1,2-Diamines as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1)

Article abstract of DOI:10.1016/j.bmcl.2009.09.110

We have identified the N¹-benzyl-N²-methylethane-1,2-diamine unit as a substitute for the (S)-alanine benzylamide moiety for the design of co-activator associated arginine methyltransferase 1 (CARM1) inhibitors. The potency of these

Full text of DOI:10.1016/j.bmcl.2009.09.110

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6725–6732
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1,2-Diamines as inhibitors of co-activator associated arginine
methyltransferase 1 (CARM1)
Eric Therrien a, Guillaume Larouche a, Sukhdev Manku a, Martin Allan a, Natalie Nguyen a, Sylvia Styhler b,
Marie-France Robert b, Anne-Christine Goulet b, Jeffrey M. Besterman b, Hannah Nguyen b, Amal Wahhab a,
*
a MethylGene Inc., Department of Medicinal Chemistry, 7220 rue Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
b MethylGene Inc., Department of Pharmacology and Cell Biology, 7220 rue Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
a r t i c l e i n f o
a b s t r a c t
We have identified the N1-benzyl-N2-methylethane-1,2-diamine unit as a substitute for the (S)-alanine
benzylamide moiety for the design of co-activator associated arginine methyltransferase 1 (CARM1)
inhibitors. The potency of these inhibitors is in the same order of magnitude as their predecessors and
their clearance, volume of distribution, and half lives were greatly improved.
Article history:
Received 16 August 2009
Revised 24 September 2009
Accepted 29 September 2009
Available online 2 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CARM1 inhibitors
PRMT4 inhibitors
Histone methyltransferase inhibitors
Co-activator associated arginine
methyltransferase 1
There is an emerging interest in the enzymes that modulate the
methylation of protein lysine and arginine residues of histones.
One such set of enzymes is the mammalian protein arginine meth-
yltransferases (PRMTs), a family of at least nine members with di-
verse biological functions.1 These enzymes catalyze the transfer of
methyl groups from S-adenosyl methionine to Arg17 and Arg26
residues in histone H3, resulting in the formation of either asym-
metric (Type I; PRMTs 1–4, 6 and 8) or symmetric (Type II; PRMTs
one peroxidase 3,5j UCP-15j), inflammation (MIP-2,5f IP-10,5f Class
II MHC) and adipogenesis (THRSP-14, FABP4/aP2; ACS5; adipsin,
APOA-1 and -IV).5j The co-activator activity of CARM1 coincides
with the arginine methylation of histone H3 and CBP/p300 on sev-
eral target promoters. Mutations of critical residues in the catalytic
domain of CARM1 has been shown to compromise transcriptional
activation, suggesting that the integrity of the methyltransferase
domain of CARM1 is important for its co-activator function.4,5c,h
Several studies demonstrated a role of CARM1 in cancer. Knock-
out or silencing of CARM1 impedes estrogen-stimulated gene
expression, cell cycle progression and growth of breast cancer
cells,3d,5l and evidence also links CARM1, cyclin E and steroid co-
activator overexpression to high-grade breast cancer tumors.6a
Genome-wide positional analyses show that overexpression gene
signatures from ER-alpha positive primary breast tumors, derived
from over 20 studies, are significantly related to enhancer-rich
clusters associated with the induction of CARM1 activity in re-
sponse to estrogen.6b Furthermore, modulation of CARM1 levels af-
fect androgen-dependent transcription and prostate cancer cell
growth,6c and elevated CARM1 levels correlate with the develop-
ment of prostate carcinoma as well as the progression of andro-
gen-independent prostate cancer.6d These studies support CARM1
as a plausible target for anti-cancer drug development.
