Design and synthesis of selective, small molecule inhibitors of coactivator-associated arginine methyltransferase 1 (CARM1)

Article abstract of DOI:10.1039/c6md00342g

Coactivator-associated arginine methyltransferase 1 (CARM1) is a type I protein arginine methyltransferase (PRMT) that catalyzes the conversion of arginine into monomethylarginine (MMA) and further into asymmetric dimethylarginine (ADMA). CARM1 methylates histone 3 arginines 17 and 26, as well as numerous non-histone proteins including CBP/p300, SRC-3, NCOA2, PABP1, and SAP49, while also functioning as a coactivator for various proteins that have been linked to cancer such as p53, NF-κβ, β-catenin, E2F1 and steroid hormone receptor ERα. As a result, CARM1 is involved in transcriptional activation, cellular differentiation, cell cycle progression, RNA splicing and DNA damage response. It has been associated with several human cancers including breast, colon, prostate and lung cancers and thus, is a potential oncological target. Herein, we present the design and synthesis of a series of CARM1 inhibitors. Based on a fragment hit, we discovered compound 9 as a potent inhibitor that displayed selectivity for CARM1 over other PRMTs.

Full text of DOI:10.1039/c6md00342g

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J. Jin, Med. Chem. Commun., 2016, DOI: 10.1039/C6MD00342G.
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DOI: 10.1039/C6MD00342G
MedChemComm
RESEARCH ARTICLE
Design and synthesis of selective, small molecule inhibitors of
coactivator-associated arginine methyltransferase 1 (CARM1)†‡
Received 00th January 20xx,
Accepted 00th January 20xx
H. Ü. Kaniskan,§,a M. S. Eram, §,b J. Liu,a D. Smil,b M. L. Martini,a Y. Shen,a V. Santhakumar,b P. J.
Brown,b C. Arrowsmith,b,c M. Vedadi,*,b,d and J. Jin*,a
DOI: 10.1039/x0xx00000x
Coactivator-associated arginine methyltransferase 1 (CARM1) is a type I protein arginine methyltransferase (PRMT) that
catalyzes the conversion of arginine into monomethylarginine (MMA) and further into asymmetric dimethylarginine
(ADMA). CARM1 methylates histone 3 arginines 17 and 26, as well as numerous non-histone proteins including CBP/p300,
SRC-3, NCOA2, PABP1, and SAP49, while also functioning as a coactivator for various proteins that have been linked to
cancer such as p53, NF-κβ, β-catenin, E2F1 and steroid hormone receptor ERα. As a result, CARM1 is involved in
transcriptional activation, cellular differentiation, cell cycle progression, RNA splicing and DNA damage response. It has
been associated with several human cancers including breast, colon, prostate and lung cancers and thus, is a potential
oncological target. Herein, we present the design and synthesis of a series of CARM1 inhibitors. Based on a fragment hit,
we discovered compound 9 as a potent inhibitor that displayed selectivity for CARM1 over other PRMTs.
www.rsc.org/medchemcomm
Several high throughput screening (HTS) campaigns resulted in
Introduction
the identification of pyrrazole-amide as well as benzo[d]imidazole
19
CARM1 inhibitors.18,
These initial reports were followed by
Coactivator-associated arginine methyltransferase 1 (CARM1,
also known as PRMT4) is a type I protein arginine methyltransferase
(PRMT) along with PRMT1, PRMT3, PRMT6 and PRMT8, which are
responsible for converting arginine into monomethylarginine
(MMA) and further into asymmetric dimethylarginine (ADMA).1
structure-activity relationship (SAR) studies, which led to the
discovery of small molecule inhibitors 1 and 2 with IC50 values
around 30 nM. These inhibitors displayed selectivity for CARM1
over PRMT1 and PRMT3 while the selectivity over other PRMTs was
not reported (Figure 1).20-23 We have recently reported a type I
PRMT chemical probe, MS023 (3), which is potent against all type I
PRMTs, but inactive against type II and type III PRMTs as well as
other methyltransferases.24 A series of adenosine-based CARM1
inhibitors with high potency and selectivity over PRMT1 and PRMT6
was also published.25 Herein we report the discovery of selective
CARM1 inhibitors and describe their design, synthesis and biological
evaluation.
