Identification and characterization of benzo[d]oxazol-2(3H)-one derivatives as the first potent and selective small-molecule inhibitors of chromodomain protein CDYL

European Journal of Medicinal Chemistry 182 (2019) 111656

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Identiﬁcation and characterization of benzo[d]oxazol-2(3H)-one

derivatives as the ﬁrst potent and selective small-molecule inhibitors

of chromodomain protein CDYL

Lixin Yang ^a, Yongqing Liu , Minghua Fan , Guiwang Zhu , Hongwei Jin , Jing Liang ,

,

1

a

,

1

a

b

a

,

**

, Liangren Zhang ^a

, *

Zhenming Liu , Zhuo Huang

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China

a

b

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Biochemistry and Molecular Biology, School of Basic

Medical Sciences, Peking University, Beijing, 100191, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 3 July 2019

Received in revised form

Chemical probes of epigenetic ‘readers’ of histone post-translational modiﬁcations (PTMs) have become

powerful tools for mechanistic and functional studies of their target proteins in physiology and pa-

thology. However, only limited ‘reader’ probes have been developed, which restricted our understanding

towards these macromolecules and their roles in cells or animals. Here, we reported a structure-guided

approach to develop and characterize benzo [d]oxazol-2(3H)-one analogs as the ﬁrst potent and selective

small-molecule inhibitors of chromodomain Y-like (CDYL), a histone methyllysine reader protein. The

binding conformation between the chromodomain of CDYL and the modiﬁed peptidomimetics was

studied via molecular docking and dynamic simulations, facilitating subsequent virtual screening of tens

24 August 2019

Accepted 28 August 2019

Available online 31 August 2019

Keywords:

CDYL

Benzo[d]oxazol-2(3H)-ones

Small-molecule inhibitors

Computer-aided drug design

SAR study

of hits from Specs chemical library validated by SPR technique (K

Further design and synthesis of 43 compounds helped to interpret the structure-activity relationship

(SAR) that lead to the discovery of novel small-molecule inhibitors of CDYL. Compound D03 (K : 0.5 M)

was discovered and showed excellent selectivity among other chromodomain proteins, including CDYL2

>140 folds), CDY1 (no observed binding) and CBX7 (>32 folds). Moreover, we demonstrated that D03

D

values: from 271.1 mM to 5.4 mM).

D

m

Dendritic morphogenesis

(

engaged with endogenous CDYL in a dose-dependent manner, and perturbed the recruitment of CDYL

onto chromatin, resulting in transcriptional derepression of its target genes. Finally, the results showed

that D03 promoted the development and branching of neurodendrites by inhibiting CDYL in hippo-

campal and cortical cultured neurons. This study not only discovers the ﬁrst selective small-molecule

inhibitors of CDYL, but provids a new chemical tool to intervene the dynamic nature of bio-

macromolecules involved in epigenetic mechanism.

©

1

. Introduction

of the genetic code is largely regulated by cell-type speciﬁc tran-

scriptional factors, chemical modiﬁcations of DNA and histone

proteins. The dynamic nature of epigenetics means that it may be

possible to alter disease-associated epigenetic states through direct

manipulation of the molecules involved in this process, which are

metaphorically called ‘writer’, ‘eraser’, and ‘reader’ proteins [1,2].

With the development of the so-called epigenome, these proteins

are increasingly in these years showing promising therapeutic

relevance in multiple diseases like cancer, metabolic dysregulation

and neuropsychiatric disorders, as well as in regenerative medicine

[3e5]. Noticeably, many inhibitors of ‘writer’ proteins like histone

methyltransferases (HMTs) have entered diverse clinical trials

Controlled regulation of gene expression is crucial for the

development and function of multicellular organisms. Expression

*

Corresponding author. State Key Laboratory of Natural and Biomimetic Drugs,

Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking

University Beijing, 100191, China.

*

* Corresponding author. State Key Laboratory of Natural and Biomimetic Drugs,

Department of Molecular and Cellular Pharmacology, School of Pharmaceutical

Sciences, Peking University, Beijing, 100191, China.

E-mail addresses: huangz@hsc.pku.edu.cn (Z. Huang), liangren@bjmu.edu.cn

(

L. Zhang).

[6e12] and inhibitors of ‘eraser’ proteins have been approved and

1

L.Y. and Y.L. contributed equally to this work.

https://doi.org/10.1016/j.ejmech.2019.111656

0

2

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

used clinically against multiple malignancies [13e15]. To date,

however, very few ‘reader’ proteins have been successfully targeted

by small-molecule ligands [16,17].

and structure-activity relationship studies that lead to the discov-

ery of novel inhibitors are described. We demonstrate that the lead

compound D03 engages with endogenous CDYL, and perturbs the

recruitment of CDYL onto chromatin, subsequently resulting in

transcriptional derepression of its target genes. Finally, D03 pro-

motes dendritic maturation in primary cultured neurons, consis-

tent with the known ability of CDYL overexpression to restrict

dendritic arborization. These small molecules bear the potential to

be modiﬁed into chemical probes that explore the functions of

CDYL and treat its related diseases.

Chromodomain Y-like (CDYL) protein is a recently identiﬁed

transcriptional corepressor that contains an N-terminal chromo-

domain and a C-terminal enoyl-coenzyme A hydrataseisomerase

catalytic domain [18,19]. The CDY family in human genome encodes

three proteins, CDY1, CDYL and CDYL2. CDYL and CDYL2 are widely

expressed in mammals, while CDY1 shows testis-speciﬁc expres-

sion [20]. In addition, CDYL contains three splicing variants (a, b, c),

among which CDYL1b is the major splicing variant. Only CDYL1b

recognizes methyl-lysines through its chromodomain, which fa-

cilitates the speciﬁc association with histone methylated modiﬁ-

cations such as H3K9me3 and H3K27me3, thereby inhibiting the

expression of downstream related genes [21e23]. CDYL was origi-

nally identiﬁed as a key player in the regulation of mammalian

spermatogenesis [24,25]. Recent years, it was put to the moonlight

that CDYL can play essential roles in the central nervous system

since a signiﬁcant number of CDYL target genes were associated

with the development and function of the nervous system, such as

the brain-derived neurotrophic factor (BDNF) [26]. CDYL negatively

regulates dendritic branching in hippocampal neurons, suggesting

that CDYL acts as a regulator during early stages of neural devel-

opment, such as neuronal migration [26,27]. Further studies by our

collaborators showed that CDYL can suppress epileptogenesis in

mice through binding to the regulatory element in the intron region

of SCN8A and repression of axonal Nav1.6 sodium channel

expression [28]. The newly released perspective disclosed that

CDYL played a critical role in regulating stress-induced depression

by repressing the expression of neuropeptide VGF [29]. All of the

mentioned correlations between CDYL and neuronal activities are

based on the molecular mechanism of its ‘reader’ function.

2. Results and discussion

2.1. Isatin derivatives are identiﬁed as hit compounds targeting

CDYL via docking-based virtual screening strategy

Given that the chromodomain of CDYL was a critical region to

drive the transcription repression (Fig. S1) and biological activities

of CDYL [25e29], we thus focused our study on its N-terminal

chromodomain. In an attempt to precisely regulate the transcrip-

tional repression activity of CDYL (CDYL1b isoform in our work), we

need to screen and identify novel CDYL modulaters. To date, the

structure of CDYL protein has not been resolved, and there is only

one NMR apo-structure (PDB ID: 2DNT) of its N-terminal chromo-

domain with 20 conformations different largely in the starting loop

region. The conformation overlay indicated that the secondary

structures were rather stable, which helped to form a hydrophobic

cage composed of its residues Trp36, Tyr39 and Tyr14 of the

starting loop (Fig. S2). The hydrophobic cage of CDYL chromodo-

main can speciﬁcally recognize H3K9/27me3 modiﬁcations by

forming a tight cation-p stacking network with the trimethylated

lysine mark [21]. This is a highly induced-ﬁt process, and thus an

energy-optimized binding model for CDYL is crucial to conduct

docking-based screening. Recently, UNC3866 was identiﬁed as the

peptidomimetic ligand of CBX7, and also exhibited micromolar

However, although CDYL has a deﬁned chromodomain and

important biological functions, no speciﬁc tools have been discov-

ered to investigate its precise role and potential to be a pharma-

cological drug target. One approach to increase our knowledge of

its regulatory mechanisms is through small-molecule perturbation

binding afﬁnity (K

isothermal titration calorimetry (ITC) [38]. We synthesized the

peptidomimetic UNC3866 and found value of 5.6 mM

D

: 0.91 mM) to CDYL chromodomain by

[

30]. Accordingly, chemical biology efforts focused on interpreting

a

K

D

the function of lysine methylation with chemical tools have gained

momentum, resulting in a number of freely available high-quality

chemical probes, such as inhibitors for heterochromatin protein 1

(Figs. S3AeB) with CDYL chromodomain via surface plasmon

resonance (SPR). However, UNC3866 didn't affect the expression of

CDYL target gene MYT1 in SH-SY5Y and HEK-293T cells, even in a

(

HP1) and chromobox homolog 7 (CBX7) [31e35]. However, little

concentration up to 200 mM (Figs. S3CeD). Due to its strong afﬁnity,

active ligands have been discovered targeting the CDY family pro-

teins, especially for the CDYL subtype, which exhibited signiﬁcant

relevance to neuropsychiatric disorders as described above.

Nevertheless, there are still some clues for potential druggability of

CDYL coming from targeting efforts of other chromodomains, like

CBX chromodomains. CBX7 is a member of PRC1 complex, which

possesses a chromodomain facilitating the recognition to certain

methylated modiﬁcations [36,37]. Recently, potent ligands for CBX7

inspired from its recognition mechanism with the H3K27me3

modiﬁcation were developed using adaptively biased molecular

dynamics simulations. The resulting peptidomimetic UNC3866

showed robust binding with CBX7 and moderate interactions with

three members of the CDY family, CDYL1b, CDYL2 and CDY1 [38].

Although the afﬁnity of optimized peptidomimetics against CDY

family was promoted several folds in subsequent combinatorial

chemistry optimization, their subtype selectivity was still barely

satisfactory [39]. Besides, their use as chemical probes in exploring

and characterizing CDYL biological functions remains unknown.

To fully elucidate the importance of CDYL in human diseases as

well as its druggable potential, especially in neuronal events,

subtype-selective chemical probes are very essential. Herein we

report a structure-based design strategy to develop ﬁrst potent and

selective small-molecule inhibitors of CDYL. The design, synthesis,

we use UNC3866 as ligand to stabilize the conformation of CDYL

chromodomain and discover novel small-molecule inhibitors

through virtual screening.

The overall in silico modeling and virtual screening workﬂow

was shown in Fig.1A and the Specs chemical library containing over

200,000 molecules was employed in this study. Firstly, We docked

peptidomimetic UNC3866 to the chromodomain functional site

containing the conserved hydrophobic cage using the induced-ﬁt

docking module in Schrodinger 2018.1. The resulting binding

model was afterwards used in a 20 ns molecular dynamic simula-

tion to see the potential binding process between CDYL and

UNC3866. As shown in Fig. 1B, the RMSD values of Ca, Lig ﬁt Prot,

and Lig ﬁt Lig gradually achieved stability after around 8 ns, which

meant that the complex had reached an equilibrium state and the

Gibbs energy of the system was low. So we selected the binding

model in 10 ns to analyze detailed interactions and construct a

relatively stable docking model (Fig. 1C).

Sixty-two compounds were picked out in the ﬁrst-round

screening (Fig. S4 and Table S1). Their binding afﬁnity to CDYL

and the transcriptional inhibition of its target genes were then

tested. Most of the hits showed about 10e20 folds weaker afﬁnity

towards CDYL than UNC3866, ranging from 14.2

mM to 271.1 mM

(Fig. 2A). Among them, those isatin derivatives like AK-778/

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

3

Fig. 1. A schematic diagram of in silico modeling and virtual screening. (A) Iterated virtual screening pipline with detailed parameter setting. IFD, induced-ﬁt docking; MD, mo-

lecular dynamics; VSW, virtual screening workﬂow; SP, standard precision; XP, extra precision. (B) Results of the 20 ns molecular dynamic simulations for CDYL-UNC3866 complex.

(

C) Energy-optimized binding model of CDYL-UNC3866 complex.

41051004 (Fig. 2B) exhibited more potent activity on gene expres-

cytotoxicity were evaluated.

sion (Fig. 2C), indicating greater potential as the hit compound to

ﬁnd better ligands with the similar scaffold.

The elimination of its hydroxyl group which yielded A01

increased the molecular rigidity, and led to undesired toxicity. Also,

For the second-round screening, we chose AK-778/41051004 as

a template to conduct rapidly shape-based 2D similarity search and

get insight into the potential for isatin scaffold to be used as CDYL

ligands. Over 200 compounds were thus screened out and around

its binding afﬁnity was slightly weakened (K

the reduction of A01 to A02 markedly increased the afﬁnity (K

1.2 M) towards CDYL while the toxicity still retained. Moreover,

when the chiral carbon atom of A02 was replaced with a nitrogen

atom to yield A03 which didn't possess a chiral center any more, the

D

: 15.4 mM). However,

D

:

m

1

/4 of which were selected based on their similarity rankings. We

also tested their binding afﬁnity towards CDYL by SPR, as well as

another three sequence highly conserved chromodomain proteins,

CDYL2, CDY1 and CBX7. The results showed that most of the active

ligands possessed similar binding afﬁnity to CDYL as the hits

identiﬁed in the ﬁrst round except AK-778/41314725, which

D

afﬁnity decreased accordingly (K : 14.8 mM), but its cytotoxicity

decreased remarkably in HEK-293T and SH-SY5Y cells. Besides, A03

retained its selectivity to CDYL among the four chromodomain

proteins compared with AK-778/41314725. The results in Table 2

indicated that the hydroxyl group at the core structure was not a

necessary moiety and can thus be removed to afford simpliﬁed lead

compound in following structural modiﬁcations.

D

exhibited comparative afﬁnity (K : 4.9 mM) with UNC3866. More-

over, it showed considerable subtype selectivity to CDYL against

other chromodomain proteins (Table 1). Also, the RT-qPCR results

demonstrated that it reversed the suppression of downstream gene

expression in a dose-dependent manner (Fig. S5). Together, these

results indicated that the isatin derivatives were competent to be

potential inhibitors of the CDY family.

