N-Benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1)

Article abstract of DOI:10.1016/j.bmcl.2008.12.075

A series of N-benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides targeting co-activator associated arginine methyltransferase 1 (CARM1) have been designed and synthesized. The potency of these inhibitors was influenced by the nature of the

Full text of DOI:10.1016/j.bmcl.2008.12.075

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1218–1223
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides
as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1)
Martin Allan a, Sukhdev Manku a, Eric Therrien a, Natalie Nguyen a, Sylvia Styhler c, Marie-France Robert c,
Anne-Christine Goulet c, Andrea J. Petschner b, Gabi Rahil b, A. Robert MacLeod c, Robert Déziel a,
Jeffrey M. Besterman c, Hannah Nguyen c, Amal Wahhab a,
*
a MethylGene Inc., Department of Medicinal Chemistry, 7220 rue Frederick-Banting, Montreal, Que., Canada H4S 2A1
b MethylGene Inc., Department of Lead Discovery, 7220 rue Frederick-Banting, Montreal, Que., Canada H4S 2A1
c MethylGene Inc., Department of Pharmacology and Cell Biology, 7220 rue Frederick-Banting, Montreal, Que., Canada H4S 2A1
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides targeting co-activa-
tor associated arginine methyltransferase 1 (CARM1) have been designed and synthesized. The potency
of these inhibitors was influenced by the nature of the heteroaryl fragment with the thiophene analogues
being superior to thiazole, pyridine, isoindoline and benzofuran based inhibitors.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 7 November 2008
Revised 16 December 2008
Accepted 17 December 2008
Available online 24 December 2008
Keywords:
CARM1
Co-activator associated arginine
methyltransferase 1
Protein arginine methyltransferases
CARM1 inhibitors
Mammalian protein arginine methyltransferases (PRMTs) com-
prise a family of at least nine members with diverse biological
functions.1 They catalyze the transfer of methyl groups from
S-adenosyl methionine to specific arginine residues, resulting in
the formation of either asymmetric (Type I; PRMTs 1–4, 6 and 8)
CARM1 has been shown to compromise transcriptional activation,
suggesting that the integrity of the methyltransferase domain
CARM1 is important for its co-activator function.4,5c,g
Knockout or silencing of CARM1 impedes estrogen-stimulated
gene expression, cell cycle progression and growth of breast cancer
cells,3d,6a and evidence also link CARM1, cyclin E and steroid co-
activator overexpression to high-grade breast cancer tumors.6b
Furthermore, modulation of CARM1 levels affects androgen-depen-
dent transcription and prostate cancer cell growth,6c and elevated
CARM1 levels correlate with the development of prostate carci-
noma as well as the progression of androgen-independent prostate
cancer.6d These studies support CARM1 as a plausible target for
anti-cancer drug development.
or symmetric (Type II; PRMTs 5, 7 and 9)
x-NG,NG-dimethylargi-
nine tails on a wide variety of protein substrates.2
PRMT4, also known as co-activator associated arginine methyl-
transferase 1 (CARM1), methylates proteins with roles encompass-
ing three key levels of gene expression: chromatin remodeling
(histone H3 and CBP/p300),3a–d RNA processing and stability
(PABP, HuR and HuD)3d–g and RNA splicing (CA150, SAP49, SmB
and U1C).3h,i CARM1 was initially identified as a secondary co-acti-
vator for the p160 steroid co-activator protein GRIP1 in transcrip-
tion mediated by nuclear hormone receptors.4 It has also been
shown to be a positive co-regulator for SRC-3, another member
of the p160 family5a as well as for a variety of other transcription
factors such as CBP/p300,3a,5b b-catenin5c p53,5d nuclear factor
F3C
O
N
N
kappa-B (NF-j
B),5e,f CIITA,5g HTLV-1 Tax,5h PPARc5i and c-fos.5j
HN
The co-activator activity of CARM1 coincides with the arginine
methylation of histone H3 and CBP/p300 on several target promot-
ers and mutations of critical residues in the catalytic domain of
R
H
N
H2N
O
* Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337 0550.
E-mail address: wahhaba@methylgene.com (A. Wahhab).
