5-Aminonaphthalene derivatives as selective nonnucleoside nuclear receptor binding SET domain-protein 2 (NSD2) inhibitors for the treatment of multiple myeloma

^aState Key Laboratory of Drug Research and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,

555 Zu Chong Zhi Road, Shanghai, 201203, China

^bUniversity of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, 100049, China

^cSchool of Chinese Materia Medica, Nanjing University of Chinese Medicine, Qixia District, Nanjing, 210023, China

^dZhongshan Institute of Drug Discovery, Institution for Drug Discovery Innovation, Chinese Academy of Science, Zhongshan, 528400, China

^eSchool of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, UCAS, Hangzhou, 310024, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Approximately 20% of multiple myeloma (MM) are caused by a chromosomal translocation t (4; 14) that

leads to the overexpression of the nuclear receptor binding SET domain-protein 2 (NSD2) histone

methyltransferase. NSD2 catalyzes the methylation of lysine 36 on histone H3 (H3K36me2) and is

associated with transcriptionally active regions. Using high-throughput screening (HTS) with biological

analyses, a series of 5-aminonaphthalene derivatives were designed and synthesized as novel NSD2

Received 20 January 2021

Received in revised form

19 May 2021

Accepted 27 May 2021

Available online 5 June 2021

inhibitors. Among all the prepared compounds, 9c displayed

a good NSD2 inhibitory activity

(IC₅₀¼ 2.7 M) and selectivity against both SET-domain-containing and non-SET-domain-containing

m

Keywords:

methyltransferases. Preliminary research indicates the inhibition mechanism of compound 9c by

signiﬁcantly suppressed the methylation of H3K36me2. Compound 9c speciﬁcally inhibits the prolifer-

ation of the human B cell precursor leukemia cell line RS4:11 and the human myeloma cell line KMS11 by

inducing cell cycle arrest and apoptosis with little cytotoxicity. It has been reported that the anti-cancer

effect of compound 9c is partly achieved by completely suppressing the transcriptional activation of

NSD2-targeted genes. When administered intraperitoneally at 25 mg/kg, compound 9c suppressed the

tumor growth of RS4:11 xenografts in vivo and no body weight loss was detected in the tested SCID mice.

NSD2

Methyltransferase

HTS

Multiple myeloma

5-Aminonaphthalene derivatives

Inhibitor

1. Introduction

Nuclear receptor binding SET domain-protein 2 (NSD2) is

known as multiple myeloma SET domain (MMSET) or

WolfeHirschhorn syndrome candidate 1 (WHSC1), which has the

lowest molecular weight among the NSD family [1e3]. An

increasing amount of evidence has shown that NSD2 is involved in

cancer pathogenesis. The overexpression of NSD2 is associated with

higher grades of bladder, oligodendroglioma, breast, prostate, head

and neck, and liver cancers. Previous research has shown a clear

role of NSD2 in the oncogenetic process among multiple myeloma

(MM) and acute lymphoblastic leukemia (ALL) cancer types. The t

(4; 14) (p16; q32) translocation is observed in approximately 20% of

* Corresponding author. State Key Laboratory of Drug Research and CAS Key

Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai, 201203, China.

** Corresponding author. State Key Laboratory of Drug Research and CAS Key

Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai, 201203, China.

*** Corresponding author. State Key Laboratory of Drug Research and CAS Key

Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai, 201203, China.

E-mail addresses: jli@simm.ac.cn (J. Li), xhuang@simm.ac.cn (X. Huang), jwang@

simm.ac.cn (J. Wang).

1

These authors contributed equally to this study.

https://doi.org/10.1016/j.ejmech.2021.113592

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

MM, which is the second most frequent translocation observed in

this disease, and closely associated with poor prognosis. This

translocation results in the overexpression of both NSD2 and

ﬁbroblast growth factor receptor 3 (FGFR3); however, approxi-

mately 30% of MM patients only overexpress the NSD2 gene, sug-

gesting its pivotal role in the progression of the disease. NSD2

knockdown leads to apoptosis, while overexpression of NSD2 can

induce oncogenic transformation [4e7]. A number of ALLs bearing a

point lysine mutation (E1099K) in the cleft between the SET and

post-SET domains of NSD2 have demonstrated an enhanced

methyltransferase activity of NSD2. Genes involved in epithelial to

mesenchymal transition (EMT), embryonic development, cell cycle

regulation and microtubule disassembly were found to be signiﬁ-

cantly upregulated in the E1099K NSD2 cell lines [8].

0.087

(IC₅₀¼ 19.4 6.5

‰

. Among them, compound 8, which potently inhibited NSD2

mM), was identiﬁed through HTS. To ﬁnd out more

potent inhibitors, we focused our design attempts on the amino-

naphthalene scaffold. In an initial effort to enhance the inhibitory

activity against NSD2, our ﬁrst round of analogs (9a-9b) were

designed by installing different bicyclic aromatic moieties in com-

pound 8. In addition, the replacement of the amide moiety in

compound 8 with a sulfamide yielded compound 9c. Next, com-

pounds 10a-10d were designed to explore the inﬂuence of the

position of the substituent (the amino and sulfamide moieties) on

the naphthalene scaffold based on compound 9c. Finally, a series of

5-aminonaphthalene-1-sulfonamides (11a-11ac) were designed to

systematically investigate the SAR involving the R¹and R²sub-

stituents (Fig. 2).

As a member of histone lysine methyltransferase (HKMT) family,

NSD2 methylates lysine 36 of histone H3 (H3K36), which is

responsible for the transcription activation of a number of genes

[9e13]. Abnormal methylation at H3K36 has been widely impli-

cated in a variety of cancers, including MM harboring the trans-

location t (4; 14) and ALL bearing the E1099K mutation [14e1 7]. In

these cancers, the accumulation of H3K36 methylation is frequently

observed and associated with oncogenic programming, including

dysregulation of genes related to cell death, EMT, cell cycle regu-

lation and DNA repair. Therefore, the enzymes involved in post-

translational modiﬁcation are regarded as potential targets in drug

discovery.

2.2. Chemistry

Four synthetic routes were designed to obtain desired com-

pounds 8e11 (Schemes 1e4). Condensation of carboxylic acid 12

and benzylamine yielded the desired compounds 9a-9b. Com-

pound 8 was obtained after saliﬁcation with hydrochloric acid

(Scheme 1). As shown in Scheme 2, naphthalene sulfonic acids 15

with amino substituents at different positions were acetylated with

acetyl chloride to afford sulfonic acids 16, which were converted to

corresponding sulfonyl chlorides 17 by treatment with phosphorus

pentachloride. Then, compounds 17 were coupled with amines 18

to provide compounds 11t and 19. Desired compounds 9c, 10a-10d

and 11a-11q were generated by hydrolysis of intermediates 11t and

19. Compounds 11r-11s could be easily prepared via a coupling

reaction of commercially available naphthalene sulfonyl chloride

20 and benzylamine followed by saliﬁcation with HCl (Scheme 3).

As shown in Scheme 4, compound 9c underwent reductive ami-

nation with aldehyde or ketones 21 and was subsequently saliﬁed

with HCl to afford desired compounds 11u-11ac.

To date, few selective nucleoside and nonnucleoside NSD2 in-

hibitors have been reported. S-Adenosyl-

a nucleoside NSD2 inhibitor with low inhibitory activity (54% @

30 M). Heightman et al. reported that the antifungal drug sine-

fungin (2) exhibited weak NSD2 inhibitory activity

(IC₅₀¼ 26 4.5 M), and N-alkyl derivatization of sinefungin

yielded the best compound in this series, 3, which showed good

binding afﬁnity (K_d¼ 1.6 M) and inhibitory effect at micromolar

level (IC₅₀¼ 3.3 1.0 M), making compound 3 more potent than

L

-homocysteine (1, SAH) is

m

SAH (1) (Fig. 1) [18]. In addition to nucleoside NSD2 inhibitors,

several nonnucleoside NSD2 inhibitors (4e7) have been reported

by Luccio and coworkers [19e21]. Although these compounds

exhibited impressive NSD2 inhibitory activities at micromolar level,

their selectivities to the other members of the NSD family are not

satisfactory. Besides, the knowledge of structural modiﬁcations are

not sufﬁciently comprehensive for the evaluation of the structure-

activity relationship (SAR) and the key pharmacophores (Fig. 1).

Therefore, the discovery and development of potent nonnucleoside

NSD2 inhibitors based on detailed SAR studies is still of great

interest.

In this study, high-throughput screening (HTS) was applied to

discover novel NSD2 inhibitors. A series of 5-aminonaphthalene

derivatives were designed, synthesized, and biologically evalu-

ated as novel selective NSD2 inhibitors. Further studies demon-

strated that the most potent compound, 9c, signiﬁcantly inhibited

cancer cell proliferation. Taken together, the results indicate that

compound 9c and its analogs are new potential NSD2-speciﬁc in-

hibitors, and provide us with new structural information for

developing more potent NSD2 inhibitors.

2.3. Structure-activity relationship of 5-aminonaphthalene

derivatives

With the hit compound 8 in hand, the scaffold was ﬁrstly

investigated. As shown in Table 1, replacement of the amino-

naphthalene scaffold in compound 8 with an indole afforded

compound 9a, which has almost no NSD2 inhibitory activity at

20 mM, and with a quinolone afforded compound 9b, which showed

activity comparable to hit compound 8. To our delight, when the

amide moiety in compound 8 was replaced with a sulfamide, the

resulting 5-aminonaphthalene-1-sulfonamide derivative 9c dis-

played a 7-fold improvement in inhibitory potency. Then, we

explored the inﬂuence of the position of the substituents (the

amino and sulfamide groups) on the naphthalene core (10a-10d)

(Table 1). Compared with compound 9c, altering the positions of

the amino and sulfamide moieties did not enhance the NSD2

inhibitory activity (IC₅₀> 20 mM).

According to the results above, we selected compound 9c as the

lead compound and systematically investigated the SAR of sub-

stituents R¹and R²on the 5-aminonaphthalene-1-sulfonamide

scaffold (Tables 2 and 3). First, compounds 11a-11q with different

R¹groups were designed to further enhance the activity. Interest-

ingly, compared with compound 9c, which possesses a benzyl

group (R¹), compounds with phenyl (11a) or phenethyl (11b) sub-

stituents displayed decreased activity. Further introduction of a

halogen (11c), an electron-donating (11d) or an electron-

withdrawing (11e-11h) substituent at the para- or meta-position

of the benzyl group resulted in a signiﬁcant decrease in potency.

The replacement of the phenyl group in compound 9c with heter-

oaromatic groups, such as pyridyl (11i) or thienyl (11j), leads to a

2. Results and discussion

2.1. Design of 5-aminonaphthalene derivatives

To develop a new structural class of potent NSD2 inhibitors, we

used HTS to identify small molecules that can inhibit NSD2. A total

of 296 080 compounds from the Chinese National Compound Li-

brary were screened. We obtained 26 compounds as hits from the

screening at a concentration of 20

mg/mL, with the hit rate of

2

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

Fig. 1. Reported nucleoside and nonnucleoside NSD2 inhibitors.

Fig. 2. Design of 5-aminonaphthalene derivatives as novel NSD2 inhibitors.

decreased inhibitory activity against NSD2 (IC₅₀> 20

addition, when the phenyl group was replaced with aliphatic

moieties, such as cyclohexyl (11k) or piperidyl (11l), reduced ac-

tivities were observed (IC₅₀> 20 mM). Interestingly, NSD2 inhibitor

11m, bearing a 1-methylpiperidinyl unit in place of the phenyl

moiety, exhibited potency comparable to compound 9c. The

m

M). In

introduction of bulky 2-phenylcyclopropyl (11n) or tert-butyl

groups (11o), on the contrary, resulted in lower potencies

compared to compound 9c. Finally, removing the hydrogen atom

from the sulfamide moiety (11p-11q) resulted in substantial losses

in NSD2 inhibitory activity. These results indicated that the

hydrogen of the sulfamide is probably a key hydrogen bond donor

3

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

Scheme 1. Synthetic route to desired compounds 8 and 9a-9b^a.

^aReaction conditions and reagents: (a) HOBt, EDCI, Et₃N, DCM, 0 ^ꢀC to r.t., 8 h; (b) 4 N HCl in dioxane, 2 h.

Scheme 2. Synthetic routes to compounds 9c, 10a-10d, 11a-11q, and 11t^a.

^aReaction conditions and reagents: (a) acetyl chloride, Et₃N, DCM, 0 ^ꢀC to r.t., 8 h; (b) PCl₅, DCM, 40 ^ꢀC, 4 h; (c) amines, Et₃N, CH₃CN, r.t., 5 h; (d) 6 N HCl, EtOH, reﬂux, 12 h.

and it forms a hydrogen bond with an amino acid residue in the

binding pocket of NSD2.

NSD2 (IC₅₀> 20 mM). Based on the aforementioned results, we

alkylated the amino acids to further enhance the potency. However,

the introduction of simple alkyl groups (compounds 11u-11v)

caused slight decreases in potency. When introducing a piperidyl

moiety to the amino group, synthesized compound 11w showed

moderate inhibitory activity against NSD2. Further alkylation of the

piperidyl (11x and 11y) could enhance or maintain the potency, and

compound 11y, with an N-ethyl-substituted piperidyl moiety,

exhibited comparative good activity against NSD2 with an IC₅₀less

Next, we focused on the SAR of the R²group and evaluated the

NSD2 inhibitory activity of the candidate compounds 11r-11ac

(Table 3). First, we removed the amino group from compound 9c to

afford compound 11r, which showed signiﬁcantly lower potency.

This result demonstrated that the amino group at the 5-position of

the naphthalene is pivotal for the inhibitory activity of the com-

pound. When R²was a dimethylamino or an acetamino group, the

corresponding compounds, 11s and 11t, showed no activity against

than 5

mM. N-Acetyl-substituted piperidyl compound 11z also

4

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

a

Table 1

SAR of scaffold and position of the substituents on the naphthalene

.