5, 7, and 9)
x-NG,NG-dimethylarginine tails on a wide variety of
protein substrates.2
The co-activator associated arginine methyltransferase
1
(CARM1), also known as PRMT4, methylates proteins with roles
in gene regulation at the level of 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-activator for the
p160 steroid co-activator protein in transcription mediated by nu-
clear hormone receptors,4 and was later found to be a positive co-
regulator for a number of other transcriptional modulators such as
SRC-3,5a CBP/p300,3a,5b b-catenin,5c p53,5d,e NF-kappa-B,5f,g CIITA,5h
HTLV-1 Tax,5i PPAR 5j and c-fos.5k As CARM1 is recruited by a di-
c
verse panel of transcription factors, it is not surprising that its tar-
get genes encompass a variety of roles including cell growth
regulation (estrogen receptor,4 pS2,5l complement C3,5j glutathi-
Early reports describing novel inhibitors of PRMTs originated
either from molecular modeling studies or high-throughput
screening.7a–e The majority of these were micromolar inhibitors
that lacked selectivity and, in some cases, did not possess drug-like
* Corresponding author.
E-mail address: wahhaba@methylgene.com (A. Wahhab).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.110

6726
E. Therrien et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6725–6732
properties. In addition, the bisubstrate inhibitor reported by Os-
borne et al.8 was over fourfold more active against PRMT1 com-
pared to CARM1. Just recently, we disclosed a new series of
thiophene-based nanomolar inhibitors of CARM1 (Fig. 1, com-
pound 1).9a The activity of these inhibitors were comparable to
those reported by Purandare et al.9b,c (Fig. 1, compound 2a–b).
Preliminary investigation of the pharmacokinetic properties of
1, and 2a proved that both the phenyl and thiophene-based inhib-
itors had poor bioavailability in rats and low exposure when dosed
ip in mouse. The recent report of Huynh et al.9d confirmed our
observations that 2a had poor permeability (PAMPA assay). How-
ever, the authors were able to improve the permeability of these
inhibitors by exchanging the amide unit on the right-hand side
with an 1,3,4-oxadiazole moiety. In addition, they reported that
the (S)-alanine benzylamide, on the left of 2 is optimal for CARM1
enzymatic activity and simple modifications such as in 3, 4, or 5
were not tolerated9b,d (Table 1). In this Letter, we disclose our ef-
forts to replace the (S)-alanine benzyl amide moiety and to identify
N1-benzyl-N2-methylethane-1,2-diamine unit for the design of
CARM1 inhibitors, as we suspected that the former unit might be
responsible for the observed poor PK of these inhibitors. Unlike
their predecessors, these diamines have improved clearance, vol-
ume of distribution, and half lives.
Table 1 (continued)
Compd
R
CARM-1 IC50
>20d
(lM)
N
4
H2N
O
H
N
5
>20d
>20
4.5
H2N
O
6
N
H
NH2
H
N
10
11
H2N
S
H
N
>20
HO
O
N
Compound 6, a homolog of 2b, was prepared starting from 2-(3-
aminophenyl)acetonitrile utilizing known procedures as depicted
in Scheme 1. Diazotization of 2-(3-aminophenyl)acetonitrile fol-
lowed by a tin chloride reduction of the intermediate diazonium
salt, and cyclocondensation of the resultant hydrazide with 4,4,4-
trifluoro-1-(furan-2-yl)butane-1,3-dione gave trisubstituted pyra-
zole 7. Borane reduction of 7 and Boc-protection of the resulting
amine, followed by the oxidative cleavage of the furan ring affor-
19
>20
>20
0.20
1.0
H2N
H2N
N
N
HN
N
20
O
H
N
12
N
H
F3C
O
N
N
H
N
HN
15a
15b
15c
15d
16
H2N
R
H
N
C
H2N
O
H
N
C
: 2,4-disubstituted thiophene
,R = OMe
7.9
1
N
C:meta-substituted phenyl;
2a
, R = H
2b
,R = OMe
H
N
Figure 1. (S)-Alanine amides as CARM1 inhibitors.
7.7
N
H
Table 1
Effect of Ala-benzylamide replacement on CARM1 inhibitory activitya
H
N
F3C
>20
21
N
H
O
N
N
HN
H
N
OMe
N
H
R
Compd
R
CARM-1 IC50
0.06
(lM)
H
N
17
>20
N
H
H
N
2bb
O
H2N
a
O
Values are means of at least two experiments.
Compound 2b (Fig. 1) CARM1 IC50 value determined in-house.