Figure 1. Pyrazole type CARM1 inhibitors. 1a, R = H, CARM1 IC50 = 0.08
1b, R = OMe.
lM, Ref. 9b;
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.075

M. Allan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1218–1223
1219
F3C
N
H2N
NH
O
e, f
S
a, b, c
d
N
CHO
(HO)2B
NC
S
4
3
2
S
CN
F3C
F3C
F3C
H
OH
N
O
N
i, j
R
N
N
g, h
N
N
N
O
O
H
H
BocHN
H
N
S
S
S
N
N
BocHN
H2N
O
O
O
6
5
7a, R=
MeO
Scheme 1. Reagents and conditions: (a) N2(Boc)2, Cu(OAc)2, THF, 86%; (b) NH2OHÁHCl, Pyr, Ac2O, 80–90 °C, 74%; (c) HCl/dioxane, iPrOH, 80 °C, 97%; (d) 4,4,4-trifluoro-1-
(furan-2-yl)butane-1,3-dione, AcOH, 80 °C, 74%; (e) BH3ÁTHF, THF; (f) Boc-Ala-OSu, Et3N, DCM, 51%; (g) NaClO2, NaH2PO4, MeCN, H2O, 60%; (h) ammonium formate, K2CO3,
10% Pd/C, ethanol/reflux, 99%; (i) (2-methoxyphenyl)methanamine, EDC, DMAP, DCM, 51.3%; (j) 1:1 TFA/DCM, 4 h, rt, 99%
Reports describing novel inhibitors of PRMTs originated either
from molecular modeling studies or high-throughput screening.7a–e
However, the majority of these are micro-molar inhibitors that lack
selectivity and in some cases they do not possess drug-like proper-
ties. Osborne et al. 8 reported the in situ synthesis of a bisubstrate
analogue inhibitor of PRMT1 that was over 4-fold more active
against PRMT1 compared to CARM1. However, no CARM1 inhibi-
tors exhibiting cellular effects have been described to date. At the
time our program began Purandare et al.9a,9b described selective
CARM1 inhibitors based on the pyrazole scaffold 1 (Fig. 1).
We envisaged the latter inhibitors as a prototype for the design of
novel proprietary CARM1 inhibitors. Our initial efforts were focused
onthereplacementof thecentral core phenylring of 19b with various
heterocycles. To that end, we prepared the 2,4-and 2,5-disubsti-
tuted-thiophene analogues shown in Schemes 1 and 2, respec-
tively.10a Reaction of 5-formylthiophen-3-ylboronic acid 2 with
(E)-di-tert-butyl diazene-1,2-dicarboxylate, followed by conversion
of the aldehyde to the nitrile using hydroxylamine and acetic anhy-
dride in pyridine, then deprotection gave hydrazine 3.
Cyclization of hydrazine 3 with 4,4,4-trifluoro-1-(furan-2-yl)-
butane-1,3-dione gave pyrazole 4 which was then subjected to
borane reduction and the resulting amine (not shown in scheme)
was reacted with Boc-Ala-OSu to yield 5. Oxidation of the furan
ring of 5 with NaClO2 produced almost a 1:1 mixture of the desired
and the 2-chloro-thiophene analogue of 6 which was readily
dehalogenated with ammonium formate and Pd/C to give acid 6.
Coupling of acid 6 with the benzyl amine and deprotection with
TFA produced 7a (Scheme 1).
Similarly, the 2,5-disubstituted-thiophene 10a was prepared
starting from 5-formylthiophen-2-ylboronic acid 8, as depicted in
Scheme 2.
The thiazole based analogue was accessible starting from the
reported intermediate 11.11 Reaction of ester 11 with ammonium
hydroxide required heating to produce the carboxamide (not
shown in scheme) which was then reduced with diborane and
the resulting amine was reacted with Boc-Ala-OSu to give 12.
Intermediate 12 was converted to 13 in three steps using method-
ologies described above for 7a and 10a (Scheme 3).
The synthesis of the pyridine scaffold started from 2-chloropyr-
idine 14 which was converted to amine 15 via a sequence of reac-
tions involving displacement of the 2-chloro group of 14 with
hydrazine hydrate, cyclization of the resulting hydrazine to the
pyrazole, conversion of the alcohol to the azide (not shown in
scheme) and reduction of the azide to furnish amine 15. The
remainder of the steps to give 16 is similar to those described for
the preparation of 10a above (Scheme 4).
For the synthesis of the pyridine regioisomer, cyclization of
hydrazine 17 with 4,4,4-trifluoro-1-(furan-2-yl)butane-1,3-dione
gave a mixture of isomers 18 and 19 in a ratio of 2:1, respectively.
