Discovery of Potent and Selective Allosteric Inhibitors of Protein Arginine Methyltransferase 3 (PRMT3)

Article abstract of DOI:10.1021/acs.jmedchem.7b01674

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is crucial for maturation of ribosomes and has been implicated in several diseases. We recently disclosed a highly potent, selective, and cell-active allosteric inhibitor of PRMT3, compound 4. Here, we report comprehensive structure-activity relationship studies that target the allosteric binding site of PRMT3. We conducted design, synthesis, and evaluation of novel compounds in biochemical, selectivity, and cellular assays that culminated in the discovery of 4 and other highly potent (IC₅₀ values: ～10-36 nM), selective, and cell-active allosteric inhibitors of PRMT3 (compounds 29, 30, 36, and 37). In addition, we generated compounds that are very close analogs of these potent inhibitors but displayed drastically reduced potency as negative controls (compounds 49-51). These inhibitors and negative controls are valuable chemical tools for the biomedical community to further investigate biological functions and disease associations of PRMT3.

Full text of DOI:10.1021/acs.jmedchem.7b01674

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Article
Discovery of Potent and Selective Allosteric Inhibitors
of Protein Arginine Methyltransferase 3 (PRMT3).
H. Ümit Kaniskan, Mohammad S. Eram, Kehao Zhao, Magdalena M Szewczyk, Xiaobao Yang, Keith
Schmidt, Xiao Luo, Sean Xiao, Miao Dai, Feng He, Irene Zang, Ying Lin, Fengling Li, Elena Dobrovetsky,
David Smil, Sun-Joon Min, Jennifer Lin-Jones, Matthieu Schapira, Peter Atadja, En Li, Dalia Barsyte-
Lovejoy, Cheryl H. Arrowsmith, Peter J. Brown, Feng Liu, Zhengtian Yu, Masoud Vedadi, and Jian Jin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01674 • Publication Date (Web): 15 Dec 2017
Downloaded from http://pubs.acs.org on December 16, 2017
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Discovery of Potent and Selective Allosteric
Inhibitors of Protein Arginine Methyltransferase 3
(PRMT3).
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2,
H. Ümit Kaniskan,1,‡ Mohammad S. Eram, Kehao Zhao,3 Magdalena M. Szewczyk,2 Xiaobao
Yang,1,4 Keith Schmidt,1 Xiao Luo,3 Sean Xiao,3 Miao Dai,3 Feng He,3 Irene Zang,3 Ying Lin,3
Fengling Li,2 Elena Dobrovetsky,2 David Smil,2 Sun-Joon Min,1,5 Jennifer Lin-Jones,6 Matthieu
Schapira,2,7 Peter Atadja,3 En Li,3 Dalia Barsyte-Lovejoy,2 Cheryl H. Arrowsmith,2,8 Peter J.
Brown,2 Feng Liu,*,9 Zhengtian Yu,*,3 Masoud Vedadi,*,2,7 and Jian Jin,*,1
‡
1Center for Chemical Biology and Drug Discovery, Departments of Pharmacological Sciences
and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai,
New York, NY 10029, USA. 2Structural Genomics Consortium, University of Toronto, Toronto,
3
ON M5G 1L7, Canada. Novartis Institutes for Biomedical Research (China), Zhangjiang Hi-
Tech Park, Pudong New Area, Shanghai 201203, China. 4Current address: Shanghai Institute for
Advanced Immunochemical Studies, Shanghai Tech University, Pudong District, Shanghai
5
201210, China. Current address: Deptartment of Chemical & Molecular Engineering/Applied
Chemistry, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu Ansan, Gyeonggi-do,
6
7
15588, South Korea. DiscoveRx Corporation, Fremont, CA 94538, USA. Department of
Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada.
8Department of Medical Biophysics, University of Toronto and Princess Margaret Cancer
Centre, 101 College St., MaRS South Tower, Suite 707, Toronto, ON, M5G 1L7, Canada.
9Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and
Department of Medicinal Chemistry, College of Pharmaceutical Sciences, Soochow University,
Suzhou, Jiangsu 215123, China.
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ABSTRACT
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PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins.
It is crucial for maturation of ribosomes, and has been implicated in several diseases. We
recently disclosed a highly potent, selective and cell-active allosteric inhibitor of PRMT3,
compound 4. Here, we report comprehensive structure−activity relationship studies that target
the allosteric binding site of PRMT3. We conducted design, synthesis and evaluation of novel
compounds in biochemical, selectivity and cellular assays that culminated in the discovery of 4
and other highly potent (IC50 values: ~10-36 nM), selective and cell-active allosteric inhibitors of
PRMT3 (compounds 29, 30, 36 and 37). In addition, we generated compounds that are very
close analogs of these potent inhibitors, but displayed drastically reduced potency as negative
controls (compounds 49-51). These inhibitors and negative controls are valuable chemical tools
for the biomedical community to further investigate biological functions and disease associations
of PRMT3.