Compd. Structure IC₅₀

(

m

M)

Compd. Structure

IC₅₀(mM)

8

19.4 6.5 10a

>20

9a

>20

10b

>20

Scheme 3. Synthetic routes to compounds 11r-11s^a.

^aReaction conditions and reagents: (a) benzylamine, Et₃N, CH₃CN, r.t., 5 h; (b) 4 N HCl

in dioxane, 2 h.

9b

9c

19.6 6.5 10c

>20

2.7 0.3

10d

SAH

e

39.1 4.1

a

IC₅₀values are reported as the mean SD deviations, n ꢁ 3.

that at least one active hydrogen atom of the amino group must be

reserved, which probably forms a key hydrogen bond with NSD2.

2.4. Compound 9c interacts with NSD2

Base on the enzymatic inhibitory potency against NSD2, we

selected compound 9c as the representation to further conﬁrm

whether these 5-aminonaphthalene derivatives as NSD2 inhibitors

could assuredly interact with the protein through NMR analysis.

And ligand observed CPMG and STD NMR experiments were per-

formed. In consistent with the result from the enzymatic inhibitory

assay, strong binding effects were clearly observed in both ligand

observed STD (Fig. 3A) and CPMG NMR spectra (Fig. 3B), which

indicating an interaction between compound 9c and NSD2.

2.5. Compound 9c selective against NSD2

Based on the NSD2 inhibitory activity, compound 9c was

selected for evaluation of its speciﬁcity for NSD2. The inhibitory

activity of compound 9c was examined against mutant NSD2

(E1099K), NSD1, SETD2, EZH2, MLL1, MLL4, DOT1L, PRMT4, and

PRMT5 (Table 4). As depicted in Table 4, compound 9c exhibited

remarkable selectivity against NSD2 over other human methyl-

transferases, including both SET-domain-containing (NSD1, SETD2,

EZH2, MLL1, and MLL4) and non-SET domain-containing methyl-

transferases (DOT1L, PRMT4, and PRMT5).

Scheme 4. Synthetic routes to compounds 11u-11ac^a.

^aReaction conditions and reagents: (a) Et₃N, AcOH, NaBH₃CN, r.t., 12 h; (b) 4 N HCl in

dioxane, 2 h.

2.6. Analysis of H3K36me2 levels in RS4:11 and KMS11 cell lines by

western blotting

showed good activity with an IC₅₀less than 10

mM. However,

Next, compound 9c was examined for its effects on the levels of

H3K36me2. As shown in Fig. 4, Western blot analysis revealed that

compound 9c signiﬁcantly suppressed the methylation of

H3K36me2 in a dose-dependent manner in RS4:11, MV4;11, KMS11,

and OPM-2 cell lines (Fig. 4A). Further analyses demonstrated that

the inhibition of H3K36me2 is highly selective relative to other

compounds with bulkier substituents, such as benzoyl (11aa) and

carbobenzyloxy (11 ab), displayed signiﬁcantly lower inhibitory

activities against NSD2. Finally, the introduction of a methylsulfonyl

unit to the piperidyl moiety afforded compound 11ac, which

showed moderate activity against NSD2. These results indicated

5

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

Table 2

Table 3

SAR of substituent R²on the 5-aminonaphthalene-1-sulfonamide scaffold^a.

a

SAR of substituent R¹on the 5-aminonaphthalene-1-sulfonamide scaffold

.

Compd. R¹

IC₅₀

(m

M)

Compd. R¹

IC₅₀

(

mM)

9c

2.7 0.3

11i

26.0 2.4

Compd. R²

IC₅₀

(

m

M)

Compd. R²

IC₅₀(mM)

11a

11b

11c

11d

11e

11f

>20

11j

22.7 7.9

9c

-NH₂

2.7 0.3

11x

6.0 1.5

3.2 0.8

7.5 3.3

15.4 6.1

11k

>20

11r

11s

H

>20

11y

11z

14.1 11.6 11l

>20

13.5 2.1

10.4 1.5

18.1 4.4

11m

3.4 0.4

19.9 4.4

18.4 6.3

11t

11u

11v

>20

11aa

18.0 12.1

11n

11o

10.7 1.6 11ab

12.6 2.1 11ac

10.2 1.3 SAH

>20

11g

11h

>20

11p

11q

>20

11.8 1.8

39.1 4.1

>20

11w

e

SAH

39.1 4.1

a

IC₅₀values are reported as the mean SD deviations, n ꢁ 3.

a

IC₅₀values are reported as the mean SD deviation, n ꢁ 3.

methylated sites (Fig. 4B). Total histone H3 and NSD2 were not

affected by compound 9c (Fig. 4B); thus, the reduction in

H3K36me2 is due to the direct inhibition of NSD2 methyltransfer-

ase activity rather than the degradation of histone H3 or NSD2.

inhibition of cell proliferation was observed for 6 consecutive days,

and net decreases in cell number were observed after 2 days. The

experiment showed that compound 9c signiﬁcantly inhibited the

growth rate of RS4:11 and KMS11 cells in a time-dependent and

dose-dependent manner (Fig. 5B).

2.7. Cell-based activity

To determine the antitumor potential of compound 9c, we

evaluated its effects on cell proliferation in a panel of human cell

lines, including gastric cancer, hepatic carcinoma, breast cancer,

lung cancer, thyroid carcinoma, and hematological tumor cells.

These cell lines with different genetic background exhibited various

levels of sensitivity to compound 9c. Based on the sources of the

cell lines, compound 9c showed relatively low IC₅₀values against

the hematological tumor cell lines (Fig. 5A). The results also indi-

cated that compound 9c remarkably inhibited cell proliferation in

the ALL cell line RS4:11 and the MM cell line KMS11 with IC₅₀values

2.8. Compound 9c induces apoptosis and cell cycle arrest

The effects of compound 9c on apoptosis and various phases of

cell cycle progression were evaluated in RS4:11, KMS11, and

MV4;11 cells. RS4:11, KMS11, and MV4;11 cells were treated with

vehicle alone or compound 9c at 5 and 10 mM for 72 h and evaluated

by ﬂow cytometry [22]. Compound 9c induced apoptosis in a dose-

dependent manner in the RS4:11 and KMS11 cells. Nevertheless, no

pronounced change was observed in the MV4:11 cells (Fig. 6A).

According to the subsequent cell cycle analysis, the proportions of

of 0.52 mM and 1.88 mM, respectively (Fig. 5A). However, a slight

proliferative effect was observed in NCIeH929, U2932, HL-60, OCI-

AML5, RPMI-8226, Pfeiffer, MV4;11, KARPAS-422, WSUCLDL2,

OPM-2, MOLT4, and Jurkat cells.

Due to the remarkable inhibition effect on the growth of the

RS4:11 and KMS11 cell lines by compound 9c, it was further

analyzed by using different timing and dosing to investigate its

effect on antiproliferation and apoptotic activities in these two

most sensitive cell lines. In the RS4:11 and KMS11 cell lines, potent

RS4:11 cells in the G0/G1 phase of the cell cycle were 47.66% at 5

mM

and 60.86% at 10 M. In contrast, the proportion of untreated cells

m

in the G0/G1 phase of the cell cycle was 41.81%. The proportions of

KMS11 cells in the G0/G1 phase of the cell cycle were 44.53% at

5 mM and 45.69% at 10 mM. In contrast, the proportion of untreated

cells in the G0/G1 phase of the cell cycle was 32.21% (Fig. 6B).

Compared with the control population, the cell cycle data clearly

demonstrated that compound 9c arrested RS4:11 and KMS11 cells

6

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

Fig. 3. Ligand observed CPMG and STD spectra conﬁrm that compound 9c could interact with NSD2. (A) STD spectrum of 200 mM compound 9c in the presence of 4 mM NSD2. (B)

CPMG spectra for 200 mM compound 9c without (red) or with (cyan) the presence of 4 mM NSD2.

2.10. In vivo antitumor efﬁcacy and preliminary PK study of

compound 9c

Table 4

Compound 9c selective against NSD2.^a.

Based on its potent effects in cell culture, compound 9c was

Enzyme

IC₅₀(mM)

selected for evaluation of its in vivo antitumor efﬁcacy due to its

good inhibitory activity against NSD2. Initially, the pharmacoki-

netic proﬁles of compound 9c were evaluated in CD-1 mice after

intraperitoneal (i.p.) at 20 mg/kg and intravenous (i.v.) injection at

2 mg/kg. As shown in Table 5, compound 9c was suitable for

intraperitoneal administration with a satisfactory area under the

curve (AUC ¼ 150 h*ng/mL) in CD-1 mice. Then, a RS4:11 xenograft

SCID mouse model was prepared according to the procedure

described previously [24]. When implanted tumors reached a vol-

ume of 100 mm³, compound 9c was administered intraperitoneally

(i.p.) at 25 mg/kg and 50 mg/kg once a day for 21 consecutive days.

Tumor volumes and body weights were monitored over the course

of treatment. The tumor growth inhibition (TGI) and relative

increment ratio (T/C) were calculated at the end of treatment. As

shown in Fig. 8A, treatment with compound 9c caused a dramatic

reduction in tumor growth (25 mg/kg: TGI ¼ 43.6%, T/C ¼ 56.3%;

50 mg/kg: TGI ¼ 37.2%, T/C ¼ 62.8%). Moreover, no body weight loss

was detected in the tested SCID mice (Fig. 8B).

NSD2

2.7 0.3

3.1 0.7

>20

Mutant NSD2 (E1009K)

NSD1

SETD2

EZH2

MLL1

MLL4

DOT1L

PRMT4

PRMT5

>20

a

IC₅₀values are reported the mean SD (

m

M) from two sepa-

rate experiments.

mainly at the G0/G1 phase. In addition, the G0/G1 fraction in the

MV4;11 cells was not signiﬁcantly increased (Fig. 6B).

2.9. Compound 9c suppresses transcriptional activation of NSD2-

3. Conclusions

target genes

In summary, a series of 5-aminonaphthalene derivatives were

designed, synthesized, and evaluated as novel NSD2 inhibitors for

the treatment of cancer. Hit compound 8 was discovered via HTS

from the compound library, and SAR studies focusing on the scaf-

fold, position of the substituents and types of substituent on the

naphthalene core of compound 8 were sequentially conducted.

Among the prepared compounds, 9c displayed the best potency

To systematically examine the cellular pathways affected by

compound 9c, we performed RNA sequencing (RNA-seq) on the

sensitive cell line RS4:11 by treating the cells with compound 9c or

DMSO as a control for 72 h. By the GO analysis of the RNA-Seq,

multiple cellular pathways that are down- or up-regulated have

been identiﬁed and the top 20 up- and down-regulated genes have

been presented between the control group and the treatment

group (Fig. 7A and Fig. 7B). According to recent studies [23], genes

such as TGFA, MET, PAK1, and RRAS2 were under control of NSD2

mediated H3K36me2. We performed a systematic quantitative

direct ChIP analysis comparing H3K36me2 levels in KMS11 cells

treated with compound 9c and DMSO in all regions spanning the

four oncogenes (TGFA, PAK1, MET, and RRAS2). The result shows a

decreased H3K36me2 signaling in compound 9c-treated cells

compared to DMSO-treated cells (Fig. 7C). These data further sup-

port the conclusion that compound 9c completely suppressed

H3K36me2 level in the promoter regions of the NSD2-target genes

TGFA, MET, PAK1, and RRAS2 in KMS11 cells.

against NSD2 (IC₅₀¼ 2.7

mM). In our study, we tested the effect of

compound 9c on various histone modiﬁcations and found that

compound 9c was able to speciﬁcally reduce H3K36me2 level

without affecting other histone methylations, such as H3K4me2,

H3K27me2 or H3K79me2. Multiple cellular assays demonstrated

the anti-tumor effect of compound 9c via proliferation inhibition,

G0/G₁arrest, and apoptosis in the human B cell precursor leukemia

cell line RS4:11 and the human myeloma cell line KMS11. The RNA

sequencing data indicated that compound 9c completely sup-

pressed the transcriptional activation of the NSD2-target genes

TGFA, MET, PAK1, and RRAS2 in KMS11 cells. This scaffold for potent

NSD2 inhibitors establishes a new research foundation for the

7

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

Fig. 4. Cellular mechanistic activity of compound 9c. (A) RS4:11, MV4;11, KMS11, and OPM-2 cell lines were treated with compound 9c at the indicated doses and time intervals, and

the expressions of NSD2 and H3K36 methylation were proﬁled. (B) RS4:11, MV4;11, KMS11, and OPM-2 cells lines were treated with compound 9c (0, 5, 10, and 50

and various histone modiﬁcations were detected by western blotting.

mM) for 4 days,

discovery of NSD2 small-molecule inhibitors and highlight poten-

tials for the development of new MM agents. We have developed a

series of compounds with activity against NSD2, compound 9c may

be useful as a tool molecule, which would require further devel-

opment and evaluation for treating cancer.

4.1.1. 5-Amino-N-benzyl-1-naphthamide hydrochloride (8)

To a stirred mixture of commercially available 5-amino-1-

naphthoic acid 12a (250 mg, 1.34 mmol) in anhydrous DCM was

successively added EDCI (384 mg, 2.0 mmol), HOBt (270 mg,

2.0 mmol) and triethylamine (278

zylamine (219 L, 2.0 mmol) was added to the reaction mixture at

^ꢀC. The reaction mixture was stirred at ambient temperature for

mL, 2.0 mmol). After 0.5 h, ben-

m

0

4. Experimental

8 h when TLC showed completion of the reaction. The resulting

mixture was sequentially washed with water (10 mL x 2), 0.1 N HCl

(10 mL x 2), 0.1 N NaOH (10 mL x 2) and brine (10 mL). The obtained

organic solution was concentrated under vacuum and puriﬁed by

silica gel column chromatography (DCM/MeOH ¼ 100/1) to afford a

white solid, which was subsequently dissolved in 4 N HCl in

dioxane (5 mL) and stirred for 2 h at room temperature. The reac-

tion mixture was concentrated to afford compound 8 as a white

solid (320 mg, yield: 86%). Mp 242e244 ^ꢀC. ¹H NMR (400 MHz,

4.1. Chemistry

The commercially available reagents and chemicals were used

without further puriﬁcation. Nuclear magnetic resonance (NMR)

spectroscopy (¹H and ¹³C) spectra was recorded on a Varian-

MERCURY Plus-400 MHz, AVANCE III 500 MHz or INVOA-600

MHz instrument. Chemical shifts were reported in parts per

million (ppm, d) downﬁeld from tetramethylsilane. Proton coupling

DMSO‑d₆)

d

9.19 (t, J ¼ 6.1 Hz, 1H), 8.18 (dt, J ¼ 8.2, 1.2 Hz, 1H), 8.08

patterns are described as singlet (s), doublet (d), triplet (t), quartet

(q), multiplet (m), and broad (br). Low-resolution mass spectra

(LRMS) was measured on Agilent Technologies 6120 LC/MS spec-

trometer. High-resolution mass spectra (HRMS) was measured on

Micromass Ultra Q-TOF spectrometer.