The two isomers were separable after the reaction with Boc-Ala-
OSu, and the major was taken to the final product 20 (Scheme 5).
The isoindoline scaffold 24 was synthesized according to
Scheme 6, starting from 4-nitroisoindoline-1,3-dione 21. Reduc-
tion of 21 with diborane, protection of the resulting amine with
Cbz and reduction of the nitro group followed by diazotization
and tin chloride reduction gave the hydrazine, which was con-
F3C
F3C
H
OH
N
HO
N
S
N
S
R
OH
N
N
B
O
O
a, b, c, d, e, f, g
BocHN
h, i
S
HN
O
HN
O
CHO
Me
O
H2N
10a, R=
8
9
Scheme 2. Reagents and conditions: (a) N2(Boc)2, Cu(OAc)2, THF, 47%; (b) NH2OHÁHCl, Pyr, Ac2O, 80–90 °C, 75%; (c) HCl/dioxane, iPrOH, 80 °C, 78%; (d) 4,4,4-trifluoro-1-
(furan-2-yl)butane-1,3-dione, AcOH, 80 °C, 37%; (e) BH3ÁTHF, THF; (f) Boc-Ala-OSu, Et3N, DCM, 49%; (g) NaClO2, NaH2PO4, MeCN, H2O, 88%; (h) (2-methoxyphenyl)meth-
anamine, POCl3, pyridine, 24%; (i) 1:1 TFA/DCM, 4 h, rt.

1220
M. Allan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1218–1223
F3C
F3C
F3C
H
N
O
O
N
N
N
N
N
N
O
O
d, e, f
S
N
O
S
N
S
N
a,b,c
H
N
O
NH
O
NHBoc
O
NH2
11
13
12
Scheme 3. Reagents and conditions: (a) NH4OH, MeOH, THF, 16 h at 50 °C; (b) BH3.THF, THF, 98% over 2 steps; (c) Boc-Ala-OSu, Et3N, DCM, 21%; (d) NaClO2, NaH2PO4, MeCN,
H2O, 90%; (e) (2-methoxyphenyl)methanamine, POCl3, pyridine, 12%; (f) 1:1 TFA/DCM, 4 h, rt, 60%.
F3C
F3C
H
N
O
Cl
N
N
N
a, b, c, d, e
OH
N
f, g, h, i
O
O
N
N
N
H
N
NH2
NH2
O
14
16
15
Scheme 4. Reagents and conditions: (a) N2H2, iPrOH, 24h, 100 °C THF, 26%; (b) 4,4,4-trifluoro-1-(furan-2-yl)butane-1,3-dione, AcOH, 80 °C, 51%; (c) methanesulfonyl
chloride, Et3N, DCM; (d) NaN3, DMF, 1 h, 60 °C, 88%; (e) SnCl2Á2H2O, MeOH, 2 h, rt., 93%; (f) Boc-Ala-OSu, Et3N, DCM, 54%; (g) NaClO2, NaH2PO4, MeCN, H2O, 77%; (h)
(2-methoxyphenyl)methanamine, POCl3, pyridine, 24%; (i) 1:1 TFA/DCM, 4 h, rt, 80%.
F3C
F3C
O
H2N
NH
H
N
O
N
N
N
d, e, f, g
N
O
a, b, c
O
N
+
N
CF3
N
N
N
H
N
NH2
Cl
NH2
N
O
NH2
17
18 major
20
19
minor
Scheme 5. Reagents and conditions: (a) 4,4,4-trifluoro-1-(furan-2-yl)butane-1,3-dione, AcOH, 80 °C, 2:1 mixture of regioisomers, 90%; (b) Zn(CN)2, Pd(PPh3)4, DMF, 12%; (c)
BH3ÁTHF, THF, 99%; (d) Boc-Ala-OSu, Et3N, DCM, 40%; (e) RuCl3, NaIO4, MeCN, CCl4, H2O, quantitative crude yield; (f) (2-methoxyphenyl)methanamine, BOP, Et3N, DCM, 59%;
(g) 1:1 TFA/DCM, 2 h, rt, 56%.