CARM1 methylates histone 3 arginines 17 and 26,2, as well as
3
numerous non-histone proteins including CBP/p300, SRC-3, NCOA2,
PABP1, SmB, HuR, HuD, CA150, SAP49, and U1C.1 Therefore, it plays
a role in cellular processes such as transcriptional activation,4 RNA
splicing,4, 5 cellular differentiation,6 cell cycle progression7 and DNA
repair.8 It has been shown that the enzymatic activity of CARM1 is
essential for the most of its in vivo functions.9 CARM1 functions as a
coactivator for various proteins that have been linked to cancer
including p53, NF-κβ, β-catenin, E2F1 and steroid hormone
10-12
receptor ERα.1,
Overexpression of CARM1 in several human
cancers including breast,7 colon,13 prostate14 and lung15 cancers has
been reported. Thus, CARM1 is a potentially attractive therapeutical
target for cancer and as a result there have been numerous studies
directed toward the discovery of small molecule CARM1
inhibitors.16, 17
Figure 1. Structures of known CARM1 inhibitors 1, 2 and the
type I PRMT chemical probe 3.
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Results and Discussion
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DOI: 10.1039/C6MD00342G
Table 1. IC50 values of compounds 5, 7-12.
The alkyl-diamino tail, shown to be bound in the substrate-binding
site of CARM1,21 is the shared feature of inhibitors 1-3 (Figure 1) as
well as of a recently published PRMT6 inhibitor.26 In a recent study,
a commercially available, diverse fragment library of compounds
mimicking this alkyl-diamino tail were tested against PRMT6 and
compound 4 was reported as a PRMT6 inhibitor with IC50 of 300 ±
40 nM (Figure 2).27 Compound 4 was 3- and 7-fold less potent for
CARM1 (IC50 = 1,000 ± 40 nM) and PRMT8 (IC50 = 2100 ± 200 nM),
respectively. It was not very potent against PRMT1 and PRMT3
(>40-fold less potent) and did not inhibit other methyltransferases.
We sought to discover potent CARM1 selective inhibitors by using
the fragment hit 4 as a starting point. Our initial SAR studies
resulted in compounds 5 and 6 as promising CARM1 inhibitors
(Figure 2). Compound 5 shares the (piperidinyl)ethan-1-amine core
of 4, but it is connected to the phenyl group via a methylene-amine
(-CH2NH-) linker instead of methylene (-CH2-) linker. It was found to
be 10-fold more potent for CARM1 (IC50 = 471 ± 36 nM) over PRMT6
(IC50 = 4,564 ± 1210 nM), exhibiting some selectivity (Figure 2). In
addition, it had no appreciable inhibitory activity against PRMT1,
PRMT3 and PRMT8 as well as PRMT5 and PRMT7 (IC50 > 50,000 nM
CARM1
PRMT6
IC50 (nM)
Cmpd
R
IC50 (nM)
471 ± 36
223 ± 57
5
7
8
H
F
4,560 ± 1210
1,890 ± 456
2,920 ± 303
Cl
157 ± 54
94 ± 23
9
Br
2,160 ± 68
10
CF3
302 ± 41
2,980 ± 298
11
12
OCF3
Ph
342 ± 48
330 ± 43
4,730 ± 174
120 ± 13
We then focused our attention on the (azetidinyl)ethan-1-amine
scaffold represented by compound 6 (Figure 2). Since this scaffold
with an oxygen linker did not show good selectivity over either
PRMT6 or PRMT8, we utilized the methylene-amine (-CH2NH-)
linker as in the piperidinyl series and synthesized substituted phenyl
derivatives 13-18 (Table 2). While there was an overall increase in
potency of these inhibitors as compared to compounds 7-12 for
CARM1, the potency for PRMT6 was even greater in comparison
and thus, the selectivity over PRMT6 suffered significantly. For
example the meta-chloro (14) and meta-bromo (15) derivatives
were potent for CARM1 with IC50 of 50 ± 14 and 67 ± 9 nM,
respectively. However, 14 and 15 displayed high potency for PRMT6
with IC50 of 133 ± 8 and 249 ± 22 nM respectively, resulting in only 3
to 4-fold selectivity for CARM1 over PRMT6.
for all). On the other hand, compound
6 contains an
(azetidinyl)ethan-1-amine core and –CH2O- connection to the aryl
ring (Figure 2). This inhibitor displayed good potency for CARM1
(IC50 = 144 ± 37 nM) and was less than 10-fold selective over PRMT6
and PRMT8 (IC50 = 1,079 ± 75 and 1,200 ± 70 nM, respectively)
while it was inactive against other PRMTs.
Since inserting a nitrogen between the piperidine and benzyl
groups of compound 4 (compound 5, Figure 2) resulted in some
selectivity for CARM1 over PRMT6, we further explored this scaffold
and synthesized derivatives of 5 by adding substituents on the
phenyl ring (Table 1). We mainly investigated meta-substitution
based on the encouraging potency of compound 6 for CARM1.
