A pan-inhibitor for protein arginine methyltransferase family enzymes

Article abstract of DOI:10.3390/biom11060854

Protein arginine methyltransferases (PRMTs) play important roles in transcription, splicing, DNA damage repair, RNA biology, and cellular metabolism. Thus, PRMTs have been attractive targets for various diseases. In this study, we reported the design and synthesis of a potent pan-inhibitor for PRMTs that tethers a thioadenosine and various substituted guanidino groups through a propyl linker. Compound II757 exhibits a half-maximal inhibition concentration (IC₅₀ ) value of 5 to 555 nM for eight tested PRMTs, with the highest inhibition for PRMT4 (IC₅₀ = 5 nM). The kinetic study demonstrated that II757 competitively binds at the SAM binding site of PRMT1. Notably, II757 is selective for PRMTs over a panel of other methyltransferases, which can serve as a general probe for PRMTs and a lead for further optimization to increase the selectivity for individual PRMT.

Full text of DOI:10.3390/biom11060854

biomolecules
Article
A Pan-Inhibitor for Protein Arginine Methyltransferase
Family Enzymes
Iredia D. Iyamu 1, Ayad A. Al-Hamashi 1,2 and Rong Huang 1,
*
1
Department of Medicinal Chemistry and Molecular Pharmacology, Institute for Drug Discovery,
Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA; iiyamu@purdue.edu (I.D.I.);
aalhamas@purdue.edu (A.A.A.-H.)
Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Bab-almoadham,
Baghdad 10047, Iraq
2
*
Correspondence: huang-r@purdue.edu; Tel.: +1-765-494-3426
Abstract: Protein arginine methyltransferases (PRMTs) play important roles in transcription, splicing,
DNA damage repair, RNA biology, and cellular metabolism. Thus, PRMTs have been attractive
targets for various diseases. In this study, we reported the design and synthesis of a potent pan-
inhibitor for PRMTs that tethers a thioadenosine and various substituted guanidino groups through
a propyl linker. Compound II757 exhibits a half-maximal inhibition concentration (IC50) value of 5
to 555 nM for eight tested PRMTs, with the highest inhibition for PRMT4 (IC50 = 5 nM). The kinetic
study demonstrated that II757 competitively binds at the SAM binding site of PRMT1. Notably, II757
is selective for PRMTs over a panel of other methyltransferases, which can serve as a general probe
for PRMTs and a lead for further optimization to increase the selectivity for individual PRMT.
Keywords: protein arginine methyltransferases; inhibitor; methyltransferase; competitive inhibitor
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Iyamu, I.D.; Al-Hamashi,
A.A.; Huang, R. A Pan-Inhibitor for
Protein Arginine Methyltransferase
Family Enzymes. Biomolecules 2021,
11, 854. https://doi.org/10.3390/
biom11060854
1. Introduction
Protein arginine methyltransferases (PRMTs) are a group of enzymes that methylate
the guanidino group of the arginine using S-adenosyl methionine (SAM) as a methyl donor,
producing mono- or dimethylated arginine residues and S-adenosyl-L-homocysteine (SAH).
To date, nine human PRMT members have been identified, which are classified into three
Academic Editor: Umesh R. Desai
types according to the characteristic of their products [1–3]. Type I PRMTs include PRMT1,
2, 3, 4, 6, and 8 that produce asymmetric dimethylated arginine, while type II PRMTs
consist of PRMT5 and PRMT9 that generate symmetric dimethylated arginine. As the
only member in Type III, PRMT7 solely produces monomethylated arginine. Arginine
methylation regulates diverse biological processes, including signal transduction, RNA
Received: 12 April 2021
Accepted: 3 June 2021
Published: 8 June 2021
splicing, DNA repair, cell proliferation, and differentiation [4]. Consequently, aberrant
levels of PRMTs have been implicated in diverse diseases including cardiovascular diseases,
cancers, inflammatory diseases, and diabetes [1,5–8].
PRMTs inhibitors have served as valuable tools to probe the biological roles of PRMTs
and even led to the development of potential therapeutic agents. Besides allosteric in-
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
hibitors for PRMT3 and PRMT5 [
9
,10], three different types of inhibitors including bisub-
strate analogues, substrate, and SAM-competitive inhibitors have been reported [11
–
14].
Generally, it is challenging to obtain highly selective inhibitors that competitively bind
to the SAM binding site. For example, Pr-SNF, a derivative of sinefungin, was devel-
oped as a PRMT4 inhibitor but it has a higher binding affinity for SETD2 (Figure 1) [15].
However, a SAM analogue JNJ-64619178 is a selective and potent PRMT5 inhibitor that is
orally bioavailable and has been developed for advanced solid tumors and non-Hodgkin
lymphoma [16].
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Biomolecules 2021, 11, 854. https://doi.org/10.3390/biom11060854
https://www.mdpi.com/journal/biomolecules

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Figure 1. Representative structures of bisubstrate inhibitors of PRMTs.
Another strategy to develop selective inhibitors is to design bisubstrate analogues
that fully or partially occupy both binding pockets. For instance, 50-(diaminobutyric acid)-
N-iodoethyl-50-deoxyadenosine ammonium hydrochloride (AAI) a SAM analogue has
been used with PRMT1 to generate its bisubstrate inhibitors in situ [17
JD-16 attached a terminal guanidine to a SAM analogue through a butyl linker, displaying
selectivity for PRMT1 (IC50 = 3.0 M) versus PRMT4 (Figure 1) [19 20]. GMS is a bisubstrate
inhibitor for PRMT6 with an IC50 of 90 nM and DS-437 is a dual PRMT5/7 inhibitor with
IC50 of 6.0 M against both enzymes [21 22]. Recently developed AH237 is a potent and
,18]. In addition,
µ
,
µ
,
dual PRMT4/5 inhibitor by tethering a thioadenosine with an RGK tripeptide through a
guanidino group [23]. Inspired by AH237, we aim to develop potent pan-inhibitors against
all PRMTs, which can be further optimized to selective inhibitors for individual PRMT in
the future.
2. Design
SAM is a common cofactor that donates the methyl group to most methyltransferases.
Thus, SAM has served as a lead compound to develop SAM-competitive inhibitors. One
successful example is the PRMT5 inhibitor JNJ-64619178 [24]. Besides, SAM mimic has
served as a building block in the development of bivalent or bisubstrate analogues to tune
the selectivity by incorporating different substrate moieties specific to the methyltransferase
of interest [17,25]. Recently, we have reported a series of bisubstrate analogues by linking
amino acid residues or peptides to a SAM analogue through a guanidino group, exhibiting
selectivity for PRMTs over protein lysine methyltransferases (PKMTs) and protein N-
terminal methyltransferase 1 (NTMT1) [23]. Among them, AH237 is highly selective for
PRMT4/5 among 41 methyltransferases, displaying an IC50 value of 2.8 and 0.42 nM for
PRMT4 and PRMT5, respectively [23]. As AH237 has limited application for cell-based
studies due to its low cell penetration, we focus on the replacement of its peptide moiety
with lipophilic groups to improve permeability. The thioadenosine and guanidino moiety
were retained to preserve the selectivity for PRMTs over other methyltransferases. Our
previous studies on PRMT inhibitors revealed that a propyl linker between the sulfur
atom and the guanidino group is optimal and modulates the binding affinity [23]. Thus,
we chose to keep the 3-C atom linker as the optimal length between the thioadenosine
and guanidino group [23]. Interestingly, bisubstrate analogues containing a propyl linker