(dt, J ¼ 8.2, 1.1 Hz, 1H), 7.73 (dd, J ¼ 7.1, 1.4 Hz, 1H), 7.69 (dd, J ¼ 8.3,

7.1 Hz, 1H), 7.61 (dd, J ¼ 7.4, 1.5 Hz, 1H), 7.56 (dd, J ¼ 8.2, 7.4 Hz, 1H),

7.43e7.35 (m, 4H), 7.28 (ddt, J ¼ 7.6, 6.2, 1.8 Hz, 1H), 4.55 (d,

J ¼ 6.0 Hz, 2H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 168.31, 139.44,

8

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

Fig. 5. Compound 9c inhibits the proliferation of the human B cell precursor leukemia cell line RS4:11 and the human myeloma cell line KMS11 in vitro. (A) The effects of compound

9c on the growth of 42 cell lines. The cell lines were treated with varying concentrations of compound 9c for 6 days before cell growth was evaluated. The x-axis represents the

growth IC₅₀value. (B) Dose-dependent and time-dependent effects of compound 9c on cell proliferation in RS4:11 and KMS11 cells. Growth is expressed as cell counts. RS4:11 and

9

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

135.16, 131.45, 130.54, 128.36, 127.22, 126.82, 126.44, 126.06, 125.84,

125.46, 123.81, 123.37, 118.60, 42.57. ESI-LRMS m/z: 277.0 [M þ H]^þ.

ESI-HRMS calcd for C₁₈H₁₇N₂O [M þ H]^þ277.1335, found 277.1336.

with DCM (40 mL x 3). The combined organic layer was washed

with brine (40 mL x 2), dried over anhydrous Na₂SO₄, then

concentrated under vacuum and puriﬁed by silica gel column

chromatography (DCM/MeOH ¼ 50/1e20/1) to afford compound

11t as a white solid (14.9 g, yield: 87%). Mp 193e194 ^ꢀC. ¹H NMR

4.1.2. N-benzyl-1H-indole-4-carboxamide (9a)

(400 MHz, DMSO‑d₆)

d 10.08 (s, 1H), 8.57e8.51 (m, 2H), 8.33 (d,

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

compound 8. Yield: 89%. Mp 111e112 ^ꢀC. ¹H NMR (400 MHz,

J ¼ 8.5 Hz, 1H), 8.14 (dd, J ¼ 7.3, 1.1 Hz, 1H), 7.76 (d, J ¼ 7.4 Hz, 1H),

7.71e7.61 (m, 2H), 7.25e7.10 (m, 5H), 4.03 (d, J ¼ 6.2 Hz, 2H), 2.20 (s,

DMSO‑d₆)

d

11.29 (s, 1H), 8.79 (t, J ¼ 6.1 Hz, 1H), 7.55 (dt, J ¼ 8.1,

3H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 169.09, 137.69, 136.14, 134.58,

1.0 Hz, 1H), 7.48 (dd, J ¼ 7.3, 0.9 Hz, 1H), 7.43 (t, J ¼ 2.8 Hz, 1H),

7.39e7.31 (m, 4H), 7.24 (ddt, J ¼ 8.6, 6.3, 1.8 Hz, 1H), 7.14 (dd, J ¼ 8.1,

7.4 Hz,1H), 6.86 (ddd, J ¼ 3.0, 2.0, 0.9 Hz,1H), 4.52 (d, J ¼ 6.1 Hz, 2H).

128.86, 128.47, 128.26, 128.06, 127.42, 127.05, 124.17, 122.93, 122.07,

45.94, 23.43. ESI-LRMS m/z: 355.1 [M þ H]^þ. ESI-HRMS calcd for

C

₁₉H₁₉N₂O₃S [M þ H]^þ355.1111, found 355.1116.

¹³C NMR (125 MHz, DMSO‑d₆)

d 167.77, 140.12, 136.54, 128.20,

127.13, 126.56, 126.41, 125.93, 120.10, 118.42, 114.14, 101.80, 42.42.

ESI-LRMS m/z: 250.9 [M þ H]^þ. ESI-HRMS calcd for C₁₆H₁₅N₂O [M þ

H]^þ251.1179, found 251.1175.

4.1.7. 5-Amino-N-benzylnaphthalene-1-sulfonamide hydrochloride

(9c)

Compound 11t (150 mg, 0.423 mmol) was dissolved in ethanol

(3 mL) and 6 N HCl (3 mL), and the mixture was reﬂuxed for 12 h.

After cooling to room temperature, a large amount of solid was

precipitated, and then ﬁltered and dried to afford compound 9c as a

white solid (131 mg, yield: 88%).

4.1.3. N-benzylquinoline-5-carboxamide (9b)

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

compound 8. Yield: 83%. Mp 149e151 ^ꢀC. ¹H NMR (400 MHz,

DMSO‑d₆)

d

9.22 (t, J ¼ 6.1 Hz, 1H), 8.95 (dd, J ¼ 4.1, 1.7 Hz, 1H), 8.66

Mp 176e178 ^ꢀC. ¹H NMR (400 MHz, DMSO‑d₆)

d 8.61 (t,

(ddd, J ¼ 8.6, 1.7, 0.9 Hz, 1H), 8.16e8.11 (m, 1H), 7.84e7.79 (m, 2H),

7.59 (dd, J ¼ 8.6, 4.1 Hz,1H), 7.43e7.35 (m, 4H), 7.28 (ddt, J ¼ 8.6, 6.2,

1.7 Hz, 1H), 4.56 (d, J ¼ 6.0 Hz, 2H). ¹³C NMR (125 MHz, DMSO‑d₆)

J ¼ 6.2 Hz, 1H), 8.49 (d, J ¼ 8.6 Hz, 1H), 8.35 (d, J ¼ 8.6 Hz, 1H), 8.18

(d, J ¼ 7.3 Hz, 1H), 7.68 (dt, J ¼ 11.6, 8.1 Hz, 2H), 7.59 (d, J ¼ 7.3 Hz,

1H), 7.22e7.12 (m, 5H), 4.03 (d, J ¼ 6.1 Hz, 2H). ¹³C NMR (125 MHz,

d

167.46,150.77,147.60, 139.37, 134.47,133.68,131.09,128.51, 128.36,

DMSO‑d₆)

d 137.64, 136.31, 133.51, 128.95, 128.48, 128.06, 127.78,

127.21, 126.82, 125.81, 125.18, 121.98, 42.61. ESI-LRMS m/z: 263.0

[M þ H]^þ. ESI-HRMS calcd for C₁₇H₁₅N₂O [M þ H]^þ263.1179, found

263.1174.

127.65, 127.43, 127.08, 126.46, 124.56, 121.74, 118.27, 45.95. ESI-

LRMS m/z: 313.0 [M þ H]^þ. ESI-HRMS calcd for C₁₇H₁₇N₂O₂S [M þ

H]^þ313.1005, found 313.1014.

4.1.4. 5-Acetamidonaphthalene-1-sulfonic acid (14)

4.1.8. 6-Amino-N-benzylnaphthalene-1-sulfonamide hydrochloride

(10a)

5-Aminonaphthalene-1-sulfonic acid 13 (15.0 g, 67.19 mmol)

was dissolved in anhydrous DCM (50 mL), and triethylamine

(10.27 mL, 73.91 mmol) was added followed by the dropwise

addition of acetylchloride (5.27 mL, 73.91 mmol) at 0 ^ꢀC. After

stirring 8 h at room temperature, TLC showed completion of the

reaction and solid was precipitated. The resulting mixture was

ﬁltered, and the ﬁlter cake was dried to afford compound 14 (16.3 g)

as a purple solid, which was used without further puriﬁcation. ESI-

LRMS m/z: 264.0 [M ꢂ H]^-

Compound 10a was prepared in a similar manner as described

for compound 9c. Yield: 82%. Mp 215e217 ^ꢀC. ¹H NMR (400 MHz,

DMSO‑d₆)

d

8.88 (s, 2H), 8.67 (d, J ¼ 9.2 Hz, 1H), 8.56 (t, J ¼ 6.2 Hz,

1H), 8.15 (dt, J ¼ 8.5, 1.0 Hz, 1H), 8.03 (dd, J ¼ 7.3, 1.2 Hz, 1H), 7.79 (d,

J ¼ 2.2 Hz, 1H), 7.60 (dd, J ¼ 8.3, 7.3 Hz, 1H), 7.54 (dd, J ¼ 9.2, 2.3 Hz,

1H), 7.21e7.10 (m, 5H), 4.02 (d, J ¼ 6.2 Hz, 2H). ¹³C NMR (125 MHz,

DMSO‑d₆)

d 138.08, 136.49, 135.07, 133.11, 128.49, 127.89, 127.56,

127.49, 126.86, 125.84, 124.88, 122.51, 117.74, 46.41. ESI-LRMS m/z:

311.1 [M ꢂ H]^-. ESI-HRMS calcd for C₁₇H₁₅N₂O₂S [M ꢂ H]^-311.0860,

found 311.0860.

4.1.5. 5-Acetamidonaphthalene-1-sulfonyl chloride (15)

Compound 14 (16.3 g, 61.44 mmol) and PCl₅(19.19 g,

92.17 mmol) were dissolved in anhydrous DCM (100 mL), and the

reaction mixture was heated at 40 ^ꢀC. After 4 h, the reaction

mixture was cooled down, and petroleum ether (100 mL) was

added and triturated under ultrasound. Then the mixture was

ﬁltered, and the resulting ﬁltrate was neutralized by saturated

NaHCO₃in an ice bath. A large amount of solid was subsequently

precipitated and these solids was ﬁltered, washed with methanol

and dried to afford compound 15 as an earthy yellow solid (13.7 g,

4.1.9. 5-Amino-N-benzylnaphthalene-2-sulfonamide hydrochloride

(10b)

Compound 10b was prepared in a similar manner as described

for compound 9c. Yield: 80%. Mp 235e237 ^ꢀC. ¹H NMR (400 MHz,

DMSO‑d₆)

d

8.86 (s, 2H), 8.44 (d, J ¼ 1.9 Hz, 1H), 8.35 (t, J ¼ 6.3 Hz,

1H), 8.27 (dt, J ¼ 9.0, 0.8 Hz, 1H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.91 (dd,

J ¼ 9.0, 2.0 Hz, 1H), 7.62 (t, J ¼ 7.7 Hz, 1H), 7.57 (dd, J ¼ 7.5, 1.4 Hz,

1H), 7.28e7.22 (m, 4H), 7.22e7.16 (m, 1H), 4.03 (d, J ¼ 6.1 Hz, 2H).

yield: 78%). ¹H NMR (400 MHz, DMSO‑d₆)

d 9.94 (s, 1H), 8.70 (d,

¹³C NMR (125 MHz, DMSO‑d₆)

d 138.28, 137.58, 134.09, 132.76,

J ¼ 8.6 Hz, 1H), 8.04 (dd, J ¼ 8.5, 1.1 Hz, 1H), 7.97 (dd, J ¼ 7.1, 1.2 Hz,

1H), 7.59 (d, J ¼ 7.2 Hz, 1H), 7.51e7.41 (m, 2H), 2.17 (s, 3H). ESI-LRMS

m/z: 282.0 [M ꢂ H]^-.

128.18, 127.63, 127.54, 127.12, 126.27, 125.14, 123.95, 121.98, 118.69,

109.51, 46.13. ESI-LRMS m/z: 311.1 [M ꢂ H]^-. ESI-HRMS calcd for

C3₇H₁₅N₂O₂S [M ꢂ H]^-311.0860, found 311.0863.

4.1.6. N-(5-(N-benzylsulfamoyl)naphthalen-1-yl)acetamide (11t)

To a stirred mixture of compound 15 (13.7 g, 48.29 mmol) and

trimethylamine (8.05 mL, 57.94 mmol) in acetonitrile (100 mL)

were added benzylamine (6.34 mL, 57.94 mmol) dropwise at room

temperature. After 5 h, TLC showed completion of the reaction, and

the reaction mixture was concentrated under vacuum. Then the

resulting mixture was diluted with water (50 mL) and extracted

4.1.10. 6-Amino-N-benzylnaphthalene-2-sulfonamide

hydrochloride (10c)

Compound 10c was prepared in a similar manner as described

for compound 9c. Yield: 82%. Mp 239e241 ^ꢀC. ¹H NMR (400 MHz,

DMSO‑d₆)

d

9.19 (s, 2H), 8.36 (d, J ¼ 1.8 Hz, 1H), 8.24 (t, J ¼ 6.4 Hz,

1H), 8.11 (d, J ¼ 8.8 Hz, 1H), 8.01 (d, J ¼ 8.7 Hz, 1H), 7.79 (dd, J ¼ 8.7,

KMS11 cells were treated with compound 9c (0, 1, 5, 10, 20, 50, and 100

mM) for 6 days, and the number of cells in each treatment group was determined daily. Growth is expressed

as cell counts. Data are reported as the mean SD.

