Design and Synthesis of Potent, Selective Inhibitors of Protein Arginine Methyltransferase 4 against Acute Myeloid Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.9b00297

PRMT4 is a type I protein arginine methyltransferase and plays important roles in various cellular processes. Overexpression of PRMT4 has been found to be involved in several types of cancers. Selective and in vivo effective PRMT4 inhibitors are needed for demonstrating PRMT4 as a promising therapeutic target. On the basis of compound 6, a weak dual PRMT4/6 inhibitor, we constructed a tetrahydroisoquinoline scaffold through a cut-and-sew scaffold hopping strategy. The subsequent SAR optimization efforts employed structure-based approach led to the identification of a novel PRMT4 inhibitor 49. Compound 49 exhibited prominently high potency and selectivity, moderate pharmacokinetic profiles, and good antitumor efficacy in acute myeloid leukemia xenograft model via oral administration, thus demonstrating this compound as a useful pharmacological tool for further target validation and drug development in cancer therapy.

Full text of DOI:10.1021/acs.jmedchem.9b00297

Subscriber access provided by UNIV OF SOUTHERN INDIANA
Article
Design and Synthesis of Potent, Selective Inhibitors of Protein
Arginine Methyltransferase 4 Against Acute Myeloid Leukemia
Zuhao Guo, Zhuqing Zhang, Hong Yang, Danyan Cao, Xiaowei Xu, Xingling
Zheng, Danqi Chen, Qi Wang, yanlian Li, jian li, Zhiyan Du, Xin Wang, Lin
Chen, Jian Ding, Jingkang Shen, Mei-Yu Geng, Xun Huang, and Bing Xiong
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2019
Downloaded from http://pubs.acs.org on May 25, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Design and Synthesis of Potent, Selective Inhibitors of Protein
Arginine Methyltransferase 4 Against Acute Myeloid Leukemia
Zuhao Guo a, c, #, Zhuqing Zhang b, c, #, Hong Yang , Danyan Cao , Xiaowei Xu
b
a
b, c
,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Xingling Zheng b, c , Danqi Chen , Qi Wang
a
a, c
, Yanlian Li , Jian Li , Zhiyan Du ,
a
a
a
Xin Wang , Lin Chen , Jian Ding , Jingkang Shen , Meiyu Geng b, c, *, Xun Huang
a
a
b, c
a
b, c, *, Bing Xiong a, c, *
a
Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, China
b
Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi
Road, Shanghai, 201203, China
c
University of Chinese Academy of Sciences, NO.19A Yuquan Road, Beijing, 100049,
China
ABSTRACT
PRMT4 is a type I protein arginine methyltransferase and plays important roles in
various cellular processes. Overexpression of PRMT4 has been found to be involved in
several types of cancers. Selective and in vivo effective PRMT4 inhibitors are needed
for demonstrating PRMT4 as a promising therapeutic target. Based on compound 6, a
weak dual PRMT4/6 inhibitor, we constructed a tetrahydroisoquinoline scaffold
through a cut-and-sew scaffold hopping strategy. The subsequent SAR optimization
efforts employed structure-based approach led to the identification of a novel PRMT4
ACS Paragon Plus Environment

Page 3 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
inhibitor 49. Compound 49 exhibited prominently high potency and selectivity,
moderate pharmacokinetic profiles and good anti-tumor efficacy in acute myeloid
leukemia xenograft model via oral administration, thus demonstrating this compound
as a useful pharmacological tool for further target validation and drug development in
cancer therapy.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Keywords: PRMT4; structure-based drug discovery; scaffold hopping; acute myeloid
leukemia
INTRODUCTION
Post-translational modification (PTM) of protein is an economical machinery in
biology to dynamically regulate a broad range of cellular processes covering cellular
1
metabolism, transcription, protein translation, and signal transduction. In the past two
2
decades, various proteins involved in PTM were discovered and the enzymes
responsible for the methylation on the guanidinium group of arginine are known as
protein arginine methyltransferases (PRMTs), which utilize S-adenosyl methionine
3
(SAM) as the methyl donor to perform the catalysis. Nine PRMTs have been identified
in human genome and divided into three subfamilies in term of the degree and position
of methylation.4, 5 Type I enzymes including PRMT1, 2, 3, 4, 6 and 8 could transfer one
methyl group to arginine to convert it into monomethylarginine (MMA) or further add
another methyl group to the same nitrogen atom to become asymmetric
dimethylarginine (aDMA). Type II enzymes, consisting of PRMT5 and 9, generate
MMA or symmetric dimethylarginine (sDMA) , which means one more methyl group
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
is added to another nitrogen atom of arginine. While PRMT7 is the sole type III enzyme
that only produces MMA. 6
A type I enzyme PRMT4, also known as coactivator-associated arginine
methyltransferase 1 (CARM1), was first identified as a transcriptional regulator.7
PRMT4 can function as a transcriptional coactivator of nuclear receptors and
methylates steroid receptor coactivators including SRC3 and CBP/P300.8,
Furthermore, PRMT4 regulates gene expression by multiple mechanisms. Specifically,
PRMT4 positively regulates transcription by methylating histone H3 at arginine 17 and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
26.10, 11 PRMT4 also methylates the RNA-binding proteins PABP1 , HuR and HuD
12
13
14
to affect their ability to bind to the transcription-related proteins, and methylates
splicing factors such as CA150 15 to regulate the exon skipping.
Overexpression of PRMT4 has been demonstrated in various cell lines of hematologic
cancers and solid tumors, such as leukemia,16, 17 breast,18 prostate,19 liver20 and
colorectal 21, 22 cancers. Wang et al. identified that PRMT4 catalyzed the methylation
of BAF155, a critical component of SWI/SNF chromatin remodeling complex, and
2
3
therefore enhanced the tumor progression and metastasis of breast cancer. Moreover,
2
4
PRMT4 has been found upregulated in grade-3 breast tumors, and the knockdown of
PRMT4 inhibited prostate cancer cell proliferation by induction of apoptosis.25
Recently, an essential role of PRMT4 in myeloid leukemogenesis has been linked to
the oncogenic transcription factors which has little effect on normal hematopoiesis.
Therefore, PRMT4 is considered to be a promising therapeutic target for anticancer
drug development.In past decade, as the increased interest in drug targeting PRMT
ACS Paragon Plus Environment