2.3. Compounds designed to form p-p stacking network

interactions with the hydrophobic cage of CDYL chromodomain

With two-round virtual screening attempts, we discovered

compound A03 as a hit ligand of CDYL which showed considerable

subtype selectivity. Then versatile structural modiﬁcations were

made based on A03 to get detailed understanding of the structure-

activity relationship (see Schemes 2e5). As suggested in the

induced-ﬁt docking model of CDYL-A03 complex (Fig. S6), the

carbonyl group of A03 formed tight hydrogen bonds with residues

Leu 13 and Tyr14 of the starting loop region and stabilized its

conformation. Although the methoxyphenyl group of A03

communicated with the hydrophobic cage residues Tyr14, Trp36

2.2. The hydroxyl group of the racemic AK-778/41314725 is not a

necessary pharmacophore for CDYL binding and function

It is worthy of attention, however, all the purchased isatin de-

rivatives were all racemic compounds, so we can't tell which

enantiomer of the pair ﬁtted better in the active site of CDYL

chromodomain. Instead of obtaining the pure enantiomer via

chemical resolution or chiral synthesis, we turned to remove the

chiral center to simplify the core structure and see the real impact

of the -OH upon binding to CDYL. As shown in Scheme 1, AK-778/

and Tyr39 mainly through

teractions, it wasn't satisfactory since only Tyr14 formed desired

stacking with A03. Besides, the 2-benzoxazolinone fragment

induced a new binding site in between of the ﬂexible loop and the

p-p stacking and hydrophobic in-

p

-

41314725 and its derivatives A01, A02 and A03 were synthesized,

p

and their binding afﬁnities to various chromodomins and their

4

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

Fig. 2. Biological evaluation of hit compounds obtained in ﬁrst round virtual screening. (A) Binding afﬁnity with CDYL (K

mean ± the standard deviation of at least two replicates each performed in duplicate. (B) The 2D structures of some representative hit compounds. (C) The mRNA expression of

MYT1 in SH-SY5Y cells treated with 100 M compounds for 24 h. Each bar represents the mean ± S.E.M. for triplicate experiments.

D D

) determined by SPR. The K values were calculated as the

m

b

-strand of CDYL, but it didn't form versatile types of interactions

that bigger and/or more rigid group substitution of 5-position

hindered their binding with CDYL and also affected the selec-

tivity, like B04, whose afﬁnity dramatically decreased when

replaced by 5-CN. However, when the oxazolinone ring was

augmented to a hexatomic ring, such as B26 and B27, their binding

afﬁnity roughly retained and showed little difference to A03.

As implied in Table 3, when the 5-Cl was replaced by 5-F (B02)

owing to its featureless aromatic structure.

Consequently, we further synthesized 29 derivatives based on

the analysis of CDYL-A03 binding model. The synthesis of com-

pounds B01eB25 was shown in Scheme 2. The benzoxazolinone

and benzothiazolone intermediates were synthesized from

commercially available substituted 2-aminophenol and 2-

aminobenzenethiol. The

a

-Br ketone derivatives were obtained

3

and 5-OCH (B03), their binding afﬁnity increased by 2 folds

via addition of a

nucleophilic substitution in the presence of K

between the two building blocks afforded compounds B01eB25.

The same reaction was also used in the synthesis of compounds

B26e29 (Scheme 3).

a

-position bromine catalyzed by CuBr. Then a

though occasionally changed their binding preference to other

chromodomain proteins like CBX7 or CDYL2. So in subsequent

modiﬁcations, we studied these substitutions one by one, and the

methoxyphenyl group that interacted with the hydrophobic cage

was also explored simultaneously. For the derivatives with a non-

substituted benzoxazolinone group (B05eB09), results in Table 4

indicated that the binding afﬁnity improved remarkably by

around 2 folds when the benzene ring was replaced with larger

aromatic rings, like the naphthalene of B07 and B08, and indole of

B09. Also, their subtype selectivity still retained accordingly. For

ꢀ

2

CO

3

at 60e70 C

At ﬁrst, the 2-benzoxazolinone scaffold of A03 was replaced by

2

-benzothiazolinone, and whose 5-Cl was removed to get com-

pound B01. Its binding afﬁnity (K : 4.9 M) slightly increased by

around 3 folds and maintained its selectivity to CDYL subtype. The

inﬂuence of 5-position was also studied. Results in Table 3 showed

D

m

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

5

Table 1

The active compounds determined in shape screening and SPR.

D

(m

M) ^a

SPR K

Cpd

R¹

3-Cl

R²

H

R³

CDYL

CDYL2

96.4 ± 32.0

CDY1

33.5 ± 4.6

CBX7

12.1 ± 5.3

AK-778/41051004

55.6 ± 12.0

100 ± 14.0

AK-778/43206467

AK-778/41314725

H

n-Butyl

H

53.6 ± 34.0

17.2 ± 2.1

8.8 ± 0.9

3-Cl

4.9 ± 0.3

n.d. ^b

n.d.

84.4 ± 25.0

AK-778/43420918

AK-778/43114937

H

i-Propyl

n-Propyl

13.1 ± 3.5

50.5 ± 6.8

54.7 ± 5.7

14.5 ± 8.6

61.6 ± 34.0

89.5 ± 26.0

41.1 ± 7.9

7.2 ± 5.7

AK-778/43114894

H

n-Propyl

23.0 ± 4.5

n.d.

21.1 ± 3.3

20.4 ± 3.2

AG-205/33681008

AK-778/43416943

H

CH

3

19.4 ± 2.1

128 ± 50.0

68.1 ± 39.0

>200 ^c

28.9 ± 1.4

24.7 ± 8.6

5.1 ± 6.7

2.4 ± 1.8

AK-778/41050965

AK-778/43465061

H

Allyl

31.7 ± 3.8

15.4 ± 0.9

51.8 ± 36.0

24.5 ± 6.4

n.d.

3-Cl

n-Propyl

n.d.

16.0 ± 7.1

a

D

The K values were calculated as the mean ± the standard deviation of at least two replicates each performed in duplicate.

Cannot be determined or not detected.

The binding constants were weaker than 200 mM.

b

c

ꢀ

Scheme 1. Synthesis of compounds A01-A03, derivatives of AK-778/41314725. Reagents and conditions: (a). HAc, concentrated HCl, EtOH, 90 C, then r.t., 0.5 h; (b). Pd/C, H

2

, 3 bar,

ꢀ

r.t., overnight; (c). CDI, DCM, r.t., 3 h; (d). Boc

r.t., 2 h.

2

O, K

2

3

CO , dry MeCN, r.t., 0.5 h, then reﬂux, 3 h; (e). 2-bromo-1-(3-methoxyphenyl)ethan-1-one, K

2

CO

3

, MeCN, 90 C, 4.5 h; (f). HCl, THF,

those with a 5-F substituted benzoxazolinone group (B10eB13),

the results showed that their afﬁnity towards CDYL was affected

and droped by around 4e5 folds when the benzene group was

further decorated (B10, B11) or replaced with larger aromatic rings

disappeared. This problem maybe came from the molecular rigidity

rooting from the short linker of the ligands and was expected to be

studied. However, situations were totally different when the 5-

position was replaced with the methoxy group such as com-

pounds B14eB17. The results showed that these ligands normally

retained their afﬁnity to CDYL and even went beyond. For example,

(B12, B13), indicating a preference binding for the original

methoxyphenyl group. At the same time, their selectivity was also

6

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

Table 2

Chemical modiﬁcations based on the racemic compound AK-778/41314725.

Cpd

SPR K

D

(

m

M) ^a

Cytotoxicity IC50

HEK-293T

(m

M) ^b

CDYL

CDYL2

CDY1

CBX7

SH-SY5Y

AK-778/41314725

4.9 ± 0.3

15.4 ± 3.7

1.2 ± 0.8

n.d.^c

4.3 ± 2.7

19.7 ± 3.2

37.4 ± 9.0

n.d.

84.4 ± 25.0

n.d.

56.1 ± 2.1

16.6 ± 1.7

36.9 ± 1.4

>200

68.1 ± 2.1

21.5 ± 1.6

43.4 ± 2.1

>200

A01

A02

A03

10.3 ± 4.6

>200 ^d

14.8 ± 3.9

70.4 ± 19.0

28.9 ± 6.9

a

D

The K values were calculated as the mean ± the standard deviation of at least two replicates each performed in duplicate.

The IC50 values were reported as the mean ± s.e.m of three biological replicates.

Cannot be determined or not detected.

The binding constants were weaker than 200 mM.

b

c

d

ꢀ

Scheme 2. Synthesis of compounds B01eB25. Reagents and conditions: (a) CDI, DCM, r.t., 2 h; (b) 2-bromo-1-ones, K

2

CO

3

, DMF, TBAI, 70 C, 5 h; (c) CuBr, CH

3

OH, reﬂux, 1 h.

retained the naphthalene or indole group further and explored the

substitutions of 5-/6-position again to enrich the SAR studies. For

the ligands with a naphthalene group in Table 5, 5-Br substituted

B18 showed good binding afﬁnity to all the chromodomains,

especially for the CDYL2 subtype with a K

B20 with a 6-Br group, its binding preference largely altered and

showed better afﬁnity to CDY1 subtype (K : 0.6 M), perhaps

D

value of 0.6 mM. But for

D

m

owing to the undesired steric hindrance. For B19, the poor binding

afﬁnity was consistent with the result got from B04, whose 5-CN

showed negative effect for CDYL binding. For the benzothiazoli-

2 2

none derivatives with a 6-substituted eNH /-NO , their afﬁnity to

CDYL can be retained or just slightly decreased, like B21 and B22,

but their selectivity was totally in chaos. Besides, when the thia-

zolinone rings were augmented to oxazinone ones, their afﬁnity

and selectivity both abruptly decreased. The similar case was also

found for the indole derivatives B23eB25 (Table 6), among which

only B24 showed considerable binding afﬁnity and preference for

CDYL chromodomain.

Scheme 3. Synthesis of compounds B26eB29. Reagents and conditions: (a) 2-bromo-

ꢀ

1-ones, K

2 3

CO , DMF, TBAI, 70 C, 5 h.

B14 (K

D

: 5.5

mM) and B17 (K

D

: 7.2

mM) showed comparable binding

afﬁnity with B03, and the afﬁnity of B16 (K

D

: 1.5 M) to CDYL even

m

increased by 5 folds, although their subtype selectivity was slightly

affected.

Taken together the results of Table 4, generally the hydrophobic

cage of CDYL preferred larger aromatic groups which may

2

.4. Compounds designed to study the effect of molecular ﬂexibility

towards CDYL binding via modifying the chemical linker

contribute to the desired

p-p stacking network. Subsequently, we

As mentioned above, the linker in the core of the ligands may

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

7

1

ꢀ

Scheme 4. Synthesis of compounds C01eC03. Reagents and conditions: (a). chloroacetyl chloride, Et

3

N, THF, ice-r.t., 8 h; (b). 2-chloro-N-R -acetamide, K CO , DMF, 70 C, 5 h.

2 3

ꢀ

Scheme 5. Synthesis of compounds D01-D08. Reagents and conditions: (a) CDI, DCM, r.t., 2 h; (b) K

2

CO

3

, DMF, 70 C, 5 h; (c) chloroacetyl chloride, Et

3

N, THF, ice-r.t., 8 h.

Table 3

Instead, some of these ligands (C01 and C03) showed exceptionally

good binding afﬁnity and preference to the CDY1 subtype. This can

be further studied in the future.

Binding afﬁnity studies for compounds B01eB04, and B26, B27.

2.5. Compounds designed to form cation-p stacking network

interactions with the hydrophobic cage of CDYL chromodomain

We concluded from the 32 derivatives discussed above that it

was not necessary to retain the trimethylated marks of histone or

diethylamine groups of UNC3866 to achieve speciﬁc binding with

the hydrophobic cage of CDYL chromodomain. Many promising li-

gands like B07, B08, B16 and B21 also showed excellent afﬁnity to

CDY proteins as well as their binding selectivity. To fully understand

the SAR of synthesized compounds, the cation-p network imitating

the interaction between the methylated lysine marks and the hy-

drophobic cage was explored simultaneously.

We designed another 8 compounds with a fatty tertiary amine

group, D01-D08. Compounds D01-D08 were synthesized as shown

in Scheme 5. The secondary amine groups of piperidine derivatives

were acetylated in ice shower by the dropwise added chloroacetyl

_M₎a

D

(m

Cpd

R

SPR K

CDYL

CDYL2

CDY1

CBX7

B01

B02

B03

B04

B26

B27

H

F

OCH

CN

…

4.9 ± 0.9

6.2 ± 1.5

7.7 ± 3.3

30.5 ± 4.8

28.7 ± 21.0

3.4 ± 1.5

9.5 ± 2.1

56.2 ± 39.0

35.8 ± 11.0

5.8 ± 3.2

n.d.

15.6 ± 6.4

42.1 ± 11.0

4.0 ± 2.0

11.3 ± 3.9

n.d.

3.4 ± 2.0

17.2 ± 2.4

19.6 ± 12.0

n.d.

3

b

n.d.

c

10.1 ± 2.7

13.0 ± 9.3

…

53.0 ± 7.6

a

The K

D

values were calculated as the mean ± the standard deviation of at least

two replicates each performed in duplicate.

b

Cannot be determined or not detected.

Structures were shown in Scheme 3.

c

chloride. Then the

a-Cl of the intermediates were substituted by the

2

-benzoxazolinone derivatives to yield the target compounds.

contribute greatly to the molecular rigidity, so we also attempted to

interpret its role upon binding to the CDYL protein. A nitrogen atom

was thus tentatively introduced in the original acetyl linker by

forming an amide bond, which not only extended the length of the

linker, but also added to its ﬂexibility. For the synthesis of com-

pounds C01eC03, aromatic amine groups were acetylated by

chloroacetyl chloride at room temperature to yield 2-Cl substituted

intermediates. Then they reacted with 2-benzoxazolinone to afford

C01eC03 by nucleophilic substitution (Scheme 4).

Noticeably, the method for separation and puriﬁcation in this

scheme were much different from the compounds described above

(see the Experimental Section).

The alkylated forms of the amine were chosen mainly in view of

the cage volume and predicted cation-

Table 8). For compounds D01-D04, most of them can bind well to

CDYL, even slightly decreased afﬁnity for D01 (K : 9.5 M) and D04

: 11.8 M) compared with B01 (K : 4.9 M). The results indi-

cated that a tailored size of these tertiary amine fragments was

necessary for potent CDYL targeting, such as D03 (K : 0.5 M). As a

contrast, D05 didn't exhibit good binding afﬁnity to the CDYL

p stacking interactions

(

D

m

(K

D

m

D

m

However, compared to B01, B07 and B08, no indications of af-

ﬁnity increase to CDYL were observed for C01eC03 (Table 7).

D

m

8

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

Table 4

Binding afﬁnity studies for compounds B05eB17.

D

(m

M) ^a

Cpd

R¹

H

R²

SPR K

CDYL

CDYL2

CDY1

CBX7

B05

14.6 ± 8.9

5.3 ± 3.2

23.8 ± 11.0

48.5 ± 9.1

B06

H

11.9 ± 5.9

n.d.^b

n.d.

36.5 ± 8.4

B07

B08

H

2.6 ± 1.0

3.0 ± 0.3

37.9 ± 1.2

24.1 ± 7.0

41.7 ± 12.1

6.5 ± 2.1

14.2 ± 4.0

29.9 ± 6.4

B09

H

1.9 ± 0.1

2.7 ± 0.4

21.0 ± 9.3

9.3 ± 3.6

B10

B11

F

20.5 ± 8.3

73.7 ± 35.6

30.2 ± 7.4

4.4 ± 1.0

28.8 ± 11.0

78.1 ± 9.2

90.1 ± 32.0

34.6 ± 12.0

B12

B13

F

20.1 ± 1.9

5.1 ± 1.1

n.d.