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INTRODUCTION
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Protein arginine methyltransferase 3 (PRMT3) is a type I PRMT that catalyzes mono-
and asymmetric dimethylation of arginine residues.1 Ribosomal protein S2 (rpS2) was identified
as the major substrate of PRMT3 via its interaction with PRMT3 zinc finger domain in
mammalian cells.2, 3 PRMT3 plays a role in ribosome biosynthesis. However, the molecular
mechanism by which PRMT3 influences ribosomal biosynthesis remains unclear.4 Very recently,
an extraribosomal complex comprising of PRMT3, rpS2 and human programmed cell-death 2-
like (PDCD2L) protein was identified.5 While PRMT3 is localized exclusively in the cytoplasm,6
it has been shown that in cells treated with palmitic acid or T0901317 (a liver X receptor α
(LXRα) agonist), PRMT3 co-localizes with LXRα in the cell nucleus, regulating hepatic
lipogenesis.7 However, this effect appears to be independent of the PRMT3 methyltransferase
activity. While rpS2 is the primary substrate of PRMT3, it is not the sole substrate. PRMT3
along with PRMT1 methylates the recombinant mammalian nuclear poly(A)-binding protein
(PABPN1) and has been implicated in oculopharyngeal muscular dystrophy, which is caused by
polyalanine expansion in PABPN1.8, 9 A protein complex comprised of the von Hippel–Lindau
(VHL) tumor suppressor protein, PRMT3 and ARF (alternative reading frame) methylates p53.10
Importantly, the tumor suppressor DAL-1 (differentially expressed in adenocarcinoma of the
lung, also known as 4.1B) interacts with PRMT3 and consequently inhibits its methyltransferase
activity, suggesting a possible role of PRMT3 regulation in tumor growth.11 The interaction
between DAL-1 and PRMT3 in the induction of apoptosis in MCF-7 cells suggests that this
interaction is likely to be an important modulator of the apoptotic pathway and can be critical to
controlling tumorigenesis in breast cancer cells.12 It has also been shown that PRMT3 methylates
a histone peptide (H4 1–24) in vitro.13 Modified histone H4R3 is associated with increased
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transcription of a number of genes, including those under control of estrogen and androgen
receptors.14-16 Furthermore, PRMT3 expression levels are elevated in myocardial tissues from
patients with atherosclerosis, potentially implicating the involvement of PRMT3.17 Additionally,
PRMT3 function has been reported to be essential for dendritic spine maturation in rats.18 A
recent study suggests that PRMT3 mediates the preventive effects of irisin against lipogenesis
and oxidative stress.19
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Our group embarked on research efforts to discover potent, selective and cell-active
inhibitors of PRMT3 as chemical tools for better understanding of biology and function of this
understudied protein methyltransferase. We previously reported the discovery of a novel
allosteric binding site of PRMT3 and the first selective, allosteric inhibitors of PRMT3
(compounds 1-3, Figure 1) and subsequently disclosed the discovery of SGC707 (4) (Figure 1), a
highly potent, selective and cell-active allosteric inhibitor of PRMT3.20-22 Here, we describe
design and synthesis of a large set of novel analogs and evaluation of these compounds in
biochemical, selectivity and cellular assays. This comprehensive structure-activity relationship
(SAR) study resulted in the identification of multiple highly potent, selective and cell-active
allosteric inhibitors of PRMT3 including compound 4.
RESULTS AND DISCUSSION
Our earlier efforts resulted in the identification of selective small-molecule inhibitors of
PRMT3 (Compounds 2, 3) starting from a hit, compound (1) (Figure 1).20, X-ray crystal
21
structures of 1 and 2 in complex with PRMT3 were obtained and showed that these inhibitors
occupied a novel allosteric binding site (PDB ID: 3SMQ and 4HSG). These co-crystal structures
revealed that the left-hand side (LHS) bicyclic benzothiadiazole moiety fits tightly in the
allosteric pocket and the middle nitrogen atom forms a hydrogen bond with T466 (Figure 2). The
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urea linker is located at the entrance of the cavity, and forms hydrogen bonds with the guanidine
of R396 and the carboxylate of E422 (Figure 2). In addition, the right-hand side (RHS) moiety
extends out of the allosteric binding pocket and makes hydrophobic interactions with a surface
composed of the side chains from two different subunits of the PRMT3 homodimer. Both
structural and SAR data clearly indicate that the middle urea region is essential for the affinity
and replacement of this group with its bioisosteres and more rigid analogs did not yield any
inhibitors with improved potency.21 Therefore, in the current study the middle urea region of this
scaffold was kept unmodified. On the other hand, further optimization of the RHS moiety led to
the discovery of 3 (Figure 1).21 Herein, we further optimized both the LHS and RHS moieties of
this scaffold to achieve improved potency for inhibiting PRMT3. First, we conducted a scaffold
hopping exercise by using the benzothiadiazole ring of 3 as a query for the allosteric pocket. A
hydrogen-bond constraint was imposed in this scaffold hopping study to preserve the important
hydrogen-bond interaction between the inhibitors and the hydroxyl group of T466. As a result of
this exercise, isoquinoline (compound 5), isobenzofuran-1-one (compound 6), and quinazoline
(compound 7) groups were selected for experimental validation (Table 1).
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RHS
Region
LHS
Bicyclic Aromatic Region
5
6
Initial hit
N
S
N
S
O
O
7
8
9
N
N
N
N
N
N
H
H
H
H
O
1 (ref. 20)
IC50 = 2.5 µM
2 (ref. 21)
IC50 = 0.48 µM
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Middle Urea
Region
structure-guided
design and
synthesis
N
S
N
O
O
N
Highly potent
N
O
N
N
N
N
N
PRMT3
inhibitors
H
H
H
H
O
3 (ref. 21)
IC50 = 0.134 µM
4 (ref. 22)
IC50 = 0.019 µM
Figure 1. Design and synthesis of highly potent inhibitors of PRMT3.
Figure 2. Reported co-crystal structure of compound 1 in complex with PRMT3 indicating key
hydrogen bonding interactions (magenta dotted lines) (PDB ID: 3SMQ).20
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The RHS of compound 3, piperidineamide, was kept exactly the same in these newly synthesized
analogs to accurately compare the effect of the different LHS bicyclic heteroaromatic rings on
potency. The isoquinoline containing analog (compound 5) with IC50 of 84 ± 5 nM, showed a small
improvement over the parent compound, 3 (IC50 = 134 ± 5 nM). On the other hand, compounds 6 and
7 were around 6-fold less potent as compared to 3 (Table 1). Therefore, the isoquinoline bicyclic ring
system was taken forward as the LHS moiety for further optimization. Compound 8 (Table 1) was
also prepared to confirm the importance of the positioning and hydrogen bonding of the isoquinoline
nitrogen with T466. This bicyclic ring is still an isoquinoline but substituted at 7-position instead of
6-position (isoquinoline numbering) in effect walking the nitrogen to the adjacent position of
compound 5. As expected, this modification resulted in ablation of inhibitory activity.
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Table 1. Inhibitors with different LHS bicyclic ring systems.