Page 5 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
enzymes, numerous PRMT4 inhibitors have been reported, and most of them are pan-
type I PRMTs inhibitors, which usually are associated with potent activities towards
PRMT1, PRMT4 and PRMT6.26-28 Recent years, selective PRMT4 inhibitors are
occasionally reported.29-31 Purandare et al. from Bristol-Myyers Squibb and Allan et al.
from MethylGene reported two series of selective PRMT4 inhibitors sharing alanine
amide motif as key pharmacophore. These compounds showed two-digit nanomolar
enzymatic activities but with very low cellular activities. Subsequent efforts on
developing selective PRMT4 inhibitors were conducted but with no obvious
improvement, probably attributed to the poor permeability and metabolic instability
that limited the further exploitation.32-35 In 2017, Drew et al. from Epizyme reported
the first oral active PRMT4 inhibitor, EZM2302, which contained 1-amino-3-
phenoxypropan-2-ol moiety as arginine mimetic and demonstrated moderate efficacy
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
36
in multiple myeloma xenograft model. Thus, developing selective PRMT4 inhibitor
with in vivo activity is still pressing, which may provide more option for target
validation and drug development.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. (A) Representative structures and profiles of reported type I PRMT inhibitors. The
common moieties of arginine mimetic are highlighted in red. (B) Structural alignment of PRMT
inhibitors in complex with PRMT4 (1: PDB code 2Y1W) and PRMT6 (2: PDB codes 4Y30):
1
(green), 2(gray).
From the chemical structural point of view, as represented in Figure 1, these inhibitors
target the substrate binding pocket by frequently using an ethylenediamino moiety as a
warhead to mimic the side chain of arginine residue. Scrutiny of cocrystal structures of
3
7
38
27
compound 1, EPZ020411 (2) nd MS023 (3) in complex with PRMT proteins, we
find that the ethylenediamino moieties stretched into a narrow subpocket and formed
critical hydrogen bonding interactions with two conserved residues (His415 and
Glu258 in PRMT4; His317 and Glu155 in PRMT6). However, the entrance of the
substrate binding pocket is dynamic and has diversified residues among different
PRMTs, which could be utilized to enhance the selectivity towards PRMT4.27, 39
In consideration of the ubiquitous expression of type I PRMTs (except for PRMT8)40
4
and distinct functions of individual PRMTs , improving the selectivity for PRMT4 can
ACS Paragon Plus Environment

Page 7 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
minimize the adverse concerns results from inhibiton of other homologous type I
PRMTs. Herein, we report the discovery of (R)-1-(methylamino)-3-(5-((4-(thiazol-2-
yl)benzyl)oxy)-3,4-dihydroisoquinolin-2(1H) yl)propan-2-ol 49 derived from a
previously reported dual PRMT4/6 inhibitor. This potent compound 49 exhibits
excellent PRMTs selectivity, moderate pharmacokinetic profiles and high inhibitory
efficacy in vitro and in vivo, which will help to validate PRMT4 as a potential
therapeutic target and provides a candidate for the treatment of acute myeloid leukemia.
RESULTS AND DISCUSSION
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Rational Design of Selective PRMT4 Inhibitor
In order to develop selective PRMT4 inhibitors, we firstly analyzed the essential moiety
of arginine mimetic and noticed that a reported alternative 1,3-diaminopropan-2-ol
compound 6 could slightly improve the selectivity to PRMT4 (PRMT4 IC = 1.22 μM;
5
0
PRMT6, IC = 3.04 μM), while the ethylenendiamino analogues (compound 4 and 5)
5
0
had better activity towards PRMT6 (Figure 2).39, 41 This implicated that the 1,3-
diaminopropan-2-ol may provide new hydrogen bonding pattern to better fit the
subpocket of PRMT4. Besides, this aminoalcohol moiety also occurred in the selective
PRMT4 inhibitor EZM2302, which further encouraged us to explore selective inhibitor
of PRMT4 based on compound 6. 36 By comparing the crystal structures of PRMT4
with PRMT6, we found that three residues (Phe153, Gln159, and Asn266 in PRMT4
and Cys50, Val56, and His163 in PRMT6) at the entrance of the arginine-binding
pocket were not conserved between the two enzymes. In particular, π-π interaction
between PRMT4’s Phe153 and aromatic ring of inhibitors can further boost the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
selectivity in favor of PRMT4 over PRMT6.34, 35 Combining the aforementioned
interaction features, we intended to use the 1,3-diaminopropan-2-ol motif as the
warhead and focus the modification on the left part of the inhibitors.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Design strategy for selective PRMT4 inhibitor. Structural alignment of PRMT inhibitors
in complex with PRMT4 (1: PDB code 2Y1W) and PRMT6 (4: PDB codes 5EGS): 1(green), 4(bule).
Following the design strategy outline in Figure 2, we initialized the project with
scaffold hopping approach by opening and recycling the ring system to generate the
bicyclic skeleton, which, as predicted, may enforce the phenyl ring to get closer to
Phe153 and form π-π interaction to improve the selectivity and potency for PRMT4
(Figure 2).
ACS Paragon Plus Environment

