Pyrazole inhibitors of coactivator associated arginine methyltransferase 1 (CARM1)

Article abstract of DOI:10.1016/j.bmcl.2008.06.026

This study reports the identification and Hits to Leads optimization of inhibitors of coactivator associated arginine methyltransferase (CARM1). Compound 7b is a potent, selective inhibitor of CARM1.

Full text of DOI:10.1016/j.bmcl.2008.06.026

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4438–4441
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazole inhibitors of coactivator associated arginine
methyltransferase 1 (CARM1)
*
Ashok V. Purandare , Zhong Chen, Tram Huynh, Suhong Pang, Jieping Geng, Wayne Vaccaro,
Michael A. Poss, Jonathan Oconnell, Kimberly Nowak, Lata Jayaraman
Bristol-Myers Squibb Pharmaceutical Research and Development, Princeton, NJ 08543, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This study reports the identification and Hits to Leads optimization of inhibitors of coactivator associated
arginine methyltransferase (CARM1). Compound 7b is a potent, selective inhibitor of CARM1.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 23 April 2008
Revised 6 June 2008
Accepted 9 June 2008
Available online 12 June 2008
Keywords:
Coactivator associated arginine
methyltransferase 1
CARM1
PRMT4
Protein arginine methyltransferase
Members of the protein arginine methyl transferase (PRMT) fam-
ily have been implicated in a wide variety of cellular processes
including nuclear hormone receptor (NHR) mediated signaling, pro-
tein–protein interactions, protein trafficking, mRNA splicing and
processing, and transcriptional regulation.1 The family, which con-
sists of at least 9 members (PRMTs 1–9), catalyzes the generation
of asymmetric (type I) or symmetric (type II) di-methyl arginine res-
idues on substrate proteins using S-adenosyl methionine (SAM) as
the methyl donor.2 CARM1 (or PRMT4), a type I enzyme, has been
shown to methylate a wide variety of substrate proteins that can
be roughly classified into three categories. In the first category,
CARM1 methylates histone H3 and p300/CBP, thereby altering chro-
matin architecture and impacting transcriptional initiation.3 Thus,
the recruitment of CARM1 to the promoter regions of genes regu-
lated by transcription factors such as nuclear hormone receptors,
during the progression of prostate cancer.6 Over-expression of
CARM1 is seen in both androgen-stimulated and castration-resis-
tant prostate cancer tumors. A convincing argument can therefore
be made to support the hypothesis that targeting CARM1 would
be a viable approach for anti-cancer therapy.
To date, there have been only a few publications describing
small molecule chemical modulators of the PRMTs,7 and none of
these report selectivity toward inhibition of CARM1. For instance,
Bedford and colleagues have identified compounds that inhibit
arginine, but not lysine methylation by multiple PRMTs in vitro,
and do not interfere with Ado-Met binding. The exact mechanism
of inhibition by these compounds is unclear especially since some
of the compounds can inhibit the methylation of some substrates,
but not others. Therefore, the identification of selective CARM1
inhibitors as tools to interrogate its function in cells would be of
significant interest. Herein, we report Hits to Lead SAR studies that
led to the identification of a selective potent pyrazole inhibitor (7b)
of CARM1.
From a high throughput screening effort, a pyrazole amide
derivative (1) and closely related analogs were identified as ‘hits’
with modest activity in the CARM1 mediated methylation assay
(Fig. 1).8 Based on these hits, we initiated Hits to Lead efforts to fur-
ther improve the in vitro potency of this series (see Table 1).
First, we embarked on exploration of the pyrazole amide (East
end of core). The required scaffold was synthesized using the re-
ported intermediate 2.9,10 SAR from this exercise indicated that
truncation of the biphenyl moiety was permitted (5d) whereas
small alkyl amides were not tolerated (5a–b) (see Scheme 1).
NF-jB, p53, etc. and the subsequent methylation of histone H3 re-
sult in increased gene expression by making the chromatin more
accessible to the transcriptional machinery. In the second category,
methylation by CARM1 of proteins involved in splicing such as SmB,
U1C, SAP49, and CA150 implicates CARM1 in regulating alternative
splicing.4 Finally, methylation of RNA-binding proteins such as HuR,
HuD, and PABP indicates a role for CARM1 in mRNA processing and
stabilization.5 Taken together, these observations point to a crucial
role for CARM1 in modulating gene expression at multiple critical
levels. More recently, CARM1 has been shown to be up-regulated
* Corresponding author. Tel.: +1 609 252 4320; fax: +1 609 252 7446.
E-mail address: ashok.purandare@bms.com (A.V. Purandare).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.026