5
6
7
8
9
O
R
N
N
N
H
H
O
10
11
12
13
14
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Compound
Structure (R)
IC50 (nM)
N
N
S
134 ± 5
3
5
6
7
8
N
84 ± 5
757 ± 1
967 ± 115
>2000
6
O
O
N
N
6
N
7
As the 6-substituted isoquinoline is an optimal LHS bicycle, we then turned our attention
to optimizing the RHS moiety of the scaffold. We first revisited saturated aliphatic groups as the
RHS functionality inspired from our initial studies (Table 2).20,
Compound 9, a
21
cyclohexenylethyl group containing analog, corresponding to the RHS region of the original hit
(compound 1) was 5-fold less potent than 5, displaying IC50 of 421 ± 29 nM. As a logical
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extension of this compound, we also synthesized the fully saturated, cyclohexylethyl analog (10)
as well as derivatives containing different ring sizes. Compound 10 (IC50 = 540 ± 54nM) was
very similar to 9 in potency. The cyclopentane-bearing analog (11) showed slight improvement
in potency with IC50 of 295 ± 43 nM while the cyclopropane-containing analog 12 was virtually
inactive (IC50 > 8,000 nM). These results indicated that hydrophobicity of the RHS moiety plays
an important role in potency, and cyclohexyl and cylopentyl rings are preferred compared to the
cyclopropyl group. Replacing the cyclohexyl group with a phenyl ring (compound 13) resulted in
a decreased potency (IC50 = 833 ± 66 nM), underlying the importance of changes to the
hydrophobicity of the RHS moiety. On the basis of these results, compound 10 was further
investigated to improve the potency. A docking study with this compound hinted that a
substituent at the C1 position of the cyclohexyl group would be a favorable position from which
E422 might be possibly reached for further interactions. Therefore, compound 14, featuring a
methyleneamine substituted cyclohexyl group was synthesized. Interestingly, compound 14
displayed a modest improvement in potency with IC50 of 291 ± 36 nM, compared to compound
10. These compounds were synthesized following the previously published synthetic route.21
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Table 2. Inhibitors with saturated aliphatic groups as the RHS moiety.
5
6
7
N
O
8
9
N
N
R
H
H
10
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12
13
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Compound
Structure (R)
IC50 (nM)
421 ± 29
9
10
540 ± 54
11
12
13
295 ± 43
>8000
833 ± 66
14
291 ± 36
NH2
Since no significant improvement in potency was achieved with compounds 9-14 featuring
aliphatic groups as the RHS moiety, we decided to keep the amide functionality as in compound
5 and further investigate the substituents on the nitrogen atom (Table 3) as the amide group could
form direct or water-mediated contacts with the side chain of K392.21 We first synthesized new
amide derivatives of compound 5, by replacing the 6-membered piperidine ring with the 4-
membered azetidine ring (compound 15), 5-membered pyrrolidine ring (compound 4) and 7-
membered azepane ring (compound 16). While the azetidine amide analog 15 displayed only a
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slight improvement in potency (IC50 = 61 ± 8 nM), the pyrrolidine amide 4 (IC50 = 19 ± 1 nM)
and azepane amide 16 (IC50 = 17 ± 2 nM) were ~5-fold more potent than 5 in inhibiting PRMT3.
The 6-membered ring analogs of 5 such as 4,4-difluoro piperidine amide 17 (IC50 = 35 ± 1 nM)
showed a more than 2-fold improvement over the unsubstituted piperidine amide 5. However,
exchanging the piperidine ring with the 4-methylpiperizine ring (compound 18), which alters the
electronic nature and polarity of the ring system, resulted in the loss of the inhibitory activity.
We also designed and synthesized non-cyclic di- and mono-substituted amide derivatives
(compounds 19-26). The dimethyl and diethyl amide derivatives 19 and 20, displayed reduced
potency with IC50 of 150 ± 11 nM and 114 ± 3 nM, respectively. The N-cyclopentyl, N-methyl
amide 21 (IC50 = 87 ± 5 nM) was as potent as compound 5. The Weinreb amide derivative 22, on
the other hand, was around 6-fold less potent than compound 5. The mono-substituted amides
were also investigated (Compounds 23-26). While N-methyl amide 23 displayed significantly
weaker inhibitory effect, the cyclopropyl (24), cyclopentyl (25) and cyclohexyl (26) amide
derivatives showed reduced potency as the ring size increased. Taken together, these results
suggest that cyclic amides are preferred RHS moieties, which possess balanced steric and
hydrophobic properties interacting with PRMT3. We, therefore, designated the pyrrolidine amide
as the RHS moiety for the rest of the SAR study.
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The compounds in Table 3 were synthesized via the general route shown in Scheme 1.
The synthesis started with reacting commercially available 6-aminoisoquinoline and ethyl
isocyanatoacetate to obtain the desired ethyl ester 27. The hydrolysis of the ethyl ester 27
resulted in the carboxylic acid 28, which was used as the key intermediate to perform amide
coupling reactions with various amines to yield the desired amide analogs (4, 5 and 15-26).
Detailed reaction conditions, yields and characterization data for final compounds are reported in
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the experimental section.
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Table 3. Inhibitors containing different RHS amide moieties.
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9
10
11
12
13
14
15
16
17
18
19
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22
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N
O
N
N
R
H
H
Compound Structure (R)
IC50 (nM)
Compound
20
Structure (R) IC50 (nM)
Et
N
N
5
84 ± 5
61 ± 8
19 ± 1
114 ± 3
Et
O
O
Me
87 ± 5
N
N
15
21
22
O
O
Me
476 ± 34
N
N
4
OMe
O
O
H
N
N
16
17
17 ± 2
35 ± 1
23
24
1938 ± 95
158 ± 9
Me
O
O
F
H
N
F
N
O
O
O
O
H
N
NMe
N
18
19
>1900
25
26
292 ± 23
477 ± 5
O
H
N
Me
N
150 ± 11
Me
O
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O
N
N
O
RT, CH2Cl2:DMF
OEt
C
N
OEt
NH2
N
H
N
5
6
7
8
O
H
O
27
LiOH,
THF:MeOH
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59
60
NHR1R2,
R1
N
O
N
O
N
OH
amide
coupling
R2
N
H
N
N
H
N
H
H
O
O
4, 5, 15-26
28
Scheme 1. General synthetic route for the preparation of compounds listed in Table 3 and 4.