Ref. 9c reported methylation of the terminal amino acid (glycine) analog
b
c
H
N
3
>20c
bearing a different amide on the right-hand side.
N
H
d
Ref. 9d reported analogues with the same modification of the amino acid ter-
O
minus but bearing an oxadiazole unit on the right-hand side of the molecule.

E. Therrien et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6725–6732
F3C F3C
6727
NH2
O
OH
N
N
N
N
c, d, e
a, b
O
N
CN
NHBoc
7
8
F3C
f, g, h, i
H
N
N
N
O
O
O
NH2
N
H
6
Scheme 1. Reagents and conditions: (a) NaNO2, HCl, SnCl2ꢀH2O, 0 °C, 65%; (b) 4,4,4-trifluoro-1-(furan-2-yl)butane-1,3-dione, AcOH, 120 °C, 46%; (c) BH3ꢀTHF, THF, crude; (d)
Boc2O, Et3N, DCM, 53% for two steps; (e) NaClO2, NaH2PO4, MeCN, H2O, crude; (f) (2-methoxyphenyl)methanamine, BOP, Et3N, DCM, 46% for two steps; (g) 1:1 TFA/DCM,
1.5 h, rt, crude; (h) Boc-Ala-OSu, Et3N, DCM, 58%; (i) 1:1 TFA/DCM, 4 h, rt, quantitative.
F3C
F3C
F3C
H
N
H
N
H
N
N
N
N
c
O
N
a, b
N
N
O
O
O
O
O
H
N
H
N
H2N
OH
NH2
d, e
O
S
9
11
10
F3C
O
N
N
HN
O
H
N
N
H
12
Scheme 2. Reagents and conditions: (a) (S)-tert-butyl 1-(6-fluoro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)-1-thioxopropan-2-ylcarbamate, DMF, 0 °C, 2 h, then rt, 18 h,
39%; (b) 1:4 TFA/DCM, 3 h, rt, 96%; (c) lactic acid, BOP, Et3N, DCM, 14%; (d) tert-butyl methyl(2-oxoethyl)carbamate, AcOH, MeOH, rt, 30 min then NaCNBH3, 16 h, 37%; (e) 1:4
TFA: DCM, rt, 1.5 h, 22%.
F3C
F3C
F3C
O
O
N
N
N
N
N
N
a, b, c, d, e
f
HN
O
HN
O
O
H
N
O
R'
N
14
15a-d
15a, R'=
13
k,l, m
g,h,i, j
NH2
N
F3C
F3C
H
N
15b
15c
, R'=
N
N
N
OMe
N
H
N
OMe
O
O
, R'=
H
H
N
N
N
N
H
NH
O
H
15d
, R'=
17
16
Scheme 3. Reagents and conditions: (a) DIBAL-H, DCM, 0 °C to rt over 2 h, crude; (b) 1,3-propanediol, p-toluene sulfonic acid, toluene, Dean-Stark, 16 h, 79%; (c) NaClO2,
NaH2PO4, MeCN, H2O, crude; (d) (2-methoxyphenyl)methanamine, BOP, Et3N, DCM, 48% for two steps; (e) 4:1 AcOH/H2O, 90 °C, 2 h, crude; (f) for 15a, tert-butyl N-(2-
aminoethyl)carbamate, DCM, AcOH, followed by NaBH(OAc)3, rt, 16 h, 62%; then 1:6.5 TFA/DCM, rt, 2.5 h, 32%; (g) for 16, MeMgBr, THF, ꢁ78 to 0 °C, 81%; (h) Dess–Martin
periodinane, DCM, rt, 87%; (i) tert-butyl 2-aminoethyl(methyl)carbamate, AcOH, DCM, 15 min, then NaBH(OAc)3, rt, 18 h, 30%; (j) 1:4 TFA/DCM, 2 h, rt, 80%; (k) for 17, 2-
methyl-2-butene, 10:1 t-BuOH/H2O, KH2PO4, NaClO2, rt, 1 h, crude, (l) tert-butyl 2-aminoethyl(methyl)carbamate, BOP, Et3N, DCM, rt, 36 h, 74%; (m) 1:4 TFA/DCM, rt, 2 h, 9%.