210 ± 41.0

23.7 ± 14.0

40.2 ± 18.0

9.8 ± 6.6

14.1 ± 3.2

B14

B15

OCH

3

5.5 ± 2.6

5.0 ± 0.6

6.2 ± 3.4

11.3 ± 0.7

2.7 ± 0.1

3.9 ± 2.9

6.1 ± 1.7

15.5 ± 1.6

B16

B17

OCH

3

1.5 ± 0.5

7.2 ± 3.7

6.3 ± 1.6

1.5 ± 0.9

59.5 ± 11.0

20.3 ± 5.7

22.0 ± 7.6

36.4 ± 16.0

a

The K

D

values were calculated as the mean ± the standard deviation of at least two replicates each performed in duplicate.

b

Cannot be determined or not detected.

family. Based on D03, the most potent and selective ligand, we

designed the 5-position substituted derivatives like D07 and D08.

Their afﬁnity and selectivity towards CDYL were signiﬁcantly

impaired. For compound D06, it surprisingly exhibited better

binding afﬁnity to all the chromodomain proteins compared with

D02, which showed good subtype selectivity to CDYL and CDYL2.

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

9

Table 5

Binding afﬁnity studies for compounds B18eB22, and B28, B29.

Table 7

Binding afﬁnity studies for compounds C01eC03.

D

(m

M) ^a

Cpd

R

SPR K

CDYL

_M₎a

D

(m

CDYL2

CDY1

CBX7

Cpd

R

X

SPR K

CDYL

C01

7.3 ± 1.0

7.2 ± 3.3

1.0 ± 0.5

14.8 ± 12.0

CDYL2

CDY1

CBX7

B18

B19

B20

B21

B22

B28

B29

5-Br

5-CN

6-Br

O

S

…

5.8 ± 0.8

0.6 ± 0.1

n.d.

13.7 ± 7.5

57.8 ± 27.0

11.9 ± 1.1

5.9 ± 1.3

14.8 ± 7.9

71.5 ± 6.8

1.7 ± 1.0

n.d.

123 ± 27.0

39.6 ± 4.7

109 ± 63.0

9.5 ± 1.1

n.d.

b

101 ± 12.2

29.0 ± 11.0

4.9 ± 1.9

26.8 ± 5.3

52.5 ± 12.0

59.8 ± 25.0

29.7 ± 17.0

c

C02

C03

18.9 ± 1.9

29.0 ± 4.3

21.5 ± 4.6

5.8 ± 4.2

20.4 ± 2.7

0.8 ± 0.2

19.6 ± 7.9

>200^b

6-NH

2

>200

6-NO

2

n.d.

26.5 ± 17.0

37.1 ± 17.0

d

…

a

D

The K values were calculated as the mean ± the standard deviation of at least

two replicates each performed in duplicate.

b

Cannot be determined or not detected.

The binding constants were weaker than 200

The structures were shown in Scheme 3.

c

mM.

a

D

The K values were calculated as the mean ± the standard deviation of at least

d

two replicates each performed in duplicate.

b

The binding constants were weaker than 200 mM.

Table 6

Binding afﬁnity studies for compounds B23eB25.

Table 8

Binding afﬁnity studies for compounds D01-D08.

D

(m

M) ^a

Cpd

R

X

SPR K

CDYL

CDYL2

CDY1

CBX7

B23

B24

B25

5-CN

6-NH

O

S

96.7 ± 38.0

3.5 ± 0.3

71.7 ± 12.0

2.8 ± 0.4

84.5 ± 19.0

3.8 ± 1.6

4.6 ± 1.6

51.1 ± 13.0

14.4 ± 3.3

46.9 ± 11.0

6.5 ± 2.1

8.8 ± 6.7

Cpd R¹

R²

SPR K

(

m

M) ^a

CDYL2

D

2

6-NO

2

CDYL

CDY1

CBX7

a

_>₂₀₀b

_n_._d_.c

The K

D

values were calculated as the mean ± the standard deviation of at least

D01

D02

H

9.5 ± 4.0

5.8 ± 2.0

33.6 ± 13.0

two replicates each performed in duplicate.

4.7 ± 1.3

n.d.

46.6 ± 2.2

2.6. The SAR summary for structural optimizations

In above structural optimizations, we divided the main structure

D03

D04

H

0.5 ± 0.2

72.3 ± 11.2 n.d.

16.2 ± 3.8

brieﬂy into three parts, namely the induced pocket binding part,

the linker part, and the hydrophobic cage binding part. To ﬁt the

hydrophobic cage, two kinds of compounds which were expected

11.8 ± 1.3 84.5 ± 3.4

25.9 ± 2.5 57.9 ± 8.6

51.1 ± 12.0 13.5 ± 0.9

to form

p-p or cation-p stacking interactions (Fig. 3A) were

designed respectively. Fig. 3B illustrated the structure-activity

relationship in terms of the molecular binding by SPR assay for

the 40 synthesized compounds. In brief, for the induced binding

part, chemical modiﬁcations in 5-/6-position of the componds were

mainly studied. The results implied that smaller substitutions at 5-

position like 5-H/F contributed to the improved binding afﬁnity

while the less explored 6-position didn't show signiﬁcant impact

on the binding. For the fragment which can bind to the hydro-

phobic cage of CDYL, the binding nature depended on the types of

the interactions. In general, bigger aromatic fragments such as

naphthalene and indole rings predominantly increased the binding

D05

D06

H

F

21.0 ± 4.6

4.5 ± 1.8

n.d.

5.6 ± 0.8

2.8 ± 1.6

8.2 ± 0.9

2.8 ± 1.2

3.4 ± 0.4

D07 OCH

3

12.5 ± 8.9 2.1 ± 0.1

16.8 ± 9.8 5.2 ± 0.6

D08 NO

2

55.8 ± 15.0 n.d.

ability of the ligands by forming a

when the interactions switched from the

well-established cation- stacking network, their binding afﬁnity

can further increase by around 5 folds. Other optimizations for the

linker part were little but still attractive since compounds with

p

-

p

stacking network. Moreover,

p-p

stacking network to a

a

p

D

The K values were calculated as the mean ± the standard deviation of at least

two replicates each performed in duplicate.

b

The binding constants were weaker than 200 mM.

Cannot be determined or not detected.

c

10

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

Fig. 3. Design rationales and the brief SAR gained from versatile structural modiﬁcations. (A) The

p-p stacking network and cation-p stacking network designed to ﬁt the hy-

drophobic cage of CDYL chromodomain. (B) The brief SAR gained from versatile structural modiﬁcations.

better binding afﬁnity and preference to CDYL may be achieved by

delicate design on this fragment, which should play a key role for

the molecular ﬂexibility of target ligands.

stepwisely increased in HEK-293T cells after 24 h treatment of B07

(Fig. 4D) or D03 (Fig. 4E). However, treatment of compound D07

performing weak binding afﬁnity to CDYL did not affect the tran-

scriptional levels of MYT1/BDNF/VGF in SH-SY5Y (Fig. 4C) or HEK-

2

93T cells (Fig. 4F). Together, these date suggest that B07 and D03

2

.7. Compounds B07 and D03 lead to transcriptional derepression

can lead to transcriptional derepression of CDYL target genes in SH-

SY5Y and HEK-293T cells.

of CDYL target genes in SH-SY5Y and HEK-293T cells

Compounds B07 and D03, the best ligands ever determined in

comprehensive consideration of their excellent binding afﬁnity and

preference to CDYL, were thus expected to be studied in a series of

biological assays related to CDYL function. As CDYL was demon-

strated to mainly function as a transcriptional repressor [19,28], we

ﬁrstly focused the effect of B07 and D03 on transcriptional activity

of CDYL. Previous research showed that deletion of CDYL chromo-

domain resulted in an increase of MYT1/BDNF/VGF transcription

levels [22,26,29]. By using real-time quantitative PCR, we indeed

observed that the mRNA levels of MYT1/BDNF/VGF gradually

increased in human SH-SY5Y cells upon dose-dependent treatment

of B07 (Fig. 4A) or D03 (Fig. 4B) for 24 h. In addition, compared with

the DMSO control, the transcript levels of MYT1/BDNF/VGF

2.8. Compound D03 disrupts the recruitment of CDYL onto

chromatin

Small-molecule inhibitors can perturb protein function and in-

crease the protein stability via forming a ligand-protein complex.

Thus, we attempted to investigate whether compound D03 could

bind to CDYL protein and increase its stability in cells. Through

cellular thermal shift assay (CETSA), we found that D03 treatment

efﬁciently protected CDYL protein from temperature-dependent

degradation in SH-SY5Y cells (Fig. 5A). Additionally, our data also

demonstrated a dose-dependent reduced CDYL degradation with

the incubation of D03 (Fig. 5B), supporting that there is a direct

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

11

Fig. 4. Compounds B07 and D03 lead to transcriptional derepression of CDYL target genes. RT-qPCR analysis shows mRNA levels of CDYL target genes in SH-SY5Y cells (AeC) and

HEK-293T cells (DeF) treated with B07, D03 and D07 respectively for 24 h. Each bar represents the mean ± S.E.M. for triplicate experiments. P-values were determined by Student's

t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

interaction between D03 and CDYL. Given that CDYL regulates its

target genes transcription through its chromodomain-dependent

association with chromatin. We next examined whether D03

could perturb the CDYL-chromatin interaction and thereby modu-

late gene transcription. Using chromatin immunoprecipitation and

quantitative PCR (ChIP-qPCR), we detected the decreases in the

abundance of CDYL at its several target genes, such as MYT1, BDNF

regulation of dendritic morphogenesis, we next examined whether

compound D03 regulates dendritic branching. After treatment with

D03 for 24 h, the mRNA levels of MYT1/BDNF/VGF was signiﬁcantly

increased in cultured mouse hippocampal neurons (Fig. 6A) and

cortical neurons (Fig. 6B), compared with the DMSO control. In

addition, we observed a signiﬁcant increase of dendritic branching

compared with the control group (Fig. 6CeF). Clearly, these results

suggest that compound D03 promotes dendritic maturation in

cultured neurons by blocking CDYL binding to its target genes.

and VGF, in SH-SY5Y cells treated with 150 mM D03 (Fig. 5C), sug-

gesting that engagement of the inhibitor could indeed prevent the

enrichment of CDYL on its targeted genes. Furthermore, signiﬁ-

cantly decreased enrichment of H3K27me3 on MYT1/BDNF/VGF

2.10. Analysis of binding models for CDYL-B07/D03 complex

promoter were found in SH-SY5Y cells treated with 150 mM D03,

whereas the enrichment of CDYL and H3K27me3 on HOXA3 pro-

moter, a non-CDYL target [23], were not altered in the above con-

dition (Fig. 5C). The observed decrease of H3K27me3 matched the

previous studies showing localized decreased H3K27me3 when

CDYL was silenced. Collectively, the above data demonstrate that

D03 can disrupt the recruitment of CDYL onto chromatin, subse-

quently resulting in transcriptional derepression of its target genes.

Compared with D03, compound B07 possesses comparable and

similar binding nature with CDY family, indicating that the tertiary

or quaternary amine groups designed for cation- stacking in-

p

teractions are not an exclusive feature for inhibition of Kme3

readers. When looking into the binding details of CDYL with B07

and D03 via induced-ﬁt docking studies, we saw the detailed

mechanism of action. As shown in Fig. 7AeC, the naphthalene

group of compound B07 bound to the hydrophobic cage in net-

2.9. Compound D03 promotes dendritic maturation in primary

worked p-p stacking interactions with Trp36; a salt bridge be-

cultured neurons

tween Glu11 and Asn 54 was then induced by the benzoxazolone

part of the compound and buried it deep into the pocket. As a

contrast, the ionized tertiary amine group of D03 was tightly bound

Previous study reported that CDYL restricted dendrite arbori-

zation by repressing BDNF expression [26]. The neuropeptide VGF

was induced by BDNF and promoted hippocampal dendritic

maturation [40,41], as well as synaptic activity [41]. And MYT1

overexpression in the dentate gyrus induced an increase of BDNF

expression, showing there was a positive correlation between

MYT1 and BDNF [42]. Based on the clear mechanism of CDYL in the

to the same hydrophobic cage via a cation-p network with residues

Tyr14, Trp36 and Tyr39. A similar salt bridge was also induced by

the benzoxazolone fragment, but it was between residues Glu11

and Hip 51 instead (Fig. 7DeF). However, to further enrich SAR

studies of CDYL inhibition, more structural modiﬁcations should be

scheduled. For example, bigger chemical space need to be enlarged

12

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

Fig. 5. D03 perturbs CDYL-chromatin interaction in SH-SY5Y cells. (A) D03 treatment (150 mM) increases the thermal stability of CDYL in cell lysates as measured by the

temperature-dependent cellular thermal shift assay. (B) D03 treatment increases the thermal stability of CDYL in cell lysates as measured by the concentration-dependent cellular

ꢀ

thermal shift assay at 55 C. (C) ChIP-qPCR analysis of CDYL target genes in SH-SY5Y cells treated with or without D03 (150

triplicate experiments. P-values were determined by Student's t-test. *P < 0.05.

mM) for 24 h. Each bar represents the mean ± S.E.M. for

in terms of volumes and interaction types to better ﬁt the hydro-

phobic cage. Besides, as depicted in Fig. 3B, the benzoxazolone part

and molecular ﬂexibility of ligands should be tailored to achieve

desired binding preference to CDYL rather than other chromodo-

mains with near-negligible pocket differences.

activity relationship for small-molecule ligands of CDYL chromo-

domain. To date, our work was the ﬁrst successful example to

design potent inhibitors of CDYL with the best binding afﬁnity, and

some of which like D03 showed excellent binding preference to the

CDYL protein.

However, there are still things we should do to further enhance

the afﬁnity and the speciﬁcity between CDYL and its inhibitors. As

mentioned above, the molecular size and ﬂexibility of the ligands

should be well designed to ﬁt the chromodomain of CDYL. Besides,

a wider range of reader chromodomains need to be included to

better evaluate the selectivity of promising ligands. Moreover, this

3

. Conclusion

In this study, we explored the possibility of speciﬁcally targeting

of several chromodomain proteins with high sequence identity by

versatile virtual screening technologies, and studied the structure-

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

13

Fig. 6. D03 increases dendritic branching in cultured mouse neurons. RT-qPCR analysis shows mRNA levels of CDYL target genes in mouse hippocampal cells (A) and cortical cells

B) treated with 150 M D03 for 24 h. (C, E) Representative images of hippocampal (C) or cortical (E) neurons treated with 150 M D03 at DIV 5 (day in vitro) for 24 h. Cells were

stained with MAP2 to visualize the morphology of the treated neurons. Scale bar, 50 m. (D, F) Sholl analysis of hippocampal (D) or cortical (F) neurons. Each bar represents the

mean ± S.E.M. for triplicate experiments. P-values were determined by Student's t-test. *P < 0.05.

(

m

study mainly focused on the chemical intervention of CDYL func-

tions in the cellular level, which ignored the therapeutic potential

of its inhibitors on neurological diseases. We should give more

attentions in the next step to the druggability of CDYL inhibitors,

such as their physio-chemical properties which reﬂect the

permeability and stability. In the future, these should be well

explored in order to improve the cellular and in vivo efﬁcacy.