Next, we focused our attention on analogs of compound 4 (Table 4), containing
substituted pyrrolidine (compounds 29-31), fused bicyclic (compounds 32 and 33) and bridged
bicyclic moieties (compounds 34 and 35). These compounds were again prepared according to
the synthetic route outlined in Scheme 1. The 3,3,4,4-tetrafluoro pyrrolidine derivative
(compound 29) as well as 2, 5-dimethylpyrrolidine (a mixture of cis and trans isomers)
(compound 30) analogs showed very similar potency compared to compound 4. On the other
hand, replacing pyrrolidine with D-proline (compound 31) resulted in around 9-fold drop in
potency. The fused 5,5-bicyclic ring system (compound 32) was also tolerated albeit with
reduced potency (IC50 = 55 ± 5 nM). Interestingly, the benzene-fused pyrrolidine ring
(isoindoline amide, compound 33) led to a complete loss of the inhibitory effect. Finally, 7-
azabicyclo[2.2.1]heptane (compound 34) and 8-azabicyclo[3.2.1]octane (compound 35), two
bridged bicyclic pyrrolidine containing moieties, did not result in any improvement of potency
compared to compound 4, with IC50 of 46 ± 5 nM and 22 ± 4 nM, respectively. These results
indicate that small substituents such as fluoro and methyl groups on the pyrrolidine ring as well
as relatively flexible hydrophobic bicyclic ring systems are tolerated.
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Table 4. Inhibitors with modified pyrrolidine amide moieties.
5
6
7
N
O
8
9
N
N
R
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
IC50
(nM)
IC50
(nM)
Compound Structure (R)
Compound
32
Structure (R)
55 ± 5
>5000
N
4
19 ± 1
24 ± 3
N
O
O
F
F
F
N
O
29
33
N
F
O
Me
N
O
30
31
22 ± 2
34
35
46 ± 5
22 ± 4
N
Me
O
N
O
N
166 ± 11
CO2Me
O
The results summarized in Table 4 and discussed above have shown that analogs of 4,
namely compounds 29, 30, 32, 34 and 35, potently inhibited PRMT3 with IC50 values of around
20-50 nM. Although these analogs did not display improved potency compared to compound 4,
these substituted pyrrolidine groups are valuable alternatives to the unsubstituted pyrrolidine
group (compound 4). After completing optimization of the RHS moiety, we further investigated
the LHS isoquinoline ring. Analysis of the crystal structure of PRMT3 in complex with
compound 4 (PDB ID: 4RYL)22 suggested that there is room in the binding pocket to tolerate a
relatively small substituent at 1, 3, 7 and 8 position of the isoquinoline ring system. Our
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structural analysis also suggested that a substituent at 4 and 5 positions of the isoquinoline ring
would not be tolerated. Therefore, we designed and synthesized the corresponding substituted
isoquinoline analogs (compounds 36-40) to determine whether potency could further be
enhanced (Table 5). For example, compounds 36 and 37 featuring small 7-fluoro and 7-methyl
substituents were prepared. Compound 36 showed similar potency as 4 while 37, which has a
slightly larger methyl substituent, was around 2-fold less potent. Compound 38, on the other
hand, displayed significant potency loss (about 10-fold), indicating that the methyl group at the
1-position of the isoquinoline ring is not preferred. Interestingly, the 3-fluoro substituted analog
39 displayed almost 2-fold higher potency with IC50 of 10 ± 1 nM. This result suggests that the
electronic modulation of the isoquinoline ring by a fluoro group does not have significant impact
on the hydrogen bonding ability of the isoquinoline with T466 and a small substituent such as the
fluoro group at the 3-position enhances potency, consistent with our structural analysis. In
addition, we synthesized the 8-chloroisoquinoline derivative 40, which exhibited similar potency
as compound 4. While the result obtained for compound 39 was consistent with our predication
based on the analysis of the crystal structure of the PRMT3 – compound 4 complex (PDB ID:
4RYL), it was surprising that a small substituent such as the fluoro group at the 7-position
(compound 36) or the chloro group at the 8-position (compound 40) did not increase potency and
a slightly larger substituent such as the methyl group at 1 and 7 positions (compounds 37 and 38)
reduced potency. More comprehensive structural analyses such as molecular dynamics
simulation are needed to explain the SAR results. Nevertheless, the fluoro or chloro substituent
could potentially improve metabolic stability of these compounds (36, 39 and 40), which are
interesting alternative PRMT3 inhibitors to compound 4. Overall, these results have
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demonstrated that a small substituent at the isoquinoline ring can be tolerated, but has limited
impact on enhancing potency.
7
8
9
Table 5. Inhibitors with substituted isoquinolines.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
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45
46
47
48
49
50
51
52
53
54
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59
60
O
R
N
N
N
H
H
O
Compound
4
Structure (R)
IC50 (nM)
1
8
7
19 ± 1
N
3
F
N
36
37
19 ± 4
36 ± 6
Me
N
Me
N
38
39
40
194 ± 8
10 ± 1
19 ± 2
N
F
Cl
N
The substituted 6-amino isoquinoline derivatives used for the synthesis of compounds 36-
40 (Table 5) were not commercially available. Therefore, we devised synthetic routes and
prepared these substituted 6-amino isoquinolines as shown in Scheme 2. The synthesis of 6-
amino-7-fluoroisoquinoline (43), 6-amino-7-methylisoquinoline (44) and 6-amino-1-
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methylisoquinoline (45) started with reductive amination reactions of amino acetaldehyde
dimethyl acetal with the corresponding 4-bromo aryl aldehyde or methyl aryl ketone to give
amino dimethyl acetals 41 (Scheme 2A). The intermediates 41 were converted to the
sulfonamides 42, via tosylation, which were then treated with aluminum chloride to yield the
desired 6-bromoisoquinolines (43). These substituted 6-bromoisoquinolines were then converted
to the desired 6-aminoisoquinolines (44-46) via aryl amination reactions (Scheme 2A). The 3-
fluoro-6-aminoisoquinoline (47) was synthesized starting from the commercially available 3-
amino-6-bromoisoquinoline in two steps via the Balz-Schiemann reaction23, 24 followed by an
aryl amination (Scheme 2B). As shown in Scheme 2C, 6-amino-8-chloroisoquinoline (48) was
prepared in six steps. The commercially available 5-aminoisoquinoline was first acetylated and
then chlorinated to install a chloro group at the 8-position. Bromination at the 6-position was
achieved by using dibromoisocyanuric acid. Deacetylation followed by reductive diazotization
resulted in 6-amino-8-chloroisoquinoline (48) (Scheme 2C). Intermediates 44 – 48 were then
used to prepare compounds 36 – 40 according to the synthetic route outlined in Scheme 1.
