Discovery and structure-activity analysis of 4-((5-nitropyrimidin-4-yl)amino)benzimidamide derivatives as novel protein arginine methyltransferase 1 (PRMT1) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.06.095

Despite a potential application of PRMT1 inhibitors in cancer treatment, very few of PRMT1 inhibitors have been reported. To obtain novel potent PRMT1 inhibitors, structure optimizations towards a hit compound, 4-((6-chloro-5-nitropyrimidin-4-yl)amino)ben

Full text of DOI:10.1016/j.bmcl.2015.06.095

Accepted Manuscript
Discovery and Structure-Activity Analysis of 4-((5-nitropyrimidin-4-yl) amino)
benzimidamide Derivatives as Novel Protein Arginine Methyltransferase 1
(
PRMT1) Inhibitors
Xu-Ri Yu, Yun Tang, Wen-Jing Wang, Sen Ji, Shuang Ma, Lei Zhong, Chun-
Hui Zhang, Jiao Yang, Xiao-Ai Wu, Zheng-Yan Fu, Lin-Li Li, Sheng-Yong
Yang
PII:
S0960-894X(15)00700-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.095
BMCL 22885
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
3 February 2015
10 June 2015
25 June 2015
Please cite this article as: Yu, X-R., Tang, Y., Wang, W-J., Ji, S., Ma, S., Zhong, L., Zhang, C-H., Yang, J., Wu, X-
A., Fu, Z-Y., Li, L-L., Yang, S-Y., Discovery and Structure-Activity Analysis of 4-((5-nitropyrimidin-4-yl) amino)
benzimidamide Derivatives as Novel Protein Arginine Methyltransferase 1 (PRMT1) Inhibitors, Bioorganic &
Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.06.095
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Discovery and Structure-Activity Analysis of 4-((5-nitropyrimidin-4-yl) amino)
benzimidamide Derivatives as Novel Protein Arginine Methyltransferase 1
(PRMT1) Inhibitors
a,†
b,†
a,†
a
a
a
Xu-Ri Yu , Yun Tang , Wen-Jing Wang , Sen Ji , Shuang Ma , Lei Zhong ,
a
a
a
a
b
Chun-Hui Zhang , Jiao Yang , Xiao-Ai Wu , Zheng-Yan Fu , Lin-Li Li , and
a,
Sheng-Yong Yang *
a
State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy,
West China Hospital, West China Medical School, Sichuan University, Chengdu,
Sichuan 610041, China
b
West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041,
China
†
These authors contributed equally.
*
Corresponding Author:
Sheng-Yong Yang. Tel: +86-28-85164063; Fax: +86-28-85164060;
E-mail: yangsy@scu.edu.cn.
Lin-Li Li. E-mail: ysylilinli@sina.com.

Abstract
Despite a potential application of PRMT1 inhibitors in cancer treatment, very few of
PRMT1 inhibitors have been reported. To obtain novel potent PRMT1 inhibitors,
structure optimizations towards a hit compound, 4-((6-chloro-5-nitropyrimidin-4-yl)
amino) benzimidamide, were carried out. A series of 4-((5-nitropyrimidin-4-yl) amino)
benzimidamide derivatives were synthesized. Structure-activity relationship analysis
led to the discovery of a number of PRMT1 inhibitors. The most potent compound
corresponds to compound 6d, which showed an IC value of 2.0 μM against PRMT1.
50
This compound also displayed a considerable anti-proliferative activity against three
tumor cell lines, DLD-1, T24 and SH-SY-5Y, with IC values of 4.4 μM, 13.1 μM
5
0
and 11.4 μM, respectively.
Keywords: PRMT1, small molecule inhibitor, epigenetics, structure-activity
relationship, protein arginine methyltransferase.
Protein arginine methyltransferase 1 (PRMT1) is an important member of PRMT
subfamily, which catalyzes the transfer of a methyl group from the co-factor
S-adenosyl methionine (SAM) onto a specific arginine residue of its substrate
1
-3
proteins. PRMT1 has been shown to be responsible for over 90% of the arginine
methylation in mammalian cells and has a wide range of substrates involved in many
biological processes, such as transcription activation, signal transduction, chromatin
4
-8
modification, RNA splicing and DNA repair.
A number of studies have
demonstrated that overexpression or dysregulation of PRMT1 appeared to be
8
-16
associated with many diseases, particularly cancer.
PRMT1 is thus thought to be a

promising target for the treatment of cancer. For example, Yoshimatsu et al. revealed
that abrogation of the expression of PRMT1 by specific siRNAs suppressed growth of
1
7
bladder cancer cells significantly. Yan et al. showed that PRMT1 inactivation could
1
8
effectively inhibit proliferation of leukemia cells. A study by Wang et al.
demonstrated that PRMT1 inhibitors could significantly suppress the growth of
1
9
castrate-resistant prostate cancer cells.
Due to the potential applications of PRMT1 inhibitors in cancer therapy,
discovery of PRMT1 inhibitors has recently attracted much attention. To date, a
number of PRMT1 inhibitors have been reported. Figure 1 shows chemical structures
2
0
21
of some typical known PRMT1 inhibitors, including AMI-1, stilbamidine,
22
18
21
23
RM65, DB75, Allantodapsone and E84. However, most of the known PRMT1
inhibitors have very weak potency against PRMT1, which restricts their application in
the disease treatment. Therefore, discovery of potent PRMT1 inhibitors is strongly
demanded at this moment.
Figure 1. Structures of several typical known PRMT1 inhibitors
During our initial screening campaign, we identified a hit compound,
4
-((6-chloro-5-nitropyrimidin-4-yl) amino) benzimidamide (1, Figure 2), which