Page 9 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
With the guidance of this design strategy, compounds 7-11 with varied saturated
heterocycle were synthesized. As shown in Table 1, these compounds displayed
moderate potency in PRMT4 enzymatic assay except compound 11 that didn’t show
any inhibition even at 10 μM. Considering the simple structure and preserved activity,
compound 7-10 could serve as applicable hit for further optimization. To verify the
design stategy and provide rational direction for optimization, we solved the co-crystal
structure of compound 10 bound with the catalytic domain of PRMT4 (aa 140-480). As
shown in Figure 3, the structure revealed that compound 10 was situated at the substrate
binding pocket, and formed critical hydrogen bonding interactions with nearby residues,
including Glu258, Met260 and His415. By aligning with the reported crystal structure
(PDB code: 2Y1W), we found that the longer tail of compound 10 extended to the
position commonly occupied by SAM. Notably, the bicyclic ring of compound 10 had
different orientation, facing the residue Met163 and pushing the residue Asn162 away.
Importantly, the bicyclic ring of compound 10 was indeed engaged in a T-shape π-π
interaction with Phe153 as expected. This distinct binding mode triggered us to further
elaborate the SAR of this series of PRMT4 inhibitors.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. PRMT4 enzymatic activity of compound 7-11
Het
Tail
a
Compd
Structure
IC (μM)
50
HO HN
7
8
2.46 ± 0.22
5.31 ± 1.08
N
N
OH
H
N
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HO HN
N
9
8.34 ± 1.16
3.19 ± 0.67
OH
N
1
0
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
N
O
OH
N
1
>10.0
H
N
a
All IC values are reported as the geometric mean from at least two determinations. The PRMT4
5
0
inhibitor MS049 was used as the positive control in our enzymatic assay, exhibiting an IC value
5
0
of 32 ± 6 nM.
Figure 3. (A) (B) Crystal structures of compounds 10 (green) in complex with PRMT4 (PDB code:
IZQ). (C) Structural alignment with compound 1 (yellow) (PDB code: 2Y1W).
6
Lead Optimization
Considering of the synthesis accessibility, we selected compound 8 as the starting point
to investigate the substitutions on the phenyl group. According to the cocrystal structure
of compound 10 in complex with PRMT4, we could find that the 5- or 6-position at the
phenyl group might be the most promising position for extension. Nevertheless, in case
of the flexibility of the PRMT4 protein, we synthesized four compounds (12-15) by
using bromide atom as a scanning group at various positions (Table 2). From the result,
ACS Paragon Plus Environment

Page 11 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
we found the 5-position of phenyl group was the possible extension site, as compound
1
2 displayed higher potency for PRMT4 with an IC value of 0.130 μM. On the other
50
hand, compound 15 (IC = 0.772μM) substituted with a bromide atom at 8-position
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
also increased the potency for PRMT4, which was probably attributed to the
introduction of halogen bonding interaction between bromide atom and the surrounding
residue Tyr154. We also synthesized the 2,3,4,5-tetrahydro-1H-benzo[c]azepine
derivative 16 with an IC value of 0.785 μM, which further confirmed the 5-position
5
0
as the preferred extension site. Furthermore, compound 12 was tested the inhibition for
PRMT1 and PRMT6 and it demonstrated excellent selectivity, with more than 450-fold
and 320-fold potent for PRMT4 over PRMT1 and PRMT6, respectively (Table 3).Thus,
compound 12 was chosen as starting scaffold for the next round SAR study.
Table 2. PRMT4 enzymatic activity of compound 12-16
5
n
6
OH
R
H
N
N
7
8
a
Compd
R
n
IC (μM)
50
1
1
1
2
3
4
5-Br
6-Br
7-Br
8-Br
5-Br
1
1
1
1
2
0.130 ± 0.012
4.48 ± 0.30
5.22 ± 0.94
15
0.772 ± 0.028
0.785 ± 0.152
1
6
a
All IC values are reported as the geometric mean from at least two determinations.
5
0
Table 3. Selectivity of compound 12
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
IC (μM)
Selectivity
Selectivity
5
0
Compd
b
b
PRMT4
0.130
PRMT1
58.8
PRMT6
41.2
Ratio (4vs.1) Ratio (4vs.6)
1
2
450
320
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
All IC values are reported as the geometric mean from at least two determinations. Selectivity
5
0
ratio = IC of PRMTx/ IC of PRMT4, x=1 or 6.
5
0
50
Taking together the information from the preliminary SAR study and cocrystal
structure, we prepared the derivatives with linkers at the 5-position to introduce diverse
functional groups (Table 4). The various linkers may direct the aromatic group to
different locations and increase potential interactions with the residues at the mouth of
the arginine-binding pocket. Without a linker, compound 17 displayed about 4-fold
potency loss against PRMT4 comparing with compound 12. Among analogs with a
one-heteroatom linker, amino linkers (18 and 19) also resulted in an adverse potency,
especially 18 (IC = 2.11 μM), while ether linkers (20 and 21) primarily retained the
5
0
potency for PRMT4. The benzyl moieties (19 and 21) had superior potency over phenyl
groups (18 and 20), which might benefit from the flexibility of benzyl group adopting
the advantageous direction. However, comparing with compound 12, the synthesized
compounds with a two-four heteroatom linker, such as amide (22, 23 and 24), urea (25),
and sulfamide (26), exhibited notably less potent against PRMT4. Nevertheless, the
loss of potency of some derivatives could be explained by the disturbance of conserved
hydrogen bonding interaction or unbefitting direction of aromatic group. Overall, the
ether linker was optimal and showed certain potential for further investigation.
Table 4. PRMT4 enzymatic activity of compound 12, 17-26
ACS Paragon Plus Environment