CF3
CF3
CF3
O-
H
N
O
N+
HO
O
O
N
N
N
a, b, c
N
g, h
N
N
d, e, f
Boc
NH
O
O
O
NH
NH2
N
NCbz
N
O
O
O
21
22
23
24
Scheme 6. Reagents and conditions: (a) BH3.THF, MeOH, crude; (b) Bn-OCOCl, Et3N, 33%; (c) SnCl2Á2H2O, EtOH (d) i—NaNO2; ii—SnCl2ÁH2O, MeOH, 93%; iii—4,4,4-trifluoro-1-
(furan-2-yl)butane-1,3-dione, AcOH, reflux, 16 h, crude.; (e) Boc-Ala-OSu, Et3N, DCM, 50%; (f) RuCl3, NaIO4, crude; (g) (2-methoxyphenyl)methanamine, BOP, Et3N, 41%; (h)
TFA, DCM, 45%.
verted to pyrazole 22 as described earlier. After removal of the Cbz
group of 22, the produced amine was converted to 24 following the
reaction sequence described for 7a.
The benzofuran scaffold 26 was obtained from 25 by the reac-
tion with dimethyl 2-bromomalonate, followed by catalytic reduc-
tion of the nitro group. Diazotization, pyrazole formation and
reduction of the ester gave 27 which was transformed to 29
employing procedures described earlier (Scheme 7).
For the construction of the pyrazole ring of compounds 7, 10,
13, 20, 24 and 29,10a we employed a synthetic methodology similar
to that reported for 1a.9a,9b As for thiazole 16 we used a reported
procedure for the synthesis of the key intermediate 11.11 To con-
firm that all the compounds above have the desired 3-CF3 isomer
we utilized 19F NMR,12a which was able to distinguish between
the 19F chemical shift of the 3-CF3 and the 5-CF3 pyrazole
regioisomers.12b

M. Allan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1218–1223
1221
F3C
O
NH2
NO2
N
OH
O
O
N
a, b
c, d
e, f
O
H
OMe
O
N
OH
25
26
27
F3C
CF3
O
H
N
N
g, h, i
N
N
O
O
O
O
O
HN
O
HN
NH2
NH
Boc
28
29
Scheme 7. Reagents and conditions: (a) dimethyl 2-bromomalonate, Bu4NBr, K2CO3, 72%; (b) Pd/C, H2, MeOH, EtOAc, 99%; (c) i—NaNO2, HCl, 91%; ii—SnCl2, iii—4,4,4-
trifluoro-1-(furan-2-yl)butane-1,3-dione, AcOH, 80 °C, 62%; (d) iBu2AlH, toluene, 86%; (e) i—Ph3P, imidazole, I2, DCM, 54%; ii—NaN3, DMF, 1 h at 60 °C, iii—SnCl2Á2H2O, MeOH,
2 h, rt, 82%; (f) Boc-Ala-OSu, Et3N, DCM, 84%; (g) NaClO2, NaH2PO4, MeCN, H2O; (h) (2-methoxyphenyl)methanamine, BOP, Et3N, DCM, 10%; (i) 1:1 TFA/DCM, 4 h, rt, 54%.
The effects of 7a, 10a, 13, 16, 20, 24 and 29 on the activity of
CARM1 was measured by means of a histone methyltransferase as-
say using recombinant CARM1 enzyme, and histone H3 as the sub-
strate10b (Table 1). Both the 2,4- and 2,5-disubstituted thiophenes,
7a and 10a, were potent inhibitors of CARM1, with IC50 of 0.06 and
Further SAR exploration centered on the thiophene based ana-
logues. Standard coupling methodologies were employed for the
synthesis of amides 7b–g and 10b–e using acids 6 and 9 and the
appropriate amine.
Table 2 shows the CARM1 inhibitory activity of the thiophene
based amides 7b–f and 10b–e. For the 2,4-series, scaffold 7, the
substituent on the benzyl group seems to have some effect on
the enzymatic activity as compared with the unsubstituted 7c.
0.3 lM, respectively. Compound 7a, was equipotent to 1b, indicat-
ing that the 2,4-substituted thiophene core is a good replacement
of the phenyl ring. The thiazole analogue 13 was 36- and 7-fold less
active than 7a and 10a, respectively, indicating that the nitrogen of
the thiazole ring of 13 is less tolerated. Also the pyridine based
inhibitors 16 and 20 were 46- and 34-fold less active than 1b, indi-
cating that the CARM1 enzyme favors a hydrophobic aryl ring over
the more polar basic pyridyl moiety. The observed inactivity of iso-
indoline analogue 24 could be due to the constriction imposed by
the less flexible isoindoline ring or the substitution at the benzylic
nitrogen. The bicyclic benzofuran core, analogue 29, was devoid of
activity indicating that mono-cyclic rings are preferred. These
results confirm that the nature of the core group influences the
inhibitory activity of these analogues.