In summary, our work on the development of chemical tools for

CDYL Kme reader provides the basis for future investigation into

the biochemical role of chromodomain-mediated signaling in hu-

man diseases.

4. Experimental Section

4.1. Chemistry

Reagents and solvents were purchased from commercial sour-

ces and used without further puriﬁcation. Reaction progress was

monitored by UV absorbance using thin-layer chromatography

(Silica gel GF254, Qingdao Haiyang Chemical Co., Ltd, Qingdao,

China). Silica column chromatography was carried out to purify

crude products (Silica gel 200e300 mesh, Shanghai Sanpont Co.,

1

13

Ltd, Shanghai, China). H NMR spectra and C NMR spectra were

14

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

Fig. 7. Induced-ﬁt binding modes for CDYL chromodomain (PDB ID: 2DNT) with B07 and D03. (A) and (B) Local interactions of CDYL with B07. (C) The overall docking model of CDYL

with B07. (D) and (E) Local interactions of CDYL with D03. (F) The overall docking model of CDYL with D03.

obtained using a Bruker Avance III spectrometer at 400 MHz

Bruker CO., Switzerland) in CDCl or DMSO‑d solution with tet-

concentrated under reduced pressure. The crude product was pu-

riﬁed by silica gel column chromatography eluting with DCM/

Methanol (20:1e10:1) to afford compounds D01-D08.

(

3

6

ramethylsilane as internal standard and chemical shift values were

given in ppm. The NMR data was processed by software MestRe-

Nova (Version 6.1.0, mestrelab research S.L.), and spectral data were

reported using the following abbreviations: (s ¼ singlet,

d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, dd ¼ doublet of

doublets); coupling constants are reported in hertz, followed by

integration. The high resolution mass (HRMS) was measured on FT-

MS-Bruker APEX IV mass spectrometer. Purity of ﬁnal compounds

4.3.1. 5-Chloro-3-hydroxy-3-(2-(3-methoxyphenyl)-2-oxoethyl)

indolin-2-one (AK-778/41314725)

1

Off-white solid. Yield: 70%. H NMR (400 MHz, DMSO‑d

6

)

d

10.38

(

5

s, 1H), 7.50 (d, J ¼ 7.6 Hz, 1H), 7.46e7.31 (m, 3H), 7.20 (td, J ¼ 6.9,

.5, 2.0 Hz, 2H), 6.81 (d, J ¼ 8.2 Hz, 1H), 6.17 (s, 1H), 4.14 (d,

13

J ¼ 18.0 Hz, 1H), 3.78 (s, 3H), 3.61 (d, J ¼ 18.0 Hz, 1H). C NMR

was determined by RP-HPLC to be >95% (UV detection (

and ELSD) using Waters Xbridge C18 column (4.6 ꢁ 250 mm 5

i.d). H O and MeCN were used as eluents (A is water, B is MeCN).

Flow rate was 0.75 mL/min, rt.

l

¼ 254 nm)

(

101 MHz, DMSO‑d 196.8, 178.5, 159.8, 142.4, 137.7, 134.4, 130.4,

6

) d

m

1

29.1, 125.6, 124.4, 121.0, 120.22, 112.6, 111.3, 73.5, 55.8, 46.3. HRMS

þ

_[_M_þ_H_]þ m/z 332.0690, found

2

(ESI-TOF ) calcd for C17

H

14ClNO

4

332.0691. Commercial source.

4

.2. General procedure 1 for the synthesis of compounds B01eB29,

4.3.2. (Z)-5-Chloro-3-(2-(3-methoxyphenyl)-2-oxoethylidene)

indolin-2-one (A01)

C01eC03

To a stirred suspension of AK-778/41314725 (0.5 mmol), glacial

To a stirred suspension of benzo [d]oxazol-2(3H)-one derivative,

or benzimidazole, or 2H-benzo [b] [1,4]oxazin-3(4H)-one

1.0 mmol), anhydrous K CO (1.2 mmol) was added in the DMF

solvent for 20 min at 70 C. Then bromoacetylated 1a-1f or 2a-2c

1.3 mmol) and TBAI (0.1 mmol) were added simultaneously. The

reaction was stirred and heated for 4 h. After completion, the

mixture was extracted with EtOAc/H O three times. The combined

organic layers were washed with brine, dried over anhydrous

Na SO , ﬁltered and concentrated under reduced pressure. The

HAc (1.0 mL) and concentrated HCl (50

neously in the EtOH solvent. The resulting mixture was heated at

90 C for 1 h. After completion, The red precipitate was ﬁltered and

mL) were added simulta-

(

2

ꢀ

3

ꢀ

washed with cold EtOH for three times. The crude product was then

1

(

dried to afford the red solid, A01. Yield: 40%. H NMR (400 MHz,

DMSO‑d₆)

d

10.95 (s, 1H), 8.15 (s, 1H), 7.79 (s, 1H), 7.69 (d, J ¼ 7.3 Hz,

2

1H), 7.55 (d, J ¼ 8.4 Hz, 2H), 7.43 (d, J ¼ 8.1 Hz,1H), 7.32 (d, J ¼ 7.9 Hz,

13

1H), 6.91 (d, J ¼ 8.2 Hz, 1H), 3.87 (s, 3H). C NMR (101 MHz,

2

4

DMSO‑d )

6

d

191.1,168.3,160.2,144.3,138.9,136.4,132.9,130.8,127.5,

þ

crude product was puriﬁed by silica gel column chromatography

eluting with EtOAc/PE (3:1e8:1) to afford compounds B01eB29,

C01eC03.

126.8, 126.0, 121.9, 121.0, 113.0, 112.3, 55.9. HRMS (ESI-TOF ) calcd

þ

for C₁₇H₁₂ClNO₃[MþH]

m/z 314.0584, found 314.0579.

HPLC:t ¼ 30.27 min, eluent A-60%, eluent B-40%.

R

4.3. General procedure 2 for the synthesis of compounds D01-D08

4.3.3. 5-Chloro-3-(2-(3-methoxyphenyl)-2-oxoethyl)indolin-2-one

(A02)

To a stirred suspension of benzo [d]oxazol-2(3H)-ones, anhy-

To the stired suspension of A01 (0.5 mmol), Pd/C (0.025 mmol)

drous K

0 min at 60 C. Then chloroacetylated 3a-3e (1.3 mmol) and TBAI

0.1 mmol) were added simultaneously. The reaction was stirred

and heated for 5 h. After completion, the mixture was extracted

with DCM/H O three times. The combined organic layers were

washed with brine, dried over anhydrous Na SO , ﬁltered and

2

CO

3

(1.2 mmol) was added in the dry DMF solvent for

was added in the anhydrous methanol solvent. The mixture was

put in the hydrogen generator under pressure (4 bars) to react

overnight in room temperature. After completion, the mixture was

ꢀ

2

(

ﬁltered and the ﬁltrate was extracted with EtOAc/H

The combined organic layers were washed with brine, dried over

anhydrous Na SO , ﬁltered and concentrated under reduced

2

O three times.

2

4

2

4

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

15

pressure. The crude product was puriﬁed by silica gel column

chromatography eluting with EtOAc/PE (1:2) to afford the off-white

4.3.8. 3-(2-(3-Methoxyphenyl)-2-oxoethyl)-2-oxo-2,3-

dihydrobenzo[d]oxazole-5-carbonitrile (B04)

1

Off-white solid. Yield: 60%. ¹H NMR (400 MHz, chloroform-d)

7.60 (d, J ¼ 7.3 Hz, 1H), 7.53e7.50 (m, 1H), 7.49 (dd, J ¼ 4.8, 3.2 Hz,

solid, A02. Yield: 90%. H NMR (400 MHz, chloroform-d)

d 8.70 (s,

1

H), 7.58e7.48 (m, 2H), 7.38 (t, J ¼ 7.9 Hz, 1H), 7.21 (s, 1H), 7.16 (dd,

d

J ¼ 16.8, 8.3 Hz, 2H), 6.84 (d, J ¼ 8.2 Hz, 1H), 4.04 (d, J ¼ 7.3 Hz, 1H),

1H), 7.34 (dd, J ¼ 8.1, 5.7 Hz, 1H), 7.24 (d, J ¼ 7.4 Hz, 2H), 7.09 (s, 1H),

13

3

.85 (s, 3H), 3.84e3.78 (m, 1H), 3.49 (dd, J ¼ 18.4, 8.4 Hz, 1H).

C

5.26 (s, 2H), 3.88 (s, 3H). C NMR (101 MHz, chloroform-d) d 189.8,

NMR (101 MHz, chloroform-d)

d

196.3, 179.8, 159.9, 140.0, 137.3,

160.2, 153.8, 145.5, 135.0, 128.0, 121.4, 120.6, 118.1, 112.4, 112.1, 111.1,

-

1

3

31.3, 129.7, 128.1, 127.9, 125.0, 120.8, 120.4, 112.2, 110.7, 55.5, 41.8,

108.0, 55.6, 48.3. HRMS (ESI-TOF ) calcd for C17

H

12

N

2

O

4

[M ꢂ H] m/

þ

9.7. HRMS (ESI-TOF ) calcd for C17

H

14ClNO

3

[MþH] m/z 316.0740,

z 304.0719, found 307.0721. HPLC:t ¼ 5.91 min, eluent A-60%,

R

found 316.0741. HPLC:t ¼ 30.28 min, eluent A-60%, eluent B-40%.

eluent B-40%.

R

4

2

.3.4. 6-Chloro-1-(2-(3-methoxyphenyl)-2-oxoethyl)-1,3-dihydro-

H-benzo[d]imidazole-2-one (A03)

To the stired suspension of 5-chloro-1,3-dihydro-2H-benzo [d]

4.3.9. 3-(2-(3,4-Dimethoxyphenyl)-2-oxoethyl)benzo[d]oxazol-

2(3H)-one (B05)

Off-white solid. Yield: 90%. ¹H NMR (400 MHz, chloroform-d)

imidazole-2-one (1.0 mmol), anhydrous K

2

CO

3

(1.0 mmol) was

d

7.69 (d, J ¼ 8.3 Hz, 1H), 7.54 (s, 1H), 7.23 (s, 1H), 7.18e7.09 (m, 2H),

added in the dry CH

3

CN solvent for 10 min. Then Boc

2

O was added

6.95 (d, J ¼ 8.4 Hz, 1H), 6.89e6.83 (m, 1H), 5.20 (s, 2H), 3.98 (s, 3H),

ꢀ

13

dropwise and reacted for 4 h at 40 C. After completion, the mixture

3.93 (s, 3H). C NMR (101 MHz, chloroform-d)

d 189.4, 154.7, 154.4,

2

was extracted with DCM/H O and the combined organic layers

149.4, 142.7, 131.2, 127.3, 124.0, 122.9, 122.7, 110.3, 110.2, 108.8, 56.2,

þ

were washed, dried. The crude intermediate was puriﬁed by silica

gel column chromatography eluting with EtOAc/PE (2:3). Then the

mono-boc form A03 was produced following the general procedure

47.7. HRMS (ESI-TOF ) calcd for C₁₇H₁₅NO₅[MþH] m/z 314.1028,

found 314.1027. HPLC:t ¼ 4.04 min, eluent A-30%, eluent B-70%.

R

1

(EtOAc/PE ¼ 1:3). At last, the boc group was removed by

4

.3.10. 3-(2-(4-(Dimethylamino)phenyl)-2-oxoethyl)benzo[d]

oxazol-2(3H)-one (B06)

Grey solid. Yield: 85%. H NMR (400 MHz, chloroform-d)

(d, J ¼ 8.8 Hz, 2H), 7.24e7.19 (m, 1H), 7.11 (p, J ¼ 7.1 Hz, 2H),

.89e6.83 (m, 1H), 6.68 (d, J ¼ 8.9 Hz, 2H), 5.14 (s, 2H), 3.09 (s, 6H).

concentrated HCl in THF at room temperature to afford the off-

white solid, A03. Yield: 30%. H NMR (400 MHz, DMSO‑d

1

6

) d 11.11

1

d

7.92

(

s, 1H), 7.68 (d, J ¼ 7.6 Hz, 1H), 7.52 (dd, J ¼ 15.8, 7.8 Hz, 2H), 7.29 (d,

13

J ¼ 8.9 Hz, 2H), 7.02 (d, J ¼ 13.8 Hz, 2H), 5.44 (s, 2H), 3.84 (s, 3H).

C

6

NMR (101 MHz, DMSO‑d

6

)

d

197.4, 179.0, 159.8, 142.5, 137.8, 132.6,

1

3

C NMR (101 MHz, chloroform-d) d 188.2, 154.8, 154.0, 142.7, 131.5,

130.4, 127.6, 125.4, 124.1, 121.0, 120.1, 112.8, 110.7, 55.8, 41.9. HRMS

þ

130.4, 123.9, 122.5, 121.9, 110.8, 110.0, 109.0, 47.4, 40.0. HRMS (ESI-

TOF ) calcd for C17

HPLC:t ¼ 6.31 min, eluent A-30%, eluent B-70%.

(

ESI-TOF ) calcd for C16

H13ClN

2

O

3

[MþH] m/z 317.0693, found

þ

H

16

N

2

O

3

[MþH] m/z 297.1239, found 297.1238.

317.1167. HPLC:t ¼ 30.20 min, eluent A-60%, eluent B-40%.

R

4

.3.5. 3-(2-(3-Methoxyphenyl)-2-oxoethyl)benzo[d]oxazol-2(3H)-

4

.3.11. 3-(2-(Naphthalen-2-yl)-2-oxoethyl)benzo[d]oxazol-2(3H)-

one (B07)

Off-white solid. Yield: 90%. ¹H NMR (400 MHz, chloroform-d)

8.59 (s, 1H), 8.09e8.00 (m, 2H), 7.94 (dd, J ¼ 17.8, 8.3 Hz, 2H), 7.64

(dt, J ¼ 22.4, 7.2 Hz, 2H), 7.27 (s, 1H), 7.15 (td, J ¼ 6.8, 5.6, 4.0 Hz, 2H),

one (B01)

Off-white solid. Yield: 80%. ¹H NMR (400 MHz, chloroform-d)

7.61 (d, J ¼ 7.6 Hz, 1H), 7.53 (s, 1H), 7.45 (t, J ¼ 7.9 Hz, 1H), 7.24 (d,

J ¼ 3.3 Hz, 1H), 7.20 (dd, J ¼ 8.2, 2.3 Hz, 1H), 7.18e7.10 (m, 2H),

d

13

6

.86e6.80 (m, 1H), 5.22 (s, 2H), 3.87 (s, 3H). C NMR (101 MHz,

13

6

.88 (dd, J ¼ 5.9, 3.0 Hz, 1H), 5.38 (s, 2H). C NMR (101 MHz,

chloroform-d)

d

190. 7, 160. 0, 142.8, 135.4, 131.1, 130.1, 124.0, 122.8,

þ

chloroform-d) 190.7, 142.8, 136.1, 132.4, 131.5, 130.2, 129.7, 129.3,

129.1, 127.9, 127.3, 124.0, 123.4, 122.8, 110.3, 108.7, 48.1. HRMS (ESI-

TOF ) calcd for C19

HPLC:t ¼ 6.11 min, eluent A-30%, eluent B-70%.

d

121.0,120.6,112.3,110.2,108.6, 55.5, 48.1. HRMS (ESI-TOF ) calcd for

þ

C

16

H

13NO

4

[MþH]

m/z

284.0923,

found

284.0922.

