Optimization of pyrazole inhibitors of Coactivator Associated Arginine Methyltransferase 1 (CARM1)

Article abstract of DOI:10.1016/j.bmcl.2009.04.075

Design, synthesis, and SAR development led to the identification of the potent, novel, and selective pyrazole based inhibitor (7f) of Coactivator Associated Arginine Methyltransferase (CARM1).

Full text of DOI:10.1016/j.bmcl.2009.04.075

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2924–2927
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of pyrazole inhibitors of Coactivator Associated Arginine
Methyltransferase 1 (CARM1)
Tram Huynh a, Zhong Chen a, Suhong Pang a, Jieping Geng a, Tiziano Bandiera b, , Simona Bindi b,
Paola Vianello b, Fulvia Roletto b, Sandrine Thieffine b, Arturo Galvani b, Wayne Vaccaro a, Michael A. Poss a,
George L. Trainor a, Matthew V. Lorenzi a, Marco Gottardis a, Lata Jayaraman a, Ashok V. Purandare a,
*
a Bristol-Myers Squibb Pharmaceutical Research and Development, Princeton, NJ 08543, United States
b Nerviano Medical Sciences Oncology Business Unit, Viale Pasteur, 20014 Nerviano, Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Design, synthesis, and SAR development led to the identification of the potent, novel, and selective pyr-
azole based inhibitor (7f) of Coactivator Associated Arginine Methyltransferase (CARM1).
Ó 2009 Elsevier Ltd. All rights reserved.
Received 21 March 2009
Revised 9 April 2009
Accepted 17 April 2009
Available online 22 April 2009
Keywords:
Coactivator associated arginine
methyltransferase 1
CARM1
PRMT4
Pyrazole
Methyltransferase
Protein arginine methyl transferase (PRMT) family members
have been shown to play a role in various cellular processes includ-
ing nuclear hormone receptor (NHR) mediated signaling, protein–
protein interactions, protein trafficking, mRNA splicing and
processing and transcriptional regulation.1 The family, consisting
of at least 9 members (PRMTs 1–9), catalyze the generation of di-
methyl arginine residues on substrate proteins.2 CARM1 (or
PRMT4), has been shown to methylate arginine residues asymmet-
rically in a wide variety of substrate proteins such as histone H3,
p300/CBP, U1C, SAP49, CA150, HuR, HuD and PABP. These events
affect chromatin architecture which impacts transcriptional initia-
tion, alternative splicing, mRNA processing and stabilization.3–5
Taken together, these observations point to a crucial role for
CARM1 in modulating gene expression at multiple critical levels.
Lately, CARM1 has been shown to be up-regulated during the pro-
gression of prostate cancer.6 Over-expression of CARM1 is seen in
both androgen-stimulated and castration resistant 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. Hence, the identification of selective
CARM1 inhibitors as tools to interrogate its function in cells may
be of significant interest.
To date, there have been only a few publications describing
small molecule chemical modulators of the PRMTs.7 Recently, we
reported8 Hits to Lead optimization studies leading to the identifi-
cation of pyrazole analogs (1 Fig. 1) as potent and selective inhib-
itors of CARM1.9
These studies had demonstrated that meta-substituted (S)-ala-
nine benzyl amide is preferred at the South end whereas the SAR
of the East end is more tolerant of substitution. Pyrazole 1 however
showed only modest permeability (PAMPA Pc < 15 nm/s).10 Herein,
F3C
H
East end
N
N
N
Core
O
1 CARM1 IC50 0.08 µM
HN
South end
NH2
* Corresponding author. Tel.: +1 609 252 4320; fax: +1 609 252 7446.
E-mail address: ashok.purandare@bms.com (A.V. Purandare).
Current address: Italian Institute of Technology, Via Morego, 30 16163 Genova,
Italy.
O
Figure 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.075