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R2
A
O
H
R1
Br
NH2
1. Toluene, reflux
with Dean-Stark trap
N
R1
Br
MeO
R2
MeO
+
OMe
2. NaBH4, EtOH, RT
41
OMe
TsCl, Et3N,
DMAP,
CH2Cl2, RT
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CuI, NH4OH,
K2CO3, L-proline
DMSO, 70 °C
AlCl3,
CH2Cl2, RT
R2
1
R2
R2
8
R1
7
Ts
R1
Br
R1
Br
N
N
N
MeO
3
6
NH2
42
OMe
44 (R1 = F ; R2 = H; for 36)
45 (R1 = Me ; R2 = H; for 37)
46 (R1 = H ; R2 = Me; for 38)
43
B
CuI, NH4OH,
K2CO3, L-proline
DMSO, 70 °C
HF.pyridine, NaNO2
H2O, 0 °C to RT
N
N
N
H2N
Br
F
Br
F
NH2
47
O
Br
Br
O
N
N
C
Cl
Cl
1. Ac2O, pyridine, RT
O
N
H
N
N
N
2. NCS, DMF, 60 °C
DMF, 65 °C
Br
NHAc
NH2
NHAc
conc. HCl,
EtOH,
reflux
CuI, NH4OH,
K2CO3, L-proline
DMSO, 70 °C
NaNO2, NaHSO3,
AcOH, Ac2O
EtOH, RT
Cl
Cl
Cl
N
N
N
NH2
Br
Br
48
NH2
Scheme 2. Synthetic routes for preparing intermediates 44 – 48 for synthesis of compounds 36 –
40.
In addition, we designed and synthesized several close analogs of compound 4 to serve as
negative controls for chemical biology studies. As described earlier, the middle urea region of
these PRMT3 inhibitors forms the key hydrogen-bonding interactions with E422 of PRMT3. We
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therefore predicted that taking either of these hydrogen-bonding interactions away by
methylating either nitrogen atom of the urea would drastically decrease PRMT3 inhibition.
Indeed, as shown in Table 6, compound 49 displayed markedly diminished inhibitory activity
(IC50 = 2594 ± 129 nM) while compound 50 was completely inactive (IC50 > 50000 nM).
Furthermore, the nitrogen atom in the isoquinoline ring of compound 4 forms a key hydrogen
bond with T466 of PRMT3 in the crystal structure of the PRMT3 – compound 4 complex (PDB
ID: 4RYL). Thus, we replaced the isoquinoline ring of 4 with the naphthalene ring (compound
51 (XY1))22, effectively removing the critical hydrogen bond with T466. As we reported
previously22, compound 51 displayed no inhibition of the PRMT3 catalytic activity in
biochemical assays.
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22
23
24
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 6. Compounds prepared as negative controls.
Compound
Structure
IC50 (nM)
N
O
2594 ± 129
N
49
50
N
H
N
Me
O
N
O
N
>50000
N
N
H
Me
O
O
No
Inhibition
N
51
N
N
H
H
O
We previously reported that compound 4 was more than 200-fold selective for PRMT3
over 31 other methyltransferases and more than 250 kinases, GPCRs, ion channels, and
transporters.22 Similarly, inhibitors 29, 30 and 36 were selective for PRMT3 over 31 other lysine
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methyltransferases, arginine methyltransferases, and DNA and RNA methyltransferases (Figure
3 and supporting information). In addition, 29, 30 and 36 were tested in a CEREP selectivity
panel consisting of 55 protein targets (47 GPCRs, 5 ion channels and 3 transporters) and did not
show any significant off-target activities (% of inhibition < 50% at 10 μM). It is of note that the
co-crystal structure of compound 4 in complex with PRMT3 reported recently (PDB ID:
4RYL)22 clearly shows that this inhibitor binds the same allosteric pocket as earlier inhibitors
(compounds 1 and 2 (PDB ID: 3SMQ and 4HSG)).
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36"
120"
100"
80"
60"
40"
20"
0"
0"µM"
1"µM"
5"µM"
20"µM"
Figure 3. Inhibitor 36 is highly selective for PRMT3 over 31 other methyltransferases. The
selectivity data for compounds 29 and 30 are shown in supporting information.
To establish the target engagement of PRMT3 inhibitors in cells (namely, inhibitors 4,
29, 36 and 37), we used an InCELL Hunter Assay, which measures intra-cellular binding of
inhibitors to the methyltransferase domain of PRMT3 in cell lines expressing the
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methyltransferase domain of PRMT3 tagged with a short fragment of β-galactosidase (ePL).
Binding of a compound to ePL-PRMT3 increases the fusion protein half-life. Inhibitors 4, 29, 36
and 37 stabilized PRMT3 in A549 cells, a human lung carcinoma cell line, with EC50 values of
2.0, 2.7, 1.6 and 4.9 μM, respectively (Figure 4, top). The same assay was performed in HEK293
cells and these compounds displayed EC50 values of 1.8, 3.1, 2.7 and 5.2 μM, respectively
(Figure 4, bottom). Compound 51 was used as a negative control in these assays. As expected, no
stabilization was observed with this compound.
