Discovery and optimization of selective inhibitors of protein arginine methyltransferase 5 by docking-based virtual screening

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Discovery and Optimization of Selective Inhibitors of Protein

Arginine Methyltransferase 5 by Docking-Based Virtual Screening

Yan Ye,‡^a,bBidong Zhang,‡^a,bRuifeng Mao,^aChenhua Zhang,^cYulan Wang,^aJing Xing,^aYu-Chih Liu,^c

Xiaomin Luo,^aHong Ding,^aYaxi Yang,^dBing Zhou,^dHualiang Jiang,^a,dKaixian Chen,^a,dCheng Luo,*^a

and Mingyue Zheng,*^a

Received 00th January 20xx,

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

www.rsc.org/

Protein arginine methyltransferase-5 (PRMT5) is a type II PRMT enzyme critical for diverse cellular processes and different

types of cancers. Many efforts have been dedicated toward discovering novel scaffold PRMT5 inhibitors. Herein, we report

the discovery of DC_P33 as a hit compound of PRMT5 inhibitor, identified by a molecular docking based virtual screening

and ³H-labeled radioactive methylation assay. Structure–activity relationship (SAR) analysis was performed on the analogs

of DC_P33, and then structure modifications were made to improve its activity. Among the derivatives, the compound

DC_C01 displayed an IC₅₀value of 2.8 µM, and good selectivity over PRMT1, EZH2 and DNMT3A. Moreover, DC_C01

exhibited anti-proliferation activities against Z-138, Maver-1, and Jeko-1 cancer cells with EC₅₀value of 12 µM, 12 µM, and

10.5 µM, respectively. Taken together, these results contribute to the development of specific inhibitors against PRMT5

and cancer therapy.

lymphoma, lung cancer, colorectal cancer and breast cancer.^13,

15-17

In addition, PRMT5 plays a role in the proliferation and

Introduction

survival of mantle cell lymphoma (MCL) and diffuse large B-cell

lymphoma (DLBCL) cells.¹³RNAi knockdown of PRMT5 in

Protein arginine methyltransferases (PRMTs) are a family of

enzymes that catalyze the transfer of a methyl group from S-

adenosylmethionine (SAM) to the guanidine nitrogen of an

arginine residue,¹involved in the regulation of cellular

processes, such as RNA processing, transcription, DNA repair

and signal transduction.^2-7Currently, nine human PRMTs

(PRMT1-9) have been reported in mammals.^{8, 9}These PRMTs

are classified into three types of arginine methylation based on

the catalytic mechanism. PRMT5 is a type II PRMT enzyme,

embryonic stem (ES) cells results in down-regulation of

pluripotency-associated genes and up-regulation of

differentiation-associated genes,⁷and shRNA knockdown of

PRMT5 in lymphoblastoid cell lines results in decreased

viability.¹⁸These observations suggest that PRMT5 is a

promising therapeutic target for cancers. Till now, many

efforts have been taken in discovering small-molecule

inhibitors against PRMT5. Sinefungin is a highly potent and

SAM-competitive PRMT5 inhibitor. However, due to its

structural similarity to SAM, sinefungin shows no selectivity

against various methyltransferases such as other PRMTs and

protein lysine methyltransferases. In the past years, high-

which is currently to be the dominant enzyme for symmetric

dimethylarginines (SDMA).^10,

PRMT5 methylates the

11

substrate of histone H3 (H3R8me2s) and H4 (H4R3me2s),

which promotes transcriptional repression by changing

chromatin structure.^12-14PRMT5 has been found to be

overexpressed in different types of cancers, including leukemia,

throughput screening (HTS) has been adopted to discover

19-22

PRMTs inhibitors.^17,

Spannhoff et al. identified

stilbamidine as a competitive inhibitor of the substrate using a

target-based virtual screening approach, which showed an IC₅₀

value of 44.1 µM against PRMT5.²³These compounds are pan-

methyltranferase inhibitors and display nonspecific interaction

to PRMT5.¹Alinari et al. discovered a first-in-class, highly

selective, small-molecule PRMT5 inhibitor CMP5.¹⁸This

compound is predicted to occupy in the SAM-binding pocket,

showing an IC₅₀value lower than 50 µM. Recently, a selective

and potent PRMT5 inhibitor EPZ015666 with an IC₅₀value of

0.022 µM was reported, and it displayed anti-tumor activity in

MCL xenograft animal models.²⁴A phase 1 clinical study has

^a.Drug Discovery and Design Center, State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555

Zuchongzhi Road, Shanghai 201203, China.

^b.University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049,

China.

^c.Shanghai ChemPartner Co., Ltd., #5 Building, 998 Halei Road, Shanghai 201203,

China.

^d.School of Life Science and Technology, Shanghai Tech University, Shanghai

200031, China.

^e.Department of Medicinal Chemistry, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, Shanghai 201203, China.

†Electronic Supplementary Information (ESI) available: Structure of the human

PRMT5 with cofactor SAM and EPZ015666. The Specs IDs of brought compounds.

HPLC analysis data of synthesized compounds. 1H and 13C NMR spectra. See been initiated by GSK to investigate the safety,

DOI: 10.1039/x0xx00000x

pharmacokinetics, pharmacodynamics of an analog of

‡These authors contributed equally to this work.

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inhibitors using virtual screening.^26-29In our group, virtual

screening methods have been used to identify inhibitors of

PRMT1, DNMT1, SET7, etc.^28-30These successful experiences

encourage us to utilize virtual screening method to discover

novel inhibitors of PRMT5. In this research, a promising hit was

first discovered by virtual screening. Similarity-based searching

and medicinal chemistry approaches were then applied in the

optimization of the hit, and to study the structure-activity

relationship (SAR) of these inhibitors.

Results and discussion

Docking-Based Virtual screening.

The PRMT5 has two ligand binding pockets: a SAM-binding site

and a substrate-binding pocket, as shown in Fig.S1. In order to

find inhibitors with better selectivity profile, we chose to use

the substrate-binding pocket for virtual screening because the

SAM-binding site is shared by various histone and DNA

methyltransferases. Recently, the cofactor SAM was reported

forming essential interactions with the methyl group-accepting

inhibitor EPZ015666 and hence contributing to the binding

affinity of the substrate-competitive inhibitors to PRMT5.²⁴

Accordingly, SAM was kept as a part of the receptor structure

in our docking-based screening. Currently, there are seven

different crystal structures of PRMT5 available in Protein Data

Bank. The crystal structure (PDB id 4X61) was used for

molecular docking because it contains both the methyl donor

SAM and a substrate-competitive inhibitor EPZ015666. To test

the “screening power” of the structure model, we collected 20

active derivatives of the inhibitor EPZ015666 from the

published patents.³¹Totally 720 decoys were compiled at a

Fig.1 The reported inhibitors against PRMT5.

EPZ015666.²⁵The structures of the published PRMT5 inhibitors

and their activities are summarized in Fig.1. Unfortunately,

there are limited inhibitors with high potency and selectivity.

The discovery of potent and selective PRMT5 inhibitors with

novel scaffolds is thus of high interest to the field of PRMT5

biology and related medicinal chemistry studies.

With the rapid development of computational methods,

many studies have successfully discovered new epigenetic

Fig.2 Virtual screening workflow and in vitro inhibitory activity assay results for the selected compounds. (A) Schematic representation of the whole screening

procedures. (B) Inhibitory activity for all compounds at the concentration of 50 μM. The red column represents the activity of the reference compound SAH.

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ratio of 1:36 with DecoyFinder V1.1.³²All the actives and In order to verify the scaffold of DC_P33 and to investigate the

decoys were docked into the model separately. Consequently, structure-activity relationship of its analogues, we performed

the area under receiver operating characteristic curve (AUC) of the second round of screening based on a chemical fingerprint

the model is 0.93, while the 2% enrichment factor (EF) of the similarity searching. Finally, 14 analogues of DC_P33 were

model is 35. Therefore, the structure model was used in our selected and purchased for activity evaluation. Five

subsequent screening procedures.

The workflow of virtual screening is shown in Fig.2A. A PRMT5 at the concentration of 50 μM, which were further

commercially available compound library SPECS evaluated for the inhibitory activity within a range of

(http://www.specs.net ), which includes 207342 compounds concentrations. The biochemical assay results and structures

compounds show more than 50% inhibitory activity against

was used as the ligand database. Filtered by “Lipinski's Rule of of these compounds are shown in Table 1

.