þ

H13NO

3

[MþH] m/z 304.0974, found 304.0973.

HPLC:t ¼ 26.31 min, eluent A-60%, eluent B-40%.

R

4

.3.6. 5-Fluoro-3-(2-(3-methoxyphenyl)-2-oxoethyl)benzo[d]

oxazol-2(3H)-one (B02)

4.3.12. 3-(2-(Naphthalen-1-yl)-2-oxoethyl)benzo[d]oxazol-2(3H)-

one (B08)

Off-white solid. Yield: 80%. ¹H NMR (400 MHz, chloroform-d)

7.59 (d, J ¼ 7.6 Hz,1H), 7.51 (s, 1H), 7.44 (t, J ¼ 7.9 Hz,1H), 7.18 (ddd,

Off-white solid. Yield: 80%. ¹H NMR (400 MHz, chloroform-d)

d

J ¼ 16.8, 8.5, 3.1 Hz, 2H), 6.82 (td, J ¼ 9.3, 2.4 Hz,1H), 6.59 (dd, J ¼ 7.7,

d

8.75 (d, J ¼ 8.4 Hz, 1H), 8.14e8.07 (m, 2H), 7.91 (d, J ¼ 7.8 Hz, 1H),

13

2

.4 Hz, 1H), 5.20 (s, 2H), 3.86 (s, 3H). C NMR (101 MHz, chloro-

7.65e7.55 (m, 3H), 7.25e7.27 (d, J ¼ 7.2 Hz, 1H), 7.15 (tt, J ¼ 7.7,

13

form-d)

d

190.3, 160.1, 158.3, 154.9, 138.6, 135.2, 132.0, 130.1, 121.1,

6.1 Hz, 2H), 6.93e6.88 (m, 1H), 5.28 (s, 2H). C NMR (101 MHz,

chloroform-d) 194.0, 154.8, 142.8, 134.6, 134.1, 131.6, 131.2, 130.4,

þ

120.6, 112.4, 110.8, 109.1, 108.9, 55.5, 48.2. HRMS (ESI-TOF ) calcd

d

þ

for

C

16

H12FNO

4

[MþH]

m/z 302.0829, found 302.0811.

128.6, 127.0, 125.6, 124.3, 124.0, 122.8, 110.3, 108.5, 49.7. HRMS (ESI-

þ

HPLC:t ¼ 26.04 min, eluent A-60%, eluent B-40%.

TOF ) calcd for C19

H13NO

3

[MþH] m/z 304.0974, found 304.0975.

R

HPLC:t ¼ 21.10 min, eluent A-60%, eluent B-40%.

R

4.3.7. 5-Methoxy-3-(2-(3-methoxyphenyl)-2-oxoethyl)benzo[d]

oxazol-2(3H)-one (B03)

4.3.13. 3-(2-(1H-indol-3-yl)-2-oxoethyl)benzo[d]oxazol-2(3H)-one

Off-white solid. Yield: 85%. ¹H NMR (400 MHz, chloroform-d)

(B09)

1

d

7.61 (d, J ¼ 7.7 Hz, 1H), 7.53 (s, 1H), 7.45 (t, J ¼ 8.0 Hz, 1H), 7.20 (dd,

Off-white solid. Yield: 70%. H NMR (400 MHz, DMSO‑d

6

)

d

12.24

J ¼ 8.1, 2.3 Hz, 1H), 7.13 (d, J ¼ 8.8 Hz, 1H), 6.63 (dd, J ¼ 8.8, 2.5 Hz,

(s, 1H), 8.61 (s, 1H), 8.07 (d, J ¼ 7.6 Hz, 1H), 7.52 (d, J ¼ 7.9 Hz, 1H),

13

1

H), 6.39 (d, J ¼ 2.5 Hz, 1H), 5.19 (s, 2H), 3.87 (s, 3H), 3.77 (s, 3H).

C

7.39 (d, J ¼ 7.4 Hz,1H), 7.19 (dt, J ¼ 16.2, 10.0 Hz, 5H), 5.35 (s, 2H).

C

NMR (101 MHz, chloroform-d)

d

190.6, 160.1, 156.8, 155.0, 136.9,

NMR (101 MHz, DMSO‑d 186.8, 154.8, 142.5, 137.0, 135.2, 132.3,

6

) d

1

4

35.4, 131.9, 130.1, 121.0, 120.6, 112.4, 110.4, 107.2, 96.0, 56.0, 55.5,

125.7, 124.4, 123.6, 122.7, 122.6, 121.4, 113.9, 112.8, 110.1, 109.9, 48.1.

þ

8.2. HRMS (ESI-TOF ) calcd for C17

H15NO

5

[MþH] m/z 314.1027,

HRMS (ESI-TOF ) calcd for C17

H

12

N

2

O

3

[MþH] m/z 293.0926,

found 314.1028. HPLC:t ¼ 26.00 min, eluent A-60%, eluent B-40%.

found 293.0921. HPLC:t ¼ 4.35 min, eluent A-30%, eluent B-70%.

R

16

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

4.3.14. 3-(2-(3,4-Dimethoxyphenyl)-2-oxoethyl)-5-ﬂuorobenzo[d]

4.3.20. 5-Methoxy-3-(2-(naphthalen-2-yl)-2-oxoethyl)benzo[d]

oxazol-2(3H)-one (B10)

oxazol-2(3H)-one (B16)

Off-white solid. Yield: 90%. ¹H NMR (400 MHz, chloroform-d)

Off-white solid. Yield: 80%. ¹H NMR (400 MHz, chloroform-d)

d

6

1

7.66 (d, J ¼ 8.2 Hz, 1H), 7.52 (s, 1H), 7.16 (dd, J ¼ 8.6, 4.0 Hz, 1H),

d

8.58 (s, 1H), 8.02 (t, J ¼ 9.8 Hz, 2H), 7.93 (dd, J ¼ 15.7, 8.4 Hz, 2H),

.94 (d, J ¼ 8.4 Hz, 1H), 6.81 (t, J ¼ 8.8 Hz, 1H), 6.62 (d, J ¼ 6.2 Hz,

7.63 (dt, J ¼ 22.8, 7.0 Hz, 2H), 7.13 (d, J ¼ 8.8 Hz, 1H), 6.63 (dd, J ¼ 8.8,

13

H), 5.18 (s, 2H), 3.97 (s, 3H), 3.93 (s, 3H). C NMR (101 MHz,

2.2 Hz,1H), 6.44 (d, J ¼ 2.2 Hz,1H), 5.34 (s, 2H), 3.76 (s, 3H). C NMR

chloroform-d)

d

189.0, 160.7, 158.3, 155.0, 154.5, 149.5, 138.6, 131.9,

(101 MHz, chloroform-d) d 190.7, 156.8, 155.3, 136.9, 136.1, 132.4,

1

27.1, 123.0, 110.7, 110.1, 109.1, 108.8, 97.7, 56.1, 47.8. HRMS (ESI-

131.9, 131.5, 130.3, 129.7, 129.2, 129.1, 127.9, 127.3, 123.4, 110.4, 107.3,

þ

TOF ) calcd for C17

HPLC:t ¼ 7.19 min, eluent A-30%, eluent B-70%.

H14FNO

5

[MþH] m/z 332.0934, found 332.0932.

96.0, 56.0, 48.1. HRMS (ESI-TOF ) calcd for C20

H

15NO

4

[MþH] m/z

334.1079, found 334.1082. HPLC:t ¼ 21.89 min, eluent A-60%,

R

eluent B-40%.

4.3.15. 3-(2-(4-(Dimethylamino)phenyl)-2-oxoethyl)-5-

ﬂuorobenzo[d]oxazol-2(3H)-one (B11)

4.3.21. 3-(2-(1H-indol-3-yl)-2-oxoethyl)-5-methoxybenzo[d]

1

Grey solid. Yield: 65%. H NMR (400 MHz, chloroform-d)

d

7.91

oxazol-2(3H)-one (B17)

1

(

2

d, J ¼ 8.9 Hz, 2H), 7.15 (dd, J ¼ 8.7, 4.1 Hz, 1H), 6.80 (td, J ¼ 9.2,

Off-white solid. Yield: 60%. H NMR (400 MHz, DMSO‑d

6

)

d

12.20

.3 Hz, 1H), 6.69 (d, J ¼ 8.9 Hz, 2H), 6.62 (dd, J ¼ 7.7, 2.3 Hz, 1H), 5.13

(s, 1H), 8.59 (s, 1H), 8.08 (d, J ¼ 7.6 Hz, 1H), 7.52 (d, J ¼ 7.8 Hz, 1H),

13

(

s, 2H), 3.10 (s, 6H). C NMR (101 MHz, chloroform-d) d 187.8, 160.7,

7.31e7.17 (m, 3H), 6.95 (s, 1H), 6.67 (d, J ¼ 8.6 Hz, 1H), 5.34 (s, 2H),

13

158.3, 155.1, 154.1, 138.6, 132.3, 130.5, 121.7, 110.9, 110.5, 108.8, 108.6,

6

3.71 (s, 3H). C NMR (101 MHz, DMSO‑d ) d 186.8, 156.9, 155.4,

þ

9

7.8, 97.5, 47.5, 40.0. HRMS (ESI-TOF ) calcd for C17

H

15FN

2

O

3

136.9, 136.5, 135.1, 125.7, 123.6, 122.6, 121.5, 113.9, 112.8, 110.5, 107.7,

þ

[MþH] m/z 315.1145, found 315.1132. HPLC:t ¼ 5.34 min, eluent

96.7, 56.3, 48.1. HRMS (ESI-TOF ) calcd for C18

14

H N

2

O

4

[MþH] m/z

R

A-30%, eluent B-70%.

323.1032, found 323.1026. HPLC:t ¼ 23.67 min, eluent A-60%,

R

eluent B-40%.

4.3.16. 5-Fluoro-3-(2-(naphthalen-2-yl)-2-oxoethyl)benzo[d]

oxazol-2(3H)-one (B12)

4.3.22. 5-Bromo-3-(2-(naphthalen-2-yl)-2-oxoethyl)benzo[d]

Off-white solid. Yield: 86%. ¹H NMR (400 MHz, chloroform-d)

oxazol-2(3H)-one (B18)

1

d

8.58 (s, 1H), 8.07e7.90 (m, 4H), 7.65 (dt, J ¼ 23.0, 7.1 Hz, 2H), 7.19

Brown solid. Yield: 90%. H NMR (400 MHz, chloroform-d)

d

8.58

(

dd, J ¼ 8.8, 4.1 Hz, 1H), 6.84 (td, J ¼ 9.2, 2.5 Hz, 1H), 6.64 (dd, J ¼ 7.7,

(s, 1H), 7.99 (ddd, J ¼ 27.8, 18.4, 8.5 Hz, 4H), 7.65 (dt, J ¼ 23.1, 6.9 Hz,

.5 Hz, 1H), 5.37 (s, 2H). ¹C NMR (101 MHz, chloroform-d)

3

d 190.4,

2H), 7.29 (d, J ¼ 1.9 Hz, 1H), 7.14 (d, J ¼ 8.5 Hz, 1H), 7.01 (d, J ¼ 1.8 Hz,

2

13

160,7, 155.0, 136.2, 132.4, 131.3, 130.3, 129.7, 129.4, 129.2, 128.0,

1H), 5.36 (s, 2H). C NMR (101 MHz, chloroform-d) d 190.2, 154.3,

þ

127.3, 123.3, 110.8, 109.1, 108.9, 97.6, 97.3, 48.2. HRMS (ESI-TOF )

141.8, 136.2, 132.5, 132.4, 131.3, 130.3, 129.7, 129.4, 129.2, 128.0,

þ

calcd for C19

H12FNO

3

[MþH] m/z 322.0879, found 322.0876.

128.0, 127.4, 125.7, 123.3, 116.6, 112.0, 111.5, 48.2. HRMS (ESI-TOF )

þ

HPLC:t ¼ 5.31 min, eluent A-30%, eluent B-70%.

calcd for C19

H

12BrNO

3

[MþH] m/z 382.0079, found 382.0078.

R

HPLC:t ¼ 23.52 min, eluent A-60%, eluent B-40%.

R

4.3.17. 3-(2-(1H-indol-3-yl)-2-oxoethyl)-5-ﬂuorobenzo[d]oxazol-

2

(3H)-one (B13)

Off-white solid. Yield: 50%. H NMR (400 MHz, DMSO‑d

4.3.23. 3-(2-(Naphthalen-2-yl)-2-oxoethyl)-2-oxo-2,3-

dihydrobenzo[d]oxazole-5-carbonitrile (B19)

1

6

)

d

12.21

(

s, 1H), 8.59 (d, J ¼ 2.5 Hz, 1H), 8.07 (d, J ¼ 7.6 Hz, 1H), 7.52 (d,

Off-white solid. Yield: 40%. ¹H NMR (400 MHz, chloroform-d)

J ¼ 7.9 Hz, 1H), 7.42 (dd, J ¼ 8.8, 4.2 Hz, 1H), 7.31e7.17 (m, 3H),

d

8.57 (s, 1H), 8.06e7.89 (m, 4H), 7.66 (dt, J ¼ 23.7, 7.1 Hz, 2H), 7.49

13

7

.01e6.92 (m, 1H), 5.35 (s, 2H). C NMR (101 MHz, DMSO‑d

6

)

(d, J ¼ 7.3 Hz,1H), 7.34 (d, J ¼ 8.3 Hz,1H), 7.15 (s,1H), 5.42 (s, 2H).

NMR (101 MHz, chloroform-d) 189.9, 153.8, 145.5, 136.2, 132.4,

132.0,131.1,130.4,129.7,129.5,129.3,128.0,127.4,123.2,118.2,112.2,

C

d

186.5, 160.5, 158.2, 155.1, 138.6, 136.9, 135.2, 125.7, 123.7, 122.6,

d

þ

121.5, 113.8, 112.8, 108.8, 108.6, 98.7, 98.4, 48.3. HRMS (ESI-TOF )

þ

calcd for C17

HPLC:t ¼ 24.27 min, eluent A-60%, eluent B-40%.

H11FN

2

O

3

[MþH] m/z 311.0833, found 311.0833.

111.0, 108.0, 48.3. HRMS (ESI-TOF ) calcd for C20

H

12

N

2

O

3

[MþH]

m/z 329.0926, found 329.0925. HPLC:t ¼ 26.06 min, eluent A-60%,

R

eluent B-40%.