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49
50
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Figure 4. InCELL Hunter Assay results of compounds 4, 29, 36, 37 and 51 in A549 and HEK
cells.
Furthermore, to establish whether these PRMT3 inhibitors can inhibit the PRMT3
catalytic activity in cells, we examined their effects on H4R3 asymmetric dimethylation. Since
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methylated arginine residues have relatively slow turnover, we overexpressed human Flag-
tagged PRMT3 and followed the methylation of both endogenous H4 and exogenously
introduced GFP-tagged H4. As we previously reported, overexpressed PRMT3 increased the
endogenous H4R3me2a from the baseline levels, and compound 4 effectively inhibited this
increase with an IC50 of 225 nM.22 The asymmetric dimethylation of exogenous H4R3 was also
inhibited by compound 4 (IC50 = 91 nM), indicating that this inhibitor has robust cellular effect.22
Similarly, as shown in Figure 5, compounds 29, 30 and 36 inhibited the exogenous asymmetric
dimethylation of H4R3 (IC50 = 240, 184, 134 nM, respectively). The dependency on the
transfected PRMT3 catalytic activity was determined by using the catalytically dead PRMT3
mutant (E335Q) that did not affect endogenous or exogenous H4R3me2a levels and therefore
was used to establish the baseline levels of the mark. It is of note that effects of 1 μM of
compounds 29, 30 and 36 matched with that of the catalytically dead PRMT3 mutant E335Q.
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Compound 29
Compound 30
Compound 36
1
0
5
0
0
0
1
0
5
0
0
0
1
0
5
0
0
0
IC50= 240 nM
IC50= 134 nM
IC50= 184 nM
1
1
0
1
0
0
1 0 0 0
1
1
0
1
0
0
1 0 0 0
1
1
0
1
0
0
1 0 0 0
( n
M )
( n
M )
( n
M )
PRMT3wt
30 100
PRMT3mt
0
PRMT3wt
30 100 300 1000
PRMT3wt
30 100 300 1000
PRMT3mt
0
PRMT3mt
300 1000
0
Comp. (nM)
H4R3me2a
0
10
0
10
0
10
GFP
overlap
FLAG
Figure 5. Cellular inhibitory activity of compounds 29, 30 and 36. HEK293 cells were
cotransfected with FLAG tagged PRMT3(wt) or its catalytic mutant(mt) and GFP-tagged
histone H4 and treated with different concentrations of compounds, as indicated. Total cell
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lysates were collected 24 h post inhibitor treatment and analyzed for H4R3me2a, GFP and
FLAG levels by western blotting. The graphs represent non-linear fits of H4R3me2a
fluorescence intensities normalized to GFP. The results are the averages of three replicates.
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CONCLUSION
In summary, we conducted comprehensive SAR studies, starting from early inhibitors 1-3
and culminating in the discovery of highly potent (IC50 values = ~10-36 nM), selective and cell-
active allosteric inhibitors of PRMT3 (inhibitors 4, 29, 30, 36 and 37). In addition, we generated
compounds that are very close analogs of these potent inhibitors, but displayed drastically
diminished potency as negative controls (compounds 49-51). The new inhibitors (compounds 29,
30 and 36) were highly selective for PRMT3 over 31 other methyltransferases and 55 other
protein targets. In cell-based assays, compounds 29, 30 and 36 engaged PRMT3 and potently
inhibited its methyltransferase activity (Figures 4 and 5). These inhibitors and negative controls
are excellent chemical tools for the biomedical community to further investigate biological
functions and disease associations of PRMT3.
EXPERIMENTAL
Chemistry General Procedures. Analytical thin-layer chromatography (TLC) was performed
employing EMD Milipore 210-270 µm 60-F254 silica gel plates. The plates were visualized by
exposure to UV light. Flash column chromatography was performed on a Teledyne ISCO
CombiFlash Rf+ system equipped with a variable wavelength UV detector and a fraction
collector using RediSep Rf normal phase silica columns. Nuclear Magnetic Resonance (NMR)
spectra were acquired on a Bruker DRX-600 spectrometer or on a Varian Mercury spectrometer
at 400 MHz. Chemical shifts are reported in parts per million (ppm, δ) scale relative to solvent
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residual peak (chloroform-d, H: 7.26 ppm; 13C: 77.16 ppm; methanol-d4, H: 3.31 ppm; 13C:
5
6
1
49.0 ppm). H NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d =
7
8
9
doublet, t = triplet, q = quartet, p = pentet, m = multiplet, app = apparent), coupling constant, and
integration. HPLC spectra for all compounds were acquired using an Agilent 6110 series system
with a UV detector set to 254 nm. Samples were injected (5 μL) onto an Agilent Eclipse Plus,
4.6 Å~ 50 mm, 1.8 μM, C18 column at room temperature. Either with a linear gradient from
50% to 100% B (MeOH + 0.1%acetic acid) in 5.0 min was followed by pumping 100% B for
another 2 min with A being H2O + 0.1% acetic acid or by a linear gradient from 10% to 100% B
(MeOH + 0.1% acetic acid) in 5.0 min was followed by pumping 100% B for another 2 min with
A being H2O + 0.1% acetic acid. The flow rate was 1.0 mL/min. Mass spectrometry (MS) data
were acquired in positive ion mode using an Agilent 6110 single-quadrupole mass spectrometer
with an electrospray ionization (ESI) source. HRMS analysis was conducted on an Agilent
Technologies G1969A high-resolution API-TOF mass spectrometer attached to an Agilent
Technologies 1200 HPLC system. Samples were ionized by electrospray ionization (ESI) in
positive mode. All biologically evaluated compounds had > 95% purity using the HPLC methods
described above.