Five” and the structural patterns of pan-assay interference

Binding Mode Prediction of DC_P33.

structures (PAINS),³³the remaining 139176 compounds were

As shown in Fig.3A, the compound DC_P33 consists of three

parts: a naphthalene ring and two substituents denoted as

chain (-CH₂NHCH₂CH₂OH) and chain (-benzyloxyl),

respectively. To analyze its interaction with PRMT5, the

putative binding mode of DC_P33 was compared with the

crystal structure of EPZ015666, which is shown in Fig.3B and

Fig.3C. Similar to the binding mode of the

prepared and docked into the substrate site of the prepared

protein. Details about the ligand preparation and molecular

docking are provided in Method section. The top ranked 1000

molecules were selected and clustered, accounting for both

structural diversity and the interactions with key residues such

as Phe327, Phe580 or Glu444. Finally, 42 molecules were

selected and purchased for the subsequent biochemical assay

evaluation.

A

B

tetrahydroisoquinoline (THIQ) in EPZ015666,

A chain of

DC_P33 is situated in the hydrophobic pocket composed by

Leu319, Tyr324, Phe327 and Trp579. This explained that

PRMT5 Inhibition Assays.

The virtual screening selected compounds were tested for increased hydrophobic interaction in R₂positon of scaffold

I

activity against PRMT5 using ³H-labeled radioactive (DC_S08, DC_S09, DC_S10 and DC_S11) improved potency,

methylation assay. Fig.2B depicted the inhibition ratio of 42 while long polar chains in the same position (DC_S04, DC_S05

compounds against PRMT5 at the concentration of 50 μM, and DC_S06) significantly reduced the activity of the

with S-adenosyl-L-homocysteine (SAH) used as a reference. compounds. Another feature of EPZ015666 is that the terminal

From the enzyme inhibition assay, DC_P33 showed the most pyrimidine forms π-π stacking interactions with Phe327 and

potent inhibitory activity against PRMT5 with the inhibition Phe580. Similar π-π interactions are observed for the phenyl

ratio of 52%. In a more quantitative analysis, we measured the group of the

B chain in DC_P33, and removing the phenyl

activity of DC_P33 against PRMT5 at a range of concentrations group (DC_S12, DC_S13 and DC_S14) resulted in markedly

and the IC₅₀is 35.6 μM. Therefore, DC_P33 was selected as a decreased activity. There are also differences between DC_P33

hit compound for further optimization.

and EPZ015666. For example, the phenyl of the THIQ in

EPZ015666 forms cation-π interaction with the partial charged

Similarity-Based Analog Searching.

Fig.3 The putative binding mode of DC_P33 in the substrate-binding site of PRMT5: (A) DC_P33 contains three parts: a naphthalene ring,

A chain (dash red box) and B

chain (dash blue box); (B) The binding mode of EPZ015666 (cyan); (C) The binding mode of DC_P33 (green). SAM is displayed as magenta color.

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methyl group of SAM, and also forms π-π stacking interaction DC_C01 was obtained from DC_P33 by adding a phenyl group

with Phe327, a conserved residue critical for directing the next to the hydroxyl group in the

A

B

chain and removing a

chain. According to the

ability of PRMT5 to catalyze SDMA.³⁴These interactions are all chloride group from the phenyl in the

missing in DC_P33, as highlighted in the red circle in Fig.3C. putative binding mode of DC_C01 shown in Fig.4C, the added

Apparently, it can be inferred that introducing an aromatic ring phenyl group is situated at the same position of THIQ, forming

into the

A

chain may help to improve the activity of DC_P33.

cation-π interaction with the positively charged SAM.

The chain is situated in a hydrophobic pocket near to the

SAM-binding pocket. As shown in Fig.4C, the hydroxyl group in

the chain has no obvious effect for the binding of compound

DC_C01 to PRMT5. Removing the hydroxyl group, DC_C08

possesses an IC₅₀value of 3.2 μM, which has comparable

potency to DC_C01. This result implies that the hydroxyl group

Hit optimization and SAR analysis.

A

To test the above inference, we designed and synthesized 19

compounds. The biochemical assay results and structures of

these compounds are shown in Table 2. Among these

compounds, DC_C01 is the most potent with an IC₅₀value of

2.8 μM (Fig.4B), and it exhibits up to 12-fold more increase in

dose potency against PRMT5 over DC_P33. As shown in Fig.4A,

A

of the

A chain is not essential to their binding to PRMT5.

Table 1 Biochemical Assays results for DC_P33 and its analogues selected by similarity-based searching.

(I)

Inhibition Rate at

50 µM (IC₅₀/µM)

Inhibition Rate at

50 µM (IC₅₀/µM)

Entry

R₁

R₂

-CH₂OH

Entry

R₁

R₂

DC_P33

DC_S01

DC_S02

DC_S03

DC_S04

DC_S05

DC_S06

DC_S07

52% (35.6)

DC_S08

DC_S09

DC_S10

DC_S11

DC_S12

DC_S13

DC_S14

-CH(CH₃)OH

-CH₂N(CH₃)₂

69% (15)

79% (8.5)

68% (22)

57% (15)

46%

H

32%

48%

-CH₂OH

22%

-CH₂CH₂NHCH₃

-CH₂NHCH₂CH₂OH

-CH₂OCH₃

10%

-OCH₂CH₃

19%

H

2%

24%

-OCH₃

38%

76% (12)

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Table 2. Biochemical Assays results for 19 synthesized analogues of DC_P33.

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(IV)

(II)

(III)

Inhibition Rate at 50 µM

Entry

Scaffold

R₁

R₂

H

R₃

IC50/µM

35.6

2.8

8.4

-

DC_P33

DC_C01

DC_C02

DC_C03

DC_C04

DC_C05

DC_C06

DC_C07

DC_C08

DC_C09

-OH

H

5%

-

25%

-

9.1

9.6

29

-

3.2

7.9

II

H

DC_C10

H

4.5

-

DC_C11

DC_C12

DC_C13

DC_C14

DC_C15

DC_C16

DC_C17

DC_C18

DC_C19

H

38

45

4.1

6.4

13

25

4.3

-

H

-

H

-

H

-

H

9%

-

III

IV

H

-OH

25

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Fig.4 The optimization of DC_P33 to DC_C01. (A) The strategy for hit optimization; (B) The IC₅₀values for DC_P33 and DC_C01; (C) The superimposition of the putative

binding modes of DC_P33 and DC_C01.

As mentioned above, the phenyl group of the

A chain is the hydrogen-bonding interaction, and therefore results in a

predicted to form cation-π interaction with SAM. To further complete loss of activity, with the inhibition ratio of 9% at the

confirm it, four analogues with the phenyl group replaced by concentration of 50 μM. This result confirms that the hydrogen

different aromatic and non-aromatic rings were synthesized. bond between the amine group and residue Ser578 is essential

As shown in Table 2, the activities of these compounds are - to the inhibitory activity against PRMT5.

phenyl > - (3-Indolyl) > - (2-pyridyl) > - (1-piperidyl) > 4-

As shown in Fig.4C, the naphthalene ring is snug into the

morpholinyl. The hetero-nonaromatic ring DC_C11 (R₂= 1- hydrophobic pocket surrounded by residue Leu312, Leu319,

piperidyl) and DC_C12 (R₂= 4-morpholinyl) exhibited 11.9-fold Phe327 and Val503. The replacement of the naphthalene ring

and 14.1-fold lower activities than DC_C08 (R₂= phenyl). These with a smaller phenyl ring decreased the hydrophobic

results agree well with our previous inference that the cation- interaction, which may explain that DC_C19 showed about 8.9-

π interaction of this moiety is very important to the activity of fold less potent PRMT5 inhibitory activity (IC₅₀= 25 μM) than

the compounds. A series of substituents such as fluorine, DC_C01 (IC₅₀= 2.8 μM). The phenyl ring on the

hydroxyl, methyl and chloride were incorporated into the π-π stacking interaction with residue Phe327 and Phe580.

para-position of the phenyl. All the compounds displayed Removing chain (DC_S13) or changing its length (DC_C03)

lower potencies than DC_C08. Particularly, the chloride completely abolished the activity, and replacing the phenyl

substituent DC_C16 showed 7.8-fold loss of potency. Since the ring on the chain by a methyl group (DC_S12) or an electron-

phenyl ring of the chain is situated at a small hydrophobic deficient pyridine (DC_C02) also showed significantly

cavity surrounding by Leu319, Tyr324, and Phe327 (Fig.4C), the decreased potency.