A. V. Purandare et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4438–4441
4439
F3C
F3 C
F3C
F
H
N
H
OH
N
N
N
SO 2 CH 3
N
a, b, c,
d
N
N
Core
SO2CH3
N
O
O
East end
1
O
CN
6
2
HN
R
HN
NH2
O
CARM1 IC50 1.8 μM
O
Scheme 2. Reagents: (a) 3-fluoro-20-(methylsulfonyl)biphenyl-4-amine, EDCI,
HOAt, Hunig’s base, CH2Cl2, 90%; (b) H2, Pd/C (10%), TFA (cat), MeOH, 80%; (c)
HCO2(CR1R2)n NHBoc.
South end
Figure 1. Initial Hit.
Table 2
SAR for South end
Table 1
Preliminary SAR for East end
F3C
F3C
F
H
N
N
R
N
SO2CH3
N
N
O
O
5a-e
6
6a-f
HN
R
HN
NH2
O
O
Compound #10
R
CARM1 IC50
16.9
(l
M)8
Compound #10
R
CARM1 IC50 (l
M)8
NH2
6a
5a
5b
5c
5d
5e
–NHCH3
–N(CH3)2
–NH-Ph
–NH-(3-SO2Me)-Ph
–NH-(4-biphenyl)
10.6
29.8
7.7
0.23
1.42
6b
0.16
NH2
6c
20.1
>30
NH2
NH2
We then examined the glycinamide portion (South end of core)
of 1. The synthesis of these analogs was readily achieved using the
6d
approach outlined in Scheme 2. As shown in Table 2,
at the South end were found to be preferred over either b or
di-substituted amino acid amides. Within the subset of analogs
prepared from -amino acids, the (S)-enantiomer (6b) was about
150-fold more active than the corresponding (R)-enantiomer (6c)
(only data for alanine are shown). Additionally, neither bulky sub-
a-amino acids
a,
a0
CF3
6e
6f
>30
>30
NH2
a
N
H
stituents on the
a-carbon nor methylation of the terminal amino
group were tolerated (see Table 2).
Next we evaluated the amideportion (East end of core) of the lead
while retaining the (S)-alanine benzylamide at the South end. New
analogs were assembled using the approach outlined in Scheme 3.
Among the various amines tried (only partial data shown), benzyl
amines were found to be superior to aniline and aliphatic amines.
F3C
F3C
OH
O
N
N
c, d
a, b
N
N
O
O
Within the set of a-methyl benzyl amines, the (R)-enantiomer was
ꢀ60-fold more active than the (S)-enantiomer (Table 3).
Next, we evaluated alternative linkages for the alanine amide
side chain, including positional isomers. These analogs were pre-
pared as per Schemes 4 and 5, respectively. An approach identical
to Scheme 5 was also used to prepare the ortho-benzylamide reg-
ioisomer (16).
The data from this exercise indicated that none of these modi-
fications were tolerated. We also examined other pyrazole amide
alternatives (East end of core), such as the benzyl carbamate (17)
(Scheme 6). Among the various substitutes (data not shown for
others), the carbamate was tolerated the best, and displayed only
an 8-fold loss of potency compared to 7b.
CN
3
2
NH2
F3C
F3C
OH
N
R
N
N
e, f
N
O
O
HN
HN
NHBoc
NH2
In order to assess the selectivity of this series versus various clo-
sely related PRMT’s, we evaluated the most active compound (7b)
for its ability to inhibit PRMT1 and PRMT3.11 Compound 7b was
found to be significantly less active against these enzymes
O
5
O
4
Scheme 1. Reagents: (a) MeOH, SOCl2, 92%; (b) H2, 10% Pd/C, MeOH, 78%; (c) N-
Boc-Gly-OSu, Et3N, CH2Cl2, 95%; (d) 1 N aq KOH, MeOH, 90%; (e) R1R2NH, DIC, HOAt,
DMF, 60–80%; (f) TFA–CH2Cl2 (1:1), 90–100%.
(IC50 > 25 lM) suggesting selectivity in binding and inhibition.