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1-(Benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-oxo-2-(piperidin-1-yl)ethyl)urea (3): Compound 3 was
prepared according to previously published procedures.21
1-(Isoquinolin-6-yl)-3-(2-oxo-2-(pyrrolidin-1-yl)ethyl)urea (4): Compound 4 was prepared
according to previously published procedures.22
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1-(Isoquinolin-6-yl)-3-(2-oxo-2-(piperidin-1-yl)ethyl)urea (5): To a solution of isoquinolin-6-
amine (50 mg, 0.347 mmol) in N,N-dimethylformamide (DMF) (1.6 mL) at room temperature
was added N,N’-carbonyldiimidazole (CDI) (84 mg, 0.520 mmol). The resulting solution was
stirred for 12 hours prior to the addition of 2-amino-1-(piperidin-1-yl)ethan-1-one (99 mg, 0.694
mmol), and stirring for a further 6 hours. Following dilution with water (20 mL), the aqueous
layer was extracted with ethyl acetate (EtOAc) (3 x 20 mL), and the combined organic extracts
dried with anhydrous sodium sulfate. After filtration, all solvents were removed under reduced
pressure, and the residue purified by column chromatography on silica gel to afford title
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1
compound (5) (31 mg, 29% yield). H NMR (500 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.35 (d, J =
5.7 Hz, 1H), 8.08 (br s, 1H), 7.98 (d, J = 8.9 Hz, 1H), 7.63 (d, J = 5.8 Hz, 1H), 7.02 (br s, 2H),
6.57 (t, J = 4.6 Hz, 1H), 4.02 (d, J = 4.7 Hz, 2H), 3.50-3.44 (m, 2H), 3.38-3.33 (m, 2H), 1.65-
1.57 (m, 2H), 1.57-1.50 (m, 2H), 1.50-1.40 (m, 2H). m/z (HRMS) [M+H]+ for C17H21N4O2+:
calculated 313.1659; found 313.1662.
1-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-3-(2-oxo-2-(piperidin-1-yl)ethyl)urea (6): To a
solution of 5-amino-3H-benzofuran-1-one (75 mg, 0.5 mmol, 1.0 eq) in DMF (1.5 mL) was
added CDI (90 mg, 0.55 mmol, 1.1 eq), and the resulting mixture was stirred for 8 h at rt. 2-
amino-1-piperidin-1-ylethanone hydrochloride salt (134 mg, 0.75 mmol, 1.5 eq) was then added
followed by Hunig’s base (131 µL, 0.75 mmol, 1.5 eq). After being stirred for 18 h at rt, the
resulting mixture was diluted with water (25 mL) and extracted with EtOAc (3 x 25 mL).
Combined organic layers were dried over sodium sulfate and concentrated under reduced
pressure to give crude product, which was then purified by flash column chromatography to
1
yield desired compound as white solid (39 mg, 25%). H NMR (600 MHz, Methanol-d4) δ 7.85
(s, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.42 (dd, J = 8.5, 1.8 Hz, 1H), 5.31 (s, 2H), 4.10 (s, 2H), 3.57
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(t, J = 5.6 Hz, 2H), 3.45 (t, J = 5.5 Hz, 2H), 1.74-1.50 (m, 6H). MS (ESI) m/z [M+H]+ for
5
6
C16H20N3O4+: calculated 318.1, found 318.1.
7
8
9
1-(2-Oxo-2-(piperidin-1-yl)ethyl)-3-(quinazolin-7-yl)urea (7): To a solution of quinazolin-7-
amine (73 mg, 0.5 mmol, 1.0 eq) in DMF (1.5 mL) was added CDI (90 mg, 0.55 mmol, 1.1 eq),
and the resulting mixture was stirred for 8 h at rt. 2-amino-1-piperidin-1-ylethanone
hydrochloride salt (134 mg, 0.75 mmol, 1.5 eq) was then added followed by Hunig’s base (131
µL, 0.75 mmol, 1.5 eq). After being stirred for 18 h at rt, the resulting mixture was diluted with
water (25 mL) and extracted with EtOAc (3 x 25 mL). Combined organic layers were dried over
sodium sulfate and concentrated under reduced pressure to give crude product, which was then
purified by flash column chromatography to yield desired compound (10 mg, 6%). 1H NMR (600
MHz, Methanol-d4) δ 9.29 (s, 1H), 9.06 (s, 1H), 8.22 (d, J = 2.1 Hz, 1H), 8.00 (d, J = 8.9 Hz,
1H), 7.71 (dd, J = 8.9, 2.1 Hz, 1H), 4.14 (s, 2H), 3.58 (t, J = 5.6 Hz, 2H), 3.47 (t, J = 5.5 Hz,
2H), 1.75-1.53 (m, 6H). MS (ESI) m/z [M+H]+ for C16H20N5O2+: calculated 314.2, found 314.2.
10
11
12
13
14
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17
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50
51
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53
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55
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58
59
60
1-(Isoquinolin-7-yl)-3-(2-oxo-2-(piperidin-1-yl)ethyl)urea (8): To a solution of isoquinolin-7-
amine (50 mg, 0.347 mmol) in DMF (1.6 mL) at room temperature was added CDI (84 mg,
0.520 mmol). The resulting solution was stirred for 12 hours prior to the addition of 2-amino-1-
(piperidin-1-yl)ethan-1-one (99 mg, 0.694 mmol), and stirring for a further 6 hours. Following
dilution with water (20 mL), the aqueous layer was extracted with EtOAc (3 x 20 mL), and the
combined organic extracts dried with anhydrous sodium sulfate. After filtration, all solvents were
removed under reduced pressure, and the residue purified by column chromatography on silica
1
gel to afford title compound (8) (45 mg, 42% yield). H NMR (500 MHz, DMSO-d6) δ 9.13 (s,
1H), 8.33 (d, J = 5.6 Hz, 1H), 8.22 (d, J = 1.9 Hz, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.69 (d, J = 5.6
Hz, 1H), 7.11 (br s, 1H), 6.92 (br s, 1H), 6.50 (t, J = 4.7 Hz, 1H), 4.03 (dd, J = 14.2, 5.9 Hz, 2H),
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3.47-3.44 (m, 2H), 3.38-3.34 (m, 2H), 1.63-1.56 (m, 2H), 1.56-1.50 (m, 2H), 1.49-1.41 (m, 2H).
5
6
m/z (HRMS) [M+H]+ for C17H21N4O2+: calculated 313.1659; found 313.1664.
