/H₂O (10 mL/5 mL) were added Fe powder

(109 mg, 1.95 mmol) and NH₄Cl (104 mg, 1.95 mmol) at room

temperature. The mixture was stirred at reﬂux for about 3 h and the

completion of the reaction was monitored by TLC. After cooling and

ﬁltration on Celite, the mixture was extracted with ethyl acetate

(20 mL ꢂ 3) and the combined organic solvents were washed with

water. The organic layer was dried with sodium sulfate and

concentrated in vacuo to give the crude product, which was used

directly for the next step. ESI-MS: calcd for [MþH]^þm/z 357.1,

found:357.2.

4.1.8.4. (S)-2,4-dimethoxy-N-(2-(2-((1-(4-methoxyphenyl)ethyl)

amino)-2-oxoethoxy) benzo[d]thiazol-6-yl)-3-methylbenzamide

(B32). The synthesis of the product B32 from 11b (30.2 mg,

0.20 mmol) and 38 (80.5 mg, 0.20 mmol) was similar to the pre-

viously reported method published in ref. 32. The residue was

puriﬁed by ﬂash column chromatography on silica gel (200e300

mesh) using petroleum ether/ethyl acetate eluent (PE/EA ¼ 3/1, v/v)

to afford the desired product as white solid (93.1 mg, 87.0% yield).

4.1.7.5. (S)-2,4-dimethoxy-N-(2-((2-((1-(4-methoxyphenyl)ethyl)

a m i n o ) - 2 - o x o e t hyl ) a m i n o ) b e n z o [ d ] t h i a z o l - 6 - yl ) - 3 -

methylbenzamide (B31). The synthesis of the product B31 from

20d (51.5 mg, 0.24 mmol) and 34 (71.3 mg, 0.20 mmol) was similar

to the previously reported method published in ref. 32. The residue

was puriﬁed by ﬂash column chromatography on silica gel

(200e300 mesh) using petroleum ether/ethyl acetate eluent (PE/

EA ¼ 3/1, v/v) to afford the desired product as white solid (80.2 mg,

¹H NMR (400 MHz, DMSO‑d₆)

d

10.26 (s, 1H), 8.63 (d, J ¼ 8.1 Hz, 1H),

8.41 (s, 1H), 7.66e7.58 (m, 2H), 7.53 (d, J ¼ 8.6 Hz, 1H), 7.26 (d,

J ¼ 8.6 Hz, 2H), 6.90 (d, J ¼ 8.7 Hz, 1H), 6.86 (d, J ¼ 8.7 Hz, 2H),

5.05e4.86 (m, 3H), 3.85 (s, 3H), 3.74 (s, 3H), 3.72 (s, 3H), 2.12 (s, 3H),

75.0% yield). ¹H NMR (400 MHz, DMSO‑d₆)

d

10.10 (s, 1H), 8.43 (d,

1.37 (d, J ¼ 7.0 Hz, 3H). ¹³C NMR (150 MHz, DMSO‑d₆)

d 170.73,

J ¼ 8.2 Hz, 1H), 8.19 (dd, J ¼ 11.1, 3.8 Hz, 2H), 7.52 (d, J ¼ 8.6 Hz, 1H),

7.48 (dd, J ¼ 8.7, 1.9 Hz, 1H), 7.34 (d, J ¼ 8.6 Hz, 1H), 7.26 (d,

J ¼ 8.6 Hz, 2H), 6.89 (d, J ¼ 8.7 Hz, 1H), 6.85 (d, J ¼ 8.7 Hz, 2H),

4.96e4.86 (m, 1H), 4.03 (d, J ¼ 5.8 Hz, 2H), 3.85 (s, 3H), 3.74 (s, 3H),

3.72 (s, 3H), 2.12 (s, 3H), 1.35 (d, J ¼ 7.0 Hz, 3H). ¹³C NMR (150 MHz,

165.00, 164.49, 159.93, 157.88, 156.31, 144.34, 135.97, 135.28, 131.82,

127.77, 127.00, 121.82, 120.33, 118.77, 113.40, 112.52, 106.17, 68.75,

61.38, 55.73, 54.88, 46.96, 22.06, 8.70. ESI-HRMS [MþH]^þcalcd for

C

₂₈H₂₉N₃O₆S: 536.1850, found: 536.1846.