10

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

Fig. 6. In RS4:11 and KMS11 cells, compound 9c induces cell apoptosis and arrests the cell cycle at the G0/G1 phase. (A) RS4:11, KMS11, and MV4;11 cells were treated with

compound 9c (at 5 and 10 M) for 72 h, and the effects on apoptosis were examined by ﬂow cytometry. (B) RS4:11, KMS11, and MV4;11 cells were incubated with compound 9c (at 5

m

and 10

mM) for 72 h, and the proportions of cells in each cell cycle phase were determined by ﬂow cytometry.

11

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

Fig. 6. (continued).

1.9 Hz, 1H), 7.65 (d, J ¼ 2.0 Hz, 1H), 7.45 (dd, J ¼ 8.7, 2.1 Hz, 1H),

4.1.13. 5-Amino-N-phenethylnaphthalene-1-sulfonamide

hydrochloride (11b)

7.26e7.20 (m, 4H), 7.20e7.14 (m, 1H), 4.00 (d, J ¼ 5.8 Hz, 2H). ¹³

C

NMR (125 MHz, DMSO‑d₆)

d

139.15, 137.65, 135.77, 135.03, 130.77,

Compound 11b was prepared in a similar manner as described

128.14, 128.07, 127.90, 127.55, 127.37, 127.05, 123.08, 121.59, 114.79,

for compound 9c. Yield: 80%. Mp 181e182 ^ꢀC. ¹H NMR (400 MHz,

46.12. ESI-LRMS m/z: 311.0 [M

C

ꢂ

H]^-. ESI-HRMS calcd for

DMSO‑d₆)

d

8.37 (dd, J ¼ 8.5, 5.4 Hz, 2H), 8.17e8.10 (m, 2H),

₁₇H₁₅N₂O₂S [M ꢂ H]^-311.0860, found 311.0866.

7.73e7.65 (m, 1H), 7.64e7.59 (m, 1H), 7.50 (d, J ¼ 7.3 Hz, 1H),

7.19e7.09 (m, 3H), 7.05e7.00 (m, 2H), 3.04e2.98 (m, 2H), 2.60 (t,

J ¼ 7.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 138.94, 136.50,

134.45, 129.33, 128.97, 128.64, 128.24, 128.13, 126.83, 126.61, 124.90,

121.77, 118.32, 44.43, 35.82. ESI-LRMS m/z: 327.2 [M þ H]^þ. ESI-

HRMS calcd for C₁₈H₁₉N₂O₂S [M þ H]^þ327.1162, found 327.1167.

4.1.11. 4-Amino-N-benzylnaphthalene-1-sulfonamide

hydrochloride (10d)

Compound 10d was prepared in a similar manner as described

for compound 9c. Yield: 85%. Mp 166e168 ^ꢀC. ¹H NMR (400 MHz,

CD₃OD)

1H), 7.85e7.77 (m, 2H), 7.49 (d, J ¼ 7.9 Hz, 1H), 7.09e6.95 (m, 5H),

4.09 (s, 2H). ¹³C NMR (125 MHz, DMSO‑d₆)

148.12, 138.09, 131.34,

129.45,128.00,127.60,127.44,126.90, 124.96, 124.70,123.07,122.79,

121.77, 106.15, 45.79. ESI-LRMS m/z: 311.1 [M ꢂ H]^-. ESI-HRMS calcd

for C₁₇H₁₅N₂O₂S [M ꢂ H]^-311.0860, found 311.0867.

d

8.83e8.77 (m, 1H), 8.17 (d, J ¼ 7.9 Hz, 1H), 8.10e8.04 (m,

4.1.14. 5-Amino-N-(4-ﬂuorobenzyl)naphthalene-1-sulfonamide

hydrochloride (11c)

d

Compound 11c was prepared in a similar manner as described

for compound 9c. Yield: 78%. Mp 191e193 ^ꢀC. ¹H NMR (400 MHz,

DMSO‑d₆)

d

8.60 (t, J ¼ 6.2 Hz, 1H), 8.40 (d, J ¼ 8.7 Hz, 1H), 8.35 (d,

J ¼ 8.6 Hz, 1H), 8.14 (dd, J ¼ 7.4, 1.1 Hz, 1H), 7.67 (dd, J ¼ 7.6, 6.4 Hz,

1H), 7.63 (dd, J ¼ 7.5, 6.4 Hz, 1H), 7.50 (d, J ¼ 7.5 Hz, 1H), 7.20e7.12

(m, 2H), 7.05e6.95 (m, 2H), 4.01 (d, J ¼ 6.1 Hz, 2H). ¹³C NMR

(125 MHz, CD₃OD)

d

163.34 (d, J ¼ 244.4 Hz), 138.86, 134.43 (d,

4.1.12. 5-Amino-N-phenylnaphthalene-1-sulfonamide

hydrochloride (11a)

J ¼ 3.1 Hz), 131.29, 130.68 (d, J ¼ 8.2 Hz), 130.38, 129.24, 128.89,

128.48, 127.39, 127.33, 127.10, 122.69, 115.75 (d, J ¼ 21.7 Hz), 46.93.

ESI-LRMS m/z: 331.2 [M þ H]^þ. ESI-HRMS calcd for C₁₇H₁₆FN₂O₂S

[M þ H]^þ331.0911, found 331.0915.

Compound 11a was prepared in a similar manner as described

for compound 9c. Yield: 77%. Mp 206e207 ^ꢀC. ¹H NMR (400 MHz,

DMSO‑d₆)

d

10.74 (s, 1H), 8.45 (d, J ¼ 8.6 Hz, 1H), 8.35 (d, J ¼ 8.5 Hz,

1H), 8.24 (d, J ¼ 7.2 Hz, 1H), 7.68e7.60 (m, 2H), 7.45 (d, J ¼ 7.4 Hz,

1H), 7.13 (t, J ¼ 7.7 Hz, 2H), 7.01 (d, J ¼ 7.9 Hz, 2H), 6.91 (t, J ¼ 7.3 Hz,

4.1.15. 5-Amino-N-(4-methoxybenzyl)naphthalene-1-sulfonamide

hydrochloride (11d)

1H), 5.28 (s, 2H). ¹³C NMR (150 MHz, CD₃OD)

d 138.42, 137.27,

132.51, 130.43, 130.06, 129.37, 128.86, 128.72, 127.73, 127.14, 127.08,

125.61, 122.85, 121.61. ESI-LRMS m/z: 299.2 [M þ H]^þ. ESI-HRMS

calcd for C₁₆H₁₅N₂O₂S [M þ H]^þ299.0849, found 299.0848.

Compound 11d was prepared in a similar manner as described

for compound 9c. Yield: 86%. Mp 172e174 ^ꢀC. ¹H NMR (400 MHz,

DMSO‑d₆)

d

8.47 (t, J ¼ 6.1 Hz, 1H), 8.39 (d, J ¼ 8.7 Hz, 1H), 8.35 (dt,

12

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

Fig. 7. Compound 9c suppresses of the NSD2-target genes by decreased H3K36me2 level in the promoter regions. (A) GO analysis of the differential genes upon compound 9c

treatment identiﬁed by RNA-seq in RS4:11 cells. (B) Volcano plot showing the differentially expressed genes in RS4:11 cells treated with DMSO or compound 9c (10 M) for 72 h, the

top 20 increased or decreased genes are labelled. (C) ChIP-qPCR analyses of H3K36me2 level in the promoter regions of the genes TGFA, MET, PAK1, and RRAS2.

m

13

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

Table 5

Pharmacokinetic proﬁles of compound 9c in CD-1 mice^a.

Compd.

T_1/2

(h)

T_max

(h)

C_max

AUC

CL

MRT

(h)

Vss

(ng/mL)

(h*ng/mL)

(mL/min/kg)

(mL/kg)

9c (i.p.)

9c (i.v.)

0.56 0.06

0.66 0.03

0.083

e

433 71.1

e

150 33.9

204 18.9

e

0.531

0.396 0.06

e

163 16.1

3854 478

a

Values are the average of three runs. T_1/2, half-life; T_max, time of maximum concentration; C_max, maximum concentration; AUC, area under the plasma concentrationꢂtime

curve; CL, clearance; MRT, mean residence time; V_ss, volume of distribution; F, oral bioavailability. Dose: i.p. at 2.0 mg/kg. Dose: i.v. at 2.0 mg/kg (n ¼ 3 per group).

Fig. 8. Compound 9c inhibits RS4:11 xenografts in vivo. (A) Tumor-bearing mice (n ¼ 8 per group) were treated with compound 9c (25 mg/kg and 50 mg/kg) daily for 21 days. The

curve shows the tumor volume after 21 days of treatment with compound 9c or vehicle. Data are shown as the mean SEM; *P ¼ 0.027. (B) The corresponding curve of body weight

after 21 days of treatment with compound 9c or vehicle was shown. Error bars represent the mean SEM from eight individuals in each group.

J ¼ 8.6, 1.1 Hz, 1H), 8.14 (dd, J ¼ 7.3, 1.1 Hz, 1H), 7.66 (dd, J ¼ 8.6,

7.4 Hz, 1H), 7.62 (dd, J ¼ 8.7, 7.5 Hz, 1H), 7.47 (d, J ¼ 7.5 Hz, 1H),

7.07e7.02 (m, 2H), 6.76e6.72 (m, 2H), 3.94 (d, J ¼ 6.0 Hz, 2H), 3.68

4.1.18. 5-Amino-N-(3-cyanobenzyl)naphthalene-1-sulfonamide

hydrochloride (11g)

Compound 11g was prepared in a similar manner as described

(s, 3H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 158.36, 136.32, 134.39,

for compound 9c. Yield: 80%. Mp 212e214 ^ꢀC. ¹H NMR (500 MHz,

129.50, 128.85, 128.82, 128.51, 127.82, 127.59, 126.20, 124.34, 120.99,

117.52, 113.48, 55.07, 45.47. ESI-LRMS m/z: 343.2 [M þ H]^þ. ESI-

HRMS calcd for C₁₈H₁₉N₂O₃S [M þ H]^þ343.1111, found 343.1119.

DMSO‑d₆)

d

8.71 (t, J ¼ 6.3 Hz, 1H), 8.37 (d, J ¼ 8.7 Hz, 1H), 8.32 (d,

J ¼ 8.5 Hz, 1H), 8.14 (dd, J ¼ 7.3, 1.0 Hz, 1H), 7.64 (ddd, J ¼ 8.7, 7.5,

3.5 Hz, 2H), 7.55 (dt, J ¼ 7.6, 1.4 Hz, 1H), 7.52 (s, 1H), 7.49 (d,

J ¼ 7.4 Hz, 1H), 7.41 (dt, J ¼ 7.8, 1.4 Hz, 1H), 7.30 (t, J ¼ 7.7 Hz, 1H),

4.10 (d, J ¼ 6.2 Hz, 2H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 139.23,

4.1.16. 5-Amino-N-(4-(triﬂuoromethyl)benzyl)naphthalene-1-

sulfonamide hydrochloride (11e)

135.83, 134.69, 132.11, 130.79, 130.69, 129.14, 129.03, 128.34, 127.92,

127.81, 126.00, 124.16, 120.42, 118.52, 117.21, 110.78, 45.01. ESI-LRMS

m/z: 336.0 [M ꢂ H]^-. ESI-HRMS calcd for C₁₈H₁₄N₃O₂S [M ꢂ H]^-

336.0812, found 336.0812.

Compound 11e was prepared in a similar manner as described

for compound 9c. Yield: 82%. Mp 178e180 ^ꢀC. ¹H NMR (400 MHz,

CD₃OD)

d

8.82 (dt, J ¼ 8.4, 1.3 Hz, 1H), 8.32 (dd, J ¼ 7.4, 1.0 Hz, 1H),

8.23 (d, J ¼ 8.5 Hz, 1H), 7.81e7.70 (m, 3H), 7.39 (d, J ¼ 8.1 Hz, 2H),

7.25 (d, J ¼ 8.1 Hz, 2H), 4.20 (s, 2H). ¹³C NMR (125 MHz, CD₃OD)

4.1.19. 4-(((5-Aminonaphthalene)-1-sulfonamido)methyl)benzoic

acid hydrochloride (11h)

d

143.21, 138.66, 131.27, 130.43 (q, J ¼ 32.2 Hz), 130.36, 129.58,

129.24, 128.87, 128.59, 127.30, 127.26, 127.14, 126.03 (q, J ¼ 3.6 Hz),

125.54 (q, J ¼ 271.1 Hz), 122.57, 47.06. ESI-LRMS m/z: 381.1 [M þ

H]^þ. ESI-HRMS calcd for C₁₈H₁₆F₃N₂O₂S [M þ H]^þ381.0879, found

381.0868.

Compound 11h was prepared in a similar manner as described

for compound 9c. Yield: 72%. Mp 259e260 ^ꢀC. ¹H NMR (400 MHz,

DMSO‑d₆)

d

8.72 (t, J ¼ 6.2 Hz, 1H), 8.47 (d, J ¼ 8.6 Hz, 1H), 8.37 (d,

J ¼ 8.6 Hz, 1H), 8.18 (dd, J ¼ 7.4, 1.0 Hz, 1H), 7.79 (d, J ¼ 8.3 Hz, 2H),

7.73e7.69 (m, 1H), 7.68e7.65 (m, 1H), 7.58 (d, J ¼ 7.5 Hz, 1H), 7.31 (d,

J ¼ 8.4 Hz, 2H), 4.10 (d, J ¼ 6.2 Hz, 2H). ¹³C NMR (125 MHz,

4.1.17. 5-Amino-N-(4-cyanobenzyl)naphthalene-1-sulfonamide

hydrochloride (11f)

DMSO‑d₆)

d 167.02, 143.02, 136.02, 134.73, 129.54, 129.14, 128.88,

128.47, 127.94, 127.81, 127.41, 126.16, 124.24, 120.62, 117.34, 45.57.

ESI-LRMS m/z: 355.0 [M ꢂ H]^-. ESI-HRMS calcd for C₁₈H₁₅N₂O₄S

[M ꢂ H]^-355.0758, found 355.0759.