B chain forms

B

A

steric hindrance may lead to their deceased activities.

Synthesis.

For DC_P33, the interaction with SAM is missing, but the

We have optimized the chemical structure in order to improve

the inhibitory activity. Initially, the treatment of the readily

available 2-hydroxy-1-naphthaldehyde 1a with (chloromethyl)-

benzene 2a using anhydrous K₂CO₃as a base at reflux to

furnish key intermediate 2-(benzyloxy)-1-naphthaldehyde

secondary amine group in

A chain can be protonated and form

cation-π interactions with Phe327 and Trp579. Moreover,

there is a hydrogen bond between the amine group and the

backbone carbonyl of Ser578, which also highlighted the

importance of the amine group in ligand binding. The

methylation of the amine group obtaining DC_C18 destroys

3a ^35,36

.

Then, the product DC_C01 was prepared by reductive

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Scheme 1 General synthesis of compounds (DC_C01 to DC_C03 and DC_C05 to DC_C18)

different aromatic-substituted alkyl chlorides (2b

-

2f) and

alkylamine compounds (4b-4l) with diverse substituents, which

is depicted in Scheme 1.

Compound DC_C04 was synthesized using the route

described in Scheme 2. As acyl chloride is very active and easily

produces much more side products, mild condition (room

temperature) was accomplished to afford the key intermediate

compound 3g in moderate yield.³⁹Subsequent reductive

amination as previously described using amine 4a afforded the

desired compound DC_C04 in 77% yield.

Additionally, in order to investigate the role of the

naphthalene ring, benzene ring was readily introduced to

provide the product DC_C19, as outlined in Scheme 3. Starting

with the monocycle 2-hydroxybenzaldehyde 1b

,

the

compound 2-(benzyloxy)benzaldehyde 3h was prepared

according to previous procedures and subsequent reductive

amination.

Reagents and conditions: (a) PhCH₂Cl (1.2 equiv), KI (0.2 eq), anhydrous K₂CO₃

(1.2 equiv), dry acetone, reflux, 4-6 h, 65-95%; (b) alkylamine 4a-l (1.2 equiv),

NaBH(OAc)₃(3.0 equiv), CH₃COOH (1.0 equiv), DCE, room temperature, 2-4 h, 83-

97%.

Methyltransferase Enzymatic Selectivity of DC_C01.

Methylation of DNA and histone proteins are mutually

involved in the epigenetic regulation of gene expression

mediated by DNA methyltransferases (DNMTs) and histone

methyltransferases (HMTs). DNMTs methylate cytosine

residues within gene promoters, whereas HMTs catalyze the

transfer of methyl groups to lysine and arginine residues of

histone proteins. To investigate the selectivity of DC_C01 for

the pre-dominant type II arginine methyltransferase PRMT5,

we also evaluated its activity on other three different types of

Scheme 2 Synthesis of compound DC_C04

methyltransferases,

including

protein

arginine

methyltransferase 1 (PRMT1), the representative of type I

arginine methyltransferase, DNA methyltransferase 3A

(DNMT3A) and enhancer of zeste homolog 2 (EZH2), one of

lysine methyltransferases. First, we conducted a multiple

sequence alignment of the substrate-binding site of PRMT5,

PRMT1, DNMT3A and EZH2. As shown in Fig.5A, the substrate-

binding sites of these methyltransferases are not highly

conserved, especially for EZH2. The sequences of PRMT1 and

DNMT3A share relatively higher homology with PRMT5. Then,

a structural alignment of PRMT5, PRMT1 and DNMT3A was

performed. As shown in Fig.5B, the substrate-pocket structure

of DNMT3A can not be aligned well with that of PRMT5, and it

is hence inaccessible to the binding of DC_C01. For PRMT1,

there are also clear differences with PRMT5, e.g., the residues

Phe327 and Phe580 of PMRT5 that form important π-π

interaction the inhibitory activity of DC_C01 against PRMT1,

with DC_C01 are replaced as Met and Lys, respectively, and

the interaction was therefore abolished. Moreover, the

substitution of Ser439 in PRMT5 with Tyr148 in PRMT1 reveals

that the tryptophan may reduce the size of the substrate-

binding pocket, and sterically hinder the binding of substrate

Reagents and conditions: (a) PhCOCl (1.5 equiv), anhydrous K₂CO₃(1.5 equiv), dry

THF, room temperature, 16 h, 75%; (b) 2-amino-1-phenylethan-1-ol 4a (1.2

equiv), NaBH(OAc)₃(3.0 equiv), CH₃COOH (1.0 equiv), DCE,room temperature, 3 h,

77%.

Scheme 3 Synthesis of compound DC_C19

Reagents and conditions: (a) PhCH₂Cl 2a (1.2 equiv), KI (0.2 equiv), anhydrous

K₂CO₃(3.0 equiv), dry acetone, reflux, 4.5 h, 88%; (b) 2-amino-1-phenylethan-1-ol

4a (1.2 equiv), NaBH(OAc)₃(3.0 equiv), CH₃COOH (1.0 equiv), DCE, room

temperature, 3.5h, 87%.

aldehydes 3a with the commercial 2-amino-1-phenylethan-1-ol

4a in the presence of acetic acid and NaBH(OAc)₃with good to

high yield.^{37, 38}Similar approach was employed for synthesis of

compounds DC_C02, DC_C03 and DC_C05 - DC_C18, utilizing

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F

ig.5 Selectivity of DC_C01 over different DNA and histone methyltransferases. (A) Sequence alignment of the substrate-binding sites of PRMT5, PRMT1, EZH2 and DNMT3A. Some

important amino acids in the substrate-binding site of PRMT5 are marked in red boxes. (B) Superimposition of the substrate-binding domains of PRMT5 (PDB id: 4X61, gray color),

PRMT1 (PDB id: 1or8, green color) and DNMT3A (PDB id: 5hyn, magenta color). DC_C01 is shown in cyan stick. (C) Inhibition ratio of DC_C01 over PRMT5, PRMT1, EZH2 and

DNMT3A at the concentration of 50 μM and 100 μM.

Fig.6 DC_C01 and DC_C08 effects on in vitro Cellular prolifertion in MCL cell lines.

and DC_C01. This effect is partially supported by a previous selective PRMT5 inhibitor. Finally, we assessed and compared

report that mutating Ser439 to a tryptophan significantly PRMT5, EZH2 and DNMT3A, using the radioactive methylation

decreased the enzymatic activity of PRMT5.³⁴Through the assay at the concentration of 50 μM and 100 μM (Fig.5C). Not

sequence and structural alignment analyses, we may find that unexpectedly, DC_C01 showed potent activity against PRMT5,

the substrate-binding sites of different methyltransferases while it is inactive to PRMT1, EZH2 and DNMT3A. Therefore,

reveal apparent diversities, which enable the design of

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Preparing the protein structure. The crystal structure of PRMT5

these results suggested that the compound DC_C01 exhibited

good selectivity toward PRMT5.

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with cofactor SAM and small inhibitor EPZ015666 was

DOI: 10.1039/C7OB00070G

retrieved from Protein Data Bank (PDB id: 4X61). The protein

structure was prepared in the Protein Preparation Wizard

Workflow of the Schrꢀdinger software package (Schrꢀdinger,

LLC, New York, NY, 2010). SAM was remained because it

formed cation-π interaction with the inhibitor. All waters were

deleted from the PRMT5 structures, and hydrogens were

added. The orientations and tautomeric states of Gln, Asn and

His were optimized to better from the hydrogen bonds. The

complex was then minimized with the OPLS_2005 force field

and a maximum rmsd of 0.30 Å to restrain steric clashes. Next,

a three-dimensional grid was generated with the Receptor

Grid Generation module in the Maestro program (Maestro,

version 9.1; Schrꢀdinger, LLC: New York, NY, 2010), on the size

and centroid of the ligand EPZ015666.