The o-OMe group is the most favored, with IC50 of 0.06 lM, intro-
duction of the o-F or o-methyl ester substituents (compounds 7b
and 7f) resulted in a 13-fold loss of activity. A meta-OMe group,
7e, was less tolerated than the ortho analogue, 7a. Replacement
of the benzyl amine with a 2-methylpyridyl, 7g, had a negative
effect reducing the CARM1 activity by 15-fold as compared to 7c.
Compounds 10a and 10c based on the 2,5-thiophene scaffold,
displayed similar trends to their counterparts 7a and 7c. An o-flu-
oro, o-CF3, or o-iso-propyl substituent (10b, 10d and 10e, respec-
tively) resulted in a marked decrease of activity as compared to
10c.
Table 1
Effect of heterocycle core on CARM1 inhibitory activitya
CF3
N
H
N
N
O
O
NH2
core
O
Compound
Core
CARM1 IC50
0.06
(l
M)
Compound
Core
CARM1 IC50
4.20
(lM)
1bb
16
N
H
N
H
N
7a
0.06
0.3
20
24
29
3.05
>10
>10
N
H
N
H
S
N
S
10a
H
N
N
S
O
N
13
2.20
HN
NH
a
Values are means of at least two experiments.
1b, Figure 1. CARM1 IC50 value determined in house.
b

1222
M. Allan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1218–1223
Table 2
SAR of thiophene scaffolds 7 and 10a
F3C
F3C
HN
O
R
H
N
N
R
N
N
N
O
S
H
H
N
S
N
NH2
O
NH2
O
10a-e
7a-g
Compound
R
CARM1 IC50
0.06
(lM)
Compound
R
CARM1 IC50
0.3
(lM)
O
F
O
F
7a
10a
7b
0.79
10b
2.31
7c
0.22
0.26
10c
10d
0.23
4.48
CF3
7d
i-Pr
O
N
O
7e
0.37
0.77
10e
7g
2.92
3.35
O
O
7f
a
Values are means of at least two experiments.
To test the specificity of our inhibitors for CARM1 versus other
arginine or lysine methyltransferases, compound 7a was tested
against the PRMT1 arginine and the Set7/9 lysine methyltransfer-
ases and was found to be significantly less active against both en-
vation of a luciferase reporter driven by three copies of the estro-
gen response element in response to estradiol,10c and a 72 h cell
count of estradiol-driven T47D breast ductal carcinoma cells, using
the estrogen modulator tamoxifen as the positive control. Andro-
gen-dependent signaling was assessed using a luciferase reporter
driven by the MMTV promoter in response to testosterone,10d
using the androgen antagonist flutamide as the positive control.
The MTT cell proliferation assay was used to measure effects on
the growth of DU145 prostate carcinoma cells, using the HDAC
inhibitor MS-275 as a positive control.13 As shown in Table 3, com-
pound 7a had no significant effect on any of the cellular endpoints
tested. Compound 1b was included in our evaluations and also did
not show any significant effect in either the methylation or the
functional cell-based assays.
There are obviously a multitude of potential explanations for
these results. One possibility is that CARM1 may be a dispensable
component of nuclear receptor-mediated transcription, as it is only
a secondary co-activator in this process. Another possibility is that
the cellular permeability of the compound may be low and, there-
fore, may require supra-physiological concentrations of compound
to see cellular effects.
zymes (IC50 > 100 lM).
To the best of our knowledge, no cellular effects using small
molecule CARM1 inhibitors have been reported. We tested com-
pound 7a for its ability to inhibit in cells the methylation of argi-
nine 26 on histone H3, a target site for CARM1. Treatment of two
different cell lines with up to 5 lM of compound 7a for 48 h did
not result in inhibition of histone methylation (data not shown).