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showed a twofold increased potency for PRMT1 when the substrate moiety changed from
a tripeptide to a single amino acid [23]. For example, AH244 showed an IC50 value of
3.0 µM for PRMT1 (Figure 2) [23]. Thus, we hypothesize that replacing the amino acid
portion with an aliphatic or aromatic group would retain the potency to PRMT1. To test
our hypothesis, we designed compounds to investigate the effect of aliphatic and aromatic
substitution on PRMT inhibition (Figure 2).
Figure 2. Design of pan-PRMT inhibitors.
3. Synthesis
The thiourea intermediates (1a–1n) were synthesized from the reaction of commer-
cially available amines with Fmoc protected isothiocyanates [23]. These amines were
selected to explore the effects of different lengths, ring sizes, and substituents on the inhi-
bition of PRMT activity. The thioadenosine intermediate was synthesized as previously
described [23]. The diol of commercially available adenosine was first protected before a
Mitsunobu reaction with thioacetic acid to afford the thioester
the thioester and subsequent thiol alkylation with phthalimide alkyl bromide produced
the phthalimide 23]. The removal of the phthalimido group by hydrazine afforded the
free amine intermediate , which was coupled with different thioureas (1a 1n) to afford
5a 5n 23]. The deprotection of the Fmoc group with piperidine followed by acid-catalyzed
deprotection of the acetal group afforded the final compounds 6a–6n (Scheme 1).
2 [23]. The hydrolysis of
3
[
4
–
–
[
Scheme 1. Cont.

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Scheme 1. Synthesis of compounds 6a
–
6n. Reagents and conditions: (
a) pTsOH, acetone, 87%, (b) thioacetic acid, PPh3,
DIAD, THF, 94%, (c) NaOCH3, MeOH 63%, (
d) hydrazine, MeOH, 81%, (
e) EDC, DIPEA, CH2Cl2, 53–79%, (f) piperidine,
HOBt, DMF, then TFA, H2O, 47–65%.
4. Biochemical Characterization
First, we evaluated the inhibitory activities of all synthesized compounds for PRMT1
in a SAH hydrolase (SAHH)-coupled fluorescence assay under the condition of the Km
values of both SAM (10 µM) and the H4-21 substrate peptide (5 µM) [23]. For those potent
compounds that displayed an IC50 value lower than half of the enzyme concentration,
we re-determined their IC50 values under the condition of 4 Km values of both SAM and
H4-21 peptide. Destruction of the alpha-amino acid moiety of AH244 (IC50 = 3.0 ± 1.1 µM
yielded an ethyl group substitution to produce 6a (IC50 = 0.62 0.08 M), exhibiting
about fivefold increased inhibition for PRMT1 compared to the parent compound AH244
Table 1). Substitution of the ethyl group with a cyclobutane yielded 6b that greatly
)
±
µ
(
improved the inhibitory activity, however, further increase in the ring size to cyclopentane
and cyclohexane led to slight reduction in the inhibitory activity. Incorporating a methylene
group between the cyclohexane and guanidino group allowed certain flexibility but did
not change the inhibitory activity, as reflected by 6d and 6e.
Table 1. SAR of synthesized compounds 6a–6n.
Ki, app (µM) b
0.31 ± 0.04
Compound
6a
R1
IC50 (µM) *
0.62 ± 0.08
6b
6c
0.23 ± 0.02 a
0.22 ± 0.016
0.11 ± 0.048
0.045 ± 0.005
0.11 ± 0.008
0.055 ± 0.024
6d

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Table 1. Cont.
Ki, app (µM) b
0.06 ± 0.013
Compound
R1
IC50 (µM) *
0.12 ± 0.026
6e
6f
0.29 ± 0.01
0.72 ± 0.15
0.21 ± 0.06
0.33 ±0.11
0.15 ± 0.005
0.36 ± 0.075
0.11 ± 0.03
0.17 ± 0.055
6g
6h
6i
6j
0.21 ± 0.01
0.44 ± 0.03
0.11 ± 0.005
0.22 ± 0.015
6k
6l (II757)
0.09 ± 0.04 a
0.018 ± 0.008
6m
6n
0.29 ± 0.04 a
0.26 ± 0.07 a
0.058 ± 0.007
0.052 ± 0.014
a
These experiments were performed with substrate concentration at 4 Km as the IC50 at Km value was less than
b
the enzyme concentration. Ki, app was calculated using the equation Ki, app = IC50/(1 + [S]/Km). * IC50 values
were performed in duplicates (n = 2) and presented as mean ± SD.
Next, we examined the effect of the aromatic group on the PRMT1 inhibition. Com-
pound 6f containing a phenyl group displayed an IC50 of 0.29
±
0.01 µM to PRMT1,
resulting in about 2.5-fold reduction compared to 6d. Surprisingly, increasing the length
between a phenyl ring and guanidino group gave comparable inhibitory effects, as reflected
by 6g and 6i. The aliphatic ring analogues 6d and 6e displayed about two- and sixfold