DMSO‑d₆)

d 167.58, 165.40, 164.10, 159.82, 157.82, 156.27, 148.32,

4.2. Biological evaluation

136.24, 132.97, 130.73, 127.77, 126.98, 121.86, 118.70, 118.11, 117.62,

113.38, 112.08, 106.15, 61.34, 55.71, 54.88, 47.10, 46.45, 22.29, 8.71.

ESI-HRMS [MþH]^þcalcd for C₂₈H₃₀N₄O₅S: 535.2010, found:

535.2010.

4.2.1. Luciferase report assay

4.2.1.1. Cell lines culture. HepG2 cells were stably transfected with

corresponding STAT-responsive ﬁreﬂy luciferase reporter plasmid

and were cultured in a-MEM supplemented with 10% FBS, penicillin

4.1.8. General procedures for the synthesis of B32

(100 U/mL), and streptomycin (100

stably transfected with NF- B-responsive ﬁreﬂy luciferase reporter

plasmid and were cultured in DMEM supplemented with 10% FBS,

mg/mL). HEK-293 cells were

4.1.8.1. N-(2-chlorobenzo[d]thiazol-6-yl)-2,4-dimethoxy-3-

methylbenzamide (36). Compounds 36 was prepared in a similar

manner as described for compound B1eB14, starting from 2-

chlorobenzo [d]thiazol-6-amine (35, 923 mg, 5.0 mmol) and 20d

(1.29 g, 6.0 mmol). The residue was puriﬁed by ﬂash column

chromatography on silica gel (200e300 mesh) using petroleum

ether/ethyl acetate eluent (PE/EA ¼ 10/1, v/v) to afford the desired

k

penicillin (100 U/mL), and streptomycin (100 mg/mL). All the cell

lines were incubated at 37 ^ꢀC in a humidiﬁed atmosphere con-

taining 5% CO₂.

product (1.27 g, 70.0% yield). ¹H NMR (400 MHz, CDCl₃)

d

10.12 (s,

4.2.1.2. Luciferase report assay. Procedure was similar to the pub-

1H), 8.68 (d, J ¼ 2.1 Hz, 1H), 8.06 (d, J ¼ 8.8 Hz, 1H), 7.36 (dd, J ¼ 8.8,

lished method in ref.32.

18

D. Gao, N. Jin, Y. Fu et al.

European Journal of Medicinal Chemistry 216 (2021) 113333

4.2.2. Cell viability assays

afﬁnity (equilibrium constants, K_D). The K_D(equilibrium constant)

values were calculated as k_d(dissociation rate constant)/k_a(asso-

ciated rate constant) for each interaction and determined by

globally ﬁtting with Biacore T200 evaluation software 2.0 (GE

Healthcare).

4.2.2.1. Cell lines culture. MDA-MB-468 cell lines were cultured in

DMEM/F-12 supplemented with 10% (v/v) fetal bovine serum,

50 mg/mL penicillin/streptomycin. HEL cell lines were maintained

in high glucose DMEM replenished with 10% fetal bovine serum

and 50 mg/mL penicillin/streptomycin. All the cell lines were

incubated at 37 ^ꢀC in a humidiﬁed atmosphere containing 5% CO₂.

4.2.8. Fluorescence polarization assay

Procedure was similar to the published method in ref. 48. Before

adding tested compounds, the 5-FAM-GpYLPQTV-NH2 probe was

incubated with STAT3 SH2 domain protein in assay buffer (Hepes

10 mM，pH ¼ 7.5，EDTA 1 mM，NaCl 50 mM，0.1% Nonidet P40)

for 30 min at 37 ^ꢀC. Then, competition experiments were per-

formed using different concentrations of compounds. The binding

rate was calculated with the following formula: binding rate (%) ¼

(maximum value - drug treatment value)/(maximum value - min-

imum value) ꢂ 100%.