Preparing the Ligand Database. In total, 207342 compounds

were obtained from SPECS database. Compounds containing

pan-assay interference structures (PAINS) were removed and

the remaining 187842 compound were then filtered by

“Lipinski's Rule of Five” to remove compounds which may have

poor absorption or permeability in Pipeline Pilot (Pipeline Pilot,

version 7.5; Accelrys Software Inc.: San Diego, CA). Finally,

139176 compounds were prepared in LigPrep module (LigPrep,

version 2.4; Schrꢀdinger, LLC: New York, NY, 2010). First these

structure were optimized with OPLS_2005 force field. The

ionization state of the structures were subsequently generated

using Epik (Epik, version 2.1; Schrꢀdinger, LLC: NewYork, NY,

Cell-based Activity.

PRMT5 is reported to have a role in MCL, as evidenced not

only by upregulation of PRMT5 in patient samples, but also by

anti-proliferative effects observed after PRMT5 knockdown in

MCL cell lines.^{12, 13, 40, 41}A panel of three MCL cell lines (Z-138,

Maver-1 and Jeko-1) were used to assess proliferation effects

upon treatment with PRMT5 inhibitors allowing for the

measurement of cell growth over 3 days. As shown in Fig.6,

DC_C01 and DC_C08 inhibit the proliferation in a dose-

dependent manner. DC_C01 exhibited potent anti-proliferative

activities against Z-138, Maver-1, and Jeko-1 cancer cells with

EC₅₀values of 12.7 µM, 12.7 µM, and 10.5 µM, respectively.

DC_C08 demonstrated that biochemical inhibition is well

correlated with proliferation, with EC₅₀values ranging from

13.7 µM to 19.5 µM. These results suggest that both of

DC_C01 and DC_C08 inhibit the proliferation of Z-138, Maver-1,

and Jeko-1 cancer cells.

Conclusions

Protein arginine methyltransferases play significant roles in the

regulation of cellular processes, such as RNA processing,

transcription, DNA repair and signal transduction. Inhibition of

PRMT5 is a novel therapeutic approach for the treatment of 2010) method at target pH 7±2.0. The specified chirality of the

ligands was retained and other chiral centers were varied. The

stereoisomers were generated at most 32 per ligand. The

generating structures were used for virtual screening.

cancer. Currently, there are limited inhibitors in reported

studies which are specific to PRMT5. In the present study, we

discovered the compound DC_P33 as a novel inhibitor of

PRMT5 with an IC₅₀value of 35.6 μM using the docking-based

virtual screening. Subsequently, 14 derivatives of DC_P33 were

obtained from SPECS database by the similarity-based

searching method. Five of them were found to show more

than 50% inhibition of PRMT5 at the concentration of 50μM.

Combining the binding mode of DC_P33 with the activity of its

analogues, we further designed and synthesized 19 analogues

of DC_P33. Among these compounds, DC_C01 showed the

highest potency against PRMT5 with an IC₅₀value of 2.8 μM.

The selectivity profile of DC_C01 on various DNA and histone

methyltransferases including of PRMT5, PRMT1, DNMT3A and

EZH2 were also investigated. By structural analyses and

biochemical assay evaluation, DC_C01 was identified to show

clear specificity for PRMT5. Finally, DC_C01 and DC_C08 were

evaluated for their anti-proliferative effects in the Z-138,

Maver-1, and Jeko-1 cancer cells, and they inhibited the

proliferation of all the three cancer cells in a dose-dependent

manner. The study presented here details the discovery of

potent, selective PRMT5 inhibitors with a novel scaffold using

a structure-guided drug design approach, which provide useful

starting point and information for the further development of

epigenetic drugs for cancer therapy and the related chemical

biology studies.

Glide Docking Procedure. The prepared receptor and ligand files

were imported in the Ligand Docking module using Glide

software (Glide, version.6; Schrꢀdinger, LLC: New York, NY,

2010) with the standard precision mode (SP). The top-ranked

1000 poses were chosen to group into 100 clusters with

Pipeline Pilot. After visual inspection of the binding poses of

the compounds, 42 molecules with structural diversity and

binding affinity were selected and purchased for biological

assay test. Table S1 lists the docking scores and ranks of 42

compounds before any manual selection.

Similarity-based analogues searching. It was shown from the

biological test that compound DC_P33 was most potent. In

order to obtain derivatives of DC_P33, the SPECS database

filtered by PAINS was searched in Pipeline Pilot with Tanimoto

coefficient and ECFP_4 fingerprints. The cutoff was set to 0.50.

A total of 14 analogues were found by the threshold of the

similarity and bought for biological test.

Commercial Compounds. Fifty-six compounds (DC_P01-42,

DC_S01-14) were purchased from SPECS. The purity of all

these compounds are reported in Table S1 and Table S2. The

purity of the active compounds is equal to or greater than 95%.

PRMT5 Inhibition Assays.

The radioactive PRMT5 methylation inhibition assay was

performed in 25 μL reaction system containing adenosyl-L-

methionine S-[methyl-³H] (³H-SAM,15.4 Ci/mmol, PerkinElmer),

biotinylated H4 derived peptide (synthesized by GL China) and

1 nM PRMT5/MEP50 (BPS, 51045) in modified Tris Buffer, pH

8.0. The protein was pre-incubated with a range of compound

concentrations for 15 min at room temperature before adding

Experimental

Virtual screening

This journal is © The Royal Society of Chemistry 20xx

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biotinylated peptide and [³H]SAM. After 60 min of incubation Data are presented as follows: chemical shift, multiplicity (s =

View Article Online

DOI: 10.1039/C7OB00070G

at room temperature, the reaction was stopped and was singlet, d = doublet, dd = doublet of doublet, t = triplet, q =

transferred to

a 384-well streptavidin-coated Flashplate quartet, m = multiplet, br = broad), coupling constant in Hz,

(PerkinElmer) and was incubated for another 1h at room and integration. Low-resolution and high-resolution mass

temperature. The Flashplate was washed with dH2O + 0.1% spectrometry (LRMS/HRMS) were recorded on a Thermo - DFS

Tween-20 for three times and the radioactivity was spectrometer. Flash and column chromatography was

determined by liquid scintillation counting (MicroBeta, performed using silica gel (200–300 mesh). Analytical TLC was

PerkinElmer). IC₅₀values were derived by fitting the data for performed silica gel plates and visualized under ultraviolet

the inhibition percentage to a dose-response curve by light (254 nm). The purity of the target compounds was

nonlinear regression in GraphPad Prism 5.0.

Methyltransferase Enzymatic Selectivity Assay.

determined to be >95% by analytical HPLC.

Procedures for synthesis of compounds. Synthesis of the presented

compounds were performed according to Schemes 1–3. Synthesis

and analytical data for representative compounds are provided

below.

PRMT1, EZH2 and DNMT3A radioactive methylation inhibition

assays were similar to that of PRMT5. The radioactive PRMT1

methylation inhibition assay was performed in 25 μL reaction

system containing ³H-SAM, biotinylated H4 derived peptide

(synthesized by GL China) and 0.5 nM PRMT1 (BPS, 51041) in

modified Tris Buffer, pH 8.0. The radioactive EZH2 methylation

inhibition assay was performed in 25 μL reaction system

The synthesis of 2-(benzyloxy)-1-naphthaldehyde. To a solution of

commercially available 2-hydroxy-1-naphthaldehyde 1a (2.58 g, 15.

00 mmol) in dry acetone (50 mL) was added anhydrous K₂CO₃(2.50

g, 18.00 mmol) and (chloromethyl)benzene 2a (2.28 g, 18.00 mmol)

containing ³H-SAM, biotinylated H3 derived peptide and KI (500 mg, 3.00 mmol) refluxed for 4h. The mixture was then

(synthesized by GL China) and 3 nM EZH2 (BPS, 51004) in

modified Tris Buffer, pH 9.0. The radioactive DNMT3A

methylation inhibition assay was performed in 30μL reaction

filtered through Celite and the filter cake was well washed with

acetone (50 mL). The filtrate was concentrated and the resulting

residue was redissolved in ethyl acetate (100 mL), washed with

water (2×100 mL) and brine(50 mL). The organic layer was dried

over anhydrous Na₂SO₄, filtered, and then concentrated in vacuo.

Purification of the crude product by silica gel column

chromatography (ethyl acetate : hexane = 1:10) afforded compound

3a (3.63 g, yield 92%).