4.3.18. 3-(2-(3,4-Dimethoxyphenyl)-2-oxoethyl)-5-methoxybenzo

[

d]oxazol-2(3H)-one (B14)

4.3.24. 6-Bromo-3-(2-(naphthalen-2-yl)-2-oxoethyl)benzo[d]

Off-white solid. Yield: 95%. ¹H NMR (400 MHz, chloroform-d)

oxazol-2(3H)-one (B20)

1

d

7.68 (dd, J ¼ 8.4, 1.6 Hz, 1H), 7.54 (d, J ¼ 1.4 Hz, 1H), 7.12 (d,

Light yellow solid. Yield: 90%. H NMR (400 MHz, chloroform-d)

J ¼ 8.8 Hz, 1H), 6.95 (d, J ¼ 8.4 Hz, 1H), 6.62 (dd, J ¼ 8.8, 2.4 Hz, 1H),

d

8.57 (s, 1H), 7.98 (dd, J ¼ 16.9, 9.1 Hz, 4H), 7.65 (dt, J ¼ 22.1, 6.9 Hz,

6

3

1

4

.43 (d, J ¼ 2.4 Hz, 1H), 5.16 (s, 2H), 3.97 (s, 3H), 3.93 (s, 3H), 3.76 (s,

2H), 7.43 (d, J ¼ 1.7 Hz, 1H), 7.30 (dd, J ¼ 8.3, 1.7 Hz, 1H), 6.75 (d,

13

H). C NMR (101 MHz, chloroform-d)

d

189.4, 156.8, 155.3, 154.3,

J ¼ 8.3 Hz, 1H), 5.37 (s, 2H). C NMR (101 MHz, chloroform-d)

49.4, 136.8, 131.9, 127.3, 122.9, 110.4, 107.2, 96.1, 56.2, 56.1, 56.0,

d 190.2, 154.3, 141.8, 136.2, 132.5, 132.4, 131.3, 130.3, 129.7, 129.4,

þ

7.8. HRMS (ESI-TOF ) calcd for C18

H17NO

6

[MþH] m/z 344.1132,

129.2,128.0,127.4, 125.7, 123.3,116.62,112.0, 111.5, 48.2. HRMS (ESI-

þ

found 344.1134. HPLC:t ¼ 24.37 min, eluent A-60%, eluent B-40%.

TOF ) calcd for

C

19

H

12BrNO

3

[MþH] m/z 382.0079, found

R

3

82.0081. HPLC:t ¼ 23.19 min, eluent A-60%, eluent B-40%.

R

4

.3.19. 3-(2-(4-(Dimethylamino)phenyl)-2-oxoethyl)-5-

methoxybenzo[d]oxazol-2(3H)-one (B15)

4.3.25. 6-Amino-3-(2-(naphthalen-2-yl)-2-oxoethyl)benzo[d]

Off-white solid. Yield: 87%. ¹H NMR (400 MHz, chloroform-d)

thiazol-2(3H)-one (B21)

1

d

2

3

7.92 (d, J ¼ 8.8 Hz, 2H), 7.10 (d, J ¼ 8.8 Hz, 1H), 6.68 (d, J ¼ 8.9 Hz,

Off-white solid. Yield: 20%. H NMR (400 MHz, DMSO‑d

6

)

d

8.89

H), 6.60 (dd, J ¼ 8.7, 2.3 Hz, 1H), 6.43 (d, J ¼ 2.2 Hz, 1H), 5.11 (s, 2H),

(s, 1H), 8.17 (s, 2H), 8.05 (dt, J ¼ 17.1, 8.7 Hz, 4H), 6.96 (d, J ¼ 8.7 Hz,

13

.75 (s, 3H), 3.09 (s, 6H). C NMR (101 MHz, chloroform-d) d 188.2,

1H), 6.82 (d, J ¼ 1.8 Hz, 1H), 6.53 (d, J ¼ 10.4 Hz, 1H), 5.65 (s, 2H),

13

1

5

3

56.8, 155.4, 154.0, 136.8, 132.2, 130.5, 121.9, 110.8, 110.2, 107.2, 96.1,

6.0, 47.4, 40.0. HRMS (ESI-TOF ) calcd for C18

6

5.05 (s, 2H). C NMR (101 MHz, DMSO‑d ) d 192.9, 168.9, 145.9,

þ

H

18

N

2

O

4

[MþH] m/z

135.9, 132.5, 132.1, 131.1, 130.1, 129.6,129.0, 128.3, 128.0, 127.7, 123.8,

þ

27.1245, found 327.1243. HPLC:t ¼ 4.31 min, eluent A-30%, eluent

122.4, 113.3, 112.4, 107.7, 49.3. HRMS (ESI-TOF ) calcd for

R

B-70%.

C

19

H

14

N

2

O

2

S

[MþH]^þ

m/z

335.0854,

found

335.0851.

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

17

HPLC:t ¼ 19.12 min, eluent A-60%, eluent B-40%.

128.8, 124.1, 122.9, 120.5, 117.1, 114.7, 112.4, 110.0, 67.5, 55.5, 48.0.

R

þ

HRMS (ESI-TOF ) calcd for C17

H

15NO

4

[MþH] m/z 298.1079, found

4.3.26. 3-(2-(Naphthalen-2-yl)-2-oxoethyl)-6-nitrobenzo[d]

298.1081. HPLC:t ¼ 19.10 min, eluent A-60%, eluent B-40%.

R

thiazol-2(3H)-one (B22)

Yellow solid. Yield: 70%. ¹H NMR (400 MHz, chloroform-d

)

4.3.32. 4-(2-(Naphthalen-2-yl)-2-oxoethyl)-2H-benzo[b] [1,4]

oxazin-3(4H)-one (B28)

6

d

8

1

8.60 (s, 1H), 8.41 (d, J ¼ 2.3 Hz, 1H), 8.19 (dd, J ¼ 8.9, 2.3 Hz, 1H),

.06e7.90 (m, 4H), 7.66 (dt, J ¼ 22.3, 7.5 Hz, 2H), 6.96 (d, J ¼ 8.9 Hz,

H), 5.59 (s, 2H). ¹³C NMR (101 MHz, chloroform-d

190.0, 169.9,

43.7, 141.9, 136.2, 132.4, 131.3, 130.3, 129.7, 129.5, 129.3, 128.0,

Off-white solid. Yield: 40%. ¹H NMR (400 MHz, chloroform-d)

6

)

d

8.58 (s, 1H), 7.98 (dd, J ¼ 18.6, 8.0 Hz, 4H), 7.62 (dd, J ¼ 15.5, 7.4 Hz,

1

2H), 7.09e6.97 (m, 2H), 6.94 (d, J ¼ 7.0 Hz, 1H), 6.67 (d, J ¼ 7.3 Hz,

-

13

1

for

27.4, 123.4, 123.3, 122.8, 118.8, 110.4, 48.9. HRMS (ESI-TOF ) calcd

1H), 5.48 (s, 2H), 4.75 (s, 2H). C NMR (101 MHz, Chloroform-d)

-

C

19

H

12

N

2

O

4

S

[M ꢂ H] m/z 363.0440, found 363.0439.

d 191.8, 165.2, 145.2, 136.0, 132.4, 131.9, 130.0, 129.7, 129.1, 129.0,

HPLC:t ¼ 25.06 min, eluent A-60%, eluent B-40%.

127.9, 127.2, 124.1, 123.5, 122.9, 117.1, 114.8, 67.6, 48.0. HRMS (ESI-

R

þ

TOF ) calcd for C20

H15NO

3

[MþH] m/z 318.1130, found 318.1129.

4.3.27. 3-(2-(1H-indol-3-yl)-2-oxoethyl)-2-oxo-2,3-dihydrobenzo

HPLC:t ¼ 20.85 min, eluent A-60%, eluent B-40%.

R

[

d]oxazole-5-carbonitrile (B23)

1

Off-white solid. Yield: 30%. H NMR (400 MHz, DMSO‑d

s,1H), 8.60 (s, 1H), 8.07 (d, J ¼ 7.6 Hz,1H), 7.88 (s,1H), 7.71e7.60 (m,

H), 7.53 (d, J ¼ 7.9 Hz, 1H), 7.22 (dt, J ¼ 20.1, 7.1 Hz, 2H), 5.40 (s, 2H).

6

)

d

12.23

4.3.33. 4-(2-(1H-indol-3-yl)-2-oxoethyl)-2H-benzo[b] [1,4]oxazin-

(

3(4H)-one (B29)

1

2

Off-white solid. Yield: 20%. H NMR (400 MHz, DMSO‑d

6

)

d

12.16

1

3

C NMR (101 MHz, DMSO‑d

6

)

d

186.3, 154.3, 145.5, 136.9, 135.3,

(s, 1H), 8.62 (d, J ¼ 3.1 Hz, 1H), 8.09 (d, J ¼ 7.6 Hz, 1H), 7.51 (d,

1

33.3, 128.2, 125.7, 123.7, 122.7, 121.4, 119.0, 113.7, 113.5, 112.8, 111.4,

J ¼ 8.0 Hz, 1H), 7.21 (dt, J ¼ 20.6, 6.8 Hz, 2H), 7.04 (d, J ¼ 8.0 Hz, 1H),

þ

13

107.0, 48.4. HRMS (ESI-TOF ) calcd for C18

H

11

N

3

O

3

[MþH] m/z

6.97 (d, J ¼ 15.5 Hz, 3H), 5.36 (s, 2H), 4.74 (s, 2H). C NMR (101 MHz,

3

18.0879, found 318.0882. HPLC:t ¼ 25.10 min, eluent A-60%,

6

DMSO‑d ) d 187.6, 165.0, 145.1, 136.9, 134.8, 129.6, 125.8, 123.9,

R

eluent B-40%.

123.5, 123.1, 122.5, 121.5, 117.0, 115.9, 114.1, 112.8, 67.4, 47.6. HRMS

þ

(

ESI-TOF ) calcd for C18

14

H N

2

O

3

[MþH] m/z 305.0026, found

4

2

.3.28. 3-(2-(1H-indol-3-yl)-2-oxoethyl)-6-aminobenzo[d]thiazol-

(3H)-one (B24)

305.0921. HPLC:t ¼ 23.73 min, eluent A-60%, eluent B-40%.

R

Light yellow solid. Yield: 60%. ¹H NMR (400 MHz, DMSO‑d

)

4.3.34. N-(3-methoxyphenyl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl)

6

d

1

12.16 (s, 1H), 8.61 (s, 1H), 8.08 (d, J ¼ 7.5 Hz, 1H), 7.51 (d, J ¼ 8.0 Hz,

acetamide (C01)

1

H), 7.21 (dt, J ¼ 19.7, 7.1 Hz, 2H), 6.86 (d, J ¼ 8.6 Hz, 1H), 6.81 (d,

Off-white solid. Yield: 70%. H NMR (400 MHz, DMSO‑d

6

)

d

10.42

J ¼ 2.1 Hz, 1H), 6.52 (dd, J ¼ 8.6, 2.1 Hz, 1H), 5.30 (s, 2H), 5.01 (s, 2H).

(s, 1H), 7.37 (d, J ¼ 7.9 Hz, 1H), 7.28 (dd, J ¼ 4.4, 2.0 Hz, 2H), 7.21 (td,

13

6

C NMR (101 MHz, DMSO‑d ) d 187.2, 168.9, 145.8, 136.9, 135.0,

J ¼ 7.8, 3.7 Hz, 2H), 7.18e7.06 (m, 2H), 6.65 (dd, J ¼ 8.2, 1.9 Hz, 1H),

13

1

4

3

28.3, 125.7, 123.6, 122.6, 122.4, 121.5, 114.1, 113.2, 112.8, 112.3, 107.7,

6

4.71 (s, 2H), 3.70 (s, 3H). C NMR (101 MHz, DMSO‑d ) d 165.2,

þ

8.7. HRMS (ESI-TOF ) calcd for

C

17

13

H N

3

O

2

S

[MþH] m/z

159.8,154.6,142.4,140.1,132.0,130.2,124.4,122.8,111.9,110.2,109.7,

105.3, 55.1, 45.1. HRMS (ESI-TOF ) calcd for C16

þ

24.0807, found 324.0801. HPLC:t ¼ 25.22 min, eluent A-60%,

H

14

N

2

O

4

[MþH] m/

R

eluent B-40%.

z 299.0561, found 299.0564. HPLC:t ¼ 25.19 min, eluent A-60%,

R

eluent B-40%.

4

2

.3.29. 3-(2-(1H-indol-3-yl)-2-oxoethyl)-6-nitrobenzo[d]thiazol-

(3H)-one (B25)

Yellow solid. Yield: 50%. H NMR (400 MHz, DMSO‑d

4.3.35. N-(naphthalen-2-yl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl)

1

6

)

d

12.24 (s,

acetamide (C02)

1

2

H), 8.79 (d, J ¼ 2.1 Hz, 1H), 8.65 (d, J ¼ 3.0 Hz, 1H), 8.22 (dd, J ¼ 9.0,

Off-white solid. Yield: 75%. H NMR (400 MHz, DMSO‑d

6

)

d

10.66

.1 Hz, 1H), 8.05 (d, J ¼ 7.7 Hz, 1H), 7.50 (dd, J ¼ 20.3, 8.5 Hz, 2H),

(s, 1H), 8.26 (s, 1H), 7.88 (d, J ¼ 8.7 Hz, 1H), 7.84 (d, J ¼ 7.8 Hz, 1H),

13

7

.22 (dt, J ¼ 22.1, 7.2 Hz, 2H), 5.58 (s, 2H). C NMR (101 MHz,

7.79 (d, J ¼ 8.3 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.50e7.43 (m, 1H),

DMSO‑d

6

)

d

186.2, 170.3, 143.4, 136.9, 135.5, 125.6, 123.7, 123.3,

7.41 (d, J ¼ 7.8 Hz, 2H), 7.32 (d, J ¼ 7.3 Hz, 1H), 7.22 (t, J ¼ 7.4 Hz, 1H),

þ

13

122.8, 122.7, 121.4, 119.8, 113.9, 112.8, 112.1, 49.3. HRMS (ESI-TOF )

7.16 (d, J ¼ 7.8 Hz, 1H), 4.79 (s, 2H). C NMR (101 MHz, DMSO‑d

6

)

þ

calcd for C17

HPLC:t ¼ 25.24 min, eluent A-60%, eluent B-40%.

11

H N

3

O

4

S [MþH] m/z 354.0549, found 354.0548.

d 168.7, 149.9, 134.5, 130.6, 129.6, 128.8, 128.6, 127.1, 126.8, 125.4,

þ

125.1, 122.6, 121.8, 121.5, 120.8, 52.0. HRMS (ESI-TOF ) calcd for

R

þ

C

19

H

14

N

2

O

3

[MþH]

m/z

319.1083,

found

319.1087.

4

.3.30. 2-(1H-benzo[d]imidazole-1-yl)-1-(3-methoxyphenyl)

HPLC:t ¼ 21.74 min, eluent A-60%, eluent B-40%.