7
8
9
General Procedures for the preparation of compounds 9-14 in table 2: Compounds 9-14
shown in table 2 were prepared according general procedures described below. To a solution of
isoquinolin-6-amine (1.0 eq.) and triethylamine (TEA) (2 eq.) in DMF (1 mL/ 0.347 mmol) was
added CDI (1.5 eq) and the reaction mixture was allowed to stir at 25 °C for 4 hours. To the
reaction mixture was then added the corresponding amine (2 eq.) and the mixture was allowed to
stir for additional one hour. Then 50 mL of water and 50 mL of EtOAc were added to the
reaction mixture. After extraction, the organic layer was washed with brine, dried over
anhydrous Na SO , filtered and concentrated in vacuo. The residue was purified by column
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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2
4
chromatography on silica gel eluting with 0~5% MeOH in DCM, to give the product.
1-(2-(Cyclohex-1-en-1-yl)ethyl)-3-(isoquinolin-6-yl)urea (9): Yellow oil (67 mg, 62% yield).
1H NMR (Chloroform-d) δ: 9.07 (s, 1H), 8.40 (d, J = 5.8 Hz, 1H), 8.04(s, 1H), 7.81 (d, J=8.8 Hz,
1H), 7.66 (s, 1H), 7.53 (d, J = 5.8 Hz, 1H), 7.44 (dd, J = 8.8, 2.0 Hz, 1H), 7.27 (s, 1H), 5.45 (s,
1H), 5.27-5.30 (m, 1H), 3.42-3.77 (m, 2H), 2.20-2.17(m, 6H), 1.97-1.92 (m, 4H), 1.627-1.47 (m,
4H). MS (ESI) m/z [M+H]+ for C18H22N3O+: calculated 296.2, found 296.1.
1-(2-Cyclohexylethyl)-3-(isoquinolin-6-yl)urea (10) : Yellow oil (69 mg, 64% yield). 1H NMR
(DMSO-d6) δ: 9.07 (s, 1H), 8.87 (s, 1H), 8.33 (d, J = 5.8 Hz, 1H), 8.06 (d, J = 1.8 Hz, 1H), 7.95
(d, J = 9.0 Hz, 1H), 7.60 (d, J = 6.0 Hz, 1H), 7.52 (dd, J = 8.8, 2.0 Hz, 1H), 6.28 (br t, J = 5.5
Hz, 1H), 3.11-3.24 (m, 2H), 1.58-1.76 (m, 5H), 1.11-1.39 (m, 6H), 0.83-0.98 (m, 2H). MS (ESI)
m/z [M+H]+ for C18H24N3O+: calculated 298.2, found 298.1.
1-(2-Cyclopentylethyl)-3-(isoquinolin-6-yl)urea (11) : Light yellow oil (30 mg, 29% yield). 1H
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9
NMR (Chloroform-d) δ: 9.04 (s, 1H), 8.36 (d, J = 5.8 Hz, 1H), 8.03 (s, 2H), 7.76 (d, J = 8.8 Hz,
1H), 7.34-7.56 (m, 2H), 7.27 (s, 1H), 3.19-3.40 (m, 2H), 1.64-1.82 (m, 3H), 1.40-1.64 (m, 6H),
0.95-1.16 (m, 2H), 0.01 (s, 1H). MS (ESI) m/z [M+H]+ for C17H22N3O+ : calculated 284.2, found
284.1.
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1-(2-Cyclopropylethyl)-3-(isoquinolin-6-yl)urea (12) : White solid (27 mg, 29% yield). H
NMR (DMSO-d6) δ: 9.59 (s, 1H), 9.46 (s, 1H), 8.34-8.51 (m, 2H), 8.28 (d, J = 9.0 Hz, 1H), 8.12
(d, J = 6.5 Hz, 1H), 7.77 (dd, J = 9.0, 2.0 Hz, 1H), 6.71 (br t, J = 5.4 Hz, 1H), 3.11-3.27 (m, 3H),
1.38 (q, J = 7.0 Hz, 2H), 0.61-0.80 (m, 1H), 0.35-0.49 (m, 2H). MS (ESI) m/z [M+H]+ for
C15H18N3O+ : calculated 256.1, found 256.1.
1
1-(Isoquinolin-6-yl)-3-phenethylurea (13): Light yellow oil (23 mg, 22% yield). H NMR
(Chloroform-d) δ: 8.95 (s, 1H), 7.89-8.47 (m, 3H), 7.74 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 5.8 Hz,
1H), 7.37 (dd, J = 8.9, 1.9 Hz, 1H), 7.20-7.30 (m, 2H), 7.09-7.20 (m, 3H), 5.63 (br s, 1H), 3.55
(q, J = 6.8 Hz, 2H), 2.84 (t, J = 6.9 Hz, 2H). MS (ESI) m/z [M+H]+ for C18H18N3O+ : calculated
292.1, found 292.1.
1-(2-(1-(Aminomethyl)cyclohexyl)ethyl)-3-(isoquinolin-6-yl)urea (14): The general procedure
was applied by using tert-butyl ((1-(2-aminoethyl)cyclohexyl)methyl)carbamate as the amine
(285
mg,
1.11
mmol)
to
give
tert-butyl
((1-(2-(3-(isoquinolin-6-
yl)ureido)ethyl)cyclohexyl)methyl)carbamate as a white solid. To the solution of tert-butyl ((1-
(2-(3-(isoquinolin-6-yl)ureido)ethyl)cyclohexyl)methyl)carbamate (130 mg, 0.305 mmol) in
DCM (1 mL) was added TFA (1.000 mL, 12.98 mmol). Then the reaction mixture was stirred at
25 °C for 0.5 hr. To the mixture was added 5 mL of toluene and was then concentrated in vacuo.
The residue was purified by preparative-HPLC to give the product as white solid (23 mg, 22%
yield). 1H NMR (Methanol-d4) δ 8.94-9.07 (m, 1H), 8.28 (br d, J = 5.8 Hz, 1H), 8.07 (br s, 1H),
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