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better inhibitory activity than their respective aromatic analogues 6f and 6g (Table 1). Next,
we introduced the bromo- or nitro substitution on the aromatic ring to evaluate the sub-
stituent effect. The selection was limited by readily available starting materials. Compound
6j with a nitro group on the para position displayed an IC50 of 0.21
was comparable to the parent analogue 6f. The substitution of an ortho-Br on the phenyl
ring generated 6k (IC50 = 0.44 0.03 M), resulting in about a twofold loss of inhibitory
±
0.01 µM, which
±
µ
activity. However, moving the bromide group to the meta and para position led to 6l and
6m, respectively. According to the results (Table 1 and Figure 3), bromo-substitution on the
meta position of the phenyl group produced the most potent inhibition. Interestingly, the
p-Br on benzyl group displayed nearly threefold increased activity when we compared 6n
to 6g, although the p-Br on phenyl ring produced comparable inhibitory activity against
PRMT1 (Table 1).
(A)
(B)
Figure 3. Biochemical characterization of compound II757
(6l). (A) SAHH-coupled fluorescence assay of compound II757 at
4 Km substrate concentration ranging in concentrations from 0 to 100
II757. All experiments were performed in duplicates (n = 2).
µM and (B) concentration–response plot for compound
5. Inhibition Mechanism Studies
To understand the mode of action, the most potent inhibitor 6l (II757) among this
series was selected for an investigation of inhibition mechanism through a kinetic analysis
using SAHH-coupled fluorescence assay with PRMT1. II757 displayed a noncompetitive
inhibition for the peptide substrate H4-21 as demonstrated by comparable IC50 values as
the peptide concentration increased from 0.5 to 8 Km (Figure 4A,B). On the other hand,
II757 showed an explicit pattern of competitive inhibition for SAM, as demonstrated by
a linear increase in the IC50 values proportional to the increased concentration of SAM
(Figure 4C,D). This result indicated that II757 act as a SAM-competitive inhibitor. Thus, its
apparent Ki value is about 18 nM (Table 1 and Figure 3). Although the guanidino group
did not reach the substrate-binding site, it may form additional interactions close to the
SAM binding site to display improved potency.

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(A)
(B)
(C)
Figure 4. Inhibition mechanism studies of II757: (
(D)
A
) IC50 curves of 6l at varying concentrations of substrate (H4-21) with
a fixed concentration of SAM at 10 M, ( ) linear regression plot of IC50 values with corresponding concentrations of
substrate (H4-21), ( ) IC50 curves of 6l at varying concentrations of SAM with a fixed concentration of substrate (H4-21)
at 5 M, and ( ) linear regression plot of IC50 values with corresponding concentrations of SAM. All experiments were
performed in duplicates (n = 2).
µ
B
C
µ
D
6. Selectivity Studies
We chose the top four potent compounds (6b and 6l–n) to investigate their selectivities
against several in-house methyltransferases including protein arginine methyltransferase
TbPRMT7, two members of the protein lysine methyltransferase PKMT (G9a and SETD7),
protein N-terminal methyltransferase 1 (NTMT1), and a small molecule methyltransferase
nicotinamide N-methyltransferase (NNMT), all of which have a SAM binding site. Addi-
tionally, SAHH was included as it is used in the coupled fluorescence assay, and it possesses
a SAH binding site.
As shown in Figure 5, all four compounds did not show any significant inhibition
to SAHH, NTMT1, SETD7, G9a, and NNMT at 33.3
pounds (6b 6m, and 6n) displayed over 50% inhibition to SETD7 at 100
6b containing a cyclobutane group showed higher selectivity over PRMT7 as no inhibitory
activity was observed against PRMT7 at 11.1 M, confirming the possibility of structural
modification of SAM analogue to achieve selectivity even among PRMTs. The most potent
PRMT1 inhibitor II757 displayed about 30% of the activity at 1.2 M against PRMT7 while
µM. Except for 6l
(II757), three com-
,
µM. For TbPRMT7,
µ
µ
demonstrating slightly higher selectivity for other enzymes such as SETD7, G9a, NNMT,
NTMT, and SAHH.

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(A)
(B)
(C)
Figure 5. Selectivity studies for compounds 6b
(D)
(A) and 6l–n (B–D) against TbPRMT7, SAHH, NTMT1, SETD7, G9a, and
NNMT with each compound at five different concentrations (100, 33, 11, 3.7, and 1.2
in duplicates (n = 2).
µM). All experiments were performed
7. Pan-PRMT Inhibition
The most potent inhibitor II757 was then sent to Reaction Biology to evaluate its selec-
tivity against all the available PRMTs using a radioisotope 3H-AdoMet methyltransferase
assay. As shown in Figure 6, II757 inhibits all eight members of the PRMTs with an IC50
value ranging from 5.05 to 555 nM. It inhibits PRMT1 with an IC50 of 16 nM, which is
similar to the value of 18 nM in our SAHH-coupled assay. Interestingly, II757 demonstrated
modest inhibition to PRMT3 and PRMT7 compared to other tested PRMT members and
was most potent against PRMT4 with an IC50 of 5.05 nM. To our knowledge, this is the
most potent SAM-competitive inhibitor for PRMT4 even compared to reported bisubstrate
analogue AH237. Future studies will focus on obtaining structural information of II757
bound to PRMTs to inform how to connect to a substrate portion to increase both potency
and selectivity for each PRMT.

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PRMT5/MEP50
Complex
18.2
PRMT5
(C449S)/MEP50
36.6
PRMT1 PRMT3 PRMT4
PRMT
IC50 (nM) 16.4 321 5.05
PRMT6 PRMT7 PRMT8
30.5 555 32.1
Figure 6. Selectivity of II757 against the panel of PRMTs. II757 was tested in 10 doses with threefold serial dilution in the
HotSpot methyltransferase assay at reaction biology.
8. Molecular Docking
To further examine the binding mode of II757, we performed a molecular docking
study on selected PRMT isoforms including PRMT1, 4, 5, and 7. The results indicated
that II757 virtually occupied the SAM binding site of the aforementioned PRMT isoforms,
which is consistent with the SAM-competitive inhibition mechanism result (Figure 4). For
PRMT 1/4/5, the m-bromophenyl substitution of II757 formed a π–π interaction with
multiple residues in the active site. Besides, multiple hydrogen bonding and van der Waals
interactions were also observed. However, II757 interacted with PRMT7 through several
water molecule bridges and the m-bromophenyl substitution located at the edge of the
binding pocket, possibly explaining the moderate inhibitory activity of II757 for PRMT7
compared to other PRMTs (Figures 7 and 8).
(A)
(B)
Figure 7. Cont.