6728
E. Therrien et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6725–6732
ded acid 8. BOP coupling of 8 with (2-methoxyphenyl)methan-
amine, followed by removal of the Boc group with TFA, reaction
of the produced amine with Boc-Ala-OSu, and further amine
deprotection gave the desired analog 6.
of NaBH(OAc)3, and further Boc-deprotection gave diamine 16. So-
dium chlorite oxidation of 14 to the corresponding acid, followed
by BOP coupling with tert-butyl 2-aminoethyl(methyl)carbamate
and subsequent Boc-deprotection provided amide 17.
Intermediate 9, prepared according to published procedures,9
was used for the synthesis of analogues 10, 11, and 12 (Scheme
2). The thioamide 10 was accessible by the reaction of 9 with (S)-
tert-butyl 1-(6-fluoro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)-1-thioxopropan-2-ylcarbamate, prepared according to the pro-
cedure of Zacharie et al.10 followed by removal of the Boc group.
Alcohol 11 was obtained in one step by coupling of 9 with lactic
acid, while diamine 12 was prepared by reductive amination of
tert-butyl methyl(2-oxoethyl)carbamate, followed by the Boc-
deprotection.
Nitrile intermediate 189 was used to access triazole 19 and oxa-
diazole 20 (Scheme 4). Reaction of 18 with HCl(gas) in MeOH, fol-
lowed by the condensation of the resulting amidate with (S)-tert-
butyl 1-hydrazinyl-1-oxopropan-2-ylcarbamate, dehydration, and
subsequent Boc-deprotection gave triazole 19. The reaction of 18
with hydroxylamine, followed by condensation of the resulting
N-hydroxyamidine with Boc-Ala-OH utilizing TBTU, dehydration,
and deprotection gave oxadiazole 20.
The synthesis of amides 23a–n is depicted in Scheme 5 below.
The nitrile 13 was converted to the bis-Boc-protected intermediate
21 following procedures described above. Oxidation of 21 to the
carboxilic acid 22, followed by a coupling of the carboxylic acid
with different amines, and subsequent Boc-deprotection gave the
desired amides in good yields.
Compound 24 was prepared according to Scheme 6 starting
from the acid 22 by cyclocondensation with 2-methoxybenzhydra-
zide utilizing 2-chloro-1,3-dimethylimidazolinium chloride as the
dehydrating agent, followed by the Boc-deprotection with TFA.
Both compounds 25 and 26 (Table 3) were prepared following
the same procedure described for 23 starting from 5-amino-2-fluo-
robenzonitrile and 5-amino-2-methylbenzonitrile, respectively.
The trifluoromethyl substituted analog 27, was prepared starting
from methyl 3-amino-5-(trifluoromethyl)benzoate as illustrated
in Scheme 7 below. The ester 28, prepared following published
The conversion of the nitrile 139 gave access to the aldehyde 14,
which was a key building block for the synthesis of analogues 15,
16 and 17 (Scheme 3). Reduction of 13 with DIBAL-H followed by
an acetal protection of the resulting aldehyde (not shown in the
scheme), oxidation of the furan ring to the acid, coupling of the
resulting acid with (2-methoxyphenyl)methanamine and finally
hydrolysis of the acetal group with acetic acid gave the aldehyde
14. Reductive amination of the aldehyde 14 with the appropriate
amine utilizing NaBH(OAc)3 as the reducing agent, followed by
the removal of the Boc-protecting groups gave diamines 15a–d
(Table 1). Reaction of the aldehyde 14 with the methyl magnesium
bromide and oxidation of the resulting alcohol with the Dess–Mar-
tin periodinane to the ketone, followed by reductive amination
with tert-butyl 2-aminoethyl(methyl)carbamate in the presence
F3C
N
F3C
H
N
H
F3C
N
N
O
N
O
H
N
N
a,b,c
d,e,f
O
O
N
O
N
O
N
N
N
NH2
N
NH2
O
NH
19
CN
18
20
Scheme 4. Reagents and conditions: (a) 1:1 MeOH/Et2O, ꢁ5 °C, HCl(gas), 30 min, then rt, 2 h, 81%; (b) (S)-tert-butyl 1-hydrazinyl-1-oxopropan-2-ylcarbamate, Et3N, reflux, 6 h,
then xylenes, 140 °C, 20 min, 18%; (c) 1:4 TFA/DCM, 1 h, rt, 54%; (d) NH2OHꢀHCl, NaHCO3, MeOH, reflux 4 h, 60%; (e) Boc-Ala, TBTU, HOBt, DIPEA, DMF, rt 1 h, then 110 °C, 2 h,
55%; (f) 1:1 TFA/DCM, rt, 1 h, quantitative.