3

system containing H-SAM, biotinylated DNA oligonucleotides

(synthesized by Life Technologies) and 12.5 nM DNMT3A (BPS,

51106) in modified Tris Buffer, pH 8.0. The proteins were

preincubated with 50 μM and 100 μM of compound DC_C01

for 15 min at room temperature before substrates and

[³H]SAM were added. After 60 min of incubation at room

temperature for PRMT1 and EZH2 assays, or after 4h of

incubation at room temperature for DNMT3A assay, the

reactions were stopped and were transferred to a 384-well

streptavidin-coated Flashplate and then incubated for 1h at

room temperature. The Flashplate was washed with dH2O +

0.1% Tween-20 for three times and the radioactivity was also

determined by liquid scintillation counting. Fit the data in Excel

to obtain inhibition percentage using equation: Inh %=( Max-

Signal)/ (Max-Min)*100.

The synthesis of 2-(((2-(benzyloxy)naphthalen-1-yl)methyl)amino)-

1-phenylethan-1-ol (DC_C01). A mixture of intermediate 3a (131

mg, 0.5 mmol), 2-amino-1-phenylethan-1-ol 4a (82 mg, 0.6 mmol),

NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol) was

stirred in dry DCE at room temperature for 2.5 h. The resulting

mixture was then diluted with water and extracted with DCM. The

combined organic layers were washed with brine, dried over

anhydrous Na₂SO₄and filtered. The solvent was evaporated under

reduced pressure to give the residue, which was purified by silica

gel column chromatography (DCM: CH₃OH =30:1) to give the

product DC_C01 as a yellow solid (174 mg, yield 91%).¹H NMR (400

MHz, CDCl₃) δ 8.08 (d, J = 8.8 Hz, 1H), 7.82 (t, J = 8.0 Hz, 2H), 7.55 (t,

J = 7.2 Hz, 1H), 7.44 (t, J = 6.8 Hz, 2H), 7.41 (d, J = 4.8 Hz, 2H), 7.39 –

7.35 (m, 2H), 7.34 (d, J = 4.0 Hz, 5H), 7.28 (q, J = 4.4 Hz, 1H), 5.25 (s,

2H), 4.73 (dd, J = 9.2, 3.6 Hz, 1H), 4.37 (q, J = 12.4 Hz, 2H), 2.87 (ddd,

J = 21.6, 12.4, 6.4 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 153.7, 142.1,

136.6, 132.8, 128.9, 128.8, 128.2, 128.1, 127.8, 127.6, 126.9, 126.9,

126.4, 125.4, 123.2, 122.6, 121.1, 114.2, 71.1, 70.9, 56.2, 42.3.

HRMS (ESI, positive) m/z calcd for C₂₆H₂₅NO₂[M + H]⁺: 384.1958.

Found: 384.1963.

In vitro proliferation assay.

For assessment of the effect of DC_C01 and DC_C08 treatment

on the cell lines, they were plated in 96-well plates at a density

of 1×105 cell/well in 100 μl media. After 6 hours of incubation,

the increasing concentrations of DC_C01 and DC_C08 (0-100

μM) were added to the medium for an additional 3 days.

Viable cell number was determined using the Cell Titer-Glo

Luminescent Cell Viability assays (Promega), and luminescence

was recorded using an EnVision Multilabel Plate Readers

(PerkinElmer) according to the manufacturer’s protocol. For

each cell line, the concentration-dependence curves were

determined at each time point using Graphpad Prism software.

Chemistry.

1-phenyl-2-(((2-(pyridin-4-ylmethoxy)naphthalen-1-

yl)methyl)amino)ethan-1-ol (DC_C02). The title compound was

synthesized from 2-(pyridin-4-ylmethoxy)-1-naphthaldehyde 3b

(131 mg, 0.5 mmol), 2-amino-1-phenylethan-1-ol 4a (82 mg, 0.6

Commercially available chemicals were used without further

purification. All products were characterized by their NMR and

MS spectra. ¹H NMR spectra were recorded with a Varian-

MERCURY Plus-400 NMR spectrometer.¹³C NMR spectra were mmol), NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol)

recorded with a Varian-MERCURY Plus-500 NMR spectrometer.

Chemical shifts are reported in ppm (δ scale) as referenced to

TMS and coupling constant (J) values are reported in hertz (Hz).

in dry DCE (15 mL) according to synthetic procedure for DC_C01.

The product DC_C02 is a yellow solid (156 mg, 85% yield). H NMR

(400 MHz, CDCl₃) δ 8.62 (d, J = 6.0 Hz, 2H), 8.07 (d, J = 8.4 Hz, 1H),

1

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7.79 (d, J = 9.2 Hz, 2H), 7.59 – 7.54 (m, 1H), 7.60 – 7.53 (m, 1H), 7.41 128.9, 128.4, 128.3, 128.2, 127.9, 127.0, 126.7, 125.3, 123.4, 122.4,

View Article Online

DOI: 10.1039/C7OB00070G

(d, J = 7.6 Hz, 1H), 7.37 (d, J = 6.0 Hz, 2H), 7.34 – 7.26 (m, 5H), 7.20 119.6, 113.8, 70.7, 70.1, 55.8, 42.1. HRMS (ESI, positive) m/z calcd

(d, J = 9.0 Hz, 1H), 5.29 (s, 2H), 4.85 (dd, J = 9.6, 3.0 Hz, 1H), 3.50 (s, for C₂₆H₂₄ClNO₂[M + H]⁺: 418.1615. Found: 418.1618.

2H), 3.29 (s, 2H), 3.10 (dd, J = 12.0, 3.0 Hz, 1H), 2.84 (dd, J = 12.0, 4-(((1-(((2-hydroxy-2-phenylethyl)amino)methyl)naphthalen-2-

9.6 Hz, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 154.3, 150.2, 146.8, yl)oxy)methyl) benzonitrile (DC_C07). The title compound was

144.3, 133.4, 130.1, 129.4, 128.8, 128.5 127.5, 127.3, 126.4, 124.2, synthesized

from

4-(((1-formylnaphthalen-2-yl)oxy)methyl)

124.0, 122.3, 115.0, 71.1, 69.1, 57.0, 42.6. HRMS (ESI, positive) m/z benzonitrile 3f (144 mg, 0.5 mmol), 2-amino-1-phenylethan-1-ol 4a

calcd for C₂₅H₂₄N₂O₂[M + H]⁺: 385.1902. Found: 384.1911.

2-(((2-phenethoxynaphthalen-1-yl)methyl)amino)-1-phenylethan-

(82 mg, 0.6 mmol), NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57

µL, 0.5 mmol) in dry DCE (15 mL) according to synthetic procedure

1-ol (DC_C03). The title compound was synthesized from 2- for DC_C01. The product DC_C07 is a white solid (186 mg, 91%

1

phenethoxy-1-naphthaldehyde 3c (138 mg, 0.5 mmol), 2-amino-1- yield). H NMR (400 MHz, CDCl₃) δ 8.06 (d, J = 8.4 Hz, 1H), 7.82 –

phenylethan-1-ol 4a (82 mg, 0.6 mmol), NaBH(OAc)₃(318 mg, 1.5 7.76 (m, 2H), 7.67 (d, J = 8.0 Hz, 2H), 7.59 – 7.54 (m, 3H), 7.42 – 7.37

mmol), CH₃COOH (57 µL, 0.5 mmol) in dry DCE (15 mL) according to (m, 1H), 7.31 (dt, J = 6.8, 5.2 Hz, 4H), 7.25 – 7.19 (m, 2H), 5.33 (s,

synthetic procedure for DC_C01. The product DC_C03 is a yellow 2H), 4.84 (d, J = 2.8 Hz, 1H), 4.49 (dd, J = 31.6, 13.2 Hz, 2H), 4.23 (s,

1

solid (163 mg, 85% yield). H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 2H), 3.09 (dd, J = 12.0, 3.2 Hz, 1H), 2.83 (dd, J = 12.0, 9.6 Hz, 1H). ¹³

C

8.8 Hz, 1H), 7.81 (t, J = 8.8 Hz, 2H), 7.56 – 7.51 (m, 1H), 7.40 – 7.35 NMR (125 MHz, CDCl₃) δ 155.5, 143.7, 143.3, 134.5, 134.0, 131.7,

(m, 2H), 7.33 (d, J = 3.6 Hz, 3H), 7.29 (ddd, J = 8.4, 6.4, 2.0 Hz, 7H), 131.0, 130.9, 130.2, 129.9, 129.1, 129.0, 127.2, 125.6, 124.2, 120.3,

4.83 (dd, J = 9.6, 3.2 Hz, 1H), 4.68 (s, 2H), 4.42 – 4.37 (m, 2H), 4.33 (t, 120.1, 116.9, 115.2, 113.4, 72.3, 71.6, 57.6, 44.0. HRMS (ESI,

J = 10.6 Hz, 2H), 3.16 (t, J = 6.8 Hz, 2H), 3.00 (dd, J = 12.2, 3.2 Hz, 1H), positive) m/z calcd for C₂₇H₂₄N₂O₂[M + H]⁺: 409.1906. Found:

2.70 (dd, J = 12.0, 9.6 Hz, 1H).¹³C NMR (125 MHz, CDCl₃) δ 156.2, 409.1908.