Page 13 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Ar
Linker
OH
H
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
a
Compd
12
Linker-Ar
IC (μM)
Compd
22
Linker-Ar
IC (μM)
50
5
0
H
N
O
O
Br
0.130 ±0.012
0.465 ± 0.055
7.98 ± 1.35
2.13 ± 0.16
17
23
24
NH
1
1
8
9
2.11 ± 0.23
6.12 ± 1.16
HN
HN
HN
O
HN
0.343 ± 0.067
25
26
3.82 ± 0.15
4.26 ± 0.12
HN
O
O
2
2
0
1
0.270 ± 0.041
0.165 ± 0.029
S
O
HN
O
O
a
All IC values are reported as the geometric mean from at least two determinations.
5
0
Firstly, we prepared analogs of compound 20 with various substitutes on the additional
phenyl group (Table 5). Incorporation of the fluorine atom from the ortho-position (27)
to meta- (28) or para-positions (29) led to a significant loss in inhibitory activity.
Unfortunately, additional substituents including 4-chlorine (30), 4-cyano (31), 3-amino
(32), 3-chloro-4-fluoro (33), 3,5-dimethyl (34) also suffered from detrimental effect on
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
inhibition. These results indicate that the phenyl ether scaffold may not be suitable for
decorating with simple moieties. On the contrary, aromatic thiazolyl-substituted
derivatives (35 and 36) maintained the potency against PRMT4, suggesting that
additional interaction might be responsible for the retention of activity. To further
confirm the influence of extension position, we synthesized the 6-phenyl ether
substituted derivative (37) of 1,2,3,4-tetrahydroisoquinoline, which showed an IC50
value of 0.900 μM （3-fold worse than 5- substituted compound 20）and indeed
exhibited the extension position is important. To understand the differential activities
of these compounds, we modeled the binding conformations of compounds 20, 35 and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7 by utilizing the molecular docking method (Figure S1). The docking result
demonstrated that the 1,3-diaminopropan-2-ol motif interacted with protein almost
exactly as in the solved co-crystal structure of compound 10. The 1,2,3,4-
tetrahydroisoquinoline scaffold was located at similar position as 2,3,4,5-tetrahydro-
1H-benzo[c]azepine of 10. The 5-substituted phenyl group of 20 extended to the
vicinity of residues Tyr150 and Phe153. However, comparing to compound 20, the 6-
substituted phenyl group of 37 reached to solvent-accessible part. Similarly, the phenyl
group of compound 35 pointed to the same direction with 20 and the thiazolyl group
extended further to the helix structure of residues Tyr150 and Phe153. These modeling
confirmed that the 5-position is the right direction for optimization and expounded the
boost in activity of compound 35 and 36.
Table 5. PRMT4 enzymatic activity of compound 20, 27-35
ACS Paragon Plus Environment