T. Huynh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2924–2927
2925
F3C
F3C
F3C
F3C
O
O
NHNH2
a, b, c
NHNH2
N
N
N
N
R
a, b
N
N
N
c, d, e
N
O
O
N
N
O
7a-f
H
H
N
H
N
NH2
N
NHBoc
NHBoc
NH2
3
3
2
O
O
O
Scheme 2. Reagent and conditions: (a) RCO2H, PyBOP, DIEA, DMF, rt or RNCO in
case of 7g; (b) DIC, DMF, microwave, 150 °C, 20 min; (c) TFA-CH2Cl2, 30–50% from 3.
F3C
O
N
N
N
N
4
additional NH-linker (as in 7g) was tolerated, however, this modi-
fication diminished the permeability (PAMPA Pc < 15 nm/s).
Next, we evaluated five-membered heterocyclic cores while
retaining the relative disposition of the East and South ends with
the same substitutions as in 4. These analogs were prepared from
the corresponding known intermediates (8a–e)12 using the general
approach as disclosed in Scheme 3.
A 3-trifluoromethyl substituted pyrazole was found to be the
preferred core from the various five-membered heterocycles (Table
3). Among the remaining cores, heterocycles with an unhindered
H
N
NH2
O
F3C
OH
N
N
g, h, e
a, f
2
O
H
N
NHBoc
5
O
F3C
Table 2
SAR for East end
N
N
N
F3C
O
N
6
O
H
N
R
N
N
NH2
N
N
7a-g
O
H
N
Scheme 1. Reagent and conditions: (a) Boc-L-Ala-OSu, CH2Cl2, Et3N, 92%; (b)
NH2
hydrazine hydrate, heat, 30 min, 80%; (c) benzoic acid, PyBOP, DIEA, DMF, rt; (d)
DIC, DMF, microwave, 150 °C, 20 min, 60% from 3; (e) TFA-CH2Cl2, 92%; (f) 1 N aq
NaOH–MeOH, rt, 65%; (g) benamidoxime, CDI, DMF, rt, 4 h; (h) CDI, 100 °C, 4 h, 45%
for combined g and h steps.
O
Compound11
R
CARM1 IC50
0.06
(lM)
9
4
we report further optimization of the core and East end that led to
the identification of a selective and potent pyrazole inhibitor (7f).
At the outset, we explored amide surrogates at the East end of
the molecule to reduce hydrogen bond donors in order to improve
the permeability. Towards this end, we first examined 1,3,4- and
1,2,4-oxadiazoles as amide surrogates. These surrogates were syn-
thesized as shown in Scheme 1.
From this exercise, the 1,3,4-oxadiazole (4) was found to be pre-
ferred over the 1,2,4-regioisomer (6). This modification not only
improved the in vitro potency versus amide (1), but also improved
the permeability as measured in the PAMPA assay (Pc < 15 nm/s for
1 to 106 nm/s for 4) (Table 1).
7a
7b
7c
0.31
1.54
0.05
N
O
O
Encouraged by these findings, we examined additional substitu-
tions at the 5-position of the 1,3,4-oxadiazole. These analogs were
readily assembled using the protocol as shown in Scheme 2.
Among the various C-5 substitutions (only partial data shown,
Table 2), aryl groups with ortho-substitution were found to be
superior to either meta- or para-substitutions. Inclusion of a nitro-
gen atom in the ring (as in 7b) was not tolerated. Interestingly, an
7d
7e
0.16
0.05
Cl
S
N
7f
0.04
0.05
Table 1
SAR for amide surrogates
Compound11
CARM1 IC50
(
lM)
9
O
1
4
6
0.08
0.06
0.21
7g
H
N

2926
T. Huynh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2924–2927
F3C
Core
O
Core
O
OH
a, b
N
NH2NH2.HCl
a, b
N
O
O
O
H
N
CN
NHBoc
Br
Br
O
9a-e
12
8a-e
11
F3C
Core
O
c, d, e, f
O
N
N
N
c, d, e, f
N
N
N
H
N
13
NH2
CHO
O
10a-e
Scheme 3. Reagent and conditions: (a) H2, 10%Pd/C, MeOH, 70–80%; (b) Boc-L-Ala-
F3C
OSu, CH2Cl2, Et3N, 85–95%; (c) hydrazine hydrate, heat, 30 min, 70–80%; (d) benzoic
acid, PyBOP, DIEA, DMF, rt; (e) DIC, DMF, microwave, 150 °C, 20 min, 40–60%
combined for d and e; (f) TFA-CH2Cl2, 80–93%.
O
N
g, h, i
N
13
N
N
hydrogen bond acceptor at the 20 position were preferred (e.g., 10c
vs 10d).
We also studied the effect of substitution of the amide nitrogen,
and removal of the amide bond itself at the South end on CARM1
inhibition. Synthesis of these analogs was achieved as outlined in
Scheme 4.
N
NH2
O
14 CARM1 IC50 >10 uM
F3C
O
N
j, i
N
13
N
N
Table 3
SAR for core
H
N
Core
O
NH2
15 CARM1 IC50 >10 uM
N
N
Scheme 4. Reagent and conditions: (a) 4,4,4-trifluoro-1-(furan-2-yl)butane-1,3-
dione, AcOH, reflux, 84%; (b) RuCl3, NaIO4, CH3CN–CH2Cl2–H2O (2:2:3); (c)
benzohydrazide, PyBOP, DIPEA, CH2Cl2, 59% for combined b and c; (d) DIC, DMF,
150 °C, 60%; (e) tributyl-(vinyl)stannane, Pd(0)(PPh3)4, toluene, 110 °C, 65%; (f)
HCOOH, H2O2, HIO4, 73%; (g) methylamine HCl, Ti(OiPr)4, NaBH4, EtOH, 50%; (h)
Boc-Ala-OH, EDCꢀHCl, HOBt, DIPEA, DMF, 91%; (i) HCl-dioxane, 49–60%; (j) Boc-
alaninal, NaH B(OAc)3, THF, 60%.
H
N
NH2
O
Compound11
Core
CARM1 IC50
0.06
(lM)
9
F3C
4
N
N
Table 4
In vitro profiling of 7f
Human microsome stability (nM/min/mg prot.)
PAMPA: Pc (nm/s)
0.026
267
>25
10a
10b
10c
10d
10e
0.13
2.20
0.4
N
N
hPXR14 (EC50
,
l
l
M)
M)
15
HHA IC50
(
All >100
N
N
Both of these modifications (compounds 14 and 15) led to a sig-
nificant loss of potency compared to 4, thereby reaffirming the ear-
lier observation of little tolerance for changes at the South end of
the molecule.
In order to assess the selectivity versus closely related PRMTs,
we evaluated the most potent compound (7f) for its ability to
inhibit PRMT1 and PRMT3.13 Compound 7f was found to be signif-
icantly less active against these enzymes (IC50 > 25 lM) suggesting
selectivity in binding and inhibition. Compound 7f showed accept-
able profile in the in vitro panel (Table 4).
N
N
N
O
N
0.36
2.1
In summary, through SAR development and optimization of the
core and East end of 1, we have identified the pyrazole derivative
7f as a potent and selective inhibitor of CARM1. Further work re-
N