Compound 11f was prepared in a similar manner as described

for compound 9c. Yield: 77%. Mp 209e211 ^ꢀC. ¹H NMR (500 MHz,

CD₃OD)

d

8.83 (ddd, J ¼ 8.1, 1.9, 1.0 Hz, 1H), 8.31 (dd, J ¼ 7.4, 1.1 Hz,

1H), 8.22 (dt, J ¼ 8.6, 1.1 Hz, 1H), 7.80e7.74 (m, 3H), 7.45e7.41 (m,

2H), 7.25e7.22 (m, 2H), 4.21 (s, 2H). ¹³C NMR (125 MHz, CD₃OD)

4.1.20. 5-Amino-N-(pyridin-4-ylmethyl)naphthalene-1-

sulfonamide dihydrochloride (11i)

d

144.42, 138.66, 133.01, 131.32, 130.31, 129.51, 129.24, 128.92,

128.61, 127.37, 127.34,127.27, 122.83, 119.45,112.03, 47.09. ESI-LRMS

m/z: 336.0 [M ꢂ H]^-. ESI-HRMS calcd for C₁₈H₁₄N₃O₂S [M ꢂ H]^-

336.0812, found 336.0818.

Compound 11i was prepared in a similar manner as described

for compound 9c. Yield: 83%. Mp 173e175 ^ꢀC. ¹H NMR (400 MHz,

DMSO‑d₆)

d

9.12 (t, J ¼ 6.3 Hz, 1H), 8.75 (d, J ¼ 6.6 Hz, 2H), 8.39 (d,

14

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

J ¼ 8.6 Hz, 1H), 8.34 (d, J ¼ 8.7 Hz, 1H), 8.16 (dd, J ¼ 7.4, 1.0 Hz, 1H),

7.86 (d, J ¼ 6.5 Hz, 2H), 7.66 (ddd, J ¼ 8.6, 7.4, 4.3 Hz, 2H), 7.49 (d,

J ¼ 7.5 Hz, 1H), 4.36 (d, J ¼ 6.3 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD)

for compound 9c. Yield: 82%. Mp 187e189 ^ꢀC. ¹H NMR (500 MHz,

DMSO‑d₆)

d

8.62 (d, J ¼ 2.7 Hz, 1H), 8.42 (t, J ¼ 8.2 Hz, 2H), 8.17 (dd,

J ¼ 7.3, 1.0 Hz, 1H), 7.68 (dd, J ¼ 8.6, 7.4 Hz, 1H), 7.63e7.56 (m, 2H),

7.18e7.13 (m, 2H), 7.11e7.06 (m, 1H), 6.76e6.70 (m, 2H), 2.26 (ddt,

J ¼ 7.3, 4.5, 2.8 Hz, 1H), 1.63 (ddd, J ¼ 9.6, 6.6, 3.1 Hz, 1H), 1.06e1.00

d

161.93, 142.34, 137.65, 131.44, 130.24, 130.20, 129.06, 128.94,

127.99, 127.25, 126.76, 126.60, 122.63, 46.44. ESI-LRMS m/z: 312.0

[M ꢂ H]^-. ESI-HRMS calcd for C₁₆H₁₄N₃O₂S [M ꢂ H]^-312.0812,

found 312.0810.

(m, 2H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 140.04, 135.71, 133.95,

129.92,128.55,128.12,128.03,127.89,126.34,125.75,125.62,124.52,

121.19, 117.96, 33.88, 23.48, 14.26. ESI-LRMS m/z: 337.0 [M ꢂ H]^-.

ESI-HRMS calcd for C₁₉H₁₇N₂O₂S [M ꢂ H]^-337.1016, found 337.1021.

4.1.21. 5-Amino-N-(thiophen-3-ylmethyl)naphthalene-1-

sulfonamide hydrochloride (11j)

Compound 11j was prepared in a similar manner as described

4.1.26. 5-Amino-N-(tert-butyl)naphthalene-1-sulfonamide

hydrochloride (11o)

for compound 9c. Yield: 78%. Mp 146e148 ^ꢀC. ¹H NMR (500 MHz,

CD₃OD)

d

8.85e8.81 (m, 1H), 8.31 (dd, J ¼ 7.4, 1.1 Hz, 1H), 8.21 (dt,

Compound 11o was prepared in a similar manner as described

J ¼ 8.6, 1.1 Hz, 1H), 7.79e7.73 (m, 3H), 7.04 (dd, J ¼ 5.0, 3.0 Hz, 1H),

for compound 9c. Yield: 85%. Mp 184e185 ^ꢀC. ¹H NMR (500 MHz,

6.93e6.91 (m,1H), 6.64 (dd, J ¼ 5.0,1.3 Hz,1H), 4.14 (s, 2H). ¹³C NMR

DMSO‑d₆)

J ¼ 7.3 Hz, 1H), 7.86 (s, 1H), 7.77e7.72 (m, 1H), 7.71e7.64 (m, 2H),

1.05 (s, 9H). ¹³C NMR (125 MHz, DMSO‑d₆)

139.45, 132.16, 128.78,

d

8.57 (d, J ¼ 7.8 Hz, 1H), 8.36 (d, J ¼ 8.5 Hz, 1H), 8.26 (d,

(125 MHz, CD₃OD)

d 139.06, 138.82, 131.25, 130.42, 129.01, 128.93,

128.39, 128.02, 127.55, 127.39, 127.07, 126.65, 123.49, 122.79, 42.74.

ESI-LRMS m/z: 317.0 [M ꢂ H]^-. ESI-HRMS calcd for C₁₅H₁₃N₂O₂S₂

[M ꢂ H]^-317.0424, found 317.0428.

d

128.53, 127.43, 127.40, 126.74, 124.96, 122.90, 119.16, 53.55, 29.66.

ESI-LRMS m/z: 277.2 [M ꢂ H]^-. ESI-HRMS calcd for C₁₄H₁₇N₂O₂S

[M ꢂ H]^-277.1016, found 277.1012.

4.1.22. 5-Amino-N-(cyclohexylmethyl)naphthalene-1-sulfonamide

hydrochloride (11k)

4.1.27. 5-Amino-N-benzyl-N-methylnaphthalene-1-sulfonamide

hydrochloride (11p)

Compound 11k was prepared in a similar manner as described

for compound 9c. Yield: 87%. Mp 218e220 ^ꢀC. ¹H NMR (500 MHz,

Compound 11p was prepared in a similar manner as described

DMSO‑d₆)

d

8.41 (d, J ¼ 8.7 Hz, 1H), 8.36 (d, J ¼ 8.5 Hz, 1H), 8.14 (d,

for compound 9c. Yield: 78%. Mp 174e175 ^ꢀC. ¹H NMR (400 MHz,

J ¼ 7.2 Hz, 1H), 7.95 (t, J ¼ 5.9 Hz, 1H), 7.69 (dd, J ¼ 8.5, 7.3 Hz, 1H),

7.63 (dd, J ¼ 8.7, 7.5 Hz,1H), 7.49 (d, J ¼ 7.5 Hz,1H), 2.60 (t, J ¼ 6.4 Hz,

2H), 1.59e1.47 (m, 5H), 1.25 (ttt, J ¼ 10.2, 6.7, 3.0 Hz, 1H), 1.07e0.96

DMSO‑d₆)

d

8.44 (d, J ¼ 8.6 Hz, 1H), 8.39 (d, J ¼ 8.9 Hz, 1H), 8.21 (d,

J ¼ 7.3 Hz, 1H), 7.74 (t, J ¼ 8.0 Hz, 1H), 7.68 (t, J ¼ 8.1 Hz, 1H), 7.51 (d,

J ¼ 7.1 Hz, 1H), 7.37e7.26 (m, 5H), 4.39 (s, 2H), 2.68 (s, 3H). ¹³C NMR

(m, 3H), 0.76e0.65 (m, 2H). ¹³C NMR (125 MHz, DMSO‑d₆)

d

136.27,

(125 MHz, CD₃OD) d 137.15, 137.12, 131.57, 130.96, 129.67, 129.51,

134.76, 128.70, 128.54, 127.77, 127.51, 126.08, 124.24, 120.63, 117.11,

48.64, 37.23, 30.02, 25.85, 25.16. ESI-LRMS m/z: 317.1 [M ꢂ H]^-. ESI-

HRMS calcd for C₁₇H₂₁N₂O₂S [M ꢂ H]^-317.1329, found 317.1331.

129.42, 129.22, 128.99, 128.69, 127.69, 127.55, 127.46, 122.90, 54.57,

34.36. ESI-LRMS m/z: 327.3 [M

þ

H]^þ. ESI-HRMS calcd for

C

₁₈H₁₉N₂O₂S [M þ H]^þ327.1162, found 327.1156.

4.1.23. 5-Amino-N-(piperidin-3-ylmethyl)naphthalene-1-

sulfonamide dihydrochloride (11l)

4.1.28. 5-((4-Benzylpiperidin-1-yl)sulfonyl)naphthalen-1-amine

hydrochloride (11q)

Compound 11l was prepared in a similar manner as described

Compound 11q was prepared in a similar manner as described

for compound 9c. Yield: 75%. Mp 272e274 ^ꢀC. ¹H NMR (500 MHz,

for compound 9c. Yield: 75%. Mp 181e183 ^ꢀC. ¹H NMR (500 MHz,

DMSO‑d₆)

d

9.17 (d, J ¼ 11.2 Hz, 1H), 8.96 (q, J ¼ 10.8 Hz, 1H), 8.44 (d,

DMSO‑d₆)

d

8.42 (d, J ¼ 8.5 Hz, 1H), 8.35 (d, J ¼ 8.8 Hz, 1H), 8.15 (dd,

J ¼ 8.8 Hz,1H), 8.41 (d, J ¼ 8.6 Hz,1H), 8.24 (t, J ¼ 6.2 Hz,1H), 8.15 (d,

J ¼ 7.2 Hz, 1H), 7.72 (dd, J ¼ 8.5, 7.4 Hz, 1H), 7.67 (t, J ¼ 8.0 Hz, 1H),

7.59 (d, J ¼ 7.5 Hz, 1H), 3.19e3.08 (m, 2H), 2.76e2.68 (m, 2H),

2.67e2.59 (m, 1H), 2.49e2.42 (m, 1H), 1.93e1.83 (m, 1H), 1.72e1.61

(m, 2H), 1.61e1.50 (m, 1H), 1.12e1.00 (m, 1H). ¹³C NMR (125 MHz,

J ¼ 7.4, 1.1 Hz, 1H), 7.72 (dd, J ¼ 8.5, 7.3 Hz, 1H), 7.63 (dd, J ¼ 8.8,

7.5 Hz, 1H), 7.48 (d, J ¼ 7.4 Hz, 1H), 7.23 (t, J ¼ 7.5 Hz, 2H), 7.16e7.12

(m, 1H), 7.11e7.07 (m, 2H), 3.70 (dt, J ¼ 12.9, 2.8 Hz, 2H), 2.55e2.47

(m, 2H), 2.43 (d, J ¼ 6.7 Hz, 2H), 1.61e1.50 (m, 3H), 1.15e1.04 (m,

2H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 139.73, 135.13, 133.38, 130.24,

DMSO‑d₆)

d

135.80, 133.96, 128.84, 128.43, 127.88, 126.42, 124.42,

129.10, 128.93, 128.33, 128.15, 128.12, 126.18, 125.82, 124.26, 120.30,

117.04, 45.42, 41.72, 36.26, 31.09. ESI-LRMS m/z: 381.0 [M þ H]^þ.

ESI-HRMS calcd for C₂₂H₂₅N₂O₂S [M þ H]^þ381.1631, found

381.1631.

121.17, 117.98, 45.75, 45.20, 43.10, 33.58, 25.59, 21.11. ESI-LRMS m/z:

318.0 [M ꢂ H]^-. ESI-HRMS calcd for C₁₆H₂₀N₃O₂S [M ꢂ H]^-318.1282,

found 318.1281.

4.1.24. 5-Amino-N-((1-methylpiperidin-4-yl)methyl)naphthalene-

1-sulfonamide dihydrochloride (11m)

4.1.29. N-benzylnaphthalene-1-sulfonamide (11r)

Compound 11r was prepared in a similar manner as described

Compound 11m was prepared in a similar manner as described

for compound 9c. Yield: 89%. Mp 131e133 ^ꢀC. ¹H NMR (500 MHz,

for compound 9c. Yield: 80%. Mp 231e232 ^ꢀC. ¹H NMR (500 MHz,

DMSO‑d₆)

d

8.68 (dd, J ¼ 8.5, 1.1 Hz, 1H), 8.51 (t, J ¼ 6.2 Hz, 1H), 8.19

CD₃OD)

d

8.87e8.84 (m, 1H), 8.36 (dd, J ¼ 7.4, 1.1 Hz, 1H), 8.30 (dt,

(d, J ¼ 8.2 Hz, 1H), 8.12 (dd, J ¼ 7.3, 1.3 Hz, 1H), 8.07 (dd, J ¼ 8.1,

1.4 Hz, 1H), 7.71 (ddd, J ¼ 8.5, 6.9, 1.6 Hz, 1H), 7.66 (ddd, J ¼ 8.0, 6.8,

1.3 Hz, 1H), 7.61 (dd, J ¼ 8.2, 7.3 Hz, 1H), 7.20e7.11 (m, 5H), 4.03 (d,

J ¼ 8.6, 1.1 Hz, 1H), 7.86 (dd, J ¼ 8.6, 7.4 Hz, 1H), 7.82e7.78 (m, 1H),

7.77 (dd, J ¼ 7.5, 1.6 Hz, 1H), 3.46 (dp, J ¼ 12.5, 1.9 Hz, 2H), 2.92 (td,

J ¼ 13.0, 2.9 Hz, 2H), 2.85e2.79 (m, 5H), 1.92 (ddd, J ¼ 13.3, 4.3,

2.4 Hz, 2H), 1.78e1.68 (m, 1H), 1.46e1.36 (m, 2H). ¹³C NMR

J ¼ 6.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 137.64, 135.82,

133.81, 133.60, 128.85, 128.35, 127.97, 127.73, 127.50, 127.37, 126.97,

126.76, 124.73, 124.43, 45.91. ESI-LRMS m/z: 296.0 [M ꢂ H]^-. ESI-

HRMS calcd for C₁₇H₁₄NO₂S [M ꢂ H]^-296.0751, found 296.0753.