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(C)
(D)
Figure 7. The in silico interaction of II757 inside the pocket of (
A) PRMT1 (PDB: 6nt2), (
B
) PRMT4 (PDB: 6s79), (
C
) PRMT5
(PDB: 6ckc), and (D) PRMT7 (PDB: 4m37). II757 is shown in the magenta stick and ball model.
(A)
(B)
(C)
(D)
Figure 8. The predicted 2D interactions of II757 inside the active site of (
A) PRMT1 (pdb: 6nt2), (B) PRMT4 (pdb: 6s79), (C)
PRMT5 (pdb: 6ckc), and (D) PRMT7 (pdb: 4m37). II757 is shown in the black line model.
9. Inhibition on Cellular Methylation Level
Furthermore, we proceeded to investigate the inhibitory effects of II757 on the methy-
lation level of histone H4 Arg3 via Western blotting to check its cellular inhibition. As

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shown in Figure 9, treatment with II757 for 48 h in HEK293 cells resulted in a decrease
in the asymmetric demethylation on Arg3 (H4R3me2a) level at 10 µM. Although cellular
inhibition was about 20- to 1000-fold lower than the value obtained from the enzymatic
assay, this preliminary study indicated that II757 is cell potent. Future studies will focus
on tethering this moiety with reported substrate-competitive inhibitors to enhance the
selectivity and cellular potency.
Figure 9. Cellular arginine methylation assay of II757 in HEK293 cells. Representative Western blot
results of effects of II757 on histone H4 Arg3 asymmetric dimethylation (H4R3me2a) level at 0, 0.1, 1,
10, and 100 µM. H4 blot is shown as a loading control.
10. Conclusions
In summary, we have designed and synthesized a pan-PRMT inhibitor II757 by
incorporating an m-Br phenyl group on a guanidino group tethered to thioadenosine.
This inhibitor showed inhibition against eight tested PRMT members with an IC50 value
ranging from 5 to 555 nM. Furthermore, it showed the highest inhibitory activity against
PRMT4 with an IC50 of 5 nM. Kinetic analysis revealed that II757 is a SAM-competitive
inhibitor for PRMT1, indicating that it can be tethered to a substrate-competitive inhibitor
to further improve its inhibitory activity and selectivity for any specific member of the
PRMT family. II757 exhibited over 1000-fold selectivity over other methyltransferases
such as SETD7, G9a, NTMT1, and NNMT. The m-Br phenyl group on the guanidino
group has the potential to be derivatized by the formation of the sp2–sp2 bond through
abundantly available chemical reactions. Compound II757 can serve as a general probe
in the screening for PRMT inhibitors and as a building block to construct cell-potent and
selective PRMT inhibitors.
11. Experimental Section
Materials and instruments: The reagents and solvents were purchased from com-
mercial sources (Fisher, Waltham, MA, USA and Sigma-Aldrich, St. Louis, MO, USA)
and used directly. Analytical thin-layer chromatography was performed on ready-to-use
plates with silica gel 60 (Merck, F254 Sigma-Aldrich, St. Louis, MO, USA). Flash column
chromatography was performed over silica gel (grade 60, 230–400 mesh, Sigma-Aldrich,
St. Louis, MO, USA) on the Teledyne ISCO CombiFlash purification system (Lincoln,
NE, USA). Final compounds were purified on preparative reversed-phase high-pressure
liquid chromatography (RP-HPLC) performed on Agilent 1260 Series system (Santa Clara,
CA, USA). Systems were run with 0–50% methanol/water gradient with 0.1% TFA. NMR
spectra were acquired on a Bruker AV500 instrument (Billerica, MA, USA) (500 MHz for
1H-NMR, 126 MHz for 13C-NMR). All the target compounds showed a purity of >95%.
11.1. General Procedure for the Synthesis of Thiourea
To a solution of amine in dichloromethane (3 mL), Fmoc-isothiocyanate (42 mg,
◦
0.15 mmol, 1.5 eq.) was added at 0 C. The reaction mixture was stirred at room tem-
perature for 1–4 h. The volatiles were removed under reduced pressure and the crude was
purified by column chromatography to afford 1a–1n.