Among the meta-fluoro (7), meta-chloro (8) and meta-bromo (9)
derivatives, 9 displayed the best potency for CARM1 with IC50 of 94
The synthetic route for the preparation of compounds 7-12 is
shown in Scheme 1. The synthesis started with a nucleophilic
displacement reaction between 4-amino-1-Boc-piperidine (19) and
± 23 nM as well as selectivity over PRMT6 (around 20–fold, IC50
=
2,160 ± 68 nM). The electron-withdrawing trifluoromethyl (10), or
electron-donating triflourometoxy (11) substitution did not result in
a more potent or selective inhibitor than 9. The phenyl-substituted
Table 2. IC50 values of compounds 13-18.
derivative (12) reversed the selectivity in favour of PRMT6 (IC50
=
120 ± 13 nM) over CARM1 (IC50 = 330 ± 43 nM).
CARM1
IC50 (nM)
PRMT6
IC50 (nM)
Cmpd
R
13
14
F
130 ± 29
50 ± 14
101 ± 7
133 ± 8
Cl
15
16
17
18
Br
CF3
OCF3
Ph
67 ± 9
94 ± 17
90 ± 25
69 ± 10
249 ± 22
469 ± 64
434 ±27
67 ± 13
Figure 2. Structures of the fragment hit 4, and initial hits 5 and
6.
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RESEARCH ARTICLE
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DOI: 10.1039/C6MD00342G
Scheme 1. General scheme for the synthesis of compounds 5, 7-
12. Conditions: a. Et3N, DCM b. Methanolic HCl (3N) c. Et3N,
NaBH(OAC)3 d. Methanolic HCl (3N).
meta-substituted benzyl bromides (20). The resulting diamines
were then treated with methanolic HCl to remove the tert-butoxy
carbamate group. The reductive amination of amines 21 with N-
Boc-(methylamino)acetaldehyde (22), followed by a deprotection
reaction, afforded the desired compounds (5 and 7-12). A similar
synthetic route was employed for the synthesis of compounds 13-
18 starting with 3-amino-1-Boc-azetidine (see supporting
information for detailed procedures).
Among all the derivatives synthesized, compound 9 emerged as
the best inhibitor with good potency and around 20-fold selectivity
toward CARM1 over PRMT6. In addition, we have tested Inhibitor 9
against other PRMTs in biochemical assays and found that it
showed excellent selectivity for CARM1 over other type I PRMTs,
PRMT1, PRMT3 (IC50 > 50,000 nM) and PRMT8 (IC50 = 9,200 ± 500
Figure 3. MOA study of compound 9 against CARM1 with
changing concentrations of the peptide substrate (top) and the
cofactor SAM (bottom).
nM) as well as the type II and type III PRMTs, PRMT 5 and 7 (IC50
50,000 nM).
>
Conclusions
To assess the mechanism of action (MOA) of inhibitor 9, we
evaluated the effect of the peptide substrate and cofactor S-
adenosyl-L-methionine (SAM) concentrations on IC50 values of 9
against CARM1. As shown in Figure 3, increasing the peptide
substrate or SAM concentrations had no effect on the IC50 values of
9 against CARM1, suggesting that this inhibitor is noncompetitive
with both the cofactor SAM and peptide substrate. It has been
previously reported that active site binding inhibitors can display
noncompetitive behavior in MOA studies.28, 29 We and others have
observed this phenomenon with PRMT inhibitors.24, 26 For example,
MOA studies of MS023 (3) suggested that the inhibitor was
noncompetitive with both SAM and peptide substrate while the X-
ray crystal structure of MS023 in complex with PRMT6 clearly
showed that it occupies the substrate binding pocket. Therefore,
based on the literature precedents, it is very likely that inhibitor 9
interacts with the substrate binding site of CARM1 to assert its
inhibitory effect.
Starting from a reported fragment hit (4), a PRMT6 inhibitor, we
discovered compound 9, a potent and selective inhibitor of CARM1,
through a SAR study. In biochemical assays, 9 displayed high
potency (IC50 = 94 ± 23 nM) for CARM1 and was around 20-fold
selective for CARM1 over PRMT6 and highly selective over PRMT1,
PRMT3, PRMT8 as well as PRMT5 and PRMT7. We believe that
inhibitor 9 could be a useful tool for studying the role of CARM1 in
health and disease. We anticipate that the work described here will
facilitate further development of CARM1 selective inhibitors and
chemical tools.
Acknowledgements
The research described here was supported by the grant
R01GM103893 (to J. J.) from the U.S. National Institutes of
Health. The Structural Genomics Consortium (SGC) is a
registered charity (number 1097737) that receives funds from
AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada
Foundation for Innovation, Eshelman Institute for Innovation,
Genome Canada, Innovative Medicines Initiative (EU/EFPIA)
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DOI: 10.1039/C6MD00342G
Structure-activity relationship studies, starting from a fragment hit, resulted in
discovery of the compound 9, an inhibitor of CARM1 with high potency and
selectivity.
Br
H
N
N
Me
N
H
9
CARM1 (IC50) = 94 ± 23 nM