F3C
F3C
N
N
N
N
a, b, c
O
O
Boc
N
N
13
21
Boc
N
d
F3C
F3C
O
N
N
e, f
CO2H
N
N
NHR
Boc
H
N
N
N
Boc
N
H
23a-n
22
Scheme 5. Reagents and conditions: (a) DIBAL-H, DCM, 0 °C to rt over 2 h, crude; (b) tert-butyl 2-aminoethyl(methyl)carbamate AcOH, MeOH, 1 h, rt, then NaBCNH3, rt, 18 h,
crude; (c) Boc2O, DCM, 0 °C to rt, 5 h, 78% over three steps; (d) NaClO2, NaH2PO4, MeCN, H2O, crude; (e) RNH2, BOP, Et3N, DCM, 18 h; (f) 1:4 TFA/DCM, rt, 1–3 h.

E. Therrien et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6725–6732
6729
periodinane. Reductive amination of 29, Boc-protection of the
resulting amine, oxidative cleavage of the furan to the carboxylic
acid, and the EDC assisted coupling with (2-methoxyphenyl)meth-
anamine, followed by the TFA removal of the Boc-protecting
groups gave analog 27.
F3C
F3C
O
OH
O
N
N
N
N
a, b
O
N N
Boc
N
H
N
The tricyclic compound 30 was prepared starting from 3-fluoro-
4-nitrobenzonitrile, as shown in the Scheme 8. Borane reduction of
the nitrile group to the amine, followed by reductive amination of
tert-butyl methyl(2-oxoethyl)carbamate and titanium(IV) isoprop-
oxide and NaBH4, followed by a subsequent Boc-protection of the
resulting amine gave the bis-Boc diamine 31. This material react-
ing with ethyl 3-(trifluoromethyl)-1H-pyrazole-5-carboxylate in
the presence of NaH, followed by catalytic reduction of the nitro
group gave the aniline 32. Cyclization of the aniline 32 to the lac-
tam 33 was achieved with Me3Al. Abstraction of the acidic proton
of lactam 33 and alkylation with 2-methoxybenzyl chloride, fol-
lowed by removal of the Boc-protecting groups gave the analog 30.
The effects of compounds 3–6, 10–13, 19, 20, 15a–d, 16, 17,
23a–n, 24–27, and 30 on the inhibitory activity of CARM1 was
measured by means of a histone methyltransferase assay using re-
combinant CARM1 enzyme, and histone H3 as the substrate.11a,d
Table 1 shows the CARM1 inhibitory activity of some of the com-
pounds we prepared to investigate replacements of the Ala-ben-
zylamide end of the molecule. The lack of enzymatic inhibitory
activity of compounds 3, 4, and 5 is consistent with the recent re-
ports of Purandare et al.9b,c and Huynh et al.9d In addition, the inac-
tivity of 6, and 11 impresses the requirement for an unsubstituted
terminal amine being a certain distance away from the core. The
amide functionality of 2b seems to be essential for activity while
the amide isosteres such as the thiourea (10), triazole (19), and
oxadiazole (20) were not active, despite the presence of the unsub-
stituted amine functionality (Table 1). The inactivity of the des-
amide analog 5 and the observed activity of analogues 12 and
15a was intriguing and prompted us to investigate the possible
binding site for this unit in order to rationalize the observed trend
in the activities of these analogues.