In the event that the absence of an effect is due to other arginine
methyltransferases targeting the same substrate, compound 7a
was also profiled in a variety of functional assays. The assays were
targeted towards some of the cancer-related processes in which
CARM1 has been shown to be involved, including estrogen-depen-
dent transcription breast cancer cell growth, androgen-dependent
signaling and androgen-independent prostate cancer growth. The
estrogen-dependent assays entailed measurement of the transacti-
In conclusion, we successfully replaced the core phenyl ring
with other heterocyclic rings and prepared novel inhibitors based
on the 2,4- and 2,5-disubstituted thiophene scaffold which dis-
played sub-micro-molar activity against CARM1. Thiazole and pyr-
idine based analogues were less potent, while the isoindoline and
benzofuran based analogues were devoid of activity. Despite the
observed potency against the CARM1 enzyme, our inhibitors did
not show measurable cellular activities.
Table 3
Cell-based activity of compound 7a
Compound Estrogen-
dependent
Estrogen-
dependent
growth T47D
Androgen-
dependent
transcription MDA IC50
MTT
DU145
transcription
T47D-KBluc IC50
(lM)
IC50
(
lM)
kb2 IC50
(l
M)
(lM)
7a
1b
>10
>10
>10
>10
1
N/A
N/A
>10
>10
N/A
2
18
18
N/A
N/A
2
Tamoxifen 0.1
Flutamide
MS-275
Acknowledgment
N/A
N/A
N/A
The authors are grateful to Dr. Arkadii Vaisburg for valuable in-
sights and for proof reading this article.
N/A, not applicable; NT, not tested.

M. Allan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1218–1223
1223
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recombinant mouse CARM1, expressed in Sf9 cells) was purchased from
Millipore (cat# is 14-575, Lot # is DAM1473541). Histone H3 (Sigma–Aldrich)
was used as the substrate, and the methylation was monitored using tritiated
S-Adenosyl-Methionine (SAM) (Amersham Pharmacia Biotech) as a methyl
donor. The reactions were performed at 30 °C for a total of 15 min, using
enzyme (CARM1), substrate (histone H3), and co-factor (SAM) in the absence
3. (a) Xu, W.; Chen, H.; Du, K.; Asahara, H.; Tini, M.; Emerson, B. M.; Montminy,
M.; Evans, R. M. Science 2001, 294, 2507; (b) Chevillard-Briet, M.; Trouche, D.;
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J.; Hu, M. C.-T.; Aldaz, C. M.; Bedford, M. T. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
6464; (e) Lee, J.; Bedford, M. T. EMBO Rep. 2002, 3, 268; (f) Li, H.; Park, S.;
Kilburn, B.; Jelinek, M. A.; Henschen-Edman, A.; Aswad, D. W.; Stallcup, M. R.;
Laird-Offringa, I. A. J. Biol. Chem. 2002, 277, 44623; (g) Fujiwara, T.; Mori, Y.;
Chu, D. L.; Koyama, Y.; Miyata, S.; Tanaka, H.; Yachi, K.; Kubo, T.; Yoshikawa, H.;
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Yaguchi, H.; Nagamura, Y.; Tsukada, T. J. Biol. Chem. 2005, 280, 28927; (i) Cheng,
D.; Côté, J.; Shaaban, S.; Bedford, M. T. Mol. Cell 2007, 25, 71.
and presence of compound. Assay protocol: 2
added to a U-Bottom of a PP 96-well plate. CARM1 enzyme was diluted in a
Tris–HCl (pH = 9) buffer (to a final enzyme concentration of 0.005 g/ l) and
l of the cold enzyme solution was immediately added to the compound and
allowed to pre-incubate for 10 min at rt. A mixture of SAM (20% [3H]SAM) and
histone H3 (10 l) was then added and incubated for 15 min at 30 °C for a final
concentration of 0.0125 g/ l histone and 2 M SAM (0.033 mCi/ml). The
reaction was stopped by adding 20 l of SAH for a final concentration of 60
and, 10 l of the quenched reaction is spotted onto P30 Filtermat paper. The
ll of the diluted compound was
4. Chen, D.; Ma, H.; Hong, H.; Koh, S. S.; Huang, S.-M.; Schurter, B. T.; Aswad, D.
W.; Stallcup, M. R. Science 1999, 284, 2174.
l
l
5. (a) Naeem, H.; Cheng, D.; Zhao, Q.; Underhill, C.; Tini, M.; Bedford, M. T.; Torchia,
J. Mol. Cell. Biol. 2007, 27, 120; (b) Chen, D.; Huang, S. M.; Stallcup, M. R. J. Biol.
Chem. 2000, 275, 40810; (c) Koh, S. S.; Li, H.; Lee, Y. H.; Widelitz, R. B.; Chuong, C.