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11.2. General Procedure for the Synthesis of Guanidine Moiety
To a solution of amine 4 (0.05 mmol, 1 eq.) in dichloromethane (2 mL), thiourea 1a
–n
(0.75 mmol, 1.5 eq.), EDC (20 mg, 0.1 mmol, 2 eq.), and DIPEA (13 mg, 0.1 mmol, 2 eq.)
were added. The reaction mixture was stirred overnight at room temperature. The mixture
was washed with dichloromethane (3 × 2 mL) to produce 5a–n.
11.3. General Procedure for Final Compounds Synthesis
The mixture 5a–n was treated with 20% piperidine in DCM for 10 min each. Then, it
was concentrated and used for the next step. Further, the crude was dissolved in TFA/H2O
(9;1). The reaction mixture was stirred at room temperature for 5 h. The mixture was
evaporated by passing N2 gas and was then washed with dry ether. The residue was
used for semi-preparatory HPLC separation using MeOH/H2O 10–40% to obtain final
compounds 6a–n. All NMR spectra, HRMS and HPLC analysis of compounds 6a–6n are
included in the supplementary materials.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
1
methyl)thio)propyl)-3-ethylguanidine (6a). H NMR (500 MHz, Methanol-d4)
δ
8.55 (s, 1H),
8.30 (s, 1H), 8.21 (s, 1H), 6.00 (d, J = 4.8 Hz, 1H), 4.80 (t, J = 5.1 Hz, 1H), 4.34 (t, J = 5.0 Hz
1H), 4.24–4.17 (m, 1H), 3.25–3.16 (m, 4H), 3.03–2.90 (m, 2H), 2.62 (t, J = 7.1 Hz, 2H), 1.87–1.76
(m, 2H), 1.19 (t, J = 7.2 Hz, 3H). 13C NMR (126 MHz, MeOD)
170.33, 157.37, 157.33, 153.94,
,
δ
150.66, 141.47, 120.58, 90.21, 85.72, 74.75, 74.10, 41.23, 37.43, 35.23, 30.60, 29.70, 14.43. HRMS
m/z calc’d for C16H26N8O3S [M + H]+: 411.1921; found: 411.1923.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl)thio)propyl)-3-cyclobutylguanidine (6b). 1H NMR (500 MHz, Methanol-d4)
δ
8.55 (s, 1H), 8.29 (s, 1H), 8.21 (s, 1H), 5.99 (d, J = 4.8 Hz, 1H), 4.80 (t, J = 5.1 Hz, 1H), 4.59 (s,
1H), 4.34 (t, J = 5.1 Hz, 1H), 4.27–4.17 (m, 1H), 4.02–3.89 (m, 1H), 3.22 (t, J = 6.9 Hz, 2H),
3.04–2.90 (m, 2H), 2.62 (t, J = 7.1 Hz, 2H), 2.44–2.30 (m, 2H), 2.08–1.93 (m, 2H), 1.86–1.70 (m,
4H). 13C NMR (126 MHz, MeOD)
δ 157.39, 156.32, 153.95, 150.66, 141.49, 120.60, 90.28, 85.72,
74.72, 74.12, 47.59, 41.28, 35.24, 31.09, 30.58, 29.70, 15.72. HRMS m/z calc’d for C18H28N8O3S
[M + H]+: 437.2078; found: 437.2079.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl)thio)propyl)-3-cyclopentylguanidine (6c). 1H NMR (500 MHz, Methanol-d4)
δ
8.53 (s, 1H), 8.30 (s, 1H), 8.21 (s, 1H), 6.00 (d, J = 4.8 Hz, 1H), 4.80 (t, J = 5.1 Hz, 1H), 4.34
(t, J = 5.0 Hz, 1H), 4.23–4.17 (m, 1H), 3.87–3.77 (m, 1H), 3.24 (t, J = 6.9 Hz, 2H), 3.04–2.90
(m, 2H), 2.62 (t, J = 7.1 Hz, 2H), 2.03–1.93 (m, 2H), 1.86–1.78 (m, 2H), 1.77–1.68 (m, 2H),
1.67–1.59 (m, 2H), 1.55–1.45 (m, 2H). 13C NMR (126 MHz, MeOD)
δ 157.38, 157.02, 153.95,
150.66, 141.47, 120.59, 90.24, 85.70, 74.74, 74.10, 54.32, 41.31, 35.24, 33.62, 30.60, 29.75, 24.44.
HRMS m/z calc’d for C19H30N8O3S [M + H]+: 451.2234; found: 451.2236.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl)thio)propyl)-3-cyclohexylguanidine (6d). 1H NMR (500 MHz, Methanol-d4)
δ
8.48 (s, 1H), 8.30 (s, 1H), 8.21 (s, 1H), 6.00 (d, J = 4.8 Hz, 1H), 4.80 (t, J = 5.1 Hz, 1H), 4.34
(t, J = 5.0 Hz, 1H), 4.24–4.17 (m, 1H), 3.38–3.32 (m, 1H), 3.23 (t, J = 6.9 Hz, 2H), 3.03–2.89
(m, 2H), 2.62 (t, J = 7.1 Hz, 2H), 1.93–1.86 (m, 2H), 1.82 (p, J = 7.0 Hz, 2H), 1.78–1.72 (m,
2H), 1.67–1.59 (m, 1H), 1.42–1.32 (m, 2H), 1.31–1.15 (m, 3H). 13C NMR (126 MHz, MeOD)
δ
157.35, 156.52, 153.92, 150.65, 141.44, 120.57, 90.17, 85.66, 74.78, 74.07, 51.80, 41.24, 35.25,
33.77, 30.62, 29.72, 26.23, 25.71. HRMS m/z calc’d for C20H32N8O3S [M + H]+: 465.2391;
found: 465.2392.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
1
methyl)thio)propyl)-3-(cyclohexylmethyl)guanidine (6e). H NMR (500 MHz, Methanol-d4)
δ
8.54 (s, 1H), 8.30 (s, 1H), 8.21 (s, 1H), 6.00 (d, J = 4.8 Hz, 1H), 4.80 (t, J = 5.1 Hz, 1H),
4.34 (t, J = 5.0 Hz, 1H), 4.24–4.16 (m, 1H), 3.23 (t, J = 6.9 Hz, 2H), 3.04–2.91 (m, 4H), 2.63 (t,
J = 7.1 Hz, 2H), 1.88–1.79 (m, 2H), 1.78–1.72 (m, 4H), 1.71–1.64 (m, 1H), 1.58–1.47 (m, 1H),
1.33–1.14 (m, 3H), 1.01–0.89 (m, 2H). 13C NMR (126 MHz, MeOD)
δ 157.60, 157.37, 153.94,
150.65, 141.46, 120.58, 90.22, 85.68, 74.75, 74.09, 41.26, 38.68, 35.25, 31.61, 30.60, 29.71, 27.38,
26.81. HRMS m/z calc’d for C21H34N8O3S [M + H]+: 479.2547; found: 479.2548.