Boc
N
NH
24
22
Scheme 6. Reagents and conditions: (a) 2-methoxybenzhydrazide, 2-chloro-1,3-
dimethylimidazolinium chloride, Et3N, DMF, rt, 24 h, 15%; (b) 1:4 TFA/DCM, rt, 1 h,
quantitative.
F3C
O
NH2
N
N
a, b
O
CF3
O
CF3
O
28
O
c, d
F3C
F3C
e, f, g, h, i
O
O
N
N
N
N
HN
OMe
H
N
O
N
H
CF3
CF3
29
27
Scheme 7. Reagents and conditions: (a) NaNO2, HCl, SnCl2ꢀH2O, 0 °C, crude; (b)
4,4,4-trifluoro-1-(furan-2-yl)butane-1,3-dione, AcOH, 120 °C, 67%; (c) DIBAL-H,
toluene, ꢁ78 °C, 1 h, then warm to rt over 1.5 h, 94%; (d) Dess–Martin periodinane,
DCM, rt, 2 h, crude; (e) tert-butyl 2-aminoethyl(methyl)carbamate AcOH, MeOH, rt,
1 h, then NaBH3CN, rt, 16 h, crude; (f) Boc2O, DCM, 0 °C to rt, 16 h, 81% over three
steps; (g) NaClO2, NaH2PO4, MeCN, H2O, crude; (d) (2-methoxyphenyl)methan-
amine, EDC, DMAP, DCM, 48 h, 10% for two steps; (n) 1:4 TFA/DCM, rt, 2 h, 84%.
A close inspection of the published crystal structure of the cat-
alytically active site of CARM1 in complex with the co-factor prod-
uct SAH, illustrated the importance of key interactions of SAH with
two active site residues, ARG169 and ASP191.12 The carboxylate
group of SAH makes a salt bridge with the arginine residues
procedures,9 was converted to the aldehyde 29 in two steps by
reduction with DIBAL-H followed by oxidation with Dess–Martin
F3C
O2N
F
O
O
F
N
N
a, b, c
d, e
O2N
H2N
Boc
N
NBoc
BocN
CN
N
Boc
31
32
f
F3C
F3C
N
O
N
O
g, h
N
N
NH
N
Boc
H
N
O
N
N
N
H
Boc
30
33
Scheme 8. Reagents and conditions: (a) BH3ꢀTHF, THF, 0 °C, then rt for 16 h, crude; (b) tert-butyl methyl(2-oxoethyl)carbamate, titanium(IV) isopropoxide, THF, rt, 5 h, then
NaBH4, EtOH, rt, 16 h, 73%; (c) Boc2O, DCM, Et3N, 0 °C to rt, 2.5 h, 22%; (d) ethyl 3-(trifluoromethyl)-1H-pyrazole-5-carboxylate, NaH, DMF, 25 min at rt, then cool and add 31
dropwise in DMF, warm to rt, 1 h, 30%; (e) 10% Pd/C, H2 (gas) 1 atm, EtOH, 1 h, rt, 94%; (f) DCM, 0 °C, Me3Al/toluene, warm up to rt for 1 h, then 2 h at 40 °C, 27%; (g) NaH, DMF,
rt, 10 min, then add 2-methoxybenzyl chloride, 0 °C, then at rt, 16 h, 70%; (h) 4:1 TFA/DCM, rt, 2 h, 39%.

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E. Therrien et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6725–6732
Table 2
SAR of diamine analogues 23a–na
F3C
O
N
N
HN
R
H
N
N
H
Compd
R
CARM-1 IC50
>10
(lM)
23a
23b
23c
13.1
1.61
Figure 2. Overlay of the conformations of 12 (yellow) and SAH (gray) docked in the
active site of CARM1. Dashed green lines indicate hydrogen bonds.
O
O
23d
23e
23f
23g
0.9
whereas the ammonium moiety is involved in a bifurcated H-
bonded interaction with ASP191 and a water molecule in the active
site. Docking experiments provided a plausible explanation for the
observed behavior of the synthesized analogues.