143.2, 139.5, 134.5, 131.7, 130.5, 130.4, 130.1, 129.8, 128.9, 128.8, N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-phenylethan-1-amine

128.2, 127.2, 125.2, 123.9, 118.7, 114.9, 71.8, 71.2, 56.6, 43.7, 37.4. (DC_C08). The title compound was synthesized from 3a (144 mg,

HRMS (ESI, positive) m/z calcd for C₂₇H₂₇NO₂[M + H]⁺: 398.2115. 0.5 mmol), 2-phenylethan-1-amine 4b (73 mg, 0.6 mmol),

Found: 398.2118.

NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol) in dry

2-(((2-((4-fluorobenzyl)oxy)naphthalen-1-yl)methyl)amino)-1-

DCE (15 mL) according to synthetic procedure for DC_C01. The

1

phenylethan-1-ol (DC_C05). The title compound was synthesized product DC_C08 is a yellow solid (173 mg, 94% yield). H NMR (400

from 2-((4-fluorobenzyl)oxy)-1-naphthaldehyde 3d (140 mg, 0.5 MHz, CDCl₃) δ 8.11 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 9.2 Hz, 1H), 7.66 (d,

mmol), 2-amino-1-phenylethan-1-ol 4a (82 mg, 0.6 mmol), J = 8.0 Hz, 1H), 7.59 – 7.53 (m, 1H), 7.44 – 7.40 (m, 2H), 7.39 – 7.32

NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol) in dry (m, 4H), 7.29 (t, J = 5.2 Hz, 2H), 7.23 – 7.21 (m, 2H), 6.98 (dd, J = 7.2,

DCE (15 mL) according to synthetic procedure for DC_C01. The 2.0 Hz, 2H), 5.26 (s, 2H), 4.53 (s, 2H), 3.04 – 2.97 (m, 2H), 2.93 –

1

product DC_C05 is a white solid (188 mg, 94% yield). H NMR (400 2.88 (m, 2H).¹³C NMR (125 MHz, CDCl₃) δ 156.9, 138.7, 137.9, 134.2,

MHz, CDCl₃) δ 8.09 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 9.2 Hz, 1H), 7.71 (d, 133.0, 130.3, 130.2, 130.1, 130.0, 129.7, 129.3, 129.2, 128.1, 125.5,

J = 8.0 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.47 (dd, J = 8.4, 5.2 Hz, 2H), 124.2, 114.8, 114.3, 72.4, 49.8, 42.9, 34.0. HRMS (ESI, positive) m/z

7.37 – 7.32 (m, 1H), 7.29 (dd, J = 6.4, 2.8 Hz, 4H), 7.18 (dd, J = 6.4, calcd for C₂₆H₂₅NO [M + H]⁺: 368.1911. Found: 368.1914.

2.4 Hz, 2H), 7.06 (t, J = 8.4 Hz, 2H), 5.51 (s, 2H), 5.31 (s, 2H), 4.99 (d, N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(pyridin-2-yl)ethan-1-

J = 8.4 Hz, 1H), 4.62 (dd, J = 29.6, 13.2 Hz, 2H), 3.02 (ddd, J = 22.4, amine (DC_C09). The title compound was synthesized from 3a (144

12.0, 6.4 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 165.1, 163.1, 156.8, mg, 0.5 mmol), 2-(pyridin-2-yl)ethan-1-amine 4c (73 mg, 0.6 mmol),

141.7, 134.1, 133.6, 133.5, 133.4, 131.3, 131.2, 130.4, 130.2, 129.8, NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol) in dry

129.5, 129.2, 127.2, 125.6, 123.7, 117.3, 117.1, 114.6, 113.5, 71.8, DCE (15 mL) according to synthetic procedure for DC_C01. The

1

70.2, 56.5, 43.6. HRMS (ESI, positive) m/z calcd for C₂₆H₂₄FNO₂[M product DC_C09 is a yellow solid (171 mg, 93% yield). H NMR (400

+H]⁺: 402.1805. Found: 402.1809.

MHz, CDCl₃) δ 8.09 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 9.2 Hz, 1H), 7.81 (d,

2-(((2-((4-chlorobenzyl)oxy)naphthalen-1-yl)methyl)amino)-1-

J = 8.0 Hz, 1H), 7.71 (d, J = 4.8 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.50

phenylethan-1-ol (DC_C06). The title compound was synthesized (td, J = 7.6, 1.6 Hz, 1H), 7.44 – 7.38 (m, 4H), 7.36 (d, J = 2.4 Hz, 1H),

from 2-((4-chlorobenzyl)oxy)-1-naphthaldehyde 3e (148 mg, 0.5 7.35 – 7.31 (m, 2H), 7.05 – 6.91 (m, 2H), 5.32 (s, 2H), 4.79 (s, 2H),

mmol), 2-amino-1-phenylethan-1-ol 4a (82 mg, 0.6 mmol), 3.34 – 3.29 (m, 2H), 3.29 – 3.24 (m, 2H). ¹³C NMR (125 MHz, CDCl₃)

NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol) in dry δ 159.1, 156.2, 148.5, 137.9, 136.8, 133.6, 132.7, 129.8, 129.7,

DCE (15 mL) according to synthetic procedure for DC_C01. The 129.6, 129.2, 128.9, 128.0, 125.0, 124.1, 122.8, 122.7, 114.4, 112.7,

1

product DC_C06 is a white solid (200 mg, 96% yield). H NMR (400 72.0, 46.4, 42.0, 31.6. HRMS (ESI, positive) m/z calcd for C₂₅H₂₄N₂O

MHz, CDCl₃) δ 8.05 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 9.2 Hz, 2H), 7.55 [M + H]⁺: 369.2013. Found: 369.2017.

(ddd, J = 8.4, 6.8, 1.2 Hz, 1H), 7.42 – 7.37 (m, 1H), 7.35 (d, J = 4.0 Hz, N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(1H-indol-3-yl)ethan-

4H), 7.34 – 7.32 (m, 1H), 7.30 (dd, J = 7.2, 4.0 Hz, 4H), 7.27 (d, J = 1.7 1-amine (DC_C10). The title compound was synthesized from 3a

Hz, 1H), 5.19 (s, 2H), 4.80 (dd, J = 9.2, 3.2 Hz, 1H), 4.52 (s, 2H), 4.41 (144 mg, 0.5 mmol), 2-(1H-indol-3-yl)ethan-1-amine 4d (96 mg, 0.6

(dd, J = 26.4, 12.8 Hz, 2H), 2.90 (ddd, J = 21.6, 12.0, 6.4 Hz, 2H).¹³C mmol), NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol)

NMR (125 MHz, CDCl₃) δ 153.7, 141.7, 134.9, 133.5, 132.7, 129.3, in dry DCE (15 mL) according to synthetic procedure for DC_C01.

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1

The product DC_C10 is a yellow solid (183 mg, 90% yield). H NMR 33.6. HRMS (ESI, positive) m/z calcd for C₂₆H₂₄FNO [M + H]⁺:

View Article Online

DOI: 10.1039/C7OB00070G

(400 MHz, DMSO-d₆) δ 10.99 (s, 1H), 8.93 (s, 1H), 8.20 (d, J = 8.4 Hz, 386.1927. Found: 386.1929.