showed an IC (the half-maximum inhibitory concentration) value of 19μM against
5
0
PRMT1. In this investigation, we shall perform structural modifications to this
compound. A series of 4-((5-nitropyrimidin-4-yl) amino) benzimidamide derivatives
will be synthesized and their structure-activity relationship (SAR) will also be
discussed. To the most active compound, in vitro anti-proliferative activities against
different tumor cell lines and mechanisms of action will be further investigated.
Figure 2. (a) The structure of hit compound 1. (b) Schematic showing regions that
were the focus of structural modifications.
The SAR analysis below will mainly focus on the 4-position of phenyl group and
the 6-position of pyrimidine ring (see Figure 2(b)). Firstly, various substituents were
used to replace the original formamidine moiety in the 4-position of phenyl ring. In
this case, the 6-position of pyrimidine was fixed as chloride. We synthesized a total of
5
compounds. These compounds (3a-e) were easily prepared through a nucleophilic
substitution reaction between commercially available 4, 6-dichloro-5-nitropyrimidine
and 4-position substituted aniline (see Scheme 1). Unfortunately, compounds with
formamidine replaced by other groups did not show an obvious inhibitory activity
against PRMT1 (IC > 100μM, Scheme 1), implying the importance of formamidine
5
0
moiety at the 4-position of phenyl group.

Scheme 1. General synthetic routes for compounds 3a-3e and bioactivities of these
compounds against PRMT1. Reagents and conditions: (a) THF, TEA, 0°C, 60-80%.
Secondly, we optimized the 6-position of pyrimidine ring. A total of 10
compounds with different substituents at the 6-position of pyrimidine and the
4
-position of phenyl group being fixed as the optimal formamidine group. Scheme 2
depicts the synthetic routes for these compounds (6a-j). In these routes, compound 1
acted as an intermediate, which was prepared through a nucleophilic substitution
reaction between commercially available 4, 6-dichloro-5-nitropyrimidine and
4
-Aminobenzamidine dihydrochloride. The intermediate then reacted with various
amines under the temperature of 55°C to afford compounds 6a-j. Bioactivities of
compounds 6a-j are shown in Table 1. Compounds 6a-e exhibited considerable
potency against PRMT1 with IC values between 2.0μM and 38μM. On the contrary,
5
0
compounds 6f-j showed a relatively poor activity; their inhibitory rates against
PRMT1 are all less than 50% at a concentration of 50μM. Among all the compounds,
6
d is the most active one, which is 10 times more potent than AMI-1 (IC : 2.0μM vs
50

2
1,22
2
2μM), an often used positive control or probe in literature
.
Scheme 2 General synthetic routes for compounds 6a-j. Reagents and conditions: (a)
THF/EtOH,TEA,0°C,15%; (b) THF/EtOH,TEA,55°C,7-25%.

Table 1 Bioactivities of compounds 6a-j and AMI-1.
a
2
IC50 (μM) or
Compound
R
a
Inhibition rate (%@50μM)
6
a
-NH2
38
6
b
9.3
6
c
13
6
d
2.0
6
e
25
6
f
28%(@50μM)
6
6
g
h
44%(@50μM)
49%(@50μM)
28%(@50μM)
6
6
i
j
9%(@50μM)
AMI-1
SAH
-
-
22
0.5
a
All assays were conducted in duplicate.
Since 6d is the most active compound, further studies including selectivity,
cellular activity and mechanistic characterization were conducted on this compound.
We first tested the selectivity of 6d for PRMT1 against several selected members of
PRMT family, including coactivator-associated arginine methyltransferase 1(CARM1,
PRMT4), PRMT5 and PRMT6. These PRMTs were chosen mostly because of the
reasons mentioned as follows. PRMTs are mainly classified into two categories, type I