4440
A. V. Purandare et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4438–4441
Table 3
SAR for East end
F3C
B(OH)2
F3C
O
a, b, c
N
d, e, f, g
F3C
N
+
N
O
N
R
N
H
N
CN
13
12
14
NH2
O
7a-g
F3C
F3C
N
NHBn
N
HN
N
NHBn
NH2
O
O
N
O
O
15
O
H2N
CARM1 IC50 >50 μM
Compound #10
R
CARM1 IC50
1.32
(l
M)8
N
H
NH2
16
CARM1 IC50 >50 μM
N
7a
H
HN
Scheme 5. Reagents and conditions: (a) Cu(OAc)2, pyridine, CH2Cl2, air, 2 days, 50%;
(b) n-BuLi, NCCO2Et, THF, 74%; (c) H2, Pd/C, PtO2 (1:1), EtOH, 2 h, 69%; (d) Boc- -Ala-
OSu, CH2Cl2, Et3N, 82%; (e) aq NaOH, 60 °C, 1 h, 78%; (f) BnNH2, EDCI, HOAt, Et3N,
NH
L
7b
7c
0.08
3.4
40%; (g) TFA–CH2Cl2 (1:4); 80%.
N
F3C
F3C
O
NH
NH
7d
7e
7.1
O
N
N
a, b, c, d
N
H
O
N
N
O
0.18
17
3
NH2
HN
CARM1 IC50 0.6 μM
NH2
7f
1.1
4.3
O
N
H
Scheme 6. Reagents: (a) Boc-L-Ala-OSu, CH2Cl2, Et3N, 92%; (b) 1 N aq KOH, MeOH,
90%; (c) DPPA, BnOH, 80–85%; (d) TFA–CH2Cl2 (1:1), 90–95% s.
7g
N
H
In summary, through a Hits to Lead approach, we have identi-
fied pyrazole amide 7b as a potent and selective inhibitor of
CARM1. Further work related to the optimization of this class of
inhibitors will be reported in due course.
F3C
N
H
N
O
N
a, b, c, d
R
N
N
Supplementary data
O
O
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.06.026.
3
7
NH2
HN
NHBoc
References and notes
O
1. Krause, C.; Yang, Z.; Kim, Y.; Lee, J.; Cook, J.; Pestka, S. Pharm. Ther. 2007, 113,
50.
2. Zhang, Y.; Reinberg, D. Genes Dev. 2001, 15, 2343.
3. Chen, D.; Ma, H.; Hong, H.; Koh, S.; Huang, S.; Schurter, B.; Aswad, D.; Stallcup,
M. Science 1999, 284, 2174; Xu, W.; Chen, H.; Du, K.; Asahara, H.; Tini, M.;
Emerson, B.; Montimony, M.; Evans, R. Science 2001, 294, 2507; Chevillard-
Briet, M.; Trouche, D.; Vandel, L. EMBO J. 2002, 21, 5457.
Scheme 3. Reagents and conditions: (a) Boc-L-Ala-OSu, CH2Cl2, Et3N, 92%; (b) 1 N
aq KOH, MeOH, 90%; (c) RNH2, DIC, HOAt, DMF, 60–75%; (d) TFA–CH2Cl2 (1:1), RT,
80–95%.
NHNH2
F3C
O
O
a, b, c
4. Cheng, D.; Cote, J.; Shaaban, S.; Bedford, M. Mol. Cell 2007, 25, 71.
5. Li, H.; Park, S.; Kilburn, B.; Jelinek, M.; Henschen-Edman, A.; Aswas, D.; Stallcup,
M.; Laird-Offringa, I. J. Biol. Chem. 2002, 277, 44623; Lee, J.; Bedford, M. EMBO
Rep. 2002, 3, 268; Fujiwara, T.; Mori, Y.; Chu, D.; Koyama, Y.; Miyata, Y.; Tanaka,
H.; Yachi, K.; Kubo, T.; Yoshikawa, H.; Tohyama, M. Mol. Cell Biol. 2006, 26,
2273.
6. (a) Hong, H.; Kao, C.; Jeng, M.; Eble, J.; Koch, M.; Gardner, T.; Zhang, S.; Li, L.;
Pan, C.; Hu, Z.; M, G.; Cheng, L. Cancer 2004, 101, 83; (b) Majumdar, S.; Liu, Y.;
Ford, O.; Mohler, J.; Whang, Y. Prostate 2006, 66, 1292.
7. (a) Cheng, D.; Yadav, N.; King, R. W.; Swanson, M. S.; Weinstein, E. J.; Bedford,
M. T. J. Biol. Chem. 2004, 279, 23892; (b) Ragno, R.; Simeoni, S.; Castellano, S.;
Vicidomini, C.; Mai, A.; Caroli, A.; Tramontano, A.; Bonaccini, C.; Trojer, P.;
Bauer, I.; Brosch, G.; Sbardella, G. J. Med. Chem. 2007, 50, 1241; (c) Spannhoff,
A.; Heinke, R.; Bauer, I.; Trojer, P.; Metzger, E.; Gust, R.; Schuele, R.; Brosch, G.;
Sippl, W.; Jung, M. J. Med. Chem. 2007, 50, 2319; (d) Spannhoff, A.; Machmur, R.;
Heinke, R.; Trojer, P.; Bauer, I.; Brosch, G.; Schule, R.; Hanefeld, W.; Sippl, W.;
Jung, M. Bioorg. Med. Chem. Lett. 2007, 17, 4150; (e) Mai, A.; Cheng, D.; Bedford,
M.; Valente, S.; Nabbioso, A.; Perrone, A.; Brosch, G.; Sbardella, G.; Bellis, F. D.;
Miceli, M.; Altucci, L. J. Med. Chem. 2008.
NHBn
+
O
N
F3C
N
NO2
O
8
9
F3C
NO
2
10
NHBn
N
d, e, f
N
O
CARM1 IC 50 >50 μM
O
NH2
N
H
11
Scheme 4. Reagents and conditions: (a) AcOH, 100 °C, 6 h, 60%; (b) KMnO4, acetone–
water, 60 °C, 3 h, 30%; (c) BnNH2, EDCI, HOAt, Et3N, DCE, 40%; (d) Zn, NH4Cl, MeOH,
1 h, 86%; (e) Boc-L-Ala-OSu, CH2Cl2, Et3N, 92%; (f) TFA–CH2Cl2 (4:1), 70%.