R

ethan-1-one (B26)

Off-white solid. Yield: 60%. ¹H NMR (400 MHz, chloroform-d)

8.00 (s, 1H), 7.86e7.80 (m, 1H), 7.55 (d, J ¼ 7.6 Hz, 1H), 7.46 (s, 1H),

4.3.36. N-(naphthalen-1-yl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl)

acetamide (C03)

d

7

5

.40 (t, J ¼ 7.9 Hz, 1H), 7.23 (d, J ¼ 3.9 Hz, 2H), 7.20e7.14 (m, 2H),

Off-white solid. Yield: 75%. ¹H NMR (400 MHz, chloroform-d)

13

.51 (s, 2H), 3.80 (s, 3H). C NMR (101 MHz, chloroform-d)

d

191.2,

d

8.01 (d, J ¼ 8.2 Hz, 1H), 7.99e7.92 (m, 1H), 7.69 (t, J ¼ 9.5 Hz, 2H),

160.1,144.1,142.6,135.4,134.1,130.1,123.6,123.3,122.7, 120.9,120.5,

7.63e7.55 (m, 3H), 7.51 (d, J ¼ 7.3 Hz, 1H), 7.23e7.20 (m, 1H), 7.10 (d,

þ

13

120.0, 112.4, 109.6, 55.5, 50.7. HRMS (ESI-TOF ) calcd for

J ¼ 8.0 Hz, 1H), 7.04 (t, J ¼ 7.7 Hz, 1H), 4.75 (q, J ¼ 17.7 Hz, 2H).

C

þ

C

H

16 14

N

2

O

2

[MþH]

m/z

267.1134,

found

267.1132.

NMR (101 MHz, chloroform-d) 168.7, 149.9, 134.5, 128.8, 128.6,

d

þ

HPLC:t ¼ 7.11 min, eluent A-30%, eluent B-70%.

127.5, 126.8, 125.4, 122.6, 121.8, 121.5, 120.8, 52.0. HRMS (ESI-TOF )

R

þ

calcd for C19

H

14

N

2

O

3

[MþH] m/z 319.1083, found 319.1086.

4

.3.31. 4-(2-(3-Methoxyphenyl)-2-oxoethyl)-2H-benzo[b] [1,4]

HPLC:t ¼ 21.70 min, eluent A-60%, eluent B-40%.

R

oxazin-3(4H)-one (B27)

Off-white solid. Yield: 40%. ¹H NMR (400 MHz, chloroform-d)

4.3.37. 3-(2-(4-(Dimethylamino)piperidin-1-yl)-2-oxoethyl)benzo

d

7.62 (d, J ¼ 7.5 Hz, 1H), 7.54 (s, 1H), 7.44 (t, J ¼ 7.9 Hz, 1H), 7.19 (d,

[d]oxazol-2(3H)-one (D01)

1

J ¼ 8.1 Hz, 1H), 7.00 (q, J ¼ 7.8 Hz, 2H), 6.93 (t, J ¼ 7.5 Hz, 1H), 6.62 (d,

Off-white solid. Yield: 40%. H NMR (400 MHz, DMSO‑d

6

)

d

7.34

13

J ¼ 7.8 Hz, 1H), 5.33 (s, 2H), 4.72 (s, 2H), 3.86 (s, 3H). C NMR

101 MHz, chloroform-d) 191.7, 165.1, 160.1, 145.1, 135.8, 130.0,

(d, J ¼ 7.7 Hz, 1H), 7.19 (d, J ¼ 0.7 Hz, 1H), 7.19e7.17 (m, 1H),

(

d

7.14e7.08 (m, 1H), 4.87e4.73 (m, 2H), 4.33e4.23 (m, 1H), 3.94 (d,

18

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

J ¼ 12.1 Hz, 1H), 3.19e3.01 (m, 2H), 2.59 (t, J ¼ 13.8 Hz, 1H), 2.48 (s,

J ¼ 24.5, 12.9 Hz, 3H), 1.06 (t, J ¼ 7.0 Hz, 6H), 0.92e0.78 (m, 1H). ¹³C

13

6

H), 1.83e1.68 (m, 2H), 1.41e1.34 (m, 2H). C NMR (101 MHz,

NMR (101 MHz, chloroform-d) d 163.0, 160.7, 158.1, 153.3, 138.5,

DMSO‑d

6

)

d

163.9, 154.7, 142.4, 132.1, 124.2, 110.0, 61.8, 50.1, 44.09,

132.1,110.6,108.8, 57.6, 44.9, 43.5, 42.2, 29.16, 28.0,13.5. HRMS (ESI-

þ

4

3

3.4, 23.5, 14.0. HRMS (ESI-TOF ) calcd for C16

04.1661, found 304.1655. HPLC:t ¼ 23.66 min, eluent A-60%,

H

21

N

3

O

3

[MþH] m/z

TOF ) calcd for C18

H24FN

3

O

3

[MþH] m/z 350.1880, found 350.1874.

HPLC:t ¼ 23.81 min, eluent A-60%, eluent B-40%.

R

eluent B-40%.

4.3.43. 5-Methoxy-3-(2-oxo-2-(4-(pyrrolidin-1-yl)piperidin-1-yl)

4

.3.38. 3-(2-(4-(Diethylamino)piperidin-1-yl)-2-oxoethyl)benzo[d]

oxazol-2(3H)-one (D02)

Off-white solid. Yield: 45%. H NMR (400 MHz, DMSO‑d

ethyl)benzo[d]oxazol-2(3H)-one (D07)

Off-white solid. Yield: 25%. ¹H NMR (400 MHz, chloroform-d)

1

6

)

d

7.34

d

7.08 (d, J ¼ 8.6 Hz, 1H), 6.61 (dd, J ¼ 8.7, 2.5 Hz, 1H), 6.58 (d,

(

d, J ¼ 7.7 Hz, 1H), 7.19 (d, J ¼ 6.8 Hz, 2H), 7.13 (q, J ¼ 8.7, 7.0 Hz, 1H),

J ¼ 2.5 Hz, 1H), 4.58 (d, J ¼ 5.6 Hz, 2H), 4.40 (d, J ¼ 13.5 Hz, 1H), 3.92

(d, J ¼ 13.7 Hz,1H), 3.79 (s, 3H), 3.39e3.29 (m,1H), 3.20 (dd, J ¼ 14.1,

11.6, 2.9 Hz, 1H), 2.89e2.78 (m, 1H), 2.63 (d, J ¼ 6.3 Hz, 4H), 2.36 (s,

4

1

.81 (q, J ¼ 17.0 Hz, 2H), 4.30 (d, J ¼ 12.3 Hz, 1H), 3.94 (d, J ¼ 12.7 Hz,

H), 3.08 (t, J ¼ 12.6 Hz, 1H), 2.73 (d, J ¼ 9.0 Hz, 1H), 2.64e2.56 (m,

H), 2.49 (s, 4H), 1.79e1.63 (m, 2H), 1.35e1.14 (m, 2H), 0.94 (d,

1H), 1.98 (dd, J ¼ 32.1, 13.1 Hz, 2H), 1.86e1.78 (m, 4H), 1.00 (t,

13

J ¼ 11.7 Hz, 6H). C NMR (101 MHz, Chloroform-d)

d

163.3, 154.6,

J ¼ 7.3 Hz, 1H). C NMR (101 MHz, chloroform-d)

d 163.4, 156.9,

142.6, 131.2, 124.0, 122.9, 110.0, 109.1, 57.6, 43.6, 42.2, 29.1, 27.9, 13.6.

155.0, 136.7, 131.9, 110.3, 107.5, 96.3, 61.12, 56.0, 51.4, 43.7, 40.9, 31.5,

þ

HRMS (ESI-TOF ) calcd for C18

found 332.1974. HPLC:t ¼ 21.73 min, eluent A-60%, eluent B-40%.

H

25

N

3

O

3

[MþH] m/z 332.1974,

23.2. HRMS (ESI-TOF ) calcd for C19

H

25

N

3

O

4

[MþH] m/z 360.1923,

found 360.1918. HPLC:t ¼ 30.14 min, eluent A-60%, eluent B-40%.

R

4.3.39. 3-(2-Oxo-2-(4-(pyrrolidin-1-yl)piperidin-1-yl)ethyl)benzo

4.3.44. 5-Nitro-3-(2-oxo-2-(4-(pyrrolidin-1-yl)piperidin-1-yl)

[

d]oxazol-2(3H)-one (D03)

ethyl)benzo[d]oxazol-2(3H)-one (D08)

Off-white solid. Yield: 30%. ¹H NMR (400 MHz, chloroform-d)

Brown solid. Yield: 30%. H NMR (400 MHz, chloroform-d)

1

d

8.17

d

7.21 (d, J ¼ 6.8 Hz, 1H), 7.18e7.13 (m, 1H), 7.13e7.08 (m, 1H), 7.00

(d, J ¼ 7.9 Hz, 1H), 8.10 (s, 1H), 7.08 (d, J ¼ 8.5 Hz, 1H), 4.76e4.64 (m,

2H), 4.36 (d, J ¼ 13.6 Hz, 1H), 3.88 (d, J ¼ 13.3 Hz, 1H), 3.35e3.19 (m,

2H), 2.88 (t, J ¼ 12.2 Hz, 1H), 2.65 (s, 4H), 2.43e2.27 (m, 2H), 2.00

(

3

d, J ¼ 7.8 Hz, 1H), 4.62 (d, J ¼ 3.1 Hz, 2H), 4.39 (d, J ¼ 12.8 Hz, 1H),

.90 (s, 1H), 3.20 (t, J ¼ 11.2 Hz, 1H), 2.85 (t, J ¼ 11.0 Hz, 1H), 2.58 (s,

H), 2.33e2.22 (m, 1H), 1.96 (dd, J ¼ 31.8, 10.5 Hz, 2H), 1.79 (d,

13

4

(dd, J ¼ 36.9,10.3 Hz, 3H),1.65 (d, J ¼ 9.1 Hz, 3H). C NMR (101 MHz,

13

J ¼ 6.4 Hz, 4H), 1.50 (dd, J ¼ 18.3, 11.7 Hz, 2H). C NMR (101 MHz,

chloroform-d)

d

162.5, 154.1, 143.3, 141.9, 136.9, 121.0, 108.8, 106.3,

þ

chloroform-d)

d

163.4, 154.6, 142.6, 131.2, 124.0, 122.7, 110.1, 109.3,

60.9, 51.4, 43.6, 41.0, 31.5, 30.5, 23.3. HRMS (ESI-TOF ) calcd for

þ

6

C

1.1, 51.5, 43.7, 41.0, 31.8, 23.2. HRMS (ESI-TOF ) calcd for

C

18

22

H N

4

O

5

[MþH]

m/z

375.1668,

found

375.1661.

þ

18

H

23

N

3

O

3

[MþH]

m/z

330.1818,

found

330.1814.

HPLC:t ¼ 23.85 min, eluent A-60%, eluent B-40%.

R

HPLC:t ¼ 5.27 min, eluent A-30%, eluent B-70%.

R

4.4. Molecular modeling and computations

0

4

2

.3.40. 3-(2-([1,4 -Bipiperidin]-1 -yl)-2-oxoethyl)benzo[d]oxazol-

(3H)-one (D04)

Off-white solid. Yield: 30%. H NMR (400 MHz, DMSO‑d

a) Molecular docking. The docking studies were carried out by

1

)

d

7.33

using the Glide module [43] of Schr o€ dinger Suite (Schr o€ dinger,

6

(

3

(

d, J ¼ 7.8 Hz, 1H), 7.21 (t, J ¼ 6.4 Hz, 1H), 7.16 (d, J ¼ 7.5 Hz, 1H), 7.11

NY, USA). The chromodomain of CDYL (PDB ID: 2DNT) was

prepared using the Protein Preparation Wizard, which assigned

bond orders, added hydrogen atoms and removed water mole-

cules. Then the protein was energy minimized using restrained

minimization using OPLS3 force ﬁeld. The grid was generated by

deﬁning the hydrophobic cage of CDYL as the box center using

Receptor Grid Generation module. For ligands preparation,

conformations were generated and energy minimized by using

LigPrep module. Extra precision (XP) was used in the Ligand

Docking module. Images depicting the proposed binding modes

were generated using Maestro 10.6. For virtual screening, the

SPECS chemical library was subjected to three levels of docking

(HTVS, SP, and XP) using the Virtual Screening Workﬂow mod-

ule in Glide. MMGBSA was then calculated after XP docking.

b) Molecular dynamic simulations. Desmond module in maestro

t, J ¼ 8.2 Hz, 1H), 4.82 (q, J ¼ 17.1 Hz, 2H), 4.29 (d, J ¼ 12.9 Hz, 1H),

.95 (d, J ¼ 13.2 Hz, 1H), 3.21e2.99 (m, 2H), 2.64e2.52 (m, 2H), 1.77

dd, J ¼ 27.2, 12.1 Hz, 2H), 1.57 (s, 3H), 1.42e1.18 (m, 6H), 0.92 (t,

13

J ¼ 7.3 Hz, 2H). C NMR (101 MHz, DMSO‑d

6

) d 163.9, 154.7, 142.4,

1

2

32.1, 124.2, 122.6, 110.1, 110.0, 61.8, 50.0, 44.1, 43.4, 41.7, 28.2, 27.5,

þ

6.2, 24.7,19.7,14.0. HRMS (ESI-TOF ) calcd for C19

H

25

N

3

O

3

[MþH]

m/z 344.1974, found 344.1971. HPLC:t ¼ 23.80 min, eluent A-60%,

R

eluent B-40%.

4

.3.41. 3-(2-(4-Morpholinopiperidin-1-yl)-2-oxoethyl)benzo[d]

oxazol-2(3H)-one (D05)

Off-white solid. Yield: 30%. ¹H NMR (400 MHz, chloroform-d)

7.21 (d, J ¼ 7.7 Hz, 1H), 7.14 (dq, J ¼ 15.0, 7.5 Hz, 2H), 7.00 (d,

d

J ¼ 7.4 Hz, 1H), 4.70e4.56 (m, 2H), 4.52 (d, J ¼ 13.1 Hz, 1H), 3.97 (d,

J ¼ 13.7 Hz, 1H), 3.75e3.67 (m, 4H), 3.16 (t, J ¼ 12.1 Hz, 1H), 2.72 (t,

J ¼ 11.6 Hz, 1H), 2.56e2.48 (m, 4H), 2.43 (t, J ¼ 10.9 Hz, 1H),

þ

ꢂ

10.6 was used. Na and Cl ions were added at physiological

concentration of 0.15 M to ensure the overall neutrality of the

systems. Simulations were then conducted in the OPLS3 force

ﬁeld and a TIP4P explicit solvent model. The ﬁnal size of the

solvated system was close to ~50,000 atoms. A 100 ps recording

interval and the NPT ensemble was employed in 300 K and

1.01 bar. The integration time step was set at 2 fs The model

systems were relaxed using a six step default protocol attached

to Desmond, and utilized to prepare systems for production-

quality simulation. Default settings were used for all other pa-

rameters, and the total simulation time was 20 ns After simu-

lations, the Simulation Interactions Diagram Analysis module

was used to monitor energy, RMSD ﬂuctuations, hydrogen bond

distances, angles, and van der Waals interactions over the

simulation trajectories.

2

.01e1.57 (m, 4H). ¹³C NMR (101 MHz, chloroform-d)

d 163.4, 154.6,

1

2

3

42.6, 131.2, 124.0, 122.7, 110.1, 109.2, 67.2, 61.4, 49.8, 44.4, 43.7, 41.6,

þ

8.7, 27.9. HRMS (ESI-TOF ) calcd for C18

H

23

N

3

O

4

[MþH] m/z

46.1767, found 346.1762. HPLC:t ¼ 30.18 min, eluent A-60%,

R

eluent B-40%.