1H), 8.09 (d, J = 9.2 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.66 – 7.60 (m, 4-(2-(((2-(benzyloxy)naphthalen-1-yl)methyl)amino)ethyl)phenol

2H), 7.55 (dd, J = 7.6, 1.6 Hz, 2H), 7.51 (d, J = 8.0 Hz, 1H), 7.46 (t, J = (DC_C14). The title compound was synthesized from 3a (144 mg,

7.2 Hz, 1H), 7.40 – 7.35 (m, 2H), 7.34 (t, J = 3.8 Hz, 2H), 7.18 (d, J = 0.5 mmol), 4-(2-aminoethyl)phenol 4h (82 mg, 0.6 mmol),

2.3 Hz, 1H), 7.14 – 7.09 (m, 1H), 7.04 – 6.99 (m, 1H), 5.37 (s, 2H), NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol) in dry

4.68 (s, 2H), 3.29 – 3.24 (m, 2H), 3.13 – 3.08 (m, 2H). ¹³C NMR (125 DCE (15 mL) according to synthetic procedure for DC_C01. The

1

MHz, CDCl₃) δ 154.5, 136.1, 135.4, 132.2, 131.3, 128.4, 128.3, 128.2, product DC_C14 is a yellow solid (167 mg, 87% yield). H NMR (400

127.8, 127.7, 126.6, 125.8, 123.8, 123.7, 121.7, 121.4, 119.1, 117.7, MHz, DMSO-d₆) δ 8.06 (d, J = 8.9 Hz, 1H), 7.85 (dd, J = 8.5, 3.0 Hz,

112.6, 111.2, 110.5, 108.2, 69.7, 45.1, 41.3, 22.0. HRMS (ESI, 2H), 7.48 (dd, J = 14.9, 7.9 Hz, 4H), 7.39 (t, J = 6.8 Hz, 2H), 7.35 (dd, J

positive) m/z calcd for C₂₈H₂₆N₂O [M + H]⁺: 407.2101. Found: = 7.1, 3.3 Hz, 2H), 6.93 (d, J = 8.3 Hz, 2H), 6.63 (d, J = 8.3 Hz, 2H),

407.2103.

5.25 (s, 2H), 4.18 (s, 2H), 3.39 (s, 2H), 2.78 (t, J = 7.3 Hz, 2H), 2.59 (t,

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(piperidin-1-yl)ethan-

J = 7.3 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 172.6, 155.9, 154.3,

1-amine (DC_C11). The title compound was synthesized from 3a 137.8, 133.5, 130.6, 129.8, 129.3, 128.9, 128.7, 128.3, 128.0, 126.9,

(144 mg, 0.5 mmol), 2-(piperidin-1-yl)ethan-1-amine 4e (77 mg, 0.6 124.2, 123.9, 121.7, 115.7, 115.5, 71.0, 51.5, 43.0, 35.2, 21.7. HRMS

mmol), NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol) (ESI, positive) m/z calcd for C₂₆H₂₅NO₂[M + H]⁺: 384.1965. Found:

in dry DCE (15 mL) according to synthetic procedure for DC_C01. 386.1972.

1

The product DC_C11 is a yellow solid (161 mg, 86% yield). H NMR N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(p-tolyl)ethan-1-

(400 MHz, CDCl₃) δ 8.00 (t, J = 8.8 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), amine(DC_C15). The title compound was synthesized from 3a (144

7.85 (d, J = 8.0 Hz, 1H), 7.65 – 7.60 (m, 1H), 7.45 (t, J = 7.2 Hz, 3H), mg, 0.5 mmol), 2-(p-tolyl)ethan-1-amine 4i (81 mg, 0.6 mmol),

7.43 – 7.39 (m, 2H), 7.38 (t, J = 3.2 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol) in dry

5.33 (s, 2H), 4.72 (s, 1H), 4.67 (s, 2H), 3.01 – 2.97 (m, 2H), 2.63 – DCE (15 mL) according to synthetic procedure for DC_C01. The

1

2.58 (m, 2H), 2.20 (s, 4H), 1.31 (s, 6H).¹³C NMR (125 MHz, CDCl₃) δ product DC_C15 is a yellow solid (185 mg, 97% yield). H NMR (400

155.1, 136.0, 132.9, 131.6, 129.4, 129.0, 129.0, 128.6, 128.3, 127.6, MHz, CDCl₃) δ 8.08 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.73 (d,

124.5, 122.0, 114.4, 113.8, 71.6, 54.1, 53.6, 42.1, 41.3, 24.8, 23.4. J = 8.0 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.43 – 7.31 (m, 6H), 7.28 (d, J

HRMS (ESI, positive) m/z calcd for C₂₅H₃₀N₂O [M + H]⁺: 375.2523. = 8.4 Hz, 1H), 7.03 (d, J = 7.2 Hz, 2H), 6.92 (d, J = 7.2 Hz, 2H), 5.52 (s,

Found: 375.2532.

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-morpholinoethan-1-

1H), 5.22 (s, 2H), 4.49 (s, 2H), 2.99 (t, J = 7.6 Hz, 2H), 2.86 (t, J = 7.2

Hz, 2H), 2.31 (s, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 156.7, 138.1,

amine (DC_C12). The title compound was synthesized from 3a (144 137.5, 136.2, 134.4, 132.5, 130.7, 130.5, 130.2, 130.1, 130.0, 129.7,

mg, 0.5 mmol), 2-morpholinoethan-1-amine 4f (78 mg, 0.6 mmol), 129.1, 125.4, 124.3, 116.2, 115.1, 72.5, 50.1, 43.1, 34.4, 22.5. HRMS

NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol) in dry (ESI, positive) m/z calcd for C₂₇H₂₇NO [M + H]⁺: 382.2287. Found:

DCE (15 mL) according to synthetic procedure for DC_C01. The 382.2293.

1

product DC_C12 is a yellow solid (163 mg, 87% yield). H NMR (400 N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(4-chlorophenyl)

MHz, CDCl₃) δ 8.01 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 9.2 Hz, 1H), 7.82 (d, ethan-1-amine (DC_C16). The title compound was synthesized from

J = 8.0 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.43 (t, J = 7.2 Hz, 5H), 7.38 (t, 3a (144 mg, 0.5 mmol), 2-(4-chlorophenyl)ethan-1-amine 4j (94 mg,

J = 4.3 Hz, 1H), 7.35 (d, J = 4.8 Hz, 1H), 5.32 (s, 2H), 4.71 (s, 2H), 3.21 0.6 mmol), NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5

(s, 4H), 2.97 – 2.90 (m, 2H), 2.56 – 2.49 (m, 2H), 1.99 (t, J = 4.4 Hz, mmol) in dry DCE (15 mL) according to synthetic procedure for

4H).¹³C NMR (125 MHz, CDCl₃) δ 156.8, 137.1, 134.2, 133.7, 130.6, DC_C01. The product DC_C16 is a yellow solid (191 mg, 95% yield).

130.6, 130.5, 130.3, 130.2, 129.1, 126.2, 123.2, 115.1, 113.8, 73.1, ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 9.2

67.8, 54.8, 54.0, 43.1, 42.5. HRMS (ESI, positive) m/z calcd for Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.55 (ddd, J = 8.4, 6.8, 1.2 Hz, 1H),

C₂₄H₂₈N₂O₂[M + H]⁺: 377.2354. Found: 377.2361.

7.43 (dt, J = 5.6, 3.2 Hz, 2H), 7.39 (d, J = 4.0 Hz, 1H), 7.37 (t, J = 1.6

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(4-fluorophenyl)

Hz, 1H), 7.35 (d, J = 5.6 Hz, 1H), 7.29 (d, J = 7.2 Hz, 2H), 7.20 – 7.15

ethan-1-amine (DC_C13). The title compound was synthesized from (m, 2H), 6.89 (d, J = 8.4 Hz, 2H), 5.25 (s, 2H), 4.48 (s, 2H), 2.97 – 2.91

3a (144 mg, 0.5 mmol), 2-(4-fluorophenyl)ethan-1-amine 4g (84 mg, (m, 2H), 2.83 – 2.77 (m, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 155.1,

0.6 mmol), NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 137.0, 136.9, 132.6, 131.1, 131.0, 130.4, 128.6, 128.5, 128.4, 128.0,

mmol) in dry DCE (15 mL) according to synthetic procedure for 127.9, 127.1, 123.8, 123.2, 114.5, 70.4, 48.6, 41.0, 31.6. HRMS (ESI,

DC_C01. The product DC_C13 is a yellow solid (160 mg, 83% yield). positive) m/z calcd for C₂₆H₂₄ClNO [M + H]⁺: 402.1852. Found:

¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 9.2 402.1874.

Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.42 (t, J = N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(3-fluorophenyl)

8.0 Hz, 2H), 7.40 – 7.34 (m, 3H), 7.34 – 7.29 (m, 2H), 6.91 (p, J = 8.8 ethan-1-amine (DC_C17). The title compound was synthesized from

Hz, 4H), 5.26 (s, 2H), 4.49 (s, 2H), 2.98 – 2.92 (m, 2H), 2.86 – 2.79 (m, 3a (144 mg, 0.5 mmol), 2-(3-fluorophenyl)ethan-1-amine 4k (84 mg,

2H). ¹³C NMR (125 MHz, CDCl₃) δ 164.1, 162.1, 156.8, 138.0, 134.7, 0.6 mmol), NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5

134.7, 134.2, 132.8, 131.7, 131.6, 130.4, 130.2, 130.0, 129.7, 129.2, mmol) in dry DCE (15 mL) according to synthetic procedure for

129.1, 125.5, 124.2, 116.8, 116.6, 115.2, 114.9, 72.5, 50.2, 43.2, DC_C01. The product DC_C17 is a yellow solid (175 mg, 91% yield).

12 | J. Name., 2012, 00, 1-3

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Organic & Biomolecular Chemistry

Page 14 of 14

Journal Name

ARTICLE

¹H NMR (400 MHz, CDCl₃) δ 8.09 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 9.2 2a (823 mg, 6.00 mmol) and KI (167 mg,1.00 mmol) in dry acetone

View Article Online

DOI: 10.1039/C7OB00070G

Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.54 (ddd, J = 8.4, 6.8, 1.2 Hz, 1H), (15 mL) according to synthetic procedure for 3a. The product 3h is a

7.45 (dd, J = 7.6, 1.6 Hz, 2H), 7.41 – 7.34 (m, 3H), 7.32 (t, J = 3.6 Hz, colorless solid (934 mg, yield 88%).

1H), 7.30 (s, 1H), 7.18 (td, J = 8.0, 6.0 Hz, 1H), 6.89 (td, J = 8.4, 2.4 Hz, 2-((2-(benzyloxy)benzyl)amino)-1-phenylethan-1-ol (DC_C19). The

1H), 6.78 (d, J = 7.6 Hz, 1H), 6.70 – 6.65 (m, 1H), 5.28 (s, 2H), 4.90 (s, title compound was synthesized from 3h (106 mg, 0.5 mmol), 2-

1H), 4.46 (s, 2H), 2.98 – 2.93 (m, 2H), 2.85 – 2.78 (m, 2H). ¹³C NMR amino-1-phenylethan-1-ol 4a (82 mg, 0.6 mmol), NaBH(OAc)₃

(125 MHz, CDCl₃) δ 165.2, 163.3, 156.8, 141.9, 141.9, 138.1, 134.3, (318mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol) in dry DCE (15 mL)

132.6, 131.4, 131.3, 130.4, 130.2, 130.0, 129.8, 129.3, 129.1, 125.9, according to synthetic procedure for DC_C01. The product DC_C19

1

125.8, 125.4, 124.3, 117.1, 117.0, 116.0, 115.1, 115.0, 114.8, 72.5, is a yellow solid (145 mg, 87% yield). H NMR (400 MHz, CDCl₃) δ

50.1, 43.3, 34.4. HRMS (ESI, positive) m/z calcd for C₂₆H₂₄FNO [M + 7.38 (dt, J = 9.2, 7.2 Hz, 6H), 7.33 – 7.29 (m, 4H), 7.28 – 7.22 (m, 2H),

H]⁺: 386.1946. Found: 386.1972.

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-N-methyl-2-

6.92 (t, J = 7.6 Hz, 2H), 5.15 (s, 2H), 5.01 (d, J = 9.6 Hz, 1H), 4.40 (s,

2H), 4.13 (dd, J = 34.0, 13.2 Hz, 2H), 3.07 (dd, J = 12.0, 2.4 Hz, 1H),

phenylethan-1-amine (DC_C18). The title compound was 2.83 (dd, J = 12.0, 10.4 Hz, 1H).¹³C NMR (125 MHz, CDCl₃) δ 156.3,

synthesized from 3a (144 mg, 0.5 mmol), N-methyl-2-phenylethan- 142.0, 136.4, 129.9, 128.4, 128.2, 127.8, 127.6, 126.9, 126.8, 126.4,

1-amine 4l (81 mg, 0.6 mmol), NaBH(OAc)₃(318 mg, 1.5 mmol), 125.4, 120.4, 111.3, 70.7, 69.6, 55.4, 48.1. HRMS (ESI, positive) m/z

CH₃COOH (57 µL, 0.5 mmol) in dry DCE (15 mL) according to calcd for C₂₂H₂₃NO₂[M + H]⁺: 334.1824. Found: 386.1846.

synthetic procedure for DC_C01. The product DC_C18 is a yellow

1

solid (183 mg, 96% yield). H NMR (400 MHz, CDCl₃) δ 8.19 (d, J =

8.4 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.60 (t, J

Author Contributions

= 8.0 Hz, 1H), 7.46 (t, J = 5.6 Hz, 2H), 7.43 – 7.34 (m, 5H), 7.29 (t, J =

7.2 Hz, 2H), 7.27 – 7.22 (m, 1H), 7.14 (d, J = 6.8 Hz, 2H), 5.31 (s, 2H),

4.57 (s, 2H), 3.14 (s, 4H), 2.58 (s, 3H).: ¹³C NMR (125 MHz, CDCl₃) δ

155.4, 135.9, 132.5, 132.2, 129.3, 129.0, 128.7, 128.6, 127.7, 124.8,

122.3, 114.0, 113.2, 71.9, 66.0, 53.2, 41.1, 20.9. HRMS (ESI, positive)

m/z calcd for C₂₇H₂₇NO [M + H]⁺: 382.2351. Found: 382.2367.

1-formylnaphthalen-2-yl benzoate (3g). To a solution of benzoyl

chloride 2g (1.23 g, 8.70 mmol) in dry THF (10 mL) was added

dropwise to a mixture of 2-hydroxynaphthaldehyde 1a (1.0 g, 5.81

mmol) and K₂CO₃(1.2 g, 8.70 mmol) in THF(40 mL) at 0 ⁰C and under

nitrogen atmosphere. The reaction mixture was warmed to room

temperature and stirred vigorously for 16 h. The mixture was

poured into water (40 mL) and extracted with ethyl acetate (3×50

mL). The combined organic phases were washed with brine (100

mL), dried (Na₂SO₄), and evaporated in vacuo. The crude product

thus obtained was purified through column chromatography over

Mingyue Zheng and Cheng Luo conceived and designed the project.

Yan Ye and Yulan Wang performed molecular modeling and virtual

screening, Bidong Zhang and Yaxi Yang performed chemical

syntheses, Ruifeng Mao and Hong Ding performed cellular assays,

Chenhua Zhang and Yu-Chih Liu measured enzyme activity and

inhibition, Yan Ye and Jing Xing analyzed the data. Yan Ye, Bidong

Zhang and Mingyue Zheng wrote the paper. All authors discussed

the results and commented on the manuscript. All authors

approved the final version of the manuscript.

Acknowledgements

We gratefully acknowledge financial support from the

Strategic Priority Research Program of the Chinese Academy of

Sciences (XDA12050201 to M.Z.), the National Basic Research

Program (2015CB910304 and 2015CB910304 to X.L.), the

silica using methylene chloride: hexanes (1:1) mixture to afford 1.2 National Natural Science Foundation of China (21210003 and

81230076 to H.J., 81430084 to K.C. and 81625022 to C.L.), and

the National Key Research Development Plan

(2016YF1201003 to M.Z.), and the Fund of State Key

Laboratory of Toxicology and Medical Countermeasures,

Academy of Military Medical Science (TMC201505 to C.L).

g (75%) of 3g as a white solid.

&

1-(((2-hydroxy-2-phenylethyl)amino)methyl)naphthalen-2-yl

benzoate (DC_C04). The title compound was synthesized from 3g

(138 mg,0.5 mmol), 2-amino-1-phenylethan-1-ol 4a (82 mg, 0.6

mmol), NaBH(OAc)₃(318 mg, 1.5 mmol), CH₃COOH (57 µL, 0.5 mmol)

in dry DCE (15 mL) according to synthetic procedure for DC_C01.

1

The product DC_C04 is a canary solid (153 mg, yield 77%). H NMR

References

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14 | J. Name., 2012, 00, 1-3

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Discovery and optimization of selective inhibitors of protein arginine methyltransferase 5 by docking-based virtual screening

DOI: 10.1039/c7ob00070g

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