O
0.71
0.71
6.5
Figure 2 shows an overlay of diamine 12 with SAH obtained
from the docking experiments using the automated docking soft-
ware FITTED 2.613 with the CARM1 crystal structure (PDB code
2v74). Analysis of the docking results highlighted the importance
of the flexible ethylene fragment of 12 for productive binding. Sub-
stituents or group modifications on this ethylene unit that con-
strain the flexibility or the size are detrimental to the activity
(see Table 1). We hypothesize that such modifications prohibit
the possibility for the ligand to adopt the required conformation
to interact with ASP191 and the water molecule in the active site,
while avoiding the positive surroundings of ARG169.13d
Having established that the unsubstituted ethylene unit of 12 is
most favorable, we turned our attention to the other parts of our
lead. Table 2, shows the CARM1 inhibitory activity of some of the
diverse amides we prepared to optimize the right-hand moiety.
We first investigated the tolerance of substitution on the benzyl
moiety of 12. However, none of the analogues prepared, 23a–
23h, were more active than 12; indicating that an o-methoxy sub-
stituent is optimal for activity. Replacement of the benzyl moiety
of the amide with an alkyl (23i) or a heterocycle (23j–l) led to more
than one log loss of inhibitory activity against the CARM1 enzyme.
A bicyclic naphthalen-1-ylmethyl group (23m) was tolerated and
resulted in a slight loss of inhibitory activity.
OMe
F
OCF3
NH2
23h
23i
>10
3.6
N
N
N
23j
4.7
4.3
However, unlike the benzyl analog, an o-methoxy substituent
off the naphthyl (23n) led to a marked decrease of activity (see Ta-
ble 2). In contrast to the observations of Huynh et al.9d replacement
of the amide group of 12 with the 1,3,4-oxadiazole moiety, com-
pound 24, resulted in a threefold loss of activity against the CARM1
NH
23k
enzyme (IC50 of 0.59 lM). Since there was no gain in potency by
NH
23l
1.8
replacement of right-hand side amide group, we next turned our
attention to the core of 12 and studied the effect of substitution
off the phenyl ring. Simple substituents such as fluoro (25), methyl
(26), or trifluoromethyl (27) groups were not tolerated leading to
substantial or total loss of activity (Table 3). Cyclization of the side
chain amide unto the core of the compound, producing the con-
strained analog 30, abolished the ability of the compound to inhibit
CARM1 activity. These results confirm that diamine 12 is the lead-
ing inhibitor in this series.14 However, despite the observed CARM1
enzymatic activity, compound 12 failed to show any cellular activ-
ities when tested in MTT assays.
23m
23n
0.32
3.35
MeO
a
Values are means of at least two experiments.

E. Therrien et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6725–6732
6731
Table 3
Acknowledgments
Substitutions on the core phenyl ringa
The authors are grateful to Dr. James Wang (PK studies), Dr. Jub-
rial Rahil and Ms. Andrea J. Petschner (enzymology), Dr. Christiane
Maroun and Ms. Claire Bonfils (Biology) for valuable discussions,
and Dr. Arkadii Vaisburg for reviewing and proof reading this
article.
H
N
R"
N
H
Compd
R00
CARM-1 IC50 (lM)
References and notes
F3C
H
N
1. (a) McBride, A. E.; Silver, P. A. Cell 2001, 106, 5; (b) Bedford, M. T.; Richard, S.
Mol. Cell. 2005, 18, 263; (c) Krause, C. D.; Yang, Z. H.; Kim, Y. S.; Lee, J. H.; Cook, J.
R.; Pestka, S. Pharmacol. Ther. 2007, 113, 50; (d) Herrmann, F.; Pably, P.;
Eckerich, C.; Bedford, M. T.; Fackelmayer, F. O. J. Cell Sci. 2009, 122, 667; (e) Lee,
Y. H.; Stallcup, M. R. Mol. Endocrinol. 2009, 23, 425; (f) Tini, M.; Naeem, H.;
Torchia, J. Methods Mol. Biol. 2009, 523, 235.