4

.3.42. 3-(2-(4-(Diethylamino)piperidin-1-yl)-2-oxoethyl)-5-

ﬂuorobenzo[d]oxazol-2(3H)-one (D06)

Off-white solid. Yield: 25%. ¹H NMR (400 MHz, chloroform-d)

d

1

7.13 (dd, J ¼ 8.6, 3.9 Hz, 1H), 6.81 (d, J ¼ 9.0 Hz, 1H), 6.78e6.72 (m,

H), 4.60 (d, J ¼ 13.4 Hz, 2H), 3.92 (d, J ¼ 13.8 Hz, 1H), 3.13 (t,

J ¼ 12.7 Hz, 1H), 2.80 (t, J ¼ 11.2 Hz, 1H), 2.66 (d, J ¼ 12.4 Hz, 1H),

2

.57 (q, J ¼ 6.9 Hz, 3H), 1.88 (dd, J ¼ 35.0, 12.6 Hz, 2H), 1.49 (dt,

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

19

c) Shape screening. For the 2D similarity search, AK-778/41051004

was used as a template. Pipeline Pilot v7.5 (PP 7.5, Accelrys, Inc.)

was used and the ﬁngerprint ECFP-6 was generated for each

structure, and similarities were calculated using Tanimoto co-

efﬁcient, and top 5% of the hits were maintained for manual

selection.

column to remove the GST tag. The collected protein was concen-

trated using Amico Ultra-15 concentrators, molecular weight 3000

cut-off (Merck Millipore). Proteins were analyzed for purity by SDS-

PAGE and stored at ꢂ80 C.

ꢀ

5.4. Cell culture

Human SH-SY5Y and HEK-293T cells were purchased from ATCC

4.5. Surface plasmon resonance (SPR)

and cultured in DMEM supplemented with 10% fetal bovine serum

ꢀ

(

FBS) in a 37 C incubator with 5% (v/v) CO

2

.

Brieﬂy, the measurement for binding afﬁnity between chro-

modomains and their ligands was conducted using Biacore T200

5.5. Cell viability experiments

ꢀ

(

GE Healthcare) at 25 C with the ﬂow rate of 30

mL/min. Recom-

binant chromodomains were immobilized at pH 4.0e4.5 on the

CM5 sensorchip by amine coupling using EDC/NHS, and the

immobilization signal ranged from 5000 to 10000 RU. All sensor

grams were reference and blank subtracted. Running buffer was

used for blank injections, and bulk effects were corrected using

solvent correction. For UNC3866, all buffers used was merely PBSP.

For small molecules, they were dissolved in PBSP containing 5%

Cell viability was detected with the CCK-8 kit (Applygen Tech,

Beijing) according to the manufacturer's instructions. Brieﬂy,

5000 cells in per well were plated in 96-well plates. After incubated

for 24 h, the cells were treated with different concentration of

tested compound or DMSO (as negative control) for 24 h. Then

10

m

L CCK-8 was added in per well (100

mL medium) and incubated

ꢀ

at 37 C for 3 h. Absorbance of each well was determined by a

microplate reader (Multiskan GO, Thermo) at a 450 nm wavelength.

The results of cytotoxicity were calculated according the for-

mula: ¼ 100% ꢁ (OD450 of tested compound - OD450 of medium

without cells)/(OD450 of DMSO - OD450 of medium without cells).

The IC₅₀values were calculated using Prism Graphpad software of

the triplicate experiment.

DMSO with 1.5-fold/2-fold dilution series starting from 100 mM,

and the running buffer was PBSP containing 5% DMSO. The asso-

ciation was followed for 60 s and the dissociation for 60 s. For data

analyses, the Biacore T200 evaluation software 2.0 (GE Healthcare)

was used, and sensor grams were directly ﬁtted to a 1:1 binding

model to calculate related k

a

, k

d D

, and K values.

5

. Chromodomain clone, expression and puriﬁcation

5.6. RT-PCR and real-time RT-PCR (qPCR)

5.1. Expression constructs

Total RNA was isolated from samples with Trizol reagents

(Invitrogen) and used for the ﬁrst strand cDNA synthesis with the

The chromodomains of CDYL (residues 1e60 of NP_004815),

Reverse Transcription System (TransGen). Any potential DNA

contamination was removed by RNase-free DNase treatment

(Promega). Relative quantitation was determined using the ABI

PRISM 7500 sequence detection system (Applied Biosystems) that

measures real-time SYBR green ﬂuorescence and then calculated by

CDYL2 (residues 1e70 of NP_689555), CDY1 (residues 1e65 of

NP_733841) and CBX7 (residues 1e62 of NP_783640) were

expressed with N-terminal GST-tags in pGEX-6P-1 expression

vector.

ꢂDDCt

means of the comparative Ct method (2

) with the expression

5.2. Protein expression and puriﬁcation

of GAPDH as an internal control. The sequences of the primers used

were provided in Supplemental Table S2.

All expression constructs were transformed into BL21 Escher-

ichia coli (TransGen Biotech, Beijing, China). Protein expression was

induced by adding isopropyl -D-1-thiogalactopyranoside (IPTG) to

5.7. Antibodies and reagents

b

a ﬁnal concentration of 0.2 mM when OD600 reached 0.6e0.8, and

the culture was further grown at 28 C for 6 h. Cell were harvested

Commercial antibodies used were: anti-CDYL (Sigma,

HPA035578), anti-b-actin (Biodragon, B1029), anti-CDYL (Abcam,

ꢀ

and resuspended in lysis buffer (1 ꢁ PBS, 5 mM DTT, 1 ꢁ EDTA free

protease inhibitor cocktail (Roche)). Following sonication and

centrifugation, the supernatant was loaded onto a GSTrap FF col-

umn (GE Healthcare) that had been pre-equilibrated with 10 col-

umn volumes of binding buffer (1x PBS, 5 mM DTT) using a AKTA

FPLC (GE Healthcare). The column was washed with 10 column

volumes of binding buffer and protein was eluted in 100% elution

buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM reduced gluta-

thione) over 10 column volumes. Peak fractions containing the

desired protein were pooled and concentrated to 2 mL in Amicon

Ultra-15 concentrators, 10,000 molecular weight cut-off (Merck

Millipore). Peak fractions were analyzed for purity by SDS-PAGE.

ab5188), anti-H3K27me3 (Abcam, ab192985), anti-MAP2 (Abcam,

ab11267) and anti-Gal 4 (Santa Cruz, sc-510). Protein A beads were

from GE Healthcare Biosciences, protease inhibitor mixture cocktail

was from Roche Applied Science. Alexa Fluor 488 was from Life

Technologies.

5.8. Luciferase reporter assay

SH-SY5Y, or HEK-293T cells in 24-well plates were transfected

with luciferase reporter, renilla, and indicated expression con-

structs. The amount of DNA in each transfection was kept constant

by addition of empty vector. 36 h after transfection, the ﬁreﬂy and

renilla luciferase were assayed according to the manufacturer's

protocol (Promega), and the ﬁreﬂy luciferase activity was normal-

ized to that of renilla luciferase. Each experiment was performed in

triplicate and repeated at last three times.

5.3. Afﬁnity tag removal

The N-terminal afﬁnity tag was removed from CDYL, CDYL2,

CDY1, and CBX7 by PreScission protease according to manufac-

turer's recommendations (Beyotime Biotech, Beijing, China).

Brieﬂy, puriﬁed protein was incubated with PreScission protease at

5.9. Western blot

a ﬁnal concentration of 2 units per 100

m

g tagged protein for 16 h at

Protein samples were resolved by SDSePAGE and transferred

onto a PVDF membrane (GE Healthcare). The membrane was

ꢀ

4

C. The cleavage reaction was then passed over a GSTrap FF

20

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

ꢀ

blocked by 5% nonfat milk in Tris-buffered saline (TBS) with 0.1%

Tween-20 (TBST) for 1 h at room temperature and incubated with

appropriate primary antibodies for overnight at 4 C. The mem-

brane was then washed by TBST for three times and incubated with

secondary antibodies (Santa Cruz Biotechnologies) in TBST for

0.25% trypsin (Life Technologies) for 30 min at 37 C, followed by

triturating with a pipette in plating medium (DMEM with 10% FBS).

Dissociated neurons were plated onto 35 mm dishes coated with

ꢀ

5

poly-D-lysine (Sigma Aldrich) at a density of 5 ꢁ 10 cells per dish.

After culturing for 4 h, the medium was changed to Neurobasal

medium supplemented with 2% B27 and 0.5 mM GlutaMAX-I (Life

Technologies). To test the role of D03 compound in dendritic

1.5 h at room temperature. After washing by TBST for three times,

the immunoreactive bands were visualized using Western Blotting

Luminal Reagent (Santa Cruz Biotechnology) according to the

manufacturer's recommendation. The bands were quantiﬁed by

densitometry with ImageJ software.

branching, neurons were treated with 150 mM D03 or DMSO at day

5 in vitro. After 24 h incubation, cells were ﬁxed with 4% para-

formaldehyde and subjected to analysis of dendritic branching.

5.10. Cellular thermal shift assay (CETSA)

5.13. Immunoﬂuorescence

The thermal shift assay was performed as previously described

44,45]. Brieﬂy, SH-SY5Y cells were collected and freeze-thawed

three times using liquid nitrogen. The lysates were diluted with

PBS and divided into two aliquots, with one aliquot being treated

Primary neurons growing on six-well chamber slides were

washed with PBS, ﬁxed in 4% paraformaldehyde, permeabilized

with 0.2% Triton X-100 in PBS, blocked with 0.8% BSA, and incu-

bated with MAP2 antibody (Abcam, ab11267) followed by staining

with 488-conjugated secondary antibody. The cells were subse-

[

with D03 compound (150

mM) and the other aliquot as control

(

DMSO). After 10 min at room temperature, the lysates were heated

quently washed four times and a ﬁnal concentration of 0.1

4,6-diamidino-2-phenylindole dihydrochloride (DAPI)

m

g/mL

was

ꢀ

individually at different temperatures (40e67 C) for 4 min fol-

lowed by cooling for 3 min at room temperature. The samples were

centrifuged at 13,000 g for 10 min at 4 C to collect the supernatant.

included in the ﬁnal wash to stain nuclei. Images were visualized

and recorded with a confocal microscope (Zeiss LSM880).

ꢀ

The supernatant was then analyzed by SDS-PAGE followed by

Western blot. For the dose-dependent thermal shift assay, the ly-

5.14. Sholl analysis

sates were incubated with various concentrations of D03 com-

ꢀ

pound (between 0 and 500

m

M) at 55 C for 4 min. The supernatant

Morphology of neurons was analyzed by Sholl analysis. Brieﬂy,

ﬂuorescent images were obtained using a confocal microscope and

imported into ImageJ software for further analysis. Concentric cir-

was isolated by centrifugation and subjected to immunoblotting

analysis of CDYL.

cles at 10 mm intervals were drawn around the soma. The number of

5.11. Chromatn immunoprecipitation (ChIP) and qChIP

dendrites crossing each circle was calculated and the data were

presented as mean ± s.e.m.

ChIP experiments were performed according to the procedure

7

described previously [28,29]. Approximate 1 ꢁ 10 SH-SY5Y cells

Author information

were treated with 150 mM D03 compound or DMSO for 24 h. Cells

were then ﬁxed with 1% formaldehyde for 10 min at room tem-

perature. The ﬁxed cells were lysed in lysis buffer (1% SDS, 5 mM

EDTA and 50 mM Tris-HCl (pH 8.1), plus protease inhibitor cocktail).

The lysates were then sonicated with 3 ꢁ 10 cycles (30 s on and off)

L.Y. and Y.L. contributed equally to this work. The manuscript

was written through contributions of all authors. All authors have

given approval to the ﬁnal version of the manuscript.

(

Bioruptor, Diagenode) to generate chromatin fragments of ~300 bp

Financial interest

in length. Cell debris was removed by centrifugation and super-

natant were collected. A dilution buffer (1% Triton X-100, 2 mM

EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl, plus protease in-

hibitor cocktail) was subsequently applied (1:10 ratio) and the

The authors declare no competing ﬁnancial interest.

Acknowledgements

resultant chromatin solution (aliquot 20e40 ml as the input) was

then incubated with control or speciﬁc antibodies (3e5

m

g) for

This study was ﬁnancially supported by National Natural Sci-

ence Foundation of China (Grants 31871083 and 81371432 to Z.H.,

Grants 21572010 and 21772005 to L.Z.). We are grateful for the

advice that Dr Jing Wang, the technician at Peking University

Health Science Center provided on SPR assays. We also thank Dr.

Jinwu Zhou, the ﬁeld application scientist at Reichert Technologies

for his advice on binding assays.

ꢀ

12 h at 4 C with constant rotation. Protein A/G Sepharose beads

(

50

mL of 50% (vol/vol)) were added for incubation of another 2 h.

ꢀ

Beads were collected by centrifugation at 500g for 5 min at 4 C.

Beads were sequentially washed with the following buffers for

ꢀ

5

min at 4 C: TSE I (1% Triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM

NaCl, 20 mM Tris-HCl (pH 8.1)); TSE II (1% Triton X-100, 0.1% SDS,

mM EDTA, 500 mM NaCl, 20 mM Tris-HCl (pH 8.1)); buffer III (1%

Nonidet P-40, 0.25 M LiCl, 1% sodium deoxycholate, 1 mM EDTA and

0 mM Tris-HCl (pH 8.1)); Tris-EDTA buffer. The input and the

2

Appendix A. Supplementary data

1

ꢀ

precipitated DNA-protein complex were decrosslinked at 65 C for

2 h in elution buffer (1% SDS, 5 mM EDTA, 50 mM NaCl, 20 mM

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2019.111656 .

1

Tris-HCl (pH 8.1), 0.1 mg/mL proteinase K), and DNA was puriﬁed

using PCR puriﬁcation kit (Qiagen). Quantiﬁcation of the precipi-

tated DNA fragments were performed with real-time PCR using

primers listed in Supplemental Table S3.

Abbreviations

CDYL

CBX7

PRC1

SAR

chromodomain Y-like

Chromobox protein homolog 7

polycomb repressive complex 1

structure-activity relationship

1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide

N-Hydroxysuccinimide

5.12. Primary neuron culture

Hippocampal or cortical explants were isolated from neonatal

EDC

NHS

(

P0) C57BL/6 mice of either sex. The explants were digested with

L. Yang et al. / European Journal of Medicinal Chemistry 182 (2019) 111656

21

TLC

CO

TBAI

EtOAc

DCM

DMF

BDNF

SPR

thin-layer chromatography

potassium carbonate

Tetra-n-butylammonium iodide

ethyl acetate

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J.E. Bradner, W. Wei, Prostate cancer-associated SPOP mutations confer

resistance to BET inhibitors through stabilization of BRD4, Nat. Med. 23 (2017)

K

2

3

1063 e 1071 .

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Article Doi

Identification and characterization of benzo[d]oxazol-2(3H)-one derivatives as the first potent and selective small-molecule inhibitors of chromodomain protein CDYL

DOI: 10.1016/j.ejmech.2019.111656

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