(125 MHz, CD₃OD)

d 138.33, 131.04, 130.39, 129.45, 129.06, 128.66,

127.47, 127.39, 127.25, 122.86, 55.28, 48.39, 43.84, 35.03, 28.42. ESI-

LRMS m/z: 332.1 [M ꢂ H]^-. ESI-HRMS calcd for C₁₇H₂₂N₃O₂S

[M ꢂ H]^-332.1438, found 332.1442.

4.1.30. N-benzyl-5-(dimethylamino)naphthalene-1-sulfonamide

hydrochloride (11s)

4.1.25. 5-Amino-N-(trans-2-phenylcyclopropyl)naphthalene-1-

sulfonamide hydrochloride (11n)

Compound 11s was prepared in a similar manner as described

for compound 9c. Yield: 88%. Mp 176e177 ^ꢀC. ¹H NMR (500 MHz,

Compound 11n was prepared in a similar manner as described

DMSO‑d₆)

d

8.77 (d, J ¼ 8.9 Hz, 1H), 8.65 (t, J ¼ 6.3 Hz, 1H), 8.60 (d,

15

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

J ¼ 8.4 Hz, 1H), 8.18 (d, J ¼ 7.3 Hz, 1H), 7.80e7.62 (m, 3H), 7.16e7.07

(m, 5H), 4.04 (d, J ¼ 6.1 Hz, 2H), 3.10 (s, 6H). ¹³C NMR (125 MHz,

J ¼ 7.4, 1.0 Hz, 1H), 7.66e7.59 (m, 2H), 7.35 (d, J ¼ 7.6 Hz, 1H),

7.09e7.00 (m, 5H), 4.06 (s, 2H), 3.96 (tt, J ¼ 11.3, 3.9 Hz, 1H),

3.67e3.62 (m, 2H), 3.18 (t, J ¼ 12.4 Hz, 2H), 2.90 (s, 3H), 2.38 (d,

J ¼ 14.2 Hz, 2H), 2.20e2.08 (m, 2H). ¹³C NMR (125 MHz, CD₃OD)

DMSO‑d₆)

d 137.48, 136.74, 128.81, 128.24, 127.93, 127.56, 127.42,

127.21, 126.98, 124.97, 123.33, 117.88, 45.97, 45.87. ESI-LRMS m/z:

341.2 [M þ H]^þ. ESI-HRMS calcd for C₁₉H₂₁N₂O₂S [M þ H]^þ

341.1318, found 341.1319.N-Benzyl-5-((cyclopropylmethyl)amino)

naphthalene-1-sulfonamide hydrochloride (11u).

d

138.40,137.97,137.78,130.91,130.82,129.11,129.03,128.74,128.24,

127.70, 127.11, 125.62, 121.54, 115.66, 54.42, 52.74, 47.70, 43.79,

29.29. ESI-LRMS m/z: 408.1 [M

C

ꢂ

H]^-. ESI-HRMS calcd for

To a stirred solution of compound 9c (150 mg, 0.430 mmol) in

₂₃H₂₆N₃O₂S [M ꢂ H]^-408.1751, found 408.1759.

anhydrous MeOH (8 mL) was added trimethylamine (60

0.430 mmol) at room temperature. After 10 min, cyclo-

propanecarboxaldehyde 19a (32 L, 0.430 mmol) and acetic acid

(25 L, 0.430 mmol) were added to the reaction mixture. Then,

mL,

4.1.34. N-benzyl-5-((1-ethylpiperidin-4-yl)amino)naphthalene-1-

sulfonamide dihydrochloride (11y)

m

Compound 11y was prepared in a similar manner as described

sodium cyanoborohydride (30 mg, 0.473 mmol) was added after

0.5 h. After 5 h, TLC showed completion of the reaction. The reac-

tion mixture was concentrated and diluted with water (10 mL),

then extracted with DCM (5 mL x 3). The combined layer was

concentrated under vacuum and puriﬁed by silica gel column

chromatography (DCM/MeOH ¼ 50/1e20/1) to afford white solid,

which was subsequently dissolved in 4 N HCl in dioxane (5 mL) and

stirred for 2 h at room temperature. Finally, the reaction mixture

was concentrated to afford compound 11u as a white solid (117 mg,

for compound 11u. Yield: 65%. Mp 192e194 ^ꢀC. ¹H NMR (500 MHz,

CD₃OD)

d

8.58 (d, J ¼ 8.7 Hz, 1H), 8.37 (d, J ¼ 8.6 Hz, 1H), 8.27 (dd,

J ¼ 7.3, 1.0 Hz, 1H), 7.71e7.65 (m, 2H), 7.51 (d, J ¼ 7.6 Hz, 1H),

7.09e7.01 (m, 5H), 4.08 (s, 2H), 4.03e3.97 (m, 1H), 3.71 (d,

J ¼ 12.9 Hz, 2H), 3.21 (q, J ¼ 7.3 Hz, 2H), 3.10 (td, J ¼ 13.1, 2.7 Hz, 2H),

2.39 (d, J ¼ 13.9 Hz, 2H), 2.22 (qd, J ¼ 13.1, 3.9 Hz, 2H), 1.38 (t,

J ¼ 7.3 Hz, 3H). ¹³C NMR (125 MHz, CD₃OD)

d 138.36, 135.69, 131.00,

130.93, 130.64, 129.12, 128.75, 128.25, 127.52, 127.42, 126.32, 124.96,

123.67, 118.31, 54.42, 53.30, 51.83, 47.71, 28.71, 9.74. ESI-LRMS m/z:

421.9 [M ꢂ H]^-. ESI-HRMS calcd for C₂₄H₂₈N₃O₂S [M ꢂ H]^-422.1908,

found 422.1917.

yield: 68%). Mp 160e161 ^ꢀC. ¹H NMR (500 MHz, DMSO‑d₆)

d 8.52

(dt, J ¼ 8.5,1.1 Hz,1H), 8.47 (t, J ¼ 6.2 Hz,1H), 8.21e8.15 (m,1H), 8.12

(dd, J ¼ 7.4,1.1 Hz,1H), 7.60e7.54 (m, 3H), 7.21e7.10 (m, 5H), 4.00 (d,

J ¼ 6.1 Hz, 2H), 3.19 (d, J ¼ 6.9 Hz, 2H), 1.21e1.14 (m, 1H), 0.54e0.49

4.1.35. 5-((1-Acetylpiperidin-4-yl)amino)-N-benzylnaphthalene-1-

sulfonamide hydrochloride (11z)

(m, 2H), 0.33e0.29 (m, 2H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 137.72,

135.96, 128.79, 128.50, 128.28, 127.99, 127.39, 127.31, 126.97, 124.72,

123.24, 59.73, 45.92, 9.18, 3.91. ESI-LRMS m/z: 365.0 [M ꢂ H]^-. ESI-

HRMS calcd for C₂₁H₂₁N₂O₂S [M ꢂ H]^-365.1329, found 365.1332.

Compound 11z was prepared in a similar manner as described

for compound 11u. Yield: 60%. Mp 162e163 ^ꢀC. ¹H NMR (400 MHz,

CD₃OD)

d

8.89 (d, J ¼ 8.0 Hz, 1H), 8.37e8.29 (m, 2H), 7.84e7.74 (m,

3H), 7.05e6.96 (m, 5H), 4.67 (d, J ¼ 13.1 Hz,1H), 4.11 (s, 2H), 3.98 (tt,

J ¼ 11.6, 4.0 Hz, 1H), 3.67e3.64 (m, 1H), 3.23e3.11 (m, 1H), 2.67 (t,

J ¼ 12.2 Hz, 1H), 2.16 (s, 3H), 2.14e2.06 (m, 2H), 1.97e1.71 (m, 2H).

4.1.31. N-benzyl-5-(cyclohexylamino)naphthalene-1-sulfonamide

hydrochloride (11v)

Compound 11v was prepared in a similar manner as described

¹³C NMR (125 MHz, CD₃OD)

d 171.84, 139.16, 138.21, 131.44, 130.87,

for compound 11u. Yield: 68%. Mp 185e187 ^ꢀC. ¹H NMR (500 MHz,

130.84, 129.07, 128.76, 128.50, 128.21, 128.19, 128.13, 127.83, 127.12,

124.58, 60.61, 47.67, 45.68, 41.00, 30.47, 29.79, 21.09. ESI-LRMS m/z:

438.4 [M þ H]^þ. ESI-HRMS calcd for C₂₄H₂₆N₃O₃S [M þ H]^þ

438.1846, found 438.1844.

DMSO‑d₆)

d

8.61e8.55 (m, 1H), 8.51 (d, J ¼ 8.6 Hz, 1H), 8.45e8.28

(m,1H), 8.15 (d, J ¼ 7.3 Hz,1H), 7.67e7.58 (m, 2H), 7.51e7.31 (m,1H),

7.19e7.04 (m, 5H), 4.03 (d, J ¼ 6.1 Hz, 2H), 3.45 (tt, J ¼ 11.2, 3.8 Hz,

1H), 2.01 (dd, J ¼ 12.8, 3.8 Hz, 2H), 1.77 (dt, J ¼ 13.3, 3.5 Hz, 2H), 1.62

(dt, J ¼ 12.9, 3.5 Hz, 1H), 1.52 (q, J ¼ 10.2 Hz, 2H), 1.29 (q, J ¼ 12.6 Hz,

2H), 1.16 (qt, J ¼ 12.6, 3.4 Hz, 1H). ¹³C NMR (125 MHz, DMSO‑d₆)

4.1.36. 5-((1-Benzoylpiperidin-4-yl)amino)-N-benzylnaphthalene-

1-sulfonamide hydrochloride (11aa)

d

137.61,136.30,128.90,128.77,127.95,127.78,127.49,127.41,126.93,

Compound 11aa was prepared in a similar manner as described

125.65, 124.06, 56.53, 45.93, 30.43, 25.10, 24.37. ESI-LRMS m/z:

395.3 [M þ H]^þ. ESI-HRMS calcd for C₂₃H₂₇N₂O₂S [M þ H]^þ

395.1788, found 395.1791.

for compound 11u. Yield: 59%. Mp 258e260 ^ꢀC. ¹H NMR (500 MHz,

DMSO‑d₆)

d

8.50 (d, J ¼ 8.6 Hz, 1H), 8.36 (t, J ¼ 6.3 Hz, 1H), 8.07 (d,

J ¼ 7.2 Hz, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.50e7.38 (m, 6H), 7.23e7.14

(m, 5H), 6.79 (d, J ¼ 7.9 Hz, 1H), 6.10 (d, J ¼ 7.5 Hz, 1H), 4.48 (s, 1H),

3.98 (d, J ¼ 6.2 Hz, 2H), 3.86e3.76 (m, 1H), 3.72e3.58 (m, 1H),

3.30e3.19 (m, 1H), 3.12e2.97 (m, 1H), 2.19e1.94 (m, 2H), 1.63e1.48

4.1.32. N-benzyl-5-(piperidin-4-ylamino)naphthalene-1-

sulfonamide dihydrochloride (11w)

Compound 11w was prepared in a similar manner as described

(m, 2H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 169.00, 143.63, 137.89,

for compound 11u. Yield: 60%. Mp > 250 ^ꢀC. ¹H NMR (500 MHz,

136.34, 135.49, 129.41, 129.09, 128.74, 128.46, 128.21, 128.03, 127.48,

127.40, 126.98, 126.60, 123.92, 121.96, 112.12, 104.85, 49.20, 46.20,

45.91, 40.70, 31.74, 31.00. ESI-LRMS m/z: 498.0 [M ꢂ H]^-. ESI-HRMS

calcd for C₂₉H₂₈N₃O₃S [M ꢂ H]^-498.1857, found 498.1862.

DMSO‑d₆)

d

9.24 (d, J ¼ 11.1 Hz, 1H), 9.17e9.07 (m, 1H), 8.58 (d,

J ¼ 8.5 Hz,1H), 8.41 (t, J ¼ 6.2 Hz,1H), 8.07 (d, J ¼ 7.2 Hz,1H), 7.92 (d,

J ¼ 8.5 Hz, 1H), 7.47 (dt, J ¼ 12.1, 8.3 Hz, 2H), 7.25e7.14 (m, 5H), 6.82

(d, J ¼ 7.8 Hz, 1H), 3.98 (d, J ¼ 6.2 Hz, 2H), 3.78 (tt, J ¼ 10.2, 3.6 Hz,

1H), 3.34 (dt, J ¼ 12.8, 3.3 Hz, 2H), 3.04 (q, J ¼ 11.6 Hz, 2H), 2.15 (dd,

J ¼ 14.2, 3.8 Hz, 2H), 1.91e1.79 (m, 2H). ¹³C NMR (125 MHz,

4.1.37. Benzyl 4-((5-(N-benzylsulfamoyl)naphthalen-1-yl)amino)

piperidine-1-carboxylate hydrochloride (11 ab)

DMSO‑d₆)

d

143.12, 137.89, 135.55, 129.07, 128.62, 128.22, 128.02,

Compound 11 ab was prepared in a similar manner as described

127.77, 127.41, 126.98, 124.06, 122.08, 112.79, 105.43, 47.22, 45.91,

for compound 11u. Yield: 62%. Mp 145e147 ^ꢀC. ¹H NMR (400 MHz,

42.15, 27.90. ESI-LRMS m/z: 396.3 [M þ H]^þ. ESI-HRMS calcd for

DMSO‑d₆)

d

8.52 (d, J ¼ 8.6 Hz,1H), 8.48 (d, J ¼ 6.1 Hz,1H), 8.16e8.14

C

₂₂H₂₆N₃O₂S [M þ H]^þ396.1740, found 396.1744.