T. Huynh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2924–2927
2927
8. Purandare, A. V.; Chen, Z.; Huynh, T.; Pang, S.; Geng, J.; Vaccaro, W.; Poss, M. A.;
Oconnell, J.; Nowak, K.; Jayaraman, L. Bioorg. Med. Chem. Lett. 2008, 18, 4438.
9. A filter based assay as described in Ref. 8 utilizing histone H3 as substrate was
used to test compounds that specifically detected CARM1 dependent
methylation, and the values reported are average of three with <10% variability.
10. PAMPA assay—10 mM stocks of compound in 100% DMSO was used.
Compounds are diluted 1:100 in pH 7.4 or pH 5.5 donor well buffer (pION
lated to the utility of this class of inhibitors will be reported in due
course.
References and notes
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.
CAT # 110151) providing a 100
diluted in donor well buffer is transferred to a Whatman Unifilter plate and
filtered prior to dispensing 200 l into the donor well of the assay plate (pION
CAT #110163). The PAMPA membrane is formed by pipetting 4 l of the lipid
solution (pION CAT #110169) onto the filter plate (VWR CAT #13503). The
membrane is then covered with 200 l of acceptor well buffer at pH 7.4 (pION
lM assay solution in 1% DMSO. Compound
l
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.
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.
l
l
CAT #110139). The PAMPA assay plate (donor side and acceptor side) is
combined and allowed to incubate at room temperature for 4 h. The plate is
then disassembled and spectrophotometer plates (VWR CAT #655801) are
filled (150 ll/well). The donor, acceptor, reference, and blank plates are read in
the SpectraMax UV plate reader. Data is captured by the pION software which
analyzes the spectra and generates Pc values.
11. (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: 1H NMR (500 MHz, MeOH-d) d
ppm 9.41 (s, 1H), 8.34 (dd, J = 8.14, 0.88 Hz, 1H), 8.01 (d, J = 7.26 Hz, 1H), 7.77
(dd, J = 8.14, 7.70 Hz, 1H), 7.72 (s, 1H), 7.65 (br s, 1H), 7.57–7.62 (m, 3H), 4.59
(A of AB, J = 15.41 Hz, 1H), 4.55 (B of AB, J = 15.19 Hz, 1H), 3.96–4.03 (m, 1H),
1.53 (d, J = 7.04 Hz, 3H); MS (ESI) m/z 514.12 (M+H).
12. Fevig, J. M.; Pinto, D. J.; Han, Q.; Quan, M. L.; Pruitt, J. R.; Jacobson, I. C.;
Galemmo, R. A., Jr.; Wang, S.; Orwat, M. J.; Bostrom, L. L.; Knabb, R. M.; Wong, P.
C.; Lam, P. Y. S.; Wexler, R. R. Bioorg. Med. Chem. Lett. 2001, 11, 641.
13. PRMT1 and PRMT3 specific methylation assays were performed using assay
conditions as reported in Ref. 8.
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) Wahhab, A.; Therrien, E.;
Allan, M.; Manku, S. WO2008104077.; (f) Allan, M.; Manku, S.; Therrien, E.;
Nguyen, N.; Styhler, S.; Robert, M.; Goulet, A.; Petschner, A. J.; Rahil, G.; Robert,
M. A.; Deziel, R.; Besterman, J. M.; Nguyen, H.; Wahhab, A. Bioorg. Med. Chem.
Lett. 2009, 19, 1218.
14. hPXR; Human pregnane-X receptor.
15. HHA: The Human Hepatocyte Assay. Compound 7f showed IC50 > 100 lM for
the parent cell line (Tc5) and all CYP transfected lines (3A4, 2C9, 2C19 and
2D6).