4.2.2.2. MTT assay. Procedure was similar to published methods in

ref. [49].

4.2.3. Western blotting

Cells were collected and lysed using pre-heated 2% SDS by

vortexing for 2e3 s at maximum speed, followed by boiling for

30 min. Protein concentrations were determined by BCA assay

(Beyotime, P0011). Proteins were subjected to SDS-PAGE, trans-

ferred to nitrocellulose membranes (Immobilon-P, Millipore) and

blocked for 1 h at room temperature with 3% milk in 1xTris-buff-

ered saline Tween-20 (TBST) (25 mM Tris, 150 mM NaCl, 2 mM KCl,

pH 7.4, supplemented with 0.1% Tween-20).

4.2.9. Molecular docking

The X-ray crystal structure of STAT3/DNA complex (PDB code:

1BG1) was used for docking studies applying Schrodinger software

package. Firstly, one monomer was deleted and the missing

hydrogen atoms were added. Also, the bond order and the correct

protonation were prepared using protein preparation wizard

model. Then the complex was submitted to a series of restrained

minimizations to relieve static clashes using the OPLS-2005 force

ﬁeld. Subsequently, the receptor grid was generated at the centroid

of selected residues (Gln644, Glu638) and the size of grid box was

20 Å ꢂ 20 Å ꢂ 20 Å. After preparing the ligands, molecular docking

was carried out using the standard precision (SP) with the default

settings. Finally, the pictures were generated using Pymol software.

Membranes were probed with a 1:1000 dilution of primary

antibodies (Cell Signaling Technology) against Phospho-STAT3

(Tyr705), Phospho-STAT3 (Ser727), STAT3, c-Myc, MCL-1, b-actin

and GAPDH at 4 ^ꢀC overnight. After washing the membranes with

TBST three times for 30 min, horseradish peroxidase-conjugated

anti-rabbit IgG (dilution, 1: 2000) or anti-mouse IgG (dilution, 1:

5000) antibodies were incubated at room temperature for 1 h. The

membranes were washed with TBST three times for 30 min, and the

antigen-antibody reaction was visualized with an enhanced

chemiluminescence assay (Thermo Scientiﬁc) or Femto chem-

iluminescence assay (Thermo Scientiﬁc).

Declaration of competing interest

4.2.4. In vitro kinases activities test

For the kinase assay based on the ELISA system, procedure was

similar to published methods in ref. [50] and ref.[51].

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.

4.2.5. Flow cytometry analysis of apoptotic cells

Cells were seeded in 12-well plates for overnight and then

treated with DMSO or the indicated compounds for 48 h. Then cells

were collected and rinsed twice with PBS. After resuspending the

Acknowledgement

Financial support was generously provided by the Signiﬁcant

New Drugs Development (2018ZX09711002-003-009), the Na-

tional Natural Science Foundation of China (Nos. 81973178 and

U1906212).

cells in 500 mL binding buffer, 5 mL Annexin V-FITC and 5 mL pro-

pidium iodide (A211-01, Vazyme) were added and mixed gently.

Finally, the mixture was incubated for 15 min at room temperature

in dark and detected by a ﬂow cytometer (BD FACs Calibur).

Appendix A. Supplementary data

4.2.6. Cell cycle effect

Cells were seeded in 12-well plates overnight and then treated

with DMSO or the indicated compounds for 48 h. The cells were

washed with PBS and ﬁxed in ice-cold 80% ethanol overnight. For

analysis, cells were incubated for 30 min at 37 ^ꢀC with PI (ST512,

Beyotime Biotechnology)-containing RNase (B600476-0001, San-

gon Biotech) and detected by a ﬂow cytometer (BD FACS CALIBUR).

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113333.

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Rational drug design of benzothiazole-based derivatives as potent signal transducer and activator of transcription 3 (STAT3) signaling pathway inhibitors

DOI: 10.1016/j.ejmech.2021.113333

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