Page 15 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
R
OH
H
N
N
a
a
Compd
R
IC (μM)
Compd
32
R
IC (μM)
50
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
0
7
0.270 ± 0.041
2.07 ± 0.26
3.49 ± 0.08
2.05 ± 0.20
O
O
NH2
F
F
33
34
O
O
Cl
F
2
2
8
9
2.31 ± 0.04
2.61 ±0.39
1.99 ± 0.12
O
O
S
F
N
N
35
0.279 ± 0.042
O
O
Cl
30
31
1.18 ± 0.30
2.31 ± 0.16
36
0.658 ± 0.105
O
O
O
S
CN
37b
0.900 ± 0.024
O
OH
H
N
N
a
b
All IC values are reported as the geometric mean from at least two determinations. Phenyl ether
5
0
substituted at 6-position of 1,2,3,4-tetrahydroisoquinoline.
We then focused our attention on the benzyl ether scaffold represented by compound
2
1 and prepared a limited number of derivatives (Table 6). Introduction of the fluorine
atom at 2-, 3-, 4-position (38-40) of the phenyl ring in compound 21 slightly impaired
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the potency, indicating that substitution on the benzyl ether scaffold was more tolerated,
respectively. Among the fluorine-substituted analogs, compound 40 with a fluorine
atom at the para-position showed almost equal potency to 21. Furthermore, we tried to
install different groups at the para-position, such as chloride, cyano, tertiary butyl,
phenyl and thiazolyl groups. The potency of compound 41 (IC = 0.037 μM) owning a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
chloride atom displayed significantly enhancement, which might profit from the
introduction of potential Van der Waals (VDW) interactions between chloride atom and
the surrounding residues (such as Tyr150 and Phe153). In contrast, incorporation of a
tertiary butyl group at 4-position (43) resulted in a loss of potency. As expected, an
enhanced potency was observed for the aromatic substituent derivatives (44 and 45),
among which thiazolyl-substituted compound 45 (IC = 0.031μM) yielded the highest
5
0
potency for PRMT4 in this series of compounds. Migration of thiazolyl group from the
para-position to meta-position generated compound 46 (IC = 0.043μM) with slight
5
0
loss of activity as compared to compound 45, which was consistent with earlier SAR
result. Similarly, 6-benzyl ether substituted derivatives (47) of 1,2,3,4-
tetrahydroisoquinoline exhibited an IC value of 0.475 μM (3-fold worse than 5-
5
0
substituted compound 21).
Encouraged by the improvement of potency, chiral separation was performed to
examine the effect of chirality on PRMT4 inhibitory activity. Eutomer 49 showed
approximately 5-fold more potent than distomer 48, suggesting that R-enantiomer was
superior to the S-enantiomer. Ultimately, 49 was selected for further profiling because
of its excellent potency for PRMT4.
ACS Paragon Plus Environment

Page 17 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 6. PRMT4 enzymatic activity of compound 21, 36-47
R
OH
H
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Comp
d
a
a
Compd
R
IC (μM)
R
IC (μM)
50
50
O
O
O
2
1
0.165 ± 0.029
0.254 ±0.011
43
44
1.95 ± 0.34
F
38
0.120 ±0.002
O
S
F
O
N
S
3
4
4
9
0
1
0.530 ± 0.031
0.228 ± 0.019
0.037 ±0.004
45
46
0.031 ± 0.010
0.043 ± 0.006
0.475 ± 0.037
O
O
O
O
F
N
47b
O
OH
Cl
H
N
N
O
4
2
0.305 ±0.042
CN
a
b
All IC values are reported as the geometric mean from at least two determinations. Benzyl ether
5
0
substituted at 6-position of 1,2,3,4-tetrahydroisoquinoline.
Table 7. PRMT4 enzymatic activity of compound 45 and its enantiomers 48, 49
O
N
OH
H
S
N
N
*
stereoa
IC (μM)
50
b
Compd
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
4
4
5
8
9
rac
S
0.031 ± 0.010
0.101 ±0.022
0.021 ± 0.005
R
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
Chirality introduced from chiral starting material. All IC values are reported as the geometric
5
0
mean from at least two determinations
Selectivity profile of compound 49
We evaluated the selectivity of compound 49 against other PRMTs. As summarized in
Table 8, compound 49 exhibited good selectivity for PRMT4 over other PRMTs (>100-
fold). Specifically, the selectivity between PRMT4 and PRMT6, which shared the most
structurally similarity with PRMT4 among the family, could reach up to 380-fold. To
further assess the selectivity of compound 49, we tested it against 7 common protein
lysine methyltransferases (PKMTs) and delightly found that compound 49 did not
significantly inhibit any of these PKMTs up to 50 μM (Figure 4).
Table 8. Selectivity evaluation of compound 49 a
Type I
Type II Type III Selectivity
Ratio b
Enzyme
PRMT4 PRMT1 PRMT3 PRMT6 PRMT8 PRMT5 PRMT7
(4vs.6)
a
IC (μM)
0.021
8.6
>10
7.9
3.7
>30
>30
380
5
0
a
b
All IC values are reported as the geometric mean from at least two determinations. Selectivity
5
0
ratio = IC of PRMT6/ IC of PRMT4
5
0
50
ACS Paragon Plus Environment

Page 19 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Inhibitory effect of compound 49 against PKMTs (n=2)
Compound 49 Inhibited Proliferation of Multiple Cancer Cell Lines
17
As known the essential role of PRMT4 for myeloid leukemogenesis , we tested 8
leukemia cell lines (MV4-11, MOLM13, THP-1, RS4.11, MOLT4, MOMOMAC6,
HEL, K562) to detect whether compound 49 was sufficient to inhibit cancer cell
proliferation (Table S2). Results showed that compound 49 was potent at inhibiting cell
proliferation in a dose-dependent manner and notably showed high potency toward
MOLM13 cell among leukemia cell lines with IC of 6.93 μM (Figure 5). Thus, we
5
0
focused on MOLM13 cell for our further research.
Figure 5. Proliferation inhibition of 8 leukemia cell lines by Compound 49 in vitro 6 days in culture,
IC values were calculated in GraphPad Prism.
5
0
Compound 49 Decreased the Arginine Methylation at Cellular Levels
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We chose MOLM13, the most sensitive cell line in the tested leukemia cells, to further
assess the cellular function of compound 49 in vitro. After treatment with compound
4
9 for 96 h, we used specific antibodies of asymmetric dimethyl BAF155 and PABP1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to detect the asymmetric methylation and an aDMA antibody to assess the overall levels
of asymmetric dimethyl arginine. Asymmetric dimethyl-BAF155 (IC =0.369 μM),
5
0
PABP1 (IC =0.545 μM) and the global aDMA levels were reduced in a concentration-
5
0
dependent manner (Figure6 and Figure S3A). We also evaluated the inhibitory effect
of 49 on ectopically expressed PRMT4 in HEK293T cells. After treatment with
compound 49, the asymmetric dimethylation of BAF155, PABP1 and the level of
aDMA in HEK293T cells with high expression of wild type PRMT4 (Figure S3B) were
also decreased in a dose-dependent manner (Figure S3C), which was in line with the
results in MOLM13 cells. These data together confirmed that 49 could inhibit the
catalysis function of PRMT4 and resulted in reduction of asymmetric dimethylation of
total aDMA, and two well-known substrate proteins BAF155 and PABP1 at cellular
levels.
Figure 6. MOLM13 cells were treated with indicated concentration of compound 49 for 4 days and
whole cell extracts were analyzed by western blotting for PABP1, BAF155 dimethylation.
According to the grey scale of Image J, PABP1me2a and BAF155me2a signal intensities
ACS Paragon Plus Environment