and type II, with PRMT1 being representative of type I and PRMT5 of type II.
CARM1 and PRMT6 were selected for determining the selectivity profile of 6d on
other type I PRMTs. The results are presented in Table 2. 6d did not show an obvious
activity against PRMT5 (IC50 > 100μM). Among the members of type I PRMTs, it
displayed almost no activity against PRMT6 (IC50 > 100μM), and a relatively weak
activity against CARM1 (IC = 10μM). These results indicate that 6d has a good
5
0
selectivity for PRMT1 against other selected PRMTs.
a
Table 2. Inhibitory activity ( IC , μM) of 6d against PRMT1 and several other
5
0
selected PRMTs
PRMT4
compound
PRMT1
2.0
PRMT5
>100
PRMT6
>100
6
d
10
a
All assays were conducted in duplicate.
Then, the in vitro antitumor activities of compound 6d and other structurally
related but weaker compounds such as 6f and 6g were tested by the MTT assay
method. In this assay, six human tumor cell lines including DLD-1(colon cancer), T24
(bladder cancer), SH-SY-5Y (neuroblastoma), A375 (melanoma), MCF-7 (breast
cancer) and UMUC-3 (bladder cancer) were chosen. Table 3 shows IC values of
5
0
compound 6d, 6f, 6g and AMI-1 against these cell lines. Compound 6d showed
considerable activity against colon cancer DLD-1 cells with an IC value of 4.4μM,
5
0
and moderate potencies against T24, SH-SY-5Y, MCF-7 and UMUC-3 cells, but no
activity against A375 cells. Compound 6f, 6g and AMI-1 displayed obviously weaker
potencies or no activity against these cancer cells compared with 6d. The reasons
might be complicated, but one of main causes should be due to their poorer activity
against PRMT1 compared with that of 6d.

Table 3. Inhibitory activities of compound 6d, 6f, 6g and AMI-1 against six selected
human cancer cell lines by MTT assay
a
IC (μM)
5
0
compound
DLD-1
4.4
T24
13.1
86.8
18.3
>100
SH-SY-5Y A375
MCF-7 UMUC-3
6
d
11.4
92.4
16.2
>100
>100
>100
87.6
50.6
>100
58.7
45.4
84.6
65.4
>100
6
f
8.5
6
g
83.8
>100
AMI-1
>100
>100
a
All assays were conducted in duplicate.
Because DLD-1 is the most sensitive cell line to compound 6d, western blot
assay was then used to examine the ability of 6d to inhibit PRMT1 in intact DLD-1
cells. As shown in Fig 3, the expression level of asymmetric dimethylation of histone
H4R3 (H4R3me2a), which is a substrate of PRMT1, significantly reduced at
concentrations of 6d larger than 3μM. This result is consistent with its anti-viability
potency against DLD-1. On the contrary, 6d did not exhibit any effect on the
symmetric dimethylation of histone H3R8 (H3R8me2). This is understandable since
H3R8me2 is not the substrate of PRMT1.

Figure 3. Western blot analysis showing the ability of 6d to inhibit PRMT1 in intact
DLD-1 cells.
To clarify the inhibition mechanism of compound 6d, steady state kinetic
characterization was performed. The initial AlphaLISA signals were measured at
several selected concentrations of 6d over a range of varied SAM concentrations
when fixing peptide substrate concentration (Figure 4). The data were used to fit with
1
8
Michaelis-Menten kinetic equation , and a K of 1.7 μM and a K of 1.5 μM were
i
m
obtained. The resemblance of the K and K values suggests that compound 6d is
i
m
classic noncompetitive inhibitor with respect to SAM.
6
d MOA on PRMT1
6
d Conc. (M)
2
50000
00000
50000
00000
0000
2
9
3
1
0
0
7
2
1
.33
1
5
0
0
2
4
6
8
10
SAM (M)
Figure 4. Kinetic analysis of PRMT1 inhibition by compound 6d.
Finally, molecular docking was performed to further understand the possible
binding mode of compound 6d with PRMT1. Since the crystal structure of human
PRMT1 (hPRMT1) had not been solved so far, we generated a homology model of
hPRMT1 structure based on the reported rat PRMT1 (rPRMT1) structures (PDB
codes: 1OR8, 1ORI, 1ORH). Details for the molecular docking and homology
modeling were given in Supplementary data. Figure 5 shows a predicted binding
mode of compound 6d with PRMT1. As can be seen, the docking program positioned
6
d in the substrate binding pocket in such a way that its formamidine moiety forms

strong hydrogen bonding interactions with residues Glu152, Glu161 and Met154. At
the same time, the nitrile group of 6d provides a nitrogen atom as a hydrogen bond
receptor to form a hydrogen bond with Lys40 residue, in addition to the electrostatic
interaction existed between nitrile nitrogen (negative charge) and Lys40 side chain
nitrogen (positive charge).
Figure 5. The predicted binding mode of compound 6d with PRMT1.
In summary, we synthesized a series of 4-((5-nitropyrimidin-4-yl) amino)
benzimidamide derivatives in this investigation. The SAR analysis led to the
discovery of a potent PRMT1 inhibitor, namely compound 6d. This compound
showed a good inhibitory activity and selectivity for PRMT1 with an IC value of
5
0
2
.0μM. Compound 6d also showed considerable potency against three tumor cell
lines, DLD-1, T24 and SH-SY-5Y. Collectively, compound 6d could be used as a
chemical probe to investigate new functions of PRMT1 in biology and also as a good
lead compound for further lead-to-candidate optimization.
Acknowledgments
This work was supported by the 863 Hi-Tech Program (2012AA020301,
2
8
012AA020308), the National Natural Science Foundation of China (81172987,
1325021, 81473140), the National Science and Technology Major Project

(2012ZX09501001-003, 2012ZX09102-101-002), and SRFDP (20100181110025).
Supporting Information: Supplementary data associated with this article can be
found in the online version.
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Graphical Abstract