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1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl)thio)propyl)-3-phenylguanidine (6f). 1H NMR (500 MHz, Methanol-d4)
δ 8.47
(s, 1H), 8.37 (s, 1H), 7.43–7.34 (m, 2H), 7.33–7.27 (m, 3H), 6.05 (d, J = 4.9 Hz, 1H), 4.73 (t,
J = 5.1 Hz, 1H), 4.41 (s, 2H), 4.32 (t, J = 5.0 Hz, 1H), 4.25–4.17 (m, 1H), 3.03–2.87 (m, 2H),
2.59 (t, J = 7.1 Hz, 2H), 1.90–1.80 (m, 2H). 13C NMR (126 MHz, MeOD)
δ 156.94, 152.71,
150.10, 146.49, 143.72, 136.40, 131.07, 128.52, 126.45, 120.62, 90.50, 85.78, 75.28, 74.01, 41.62,
35.28, 30.59, 29.61. HRMS m/z calc’d for C20H26N8O3S [M + H]+: 459.1921; found: 459.1921.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl)thio)propyl)-3-benzylguanidine (6g). 1H NMR (500 MHz, Methanol-d4)
δ 8.47
(s, 1H), 8.37 (s, 1H), 7.40–7.35 (m, 2H), 7.33–7.26 (m, 3H), 6.05 (d, J = 4.9 Hz, 1H), 4.73 (t,
J = 5.1 Hz, 1H), 4.41 (s, 2H), 4.32 (t, J = 5.0 Hz, 1H), 4.24–4.18 (m, 1H), 3.29 (d, J = 6.9 Hz,
2H), 3.02–2.86 (m, 2H), 2.59 (t, J = 7.1 Hz, 2H), 1.89–1.79 (m, 2H). 13C NMR (126 MHz,
MeOD) δ 157.57, 153.32, 150.22, 147.64, 143.40, 137.74, 129.93, 129.01, 128.21, 90.45, 85.71,
75.19, 74.02, 45.91, 41.36, 35.27, 30.47, 29.65. HRMS m/z calc’d for C21H28N8O3S [M + H]+:
473.2078; found: 473.2079.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
1
methyl)thio)propyl)-3-phenethylguanidine (6h). H NMR (500 MHz, Methanol-d4)
δ
8.36
(s, 1H), 8.30 (s, 1H), 8.20 (s, 1H), 7.32–7.26 (m, 2H), 7.25–7.17 (m, 3H), 6.00 (d, J = 4.8 Hz,
1H), 4.80 (t, J = 5.1 Hz, 1H), 4.34 (t, J = 5.0 Hz, 1H), 4.24–4.17 (m, 1H), 3.43 (t, J = 7.1 Hz,
2H), 3.17 (t, J = 6.9 Hz, 2H), 3.02–2.89 (m, 2H), 2.85 (t, J = 7.1 Hz, 2H), 2.58 (t, J = 7.1 Hz,
2H), 1.81–1.70 (m, 2H). 13C NMR (126 MHz, MeOD)
δ 168.06, 157.43, 153.90, 150.64, 141.46,
139.38, 129.90, 129.68, 127.78, 120.57, 90.20, 85.68, 74.76, 74.08, 43.76, 41.20, 36.01, 35.23,
30.58, 29.59. HRMS m/z calc’d for C22H30N8O3S [M + H]+: 487.2234; found: 487.2235.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl)thio)propyl)-3-(3-phenylpropyl)guanidine (6i). 1H NMR (500 MHz, Methanol-
d4) δ 8.29 (s, 1H), 8.20 (s, 1H), 7.25 (t, J = 7.6 Hz, 2H), 7.19–7.14 (m, 3H), 6.00 (d, J = 4.9 Hz,
1H), 4.80 (t, J = 5.1 Hz, 1H), 4.34 (t, J = 5.0 Hz, 1H), 4.25–4.18 (m, 1H), 3.24–3.12 (m,
4H), 3.02–2.90 (m, 2H), 2.68–2.58 (m, 4H), 1.94–1.85 (m, 2H), 1.84–1.77 (m, 2H). 13C NMR
(
126 MHz, MeOD) δ 157.45, 157.32, 153.92, 150.62, 142.30, 141.39, 129.50, 129.38, 127.11,
120.55, 90.13, 85.65, 74.78, 74.06, 42.00, 41.25, 35.25, 33.68, 31.55, 30.60, 29.66. HRMS m/z
calc’d for C23H32N8O3S [M + H]+: 501.2391; found: 501.2394.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
1
methyl)thio)propyl)-3-(2-nitrophenyl)guanidine (6j). H NMR (500 MHz, Methanol-d4)
δ
8.56 (s, 1H), 8.30 (s, 1H), 8.21 (s, 1H), 8.11 (dd, J = 8.2, 1.5 Hz, 1H), 7.75 (td, J = 7.8, 1.5 Hz
1H), 7.56–7.47 (m, 2H), 6.00 (d, J = 4.8 Hz, 1H), 4.35 (t, J = 5.1 Hz, 1H), 4.26–4.17 (m, 1H),
3.06–2.91 (m, 2H), 2.70–2.62 (m, 2H), 1.92–1.83 (m, 2H). 13C NMR (126 MHz, MeOD)
,
δ
157.20, 156.61, 153.78, 150.48, 141.30, 138.16, 132.30, 130.99, 128.83, 124.61, 123.86, 120.43,
90.09, 85.55, 74.58, 73.94, 41.55, 35.09, 30.49, 29.29. HRMS m/z calc’d for C20H25N9O3S
[M + H]+: 504.1772; found: 504.1774.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
1
methyl)thio)propyl)-3-(2-bromophenyl)guanidine (6k). H NMR (500 MHz, Methanol-d4)
δ
8.55 (s, 1H), 8.30 (s, 1H), 8.21 (s, 1H), 7.75 (dd, J = 8.1, 1.4 Hz, 1H), 7.49–7.44 (m, 1H),
7.39 (dd, J = 7.9, 1.7 Hz, 1H), 7.36–7.30 (m, 1H), 6.00 (d, J = 4.8 Hz, 1H), 4.59 (s, 2H), 4.34
(t, J = 5.1 Hz, 1H), 4.24–4.17 (m, 1H), 3.04–2.91 (m, 2H), 2.65 (t, J = 7.1 Hz, 2H), 1.92–1.83
(m, 2H). 13C NMR (126 MHz, MeOD)
δ 157.71, 157.12, 154.29, 150.99, 141.81, 138.67, 132.81,
131.50, 129.34, 125.12, 124.37, 120.94, 90.60, 86.06, 75.09, 74.45, 42.06, 35.60, 31.00, 29.80.
HRMS m/z calc’d for C20H25BrN8O3S [M + H]+: 537.1026; found: 537.1026.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
methyl)thio)propyl)-3-(3-bromophenyl)guanidine (6l). 1H NMR (500 MHz, Methanol-
d4)
(dd, J = 8.4, 1.8 Hz, 1H), 6.00 (d, J = 4.8 Hz, 1H), 4.80 (t, J = 5.1 Hz, 1H), 4.35 (t, J = 5.1 Hz
1H), 4.28–4.17 (m, 1H), 3.33 (d, J = 6.9 Hz, 2H), 3.09–2.91 (m, 2H), 2.66 (t, J = 7.1 Hz, 2H),
1.96–1.81 (m, 2H). 13C NMR (126 MHz, MeOD)
157.37, 156.78, 153.95, 150.65, 141.47,
δ 8.51 (s, 1H), 8.30 (s, 1H), 8.21 (s, 1H), 7.54–7.43 (m, 2H), 7.35 (t, J = 8.0 Hz, 1H), 7.22
,
δ