M.; Stallcup, M. R. J. Biol. Chem. 2002, 277, 26031; (d) An, W.; Kim, J.; Roeder, R. G.
Cell 2004, 117, 735; (e) Covic, M.; Hassa, P. O.; Saccani, S.; Buerki, C.; Meier, N. I.;
Lombardi, C.; Imhof, R.; Bedford, M. T.; Natoli, G.; Hottiger, N. O. EMBO J. 2005, 24,
85; (f) Miao, F.; Li, S.; Chavez, V.; Lanting, L.; Natarajan, R. Mol. Endocrinol. 2006,
20, 1562; (g) Zika, E.; Fauquier, L.; Vandel, L.; Ting, J. P. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 16321; (h) Jeong, S. J.; Lu, H.; Cho, W. K.; Park, H. U.; Pise-Masison, C.;
Brady, J. N. J. Virol. 2006, 80, 10036; (i) Fauquier, L.; Duboé, C.; Joré, C.; Trouche, D.;
Vandel, L. FASEB J. 2008, 22, 3337; (j) Yadav, N.; Cheng, D.; Richard, S.; Morel, M.;
Iyer, V. R.; Aldaz, C. M.; Bedford, M. T. EMBO Rep. 2008, 9, 193.
6. (a) Frietze, S.; Lupien, M.; Silver, P. A.; Brown, M. Cancer Res. 2008, 68, 301; (b)
El, ; Messaoudi, S.; Fabbrizio, E.; Rodriguez, C.; Chuchana, P.; Fauquier, L.;
Cheng, D.; Theillet, C.; Vandel, L.; Bedford, M. T.; Sardet, C. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 13351; (c) Majumder, S.; Liu, Y.; Ford, O. H., 3rd; Mohler, J. L.;
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N.; Koch, M. O.; Gardner, T. A.; Zhang, S.; Li, L.; Pan, C. X.; Hu, Z.; MacLennan, G.
T.; Cheng, L. Cancer 2004, 101, 83.
8
l
l
l
l
l
l
lM
l
Filtermat is washed twice for 15 min with 10% TCA solution and then once for
5 min with 95% ethanol. A wax scintillant is used with the filtermat and the
radioactivity was read using a Wallac Microbeta counter (CCPM).; (c) The cell
line and methods used for the estrogen-dependent transcriptional assay are
described in Wilson, V. S.; Bobseine, K.; Gray, L. E. Jr. Toxicol. Sci. 2004, 81, 69.;
(d) The cell line and methods used for the androgen-dependent transcriptional
assay are described in Wilson, V. S.; Bobseine, K.; Lambright, C. R.; Gray, L. E. Jr.
Toxicol. Sci. 2002, 66, 69.
11. Denisova, A. B.; Sosnovskikh, V. Y.; Dehaen, W.; Toppet, S.; Van Meervelt, L.;
Bakulev, V. A. J. Fluorine Chem. 2002, 115, 183.
12. (a) The chemical shift of the trifluoromethyl group appeared at 19F NMR
(DMSO-d6) d ppm: À61.3, À61.32, À61.45, À61.71, À61.37, À61.4, À61.3 and
À61.39 for 1b, 7a, 10a, 13, 16, 20, 24 and 29, respectively, supporting the
assumption that the compounds have the same pyrazole regioisomer.; (b)
Fustero, S.; Roman, R.; Sanz-Cervera, J. F.; Simon-Fuentes, A.; Cunat, A. C.;
Villanova, S.; Murguia, M. J. Org. Chem. 2008, 73, 3523.
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) Spannhoff, A.; Heinke, R.; Bauer, I.;
Trojer, P.; Metzger, E.; Gust, R.; Schüle, R.; Brosch, G.; Sippl, W.; Jung, M. J. Med.
Chem. 2007, 50, 2319; (c) Spannhoff, A.; Machmur, R.; Heinke, R.; Trojer, P.;
Bauer, I.; Brosch, G.; Schüle, R.; Hanefeld, W.; Sippl, W.; Jung, M. Bioorg. Med.
13. Zhang, H.; Chen, P.; Bai, S.; Huang, C. Drug Dev. Res. 2007, 68, 61.