Page 21 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
normalized to total PABP1 and total BAF155, respectively. IC values were calculated in GraphPad
5
0
Prism.
Although the roughly 10-fold difference between the antiproliferative activity and the
cellular methylation inhibition is consistent with previous reported PRMT4 inhibitors,
the slightly large gap between enzymatic activity and cellular methylation inhibition of
49 may stem from other factors rather than target relevant.35, 36 By investigating the
reported PRMT4 inhibitors, such as EZM2302 and TP-064, we found that different cell
lines could affect the methylation inhibition activity and different molecules also harbor
different physicochemical characteristics leading various cellular effects. Therefore, we
thought that these two factors may account for the slightly large discrepancy between
enzymatic activity and cellular methylation inhibition of 49. Nevertheless, giving the
similar antiproliferative activity of compound 49 with those of other PRMT4 inhibitors,
we further evaluate its in vitro and vivo properties.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 49 Induced G1 Cell Cycle Arrest and Apoptosis in MOLM13
To investigate the mechanism of antiproliferation in MOLM13 cells, we performed cell
cycle analyses and apoptosis analyses by flow cytometry. Results showed that
compound 49 decreased the proportion of MOLM13 cells in S phase, while increased
the percentage of cells in G /G phase in a dose-dependent manner (Figure 7A). As
0
1
shown in Figure 7B, compound 49 also induced cell apoptosis in a concentration-
dependent manner. Once MOLM13 cells were treated with compound 49 at 12 μM, the
proportion of total apoptosis including the early stage apoptosis and the later stage of
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
apoptosis was 96.4%. These data indicated that the antiproliferative effect of compound
4
9 was dependent on arresting cell cycle and inducing apoptosis.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Cellular responses of MOLM13 cells treated with 49. (A) Cell cycle of MOLM13 cells
was arrested at G1 phase, after treatment with 49 from 3 to 12 μM for 72 h . (B) MOLM13 was
induced apoptosis, after treatment with 49 from 3 to 12 μM for 96 h.
In vivo Pharmacokinetic Properties and Anti-tumor Effect in Xenografts
As compound 49 showed high potency in vitro, the pharmacokinetic (PK) profiles were
next evaluated in ICR mice by intravenous and oral administration. A single dose of
compound 49 at 3 mg/kg i.v. administration showed high volume of distribution at
steady state (Vss) of 42.6 L/kg and moderate clearance (CL) of 44.9 mL/min/kg (the
curve of drug concentrations in plasma was shown in Figure S2A). Besides, a slow oral
absorption (Tmax = 8.0 h), long half-life (t1/2 = 24.1 h) and good oral bioavailability (F
=
48.8%) at an oral dose of 10 mg/kg were observed. From detailed analysis of the
plasma concentration at various time points, we could find that the inhibitor could retain
about 120 nM concentration at most of time in the plasma after a single oral dosage of
10 mg/kg. We also determined the plasma protein binding of 49 (PPB, about 98%,
Table S4), together with the high volume of distribution at steady state, indicating that
ACS Paragon Plus Environment