A. V. Purandare et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4438–4441
4441
8. CARM1 assay. A methylation based filter assay was devised to test compounds
that specifically inhibited CARM1 dependent methylation. Human full length
GST-tagged CARM1 purified from baculovirus infected insect cells was used as
the source for enzyme. Histone H3 (Roche Applied Science) and tritiated S-
9. Pinto, D. J. P.; Orwat, M. J.; Wang, S.; Fevig, J. M.; Quan, M. L.; Amparo, E.;
Cacciola, J.; Rossi, K. A.; Alexander, R. S.; Smallwood, A. M.; Luettgen, J. M.;
Liang, L.; Aungst, B. J.; Wright, M. R.; Knabb, R. M.; Wong, P. C.; Wexler, R. R.;
Lam, P. Y. S. J. Med. Chem. 2001, 444, 566.
adenosyl-
L
-methionine (SAM) (Amersham Pharmacia Biotech) were used as
10. a) All compounds were characterized by LC–MS and NMR analysis. In addition,
the yields were based on the weight of pure product unless mentioned
otherwise. (b) Analytical data for compound 7b: NMR (CD3OD, d): 8.2 (s, 1H),
7.96 (s, 1H), 7.71 (d, 1H), 7.33 (m, 5H), 7.2 (m, 1H), 7.1 (d, 1H), 4.6 (s, 1H), 4.4 (s,
1H), 3.9 (m, 1H), 1.3 (d, 3H); MS (ESI) m/z 446.18 (M+H).
11. PRMT1 and PRMT3 specific methylation assays were performed using assay
conditions identical to the CARM1 assay8 with the following exceptions:
GST-PRMT1 or GST-PRMT3, expressed and purified from bacteria, were
used as the enzymes instead of CARM1. Histone H4 (Roche Applied
Science) was used as the substrate in the PRMT1 assay. GST-GAR,
expressed and purified from bacteria, was used as the substrate in the
PRMT3 assay.
the substrate and cofactor, respectively, for the assay. Methylation reactions
were performed for 75–90 min at room temperature using enzyme (CARM1),
substrate (histone H3) and cofactor (SAM) in the presence of methylation
buffer (20 mM TrisꢁHCl, pH 8.0, 200 mM NaCl, 0.4 mM EDTA) and in the
presence or absence of compound. Reactions were terminated using TCA and
the reaction mixture was precipitated with BSA overnight. Reaction mixtures
were then harvested the next morning by filtering the samples, and the filters
washed. The signal on the filters was read in a Top Count after addition of
MicroScint-20. Three independent IC50 determinations were done. Values are
reported as the average of three IC50 values. The difference between the three
IC50 determinations was less than 25% in all cases.