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138.33, 132.47, 131.16, 129.00, 124.78, 124.03, 120.60, 90.26, 85.72, 74.75, 74.11, 41.72, 35.26,
30.66, 29.46. HRMS m/z calc’d for C20H25BrN8O3S [M + H]+: 537.1026; found: 537.1027.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
1
methyl)thio)propyl)-3-(4-bromophenyl)guanidine (6m). H NMR (500 MHz, Methanol-d4)
δ
8.34 (s, 1H), 8.30 (s, 1H), 8.21 (s, 1H), 7.62–7.55 (m, 2H), 7.16 (d, J = 8.3 Hz, 2H), 6.00 (d,
J = 4.8 Hz, 1H), 4.80 (t, J = 5.1 Hz, 1H), 4.34 (t, J = 5.1 Hz, 1H), 4.26–4.16 (m, 1H), 3.33 (s, 2H),
3.05–2.91 (m, 2H), 2.65 (t, J = 7.1 Hz, 2H), 1.93–1.81 (m, 2H). 13C NMR (126 MHz, MeOD)
δ
157.37, 156.81, 153.94, 150.66, 141.48, 135.91, 134.11, 128.09, 121.53, 120.60, 90.25, 85.73, 74.75,
74.11, 41.67, 35.25, 30.63, 29.51. HRMS m/z calc’d for C20H25BrN8O3S [M + H]+: 537.1026;
found: 537.1028.
1-(3-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)
1
methyl)thio)propyl)-3-(4-bromobenzyl)guanidine (6n). H NMR (500 MHz, Methanol-d4)
δ
8.61–8.48 (m, 1H), 8.29 (s, 1H), 8.21 (s, 1H), 7.56–7.49 (m, 2H), 7.27–7.20 (m, 2H), 6.00 (d,
J = 4.9 Hz, 1H), 4.80 (t, J = 5.1 Hz, 1H), 4.63 (d, J = 26.9 Hz, 2H), 4.37 (s, 2H), 4.34 (t, J = 5.1 Hz
1H), 4.23–4.17 (m, 1H), 3.26 (t, J = 6.8 Hz, 2H), 3.01–2.89 (m, 2H), 2.58 (t, J = 7.1 Hz, 2H),
1.85–1.76 (m, 2H). 13C NMR (126 MHz, MeOD)
157.55, 157.37, 153.95, 150.65, 141.46,
,
δ
137.13, 132.99, 130.09, 122.68, 120.59, 90.23, 85.67, 74.74, 74.10, 45.23, 41.37, 35.25, 30.54,
29.59. HRMS m/z calc’d for C21H27BrN8O3S [M + H]+: 551.1183; found: 551.1185.
11.4. PRMT1 Biochemical Assays and Enzyme Kinetics Study
A fluorescence-based SAHH-coupled assay was used to evaluate the IC50 values
of inhibitors by monitoring the production of SAH from SAM after methylation. The
condition of the assay in a final well volume of 100
25 mM NaCl, 25 M EDTA, 50 M TCEP, 0.01% Triton X-100, 5
10 M AdoMet, and 10 M ThioGlo4. After incubating with the inhibitors for 10 min at
37 ◦C, reactions were initiated by the addition of 5
M H4-21 peptide (Km value). The
µL was: 2.5 mM HEPES (pH = 7.0),
µ
µ
µ
M SAHH, 0.1 M PRMT1,
µ
µ
µ
µ
fluorescence signal was monitored on a BMG CLARIOstar microplate reader with excitation
at 400 nm and emission at 465 nm for 18 min. Data were processed using GraphPad Prism
software 7.0. All experiments were performed in duplicate.
Where the IC50 at Km value was less than the enzyme concentration, the condition
of the assay was modified to a final well volume of 100
25 mM NaCl, 25 M EDTA, 50 M TCEP, 0.01% Triton X-100, 5
M ThioGlo4. After incubating with the inhibitors for 10 min
M H4-21 peptide (4 Km value).
µL: 2.5 mM HEPES (pH = 7.0),
µ
µ
µ
M SAHH, 0.1 M PRMT1,
µ
10
µ
M AdoMet, and 10
µ
◦
at 37 C, reactions were initiated by the addition of 20
µ
The fluorescence signal was monitored on a BMG CLARIOstar microplate reader with
excitation at 400 nm and emission at 465 nm for 18 min. Data were processed using
GraphPad Prism software 7.0. All experiments were performed in duplicate. Data were
processed using GraphPad Prism software 7.0. Ki, app was calculated using the equation
Ki, app = IC50/(1 + [S]/Km).
11.5. Selectivity Assays
A fluorescence-based SAHH-coupled assay was applied to study the effect of the
compound on methyltransferase activity of PRMT7, NTMT1, SETD7, G9a, NNMT, and
SAHH. For PRMT7, the assay was performed in a final well volume of 100
(pH = 7.5), 50 mM KCl, 0.01% Triton X-100, 5 M SAHH, 0.2 M PRMT7, 3
and 15 M ThioGlo1. After incubation for 10 min with the inhibitor, reactions were initiated
by the addition of 60 M H4-21 peptide, and the reaction was monitored for 15 min. For
NTMT1, the assay was performed in a final well volume of 100 L: 25 mM Tris (pH = 7.5),
50 mM KCl, 0.01% Triton X-100, 5 M SAHH, 0.1 M NTMT1, 3 M AdoMet, and 10
ThioGlo4. After incubation for 10 min with the inhibitor, reactions were initiated by the
addition of 0.5 M GPKRIA peptide, and the reaction was monitored for 15 min. For SETD7,
the assay was performed in a final well volume of 100 L: 25 mM potassium phosphate
buffer (pH = 7.6), 0.01% Triton X-100, 5 M SAHH, 1 M SETD7, 2 M AdoMet, and 10
ThioGlo1. After incubation for 10 min with the inhibitor, reactions were initiated by the
µL: 25 mM Tris
µ
µ
µM AdoMet,
µ
µ
µ
µ
µ
µ
µM
µ
µ
µ
µ
µ
µM