Page 23 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compound 49 had a capability to dissociate from plasma proteins and distributed into
the tissue. By comparing the cellular activities, we presumed that a higher dosage is
needed to offer sufficient drug concentration to exhibit antitumor efficacy. Therefore,
we further studied the in vivo activity of compound 49.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 9. Pharmacokinetic parameters for compound 49 in ICR (CD-1) mice a
Parameter
3 mg/kg i.v.
44.9
10 mg/kg p.o.
CL(mL/min/kg)
V (L/kg)
ss
42.6
t (h)
12.1
24.1
8.0
1
/2
Tmax(h)
Cmax(ng/mL)
AUC0-last(ng.h/mL)
AUC0-inf(ng.h/mL)
F (%)
75.4
1412
3032
48.8
869
1165
a
Mice (n = 3); Parameters were calculated from composite mean plasma concentration–time data.
Compound dissolved in 1% Tween80/water.
To further evaluate the antitumor effect of compound 49, we performed tumor growth
inhibition studies in BALB/c nude mice bearing subcutaneous MOLM13 xenografts
with daily oral dose of 100 mg/kg (Figure 8A-D). As demonstrated in Table 10,
compound 49 showed potent therapeutic effect with tumor growth inhibition (TGI) rate
of 53.5%. After sacrificing the xenograft mice, we analysized the intratumoral level of
aDMA, BAF155me2a, and PABP1me2a, and the result showed a remarkably decrease
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of asymmetrical dimethylation (Figure 8E), which was consistent with cellular results.
We also tested the drug concentration in tumor tissues collected from in vivo
pharmacological study. The result showed that the average of drug concentration in
tumor tissues was about 80724 ng/g (Table S3). As compound 49 inhibited the
proliferation of MOLM13 with an IC of 6.93 μM (equal to 2835 ng/mL), it meant the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
drug concentration in tumor tissues was about 20-30 folds higher than IC of the
5
0
antiproliferative activities, which may explain the fact that compound 49 displayed the
good anti-tumor activity in xenograft model.
Table 10. Summary of tumor growth inhibition of compound 49
Administration
Tumor
model
Survivors
(day)
Tumor growth
inhibition (%)
Compd
Dose
mg/kg)
100
Route
po
(
MOLM13
49
18
53.5(p<0.01)
Figure 8. (A-E) Antitumor efficacy of 49 in vivo. Mice carrying MOLM13 xenograft model were
treated with 49 in a dose of 100 mg/kg daily for indicated days. Error bars represent mean ± SD, n
ACS Paragon Plus Environment

Page 25 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
≥5 mice per group for (A) and (C). (A) and (C) were analyzed by unpaired two tailed t test. **p <
0
.01, ***p < 0.001. (B) Mean body weight of MOLM13 xengraft mice under the administration of
compound 49 (data shown as mean values, n=5 mice per group). (D) Visual pictures showing
representative tumor volumes of control and treatment group of MOLM13 model. (E) the
asymmetric dimethylation of BAF155, PABP1 and the level of aDMA were detected by Western
Blot. Beta-actin was used as loading controls.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Chemistry
Scheme 1. Synthesis of compounds 7-16 a
n1
n1
a,b
OH
N Boc
7a, 7 (n = 0, n = 1, R = H)
1 2
8a, 8 (n1 = 1, n2 = 1, R = H)
O
R
H
R
+
N
n2
N
NH
n2
9
a, 9 (n = 1, n = 2, R = H)
1 2
1
0a, 10 (n = 2, n = 1, R = H)
1
2
7
a-10a, 12a-16a
50
7-10, 12-16
1
1
1
2a, 12 (n = 1, n = 1, R = 5-Br)
1 2
3a, 13 (n = 1, n = 1, R = 6-Br)
1
2
O
4a, 14 (n = 1, n = 1, R = 7-Br)
O
1
2
a,b
N Boc
O
15a, 15 (n = 1, n = 1, R = 8-Br)
1 2
+
OH
N
16a, 16 (n1 = 2, n2 = 1, R = 5-Br)
NH
H
N
11a
50
11
a
Reagents and conditions: (a) i-PrOH, 80 °C; (b) MeOH/HCl.
The preparation of compounds 7-16, as depicted in Scheme 1, began with a nucleophilic
substitution reaction between different commercially available cyclic secondary amine
(7a-16a) and tert-butyl methyl(oxiran-2-ylmethyl) carbamate (50), followed by a
deprotection reaction.
Scheme 2. Synthesis of compounds 17-19 and 22-26 a
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Br
Br
Br
b
N Boc
a
O
OH
OTBS
+
N
N
N
N
NH
Boc
Boc
1
2a
50
51
52
Ar Linker
Ar Linker
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
d
OTBS
OH
H
N
N
N
N
Boc
17a-19a, 22a-26a
17-19, 22-26
Ar
Linker
H
O
N
HN
O
NH
HN
1
7a, 17
18a, 18
19a, 19
22a, 22
23a, 23
HN
O
S
HN
HN
O
HN
O
O
2
4a, 24
25a, 25
26a, 26
a
Reagents and conditions: (a) i-PrOH, 80 °C; (b) TBSCl, imidazole, DCM; (c) Coupling reaction
or carbonyl extrusion condensation reaction; (d) MeOH/HCl.
Synthetic routes for preparing tetrahydroisoquinoline derivatives 17-19 and 22-26 with
various linkers are outlined in Scheme 2. Intermediate 52 was prepared via a two-step
sequence of reactions, involving a nucleophilic substitution reaction and addition of
TBS protecting group. The resulting intermediate 52 was then subjected palladium or
copper catalyzed coupling reaction or carbonyl extrusion condensation reaction and
subsequent deprotection reaction to afford the corresponding end-product.
Scheme 3. Synthesis of phenyl ether derivatives 20 and 27-37 a
ACS Paragon Plus Environment