(m, 1H), 8.11 (d, J ¼ 7.2 Hz, 1H), 7.55 (td, J ¼ 8.3, 3.5 Hz, 2H),

7.41e7.35 (m, 4H), 7.35e7.28 (m, 1H), 7.21e7.11 (m, 6H), 5.10 (s, 2H),

4.07 (d, J ¼ 13.4 Hz, 2H), 4.00 (d, J ¼ 6.0 Hz, 2H), 3.73 (tt, J ¼ 10.2,

3.4 Hz, 1H), 2.98 (s, 2H), 2.02 (d, J ¼ 10.6 Hz, 2H), 1.60 (q, J ¼ 11.4 Hz,

4.1.33. N-benzyl-5-((1-methylpiperidin-4-yl)amino)naphthalene-

1-sulfonamide dihydrochloride (11x)

Compound 11x was prepared in a similar manner as described

2H). ¹³C NMR (125 MHz, DMSO‑d₆)

d 154.41, 137.77, 137.01, 135.93,

for compound 11u. Yield: 66%. Mp 159e160 ^ꢀC. ¹H NMR (500 MHz,

129.01, 128.51, 128.45, 128.25, 128.01, 127.84, 127.52, 127.42, 126.99,

CD₃OD)

d

8.44 (d, J ¼ 8.7 Hz, 1H), 8.37 (d, J ¼ 8.5 Hz, 1H), 8.23 (dd,

124.81, 123.08, 66.20, 51.77, 45.92, 42.52, 30.28. ESI-LRMS m/z:

16

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

530.3 [M þ H]^þ. ESI-HRMS calcd for C₃₀H₃₂N₃O₄S [M þ H]^þ

Dilute enzyme, SAM(S-(5⁰-Adenosyl)-

compounds and peptide substrate in Assay Buffer just before use.

Add 4 L of inhibitor, 2 L of enzyme to the wells of a white

OptiPlate-384 and incubate for 10 min at room temperature. Sub-

sequently, add 4 L of peptide/SAM mix to the reaction system.

Cover the plate with TopSeal-A ﬁlm and incubate at room tem-

perature for h. Prepare 2X mix of antibody and SA-

XL665,respectively, in Detection Buffer. Add 10 L of detection

mixture (2X) to the plate. Cover with TopSeal-A ﬁlm and incubate in

subdued light for 1 h at room temperature. Remove plate sealer and

read ﬂuorescence emission at 665 nm and 620 nm wavelengths on

an HTRF compatible reader. The resultant data were analyzed with

GraphPad Prism.

L-methionine chloride),

530.2108, found 530.2118.

m

4.1.38. N-benzyl-5-((1-(methylsulfonyl)piperidin-4-yl)amino)

naphthalene-1-sulfonamide hydrochloride (11ac)

m

Compound 11ac was prepared in a similar manner as described

for compound 11u. Yield: 71%. Mp 159e161 ^ꢀC. ¹H NMR (500 MHz,

4

a

DMSO‑d₆)

d

8.52 (d, J ¼ 8.6 Hz, 1H), 8.43 (t, J ¼ 6.3 Hz, 1H), 8.10 (dd,

m

J ¼ 7.3, 1.1 Hz, 1H), 8.04 (d, J ¼ 8.6 Hz, 1H), 7.57e7.47 (m, 2H),

7.23e7.13 (m, 5H), 7.04e6.88 (m, 1H), 3.99 (d, J ¼ 6.0 Hz, 2H),

3.69e3.59 (m, 3H), 2.93 (dd, J ¼ 12.2, 2.6 Hz, 2H), 2.90 (s, 3H), 2.10

(dd, J ¼ 13.5, 3.6 Hz, 2H), 1.71 (qd, J ¼ 11.7, 4.0 Hz, 2H). ¹³C NMR

(125 MHz, DMSO‑d₆) d 137.80,135.77,129.03,128.42,128.38,128.00,

127.53, 127.40, 126.97, 124.47, 122.63, 50.10, 45.91, 44.57, 34.48,

30.11. ESI-LRMS m/z: 472.1 [M

C

ꢂ

H]^-. ESI-HRMS calcd for

4.2.4. Cell lines

₂₃H₂₆N₃O₄S₂[M ꢂ H]^-472.1370, found 472.1378.

The human B cell precursor leukemia cell line RS4:11, OPM-2

and human myeloma cell line KMS11 were cultured in the altered

RPMI-1640 medium (Invitrogen) containing 10% selected FBS

(Gibco) and 2 mM Glutamine. The MV4:11 cell line was maintained

in IMDM (Iscove's Modiﬁed Dulbecco's Medium) supplemented

with 10% selected FBS (Gibco).

4.2. Biological evaluation

4.2.1. HTS screening for NSD2 inhibitors

The compound library used in this study consisted of 296 080

compounds, synthetic and natural product-derived compounds

solubilized in 100% DMSO prior to application in the HTS campaign.

Recombinant human NSD2 (aa941-1240) was purchased from BPS

Bioscience. The reaction was carried out in white 384-well plates

(ProxiPlateTM-384 Plus, PerkinElmer), using AlphaLISA method. In

brief, the assay were diluted in assay buffer (50 mM Tris-HCl, 0.01%

Tween-20, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, pH8.5) for 3 h at

4.2.5. Western blot analysis

The cells were lysed in buffer containing 2% SDS and quantiﬁed

by performing a BCA assay. An equal amount of protein was sub-

jected to electrophoresis on SDSePAGE and transferred to nitro-

cellulose membranes, incubated with primary antibodies and then

incubated with horseradish peroxidase-conjugated secondary an-

tibodies. Primary antibodies used were as follows: anti-beta-actin

(Proteintech Group, 66 009-1-lg), anti-histone H3 (Cell Signaling

Technology, #9715), anti-WHSC1/NSD2 (abcam, ab75359),anti-Di-

Methyl-Histone H3 (Lys27) (Cell Signaling Technology, #9728),

anti-Tri-Methyl-Histone H3 (Lys27) (Cell Signaling Technology,

#9733), anti-Tri-Methyl-Histone H3 (Lys36) (Cell Signaling Tech-

nology, #4909), anti-Di-Methyl-Histone H3 (Lys36) (Cell Signaling

Technology, #2901), anti-Di-Methyl-Histone H3 (Lys4) (Cell

Signaling Technology, #9725), anti-Di-Methyl-Histone H3 (Lys9)

(Cell Signaling Technology, #4658), anti-Tri-Methyl-Histone H3

(Lys79) (Cell Signaling Technology, #4260). The detection was

performed using a SuperSignal west pico stable peroxide solution

(Thermo Fisher Scientiﬁc).

room temperature, containing 2

L substrate (20 M SAM, 0.15 ng/mL Nucleosomes) and 1

compound (20 g/mL) or control (2% DMSO as positive control, SAH

as negative control), followed by the addition of 2.5 L High Salt

buffer (50 mM Tris-HCl, 1 M NaCl, 0.1% Tween-20, 0.3% poly-

mL NSD2 enzyme solution (10 nM),

2

m

mL

m

L-

lysine, pH7.4) to stop the reaction. The activity of NSD2 was

detected by using AlphaLISA NSD2 Histone H3 Lysine-N-methyl-

transferase assay (PerkinElmer). The ﬂuorescence intensity was

measured with excitation and emission wavelengths at 680 and

615 nm using Envision (PerkinElmer). Hits identiﬁed from the

primary screen were subsequently rescreened against NSD2 at two

points (20 mg/mL and 2 mg/mL) in duplicate wells. And IC₅₀values

were determined using the AlphaLISA assay described above.

Selected hits were dissolved and serially diluted in 100% DMSO,

with a ﬁnal in-assay concentration of 2% DMSO in duplicate wells.

The results were obtained using an Envision (PerkinElmer) and the

IC₅₀data was calculated using the software GraphPad Prism 5, and

chosen the equation “sigmoidal dose-response (variable slope)” for

curve ﬁtting.

4.2.6. Cell proliferation assay

Cells were seeded in 96-well plates at 6000e20000 cells per

well in triplicate for 24 h. Then the cells were treated with

increasing doses of compound 9c and were cultured for another 6 d.

Cell viability was examined using the cell counting kit-8 (Life

Technologies) according to the manufacturer's instructions. The

optical density measured at a wavelength of 450 nm was used as an

indicator of cell viability.

4.2.2. NMR experiments

Ligand observed CPMG and saturation transfer difference (STD)

NMR experiments were applied to investigate ligand-protein in-

teractions. All NMR spectra were acquired at 25 ^ꢀC on a Bruker

Avance III 600 MHz NMR spectrometer equipped with a cryoprobe

(Bruker biospin, Germany). Samples containing 200 mM compound

4.2.7. Cell cycle analysis and annexin V staining

Cell cycle analysis was conducted by incorporating propidium

iodide (PI). Leukemic cells were treated with indicated does of

compound 9c for 72 h. Then cells were washed twice and ﬁxed by

70% pre-cooled ethanol overnight. Cells were resuspended in stain

9c, 200 mM compound 9c in the presence of 4

mM NSD2 were

dissolved in phosphate buffer (20 mM NaH₂PO₄, 20 mM Na₂HPO₄,

100 mM NaCl, 5% DMSO‑d₆, pH 7.4) and then used in NMR data

acquisition.

buffer containing 5 mg/mL ribonuclease A (Sigma-Aldrich) and 5 mg/

mL PI for 45 min in room tempreture. Acquisition was performed

on FACSCanto (BD Biosciences), and results were analyzed using

modiﬁt software.

4.2.3. Selectivity assays

The selectivity of compound 9c against NSD1, SETD2, DOT1L,

PRMT4, PRMT5 was determined using AlphaLISA immunodetection

assay as described previously. The selectivity against EZH2, MLL1,

and MLL4 methylases were assessed with homogenous time-

resolved ﬂuorescence resonance energy transfer (HTRF) assays.

For apoptosis assay,10⁵cells were collected and re-suspended in

100

mL annexin V binding solution containing 0.25

m

g/mL anti-

g/mL PI in the dark for 15 min. Samples

L annexin V binding solution and analyzed

annexin V-FITC and 1

were diluted with 400

with the FACS.

m

17

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

4.2.8. RNA-seq

5⁰-TTTCCTGTTCCTGCTTGCTT-3’

RNA-seq data was generated by novogene. RS4:11 cells plated in

10 cm dishes were treated with DMSO or 10 mM compound 9c for 6

5⁰-AATTAGAGCCACGTGCCAAG-3’

MET promoter:

days respectively. Then cells were washed by PBS for 3 times and

then lysed by trizol at room temperature. Samples were sent to

novogene on dry ice. A total amount of 3 mg RNA per sample was

used as input material for the RNA sample preparations.

Sequencing libraries were generated using NEBNext UltraTM RNA

library prep it for illumina (NEB, USA) following manufacturer's

recommendations and index codes were added to attribute se-

quences to each sample. The clustering of the index-coded samples

was performed on a cBot cluster generation system using TruSeq PE

cluster kit v3-cBot-HS (Illumia) according to the manufacturer's

instructions. After cluster generation, the library preparations were

sequenced on an illumina platform and 125 bp/150 bp paired-end

reads were generated. GO was used for cellular pathway analysis.

5⁰-GGAGACTCGGTCCCGCTTAT-3'

5⁰-CCCAGCTCAGGCAGTCTGA-3'

MET TSS:

5⁰-TGACACTCGCCTCCCAAG-3’

5⁰-AAGTTAGCACAGCCGGAGATA-3’

MET (1) 3 kb:

5⁰-CGTTTCTTCTTTAGGCATTAGGC-3’

5⁰-ACCACGGAAAAGAAAGCGTA-3’

MET (2) 26 kb:

5⁰-TGGTGCAGAGGAGCAATGG-3’

5⁰-CCCAGTCTTGTACTCAGCAAC-3’

MET (3) 45 kb:

5⁰-GTCATCACCACGAGGCTGT-3’

5⁰-GTGATTCACAAGGTGATGGAAG-3’

MET (4) 71 kb:

4.2.9. ChIP-qPCR

ChIP-qPCR assay was performed using SimpleChIP plus enzy-

matic chromatin IP kit (Magnetic Beads) (Cell Signaling Technology,

#9005) according to the procedures provided by the manufacturer.

Antibodies used for ChIP were as follows: anti-immunoglobulin G

(Cell Signaling Technology, #2729), anti-Histone H3 (Cell Signaling

Technology, #4620), anti-Histone H3K36me2 (Invitrogen, # MA5-

14867). Puriﬁed DNA was subject to quantitative real-time PCR

analysis, using primers that encompass genes’ promoter region. All

samples were normalized to input as control. The sequences of the

primers were as follows:

5⁰-GCTCGCAGCAAGATCAGTG-3’

5⁰-CCGTGTACCTCTGTTGGACA-3’

MET (5) 114 kb:

5⁰-GGAGAAATTGGATGCTCAACA-3’

5⁰-TTCCAAGACCCTTCTGGTGT-3’

RRAS2 promoter:

5⁰-AGTGGGTGTCAGTTGGGAGT-3’

5⁰-CCACACAATCCCTTACATAGACAA-3’

RRAS2 TSS:

5⁰-CCAAGTTGCCACCGCTAT-3’

5⁰-AGCCGGGCTTTACTGCTC-3’

RRAS2 (1) 5 kb:

TGFA promoter:

5⁰-GGAGCTCACGGACTGACC-3’

5⁰-GATCTACCCGGAGCACGTAG-3’

TGFA TSS:

5⁰-CAAATCTCCTGAAATCTCTTCTCG-3’

5⁰-TGCTGAGTACTTTTTCATTGCTTT-3’

RRAS2 (2) 15 kb:

5⁰-CGTAGCCGGATTGTCCTG-3’

5⁰-GCCCCTCGGTGTAGGTAAC-3’

TGFA (1) 6 kb:

5⁰-CACGCCTGTACTCCCAGTTAC-3’

5⁰-TGTCCCCGGCTTAAGTGAT-3’

RRAS2 (3) 24 kb:

5⁰-CAAGATGAAAACAGGTGAAGC A-3’

5⁰-TCAAGACGGAACCCCTTTAG-3’

TGFA (2) 22 kb:

5⁰-GGTCCTTGCCATCAACAACT-3’

5⁰-TGAGGAAAAAGCTAGTACAATAGGG-3’

RRAS2 (4) 50 kb:

5⁰-GTTGAAAGCGACGAAACCAT-3’

5⁰-GTGCTGGTTTGGGTTTTCTC-3’

TGFA (3) 57 kb:

5⁰-AATGTGCCAAGCATTGTGTC-3’

5⁰-GAAACTGCCAGCCCTCTAAG-3’

RRAS2 (5) 74 kb:

5⁰-CTGGAAAAACTGGGGAAAGA-3’

5⁰-TTCTAGGCTTTGCACTTGCTC-3’

TGFA (4) 73 kb:

5⁰-CCATACCAATTCAAGTATGGTTTAAGA-3’

5⁰-GGGACTTTAGGCATACACCACT-3’

5⁰-TCCTGCCTCTTCCATGATCT-3’

5⁰-AAACCAGGAATTCCCAACTTTT-3’

TGFA (5) 101 kb:

4.2.10. Tumor xenograft experiment

RS4:11 cells at a density of 1 ꢃ 10⁷in 0.2 mL of phosphate-

buffered saline were injected subcutaneously into the ﬂank of 4-

week-old SCID female mice. When the tumor volume reached

approximately 100 mm³, mice were distributed into three groups

(n ¼ 8 per group) and treated with 25 mg/kg compound 9c, 50 mg/

kg compound 9c or vehicle by intravenous and intraperitoneal in-

jection every day. The tumors and body weight of the mice were

measured individually twice weekly. This study was conducted in

strict accordance with the recommendations in the guide for the

care and use of laboratory animals.