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addition of 90
µM H3-21 peptide, and the reaction was monitored for 15 min. For G9a, the
assay was performed in a final well volume of 100
(pH = 7.6), 1 mM EDTA, 2 mM MgCl2, 0.01% Triton X-100, 5
10 M AdoMet, and 10 M ThioGlo4. For NNMT, the assay was performed in a final well
volume of 100 L: 25 mM Tris (pH = 7.5), 50 mM KCl, 0.01% Triton X- 100, 5 M SAHH,
0.1 µM NNMT, 10 M AdoMet, and 10 M ThioGlo1. After incubation for 10 min with the
inhibitor, reactions were initiated by the addition of 10 M nicotinamide, and the reaction
µL: 25 mM potassium phosphate buffer
µ
M SAHH, 0.1 M His-G9a,
µ
µ
µ
µ
µ
µ
µ
µ
was monitored for 18 min. The inhibitors were added at five concentrations: 100, 33.3, 11.1,
3.7, and 1.2 µM.
The effect of the inhibitors on the coupled enzyme, SAHH, activity was also evaluated.
The assay was performed in a final well volume of 100
KCl, 0.01% Triton X-100, 0.1 M SAHH, and 15 M ThioGlo1. After incubation for 10 min
with the compound, 0.5 M SAH was added to initiate the reactions. All experiments were
µL: 25 mM Tris (pH = 7.5), 50 mM
µ
µ
µ
performed in duplicate. Fluorescence was monitored on a BMG CLARIOstar microplate
reader with excitation at 380 nm and emission at 505 nm.
11.6. Inhibition Mechanism Studies
The fluorescence-based SAHH-coupled assay was employed for the enzyme kinetic
study. Varying concentrations of SAM (from 0.5 to 8 Km) with 5
µM fixed concentration of
H4-21 or varying concentration of H4-21 (from 0.5 to 8 Km) with 10
µM fixed concentration
of SAM was included in reactions with a series concentration of compounds. All the IC50
values were determined in triplicate. Fluorescence was monitored on a BMG CLARIOstar
microplate with excitation at 380 nm and emission at 505 nm. Data were processed using
GraphPad Prism software 7.0.
11.7. Molecular Docking
To evaluate the binding of II757 on selected PRMT isoforms, a Glide software of
Schrödinger Maestro 2021-1 release was implicated. The crystal structures of PRMT1,
PRMT4, PRMT5, and PRMT7 were downloaded from protein data bank (www.rcsb.org)
with PDB code of 6nt2, 6s79, 6ckc, and 4m37 (accessed on 26 April 2021), respectively.
The proteins were prepared by minimizing energy with OPLS_2005 force field. LigPrep
was used to sketch the chemical structure of II757 as the ionization state was generated
at pH 7.0
±
2.0. Glide is applied to generate grids for PRMT subtypes using the default
parameters without any protein constraint. A cubic box of specific dimensions centered
around the active site residues was generated for the proteins. The bounding box was set
to 12 Å
×
12 Å
×
12 Å. Flexible docking was performed into the prepared proteins using
extra precision mode by keeping all docking parameters as default. OPLS_2005 force field
was used to calculate the binding free energy, and 10 poses per docked ligand were saved.
11.8. Cellular Methylation Level
HEK293 cell line was cultured in DMEM media supplemented with 10% FBS and 1%
antibiotic-antimycotic (Gibco, Fisher, Waltham, MA, USA). The cells were maintained in a
tissue culture dish (Falcon 353003, Fisher, Waltham, MA, USA) until seeding at a density
of 0.03
×
106 cells/mL into a 24-well tissue culture plate (Falcon 353047, Fisher, Waltham,
MA, USA). The cells were incubated overnight at 37 ◦C in 5% CO2 with the lid on. They
were then treated with the inhibitors at different concentrations for 48 h. The media was
removed, the cells were washed three times with cold 1X PBS, and lysed on ice in cold
SDS-containing buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium
deoxycholate, 0.1% SDS, and Halt protease and phosphate inhibitor, 1861280) for 10 min.
The cell lysate was sonicated and centrifuged at 14,000× g for 10 min to collect the debris.
The protein was quantified using BCA (Pierce BCA Protein Assay Kit, 23225, Fisher,
Waltham, MA, USA) and equal amounts of protein were resolved by SDS-PAGE elec-
trophoresis and blotted on Transfer membrane (Immun-Blot PVDF Membranes, Bio-Rad,
Hercules, CA, USA Bio-Rad, 1620177). The membrane was blocked in blocking buffer (5%

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milk in 1X TBST) for 1 h at RT followed by overnight incubation with primary antibodies
(Histone H4R3me2a,◦ABclonal, Woburn, MA, USA, ABclonal, A2376, and Histone H4,
ABclonal, A1131) at 4 C. After washing three times with 1X TBST for 5 min each, the mem-
brane was incubated with HRP-linked secondary antibodies (Cell Signaling Technologies,
Danvers, MA, USA. Cell Signaling Technology, 7074S) at RT for 1 h. The membranes were
washed three times with 1X TBST for 5 min each, and the proteins were detected by ECL
(Bio-Rad, Hercules, CA, USA).
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/biom11060854/s1, NMR spectra, HRMS and HPLC analysis of compounds 6a–n.
Author Contributions: R.H. devised the project and conceived the original ideas. A.A.A.-H. prepared
and characterized compounds 6f and 6g. A.A.A.-H. also performed the molecular docking study.
I.D.I. synthesized, purified, and characterized the rest compounds. I.D.I. performed the selectivity
study, inhibition mechanism, and cellular methylation study. I.D.I. took the lead in writing the
manuscript, and R.H. edited the manuscript. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors acknowledge the support from the Department of Medicinal Chem-
istry and Molecular Pharmacology (RH) at Purdue University, West Lafayette, IN, USA.
Conflicts of Interest: The authors declare no competing financial interest.
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