Page 27 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
R
R
R
5
5
O
Br
6
O 5
b
c
O
5
6
a
6
6
O
F
OH
N
N
H
N
Boc
Boc
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
3-a (5-Br)
53-b (6-Br)
20a, 27a-37a
2
0b, 27b-37b
20, 27- 37
F
5
-
5-
5-
5-
5-
F
Cl
O
O
O
O
O
2
0a, 20b, 20
27a, 27b, 27
28a, 28b, 28
5-
29a, 29b, 29
30a, 30b, 30
S
CN 5-
F
5-
5-
5
-
N
O
O
NH2
O
Cl
O
O
3
1a, 31b, 31
32a, 32b, 32
33a, 33b, 33
34a, 34b, 34
35a, 35b, 35
6-
5
-
N
O
O
S
36a, 36b, 36
37a, 37b, 37
a
Reagents and conditions: (a) CuI, 2-picolinic acid, K PO , phenol, DMSO, 90 °C; (b) MeOH/HCl
3
4
and then THF, epibromohydrin, KF, 45 °C; (c) MeOH/MeNH2.
The synthesis of phenyl ether derivatives 20 and 27-37 is summarized in Scheme 3. A
copper catalyzed Ullmann reaction between aryl bromide (53a or 53b) and phenol
afforded the intermediate 20a and 27a-37a. After removing the Boc protecting group
and a nucleophilic substitution reaction, the intermediate 20b and 27b-37b were
obtained. Sequential ethylene oxide ring opening reaction under the attack of
methylamine produced the compounds 20 and 27-37.
Scheme 4. Synthesis of benzyl ether derivatives 21, 38-44 and 47 a
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
R
R
N
R
N
5
HO
6
5
5
O
O
5
O
a
6
b
c
6
OH
6
O
H
N
N
N
Boc
Boc
5
5
4a (5-OH)
4b (6-OH)
21a, 38a-44a, 47a
21b, 38b-44b, 47b
21, 38-44, 47
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
-
F
5-
5-
5-
5-
F
O
O
O
O
O
F
Cl
38a, 38b, 38
3
9a, 39b, 39
40a, 40b, 40
6-
41a, 41b, 41
2
1a, 21b, 21
5
-
5
-
5-
O
O
O
O
CN
42a, 42b, 42
43a, 43b, 43
4
4a, 44b, 44
47a, 47b, 47
a
Reagents and conditions: (a) K CO , benzyl bromide derivatives, acetonitrile; (b) MeOH/HCl and
2
3
then THF, epibromohydrin, KF, 45 °C; (c) MeOH/MeNH2.
Besides, the benzyl ether derivatives 21, 38-44 and 47 were synthesized in a similar
route to phenyl ether derivatives starting from a nucleophilic substitution reaction
between Boc-protected 1,2,3,4-tetra-hydroisoquinolin-5-ol (54a) or 1,2,3,4-tetra-
hydroisoquinolin-6-ol (54b) and the corresponding benzyl bromide derivatives.
Scheme 5. Synthesis of compound 45,46, 48 and 49 a
4
4
4
3
B
O
O
N
S
3
S
N
3
OH
a
b
c
O
O
O
N
Boc
4
O
N
N
54a
N
Boc
Boc
*
5
5
5a (4-boronic acid ester)
5b (3-boronic acid ester)
56a (4-thiazoly)
56b (3-thiazoly)
45a, 48a, 49a (from 56a)
46a (from 56b)
3
S
N
d
O
OH
*
45a, 45 (rac, 4-thiazolyl)
6a, 46 (rac, 3-thiazolyl)
48a, 48 (S, 4-thiazolyl)
49a, 49 (R, 4-thiazolyl)
H
4
N
N
45, 46, 48, 49
ACS Paragon Plus Environment

Page 29 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Reagents and conditions: (a) K CO , benzyl bromide derivatives, acetonitrile；(b) Ph P, Pd(Ph P) ,
2
3
3
3
4
K PO , 2-bromothiazole, 1,4-dioxane/H O, 90 °C; (c) MeOH/HCl and then THF, epibromohydrin
3
4
2
or S-glycidylnosylate or R-glycidylnosylate, KF, 45 °C; (d) MeOH/MeNH2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In consideration of the commercially unavailable of thiazolyl substituted benzyl
bromide, compound 45, 46, 48 and 49 were synthesized in a slightly altered route. Boc-
protected 1,2,3,4-tetrahydroisoquinolin-5-ol (54a) and boronic acid pinacol ester
substituted benzyl bromide underwent a nucleophilic substitution reaction, followed by
a Suzuki-coupling reaction with 2-bromothiazole to afford intermediate 56a or 56b.
Then 56a or 56b subjected a deprotected reaction and another nucleophilic substitution
reaction with epibromohydrin or S-glycidylnosylate or R-glycidylnosylate to yield the
corresponding racemic (45a and 46a) or enantiomeric (48a and 49a) intermediate.
Methylamine facilitated the SN ring opening reaction to produce the end-product with
2
chiral inversion.
CONCLUSIONS
Starting from a reported dual PRMT4/6 inhibitor 6, we discovered a selective and in
vivo effective PRMT4 inhibitor 49 through scaffold hopping strategy and subsequent
structure-based optimization. Compound 49 exhibited high potency against PRMT4
(
IC = 21 nM) and excellent selectivity over other PRMTs and PKMTs (>100-fold).
50
Compound 49 could induce anti-proliferative effect on a panel of leukemia cancer cell
lines by inducing cell cycle arrest at G1 phase and apoptosis. Oral administration of
compound 49 demonstrated good pharmacokinetic profiles and significant anti-tumor
activity in acute myeloid leukemia MOLM13 xenograft model, without the obvious
ACS Paragon Plus Environment