5⁰-AGAGGTGAGTAACTTGTCAAAGGTC-3’

5⁰-GTAGGGACCCCTTACATCCTG-3’

PAK1 promoter:

5⁰-TGTTTTCTTTCTGTGGGAGAGG-3’

5⁰-CACATTAGTTCAAACATCTCCGTTA-3’

PAK1 TSS:

5⁰-GAGGGGGCGTCTACTGTG-3’

5⁰-GTACAATAGCGCGGCTGTG-3’

PAK1 (1) 7 kb:

5⁰-CACCCCCATTTCAAGTGC-3’

5⁰-TAAGTGCAAGTGCCATGTGAA-3’

PAK1 (2) 47 kb:

4.2.11. Statistical analysis

Meaningful differences and changes were performed using

student's t-test with Graphpad prism 6 software. Results and

averaged data are presented as mean standard deviation (SD).

When P < 0.05, results were considered as statistically signiﬁcant.

5⁰-CAGAACAAGAAAAGTTCAAGATGC-3’

5⁰-ATCCCACTACCAACCCCATT-3’

PAK1 (3) 90 kb:

5⁰-CCAGATTCACAAAGCACATGA-3’

5⁰-TCCCTCAGTCCGAAGCTCT-3’

4.2.12. Pharmacokinetic proﬁles in CD-1 mice

PAK1 (4) 124 kb:

Compound 9c (PEG300/EtOH/NaCl ¼ 40/10/50, v/v/v) was

18

S. Wang, H. Yang, M. Su et al.

European Journal of Medicinal Chemistry 222 (2021) 113592

[8] K. Nimura, K. Ura, H. Shiratori, M. Ikawa, M. Okabe, R.J. Schwartz, Y. Kaneda,

A histone H3 lysine 36 trimethyltransferase links Nkx2-5 to Wolf-Hirschhorn

syndrome, Nature 460 (2009) 287e291.

[9] E. Martinez-Garcia, R. Popovic, D.J. Min, S.M. Sweet, P.M. Thomas, L. Zamdborg,

A. Heffner, C. Will, L. Lamy, L.M. Staudt, D.L. Levens, N.L. Kelleher, J.D. Licht,

The MMSET histone methyl transferase switches global histone methylation

and alters gene expression in t(4;14) multiple myeloma cells, Blood 117

(2011) 211e220.

[10] J.D. Jaffe, Y. Wang, H.M. Chan, J. Zhang, R. Huether, G.V. Kryukov, H.E. Bhang,

J.E. Taylor, M. Hu, N.P. Englund, F. Yan, Z. Wang, E. Robert McDonald 3rd,

L. Wei, J. Ma, J. Easton, Z. Yu, R. deBeaumount, V. Gibaja, K. Venkatesan,

R. Schlegel, W.R. Sellers, N. Keen, J. Liu, G. Caponigro, J. Barretina, V.G. Cooke,

C. Mullighan, S.A. Carr, J.R. Downing, L.A. Garraway, F. Stegmeier, Global

chromatin pro ﬁ ling reveals NSD2 mutations in pediatric acute lymphoblastic

leukemia, Nat. Genet. 45 (2013) 1386e1391.

[11] M. Morishita, D. Mevius, E. di Luccio, In vitro histone lysine methylation by

NSD1, NSD2/MMSET/WHSC1 and NSD3/WHSC1L, BMC Struct. Biol. 14 (2014)

25.

subjected to PK studies in CD-1 mice. Compound 9c was adminis-

tered via the intraperitoneal and intravenous route at 2 mg/kg in

CD-1 mice, respectively. After administration, blood samples were

orderly collected at 0 h, 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h,

and 24 h. The blood samples were centrifuged at 11 000 rpm for

5 min to obtain the plasma fraction and added PK-IS immediately.

The plasma samples were deproteinized with methanol and

acetonitrile (1:1, v/v). After centrifugation, the supernatant was

diluted with acetonitrile and water. The compound concentrations

in the supernatant were measured by LC/MS/MS.

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

[12] D.J. Min, T. Ezponda, M.K. Kim, C.M. Will, E. Martinez-Garcia, R. Popovic,

V. Basrur, K.S. Elenitoba-Johnson, J.D. Licht, MMSET stimulates myeloma cell

growth through microRNA-mediated modulation of c-MYC, Leukemia 27

(2013) 686e694.

[13] T. Ezponda, R. Popovic, M.Y. Shah, E. Martinez-Garcia, Y. Zheng, D.J. Min,

C. Will, A. Neri, N.L. Kelleher, J. Yu, J.D. Licht, The histone methyltransferase

MMSET/WHSC1 activates TWIST1 to promote an epithelial-mesenchymal

transition and invasive properties of prostate cancer, Oncogene 32 (2013)

2882e2890.

[14] V. Saloura, H.S. Cho, K. Kiyotani, H. Alachkar, Z. Zuo, M. Nakakido, T. Tsunoda,

T. Seiwert, M. Lingen, J. Licht, Y. Nakamura, R. Hamamoto, WHSC1 promotes

oncogenesis through regulation of NIMA-related kinase-7 in squamous cell

carcinoma of the head and neck, Mol. Canc. Res. 13 (2015) 293e304.

[15] G. Toyokawa, H.S. Cho, K. Masuda, Y. Yamane, M. Yoshimatsu, S. Hayami,

M. Takawa, Y. Iwai, Y. Daigo, E. Tsuchiya, T. Tsunoda, H.I. Field, J.D. Kelly,

D.E. Neal, Y. Maehara, B.A.J. Ponder, Y. Nakamura, R. Hamamoto, Histone

lysine methyltransferase wolf-Hirschhorn syndrome candidate 1 is involved

in human carcinogenesis through regulation of the wnt pathway, Neoplasia

13 (2011) 887.

[16] P. Yang, L. Guo, Z.J. Duan, C.G. Tepper, L. Xue, X. Chen, H.J. Kung, A.C. Gao,

J.X. Zou, H.W. Chen, Histone methyltransferase NSD2/MMSET mediates

constitutive NF-kappaB signaling for cancer cell proliferation, survival, and

tumor growth via a feed-forward loop, Mol. Cell Biol. 32 (2012) 3121e3131.

[17] I.A. Asangani, B. Ateeq, Q. Cao, L. Dodson, M. Pandhi, L.P. Kunju, R. Mehra,

R.J. Lonigro, J. Siddiqui, N. Palanisamy, Y.M. Wu, X. Cao, J.H. Kim, M. Zhao,

Z.S. Qin, M.K. Iyer, C.A. Maher, C. Kumar-Sinha, S. Varambally,

A.M. Chinnaiyan, Characterization of the EZH2-MMSET histone methyl-

transferase regulatory axis in cancer, Mol. Cell. 49 (2013) 80e93.

[18] D. Tisi, E. Chiarparin, E. Tamanini, P. Pathuri, J.E. Coyle, A. Hold, F.P. Holding,

N. Amin, A.C. Martin, S.J. Rich, V. Berdini, J. Yon, P. Acklam, R. Burke, L. Drouin,

J.E. Harmer, F. Jeganathan, R.L. van Montfort, Y. Newbatt, M. Tortorici,

M. Westlake, A. Wood, S. Hoelder, T.D. Heightman, Structure of the epigenetic

oncogene MMSET and inhibition by N -alkyl sinefungin derivatives, ACS Chem.

Biol. 11 (2016) 3093e3105.

Acknowledgement

We are grateful for the National Natural Science Foundation of

China (Grant 21922707, 21632008, 21877118, 81620108027,

81821005 and 91953108), the Major projects of National Natural

Science Foundation of China (Grant 81991523), the Collaborative

Innovation Cluster Project of Shanghai Municipal Commission of

Health and Family Planning (Grant 2019CXJQ02), the Science and

Technology Commission of Shanghai Municipality (Grant

18JC1411304, 19430750100, and 18431907100).

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.1 13592.

References

[1] M. Morishita, D. Mevius, E. di Luccio, In vitro histone lysine methylation by

NSD1, NSD2/MMSET/WHSC1 and NSD3/WHSC1L, BMC Struct. Biol. 14 (2014)

25.

[2] M. Morishita, E. di Luccio, Cancers and the NSD family of histone lysine

methyltransferases, Biochim. Biophys. Acta 1816 (2011) 158e163.

[3] I. tec, T.J. Wright, G.J. van Ommen, P.A. de Boer, A. van Haeringen,

A.F. Moorman, M.R. Altherr, J.T. den Dunnen, WHSC1, a 90 kb SET domain-

[19] M. Morishita, D.E.H.F. Mevius, Y. Shen, S. Zhao, E. di Luccio, BIX-01294 inhibits

oncoproteins NSD1, NSD2 and NSD3, Med. Chem. Res. 26 (2017) 2038e2047.

[20] E. di Luccio, Inhibition of nuclear receptor binding SET domain 2/multiple

myeloma SET domain by LEM-06 implication for epigenetic cancer therapies,

J. Cancer Prev 20 (2015) 113.

containing gene, expressed in early development and homologous to

a

Drosophila dysmorphy gene maps in the Wolf-Hirschhorn syndrome critical

region and is fused to IgH in t(4;14) multiple myeloma, Hum. Mol. Genet. 7

(1998) 1071e1082.

[4] T. Vougiouklakis, R. Hamamoto, Y. Nakamura, V. Saloura, The NSD family of

protein methyltransferases in human cancer, Epigenomics 7 (2015) 863e874.

[5] N.P. Coussens, S.C. Kales, M.J. Henderson, O.W. Lee, K.Y. Horiuchi, Y. Wang,

Q. Chen, E. Kuznetsova, J. Wu, S. Chakka, D.M. Cheff, K.C. Cheng, P. Shinn,

K.R. Brimacombe, M. Shen, A. Simeonov, M. Lal-Nag, H. Ma, A. Jadhav,

M.D. Hall, High-throughput screening with nucleosome substrate identi ﬁ es

small-molecule inhibitors of the human histone lysine methyltransferase

NSD2, J. Biol. Chem. 293 (2018) 13750e13765.

[6] Y. Li, P. Trojer, C.F. Xu, P. Cheung, A. Kuo, W.J. Drury 3rd, Q. Qiao, T.A. Neubert,

R.M. Xu, O. Gozani, D. Reinberg, The target of the NSD family of histone lysine

methyltransferases depends on the nature of the substrate, J. Biol. Chem. 284

(2009) 34283e34295.

[21] J. Baell, M.A. Walters, Chemistry: chemical con artists foil drug discovery,

Nature 513 (2014) 481e483.

[22] X. Zhang, X.L. Zheng, H. Yang, J. Yan, X.H. Fu, R.R. Wei, X.W. Xu, Z.Q. Zhang,

A.S. Yu, K.X. Zhou, J. Ding, M.Y. Geng, X. Huang, Piribedil disrupts the MLL1-

WDR5 interaction and sensitizes MLL-rearranged acute myeloid leukemia

(AML) to doxorubicin-induced apoptosis, Canc. Lett. 431 (2018) 150e156.

[23] A.J. Kuo, P. Cheung, K. Chen, B.M. Zee, M. Kioi, J. Lauring, Y. Xi, B.H. Park, X. Shi,

B.A. Garcia, W. Li, O. Gozani, NSD2 links the dimethylation of histone H3 at

lysine 36 to oncogenic programming, Mol. Cell. 44 (2011) 609.

[24] R. Stein, M.R. Smith, S. Chen, M. Zalath, D.M. Goldenberg, Goldenberg,

Combining milatuzumab with bortezomib, doxorubicin, or dexamethasone

improves responses in multiple myeloma cell lines, Clin. Canc. Res. 15 (2009)

2808e2817.

[7] Q. Qiao, Y. Li, Z. Chen, M. Wang, D. Reinberg, R.M. Xu, The structure of NSD1

reveals an autoregulatory mechanism underlying histone H3K36 methylation,

J. Biol. Chem. 286 (2011) 8361e8368.

19

Article Doi

5-Aminonaphthalene derivatives as selective nonnucleoside nuclear receptor binding SET domain-protein 2 (NSD2) inhibitors for the treatment of multiple myeloma

DOI: 10.1016/j.ejmech.2021.113592

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2021.113592

Full text of DOI:10.1016/j.ejmech.2021.113592

Products guided by the article

R&D Labs maybe for 1153849-22-9

Relevant to this article

Hot Product