N
N
F
O
O
25
5.6
2. (a) Gary, J. D.; Clarke, S. Prog. Nucleic Acid Res. Mol. Biol. 1998, 61, 65; (b) Zhang,
Y.; Reinberg, D. Genes Dev. 2001, 15, 2343.
F3C
H
N
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.;
Vandel, L. EMBO J. 2002, 21, 5457; (c) Lee, Y. H.; Koh, S. S.; Zhang, X.; Cheng, X.;
Stallcup, M. R. Mol. Cell Biol. 2002, 22, 3621; (d) Yadav, N.; Lee, J.; Kim, J.; Shen,
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.;
Tohyama, M. Mol. Cell. Biol. 2006, 26, 2273; (h) Ohkura, N.; Takahashi, M.;
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.
N
N
26
27
30
7.94
O
O
O
F3C
H
N
N
N
N
>20
O
4. Chen, D.; Ma, H.; Hong, H.; Koh, S. S.; Huang, S.-M.; Schurter, B. T.; Aswad, D.
CF3
W.; Stallcup, M. R. Science 1999, 284, 2174.
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) Shirley, S. H.; Rundhaug, J. E.; Tian, J.;
Cullinan-Ammann, N.; Lambertz, I.; Conti, C. J.; Fuchs-Young, R. Cancer Res.
2009, 69, 3405; (f) 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; (g) Miao, F.; Li, S.; Chavez, V.; Lanting, L.; Natarajan, R. Mol. Endocrinol.
2006, 20, 1562; (h) Zika, E.; Fauquier, L.; Vandel, L.; Ting, J. P. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 16321; (i) Jeong, S. J.; Lu, H.; Cho, W. K.; Park, H. U.; Pise-
Masison, C.; Brady, J. N. J. Virol. 2006, 80, 10036; (j) Fauquier, L.; Duboé, C.; Joré,
C.; Trouche, D.; Vandel, L. FASEB J. 2008, 22, 3337; (k) Yadav, N.; Cheng, D.;
Richard, S.; Morel, M.; Iyer, V. R.; Aldaz, C. M.; Bedford, M. T. EMBO Rep. 2008, 9,
193; (l) Frietze, S.; Lupien, M.; Silver, P. A.; Brown, M. Cancer Res. 2008, 68, 301.
6. (a) 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; (b) Lupien, M.; Eeckhoute, J.; Meyer, C. A.; Krum, S. A.;
Rhodes, D. R.; Liu, X. S.; Brown, M. Mol. Cell. Biol. 2009, 29, 3413; (c) Majumder,
S.; Liu, Y.; Ford, O. H., 3rd; Mohler, J. L.; Whang, Y. E. Prostate 2006, 66, 1292; (d)
Hong, H.; Kao, C.; Jeng, M. H.; Eble, J. 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.
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.
Chem. Lett. 2007, 17, 4150; (d) Mai, A.; Valente, S.; Cheng, D.; Ragno, R.;
Simeoni, S.; Sbardella, G.; Brosch, G.; Nebbioso, A.; Conte, M.; Altucci, L.;
Bedford, M. T. Chem. Med. Chem. 2007, 2, 987; (e) Mai, A.; Cheng, D.; Bedford, M.
T.; Valente, S.; Nebbioso, A.; Perrone, A.; Brosch, G.; Sbardella, G.; De Bellis, F.;
Miceli, M.; Altucci, L. J. Med. Chem. 2008, 51, 2279.
F3C
N
O
>10
N
O
a
Values are means of at least two experiments.
To test whether our inhibitors had better PK properties than the
previously reported compounds, we evaluated the PK profiles of
12, 23d, 23m, and 24 in rats (Table 4). The results revealed that
these diamine inhibitors have lower clearance, longer half lives
and smaller volumes of distribution as compared to 2b (Table 4).
However, they still suffered from poor oral absorption as indicated
by the low AUC values and require further optimization.
In conclusion, we identified the N1-benzyl-N2-methylethane-
1,2-diamine unit as a substitute for the (S)-alanine benzylamide
moiety for the design of CARM1 small molecule inhibitors. The
inhibitory activities of the diamines were of the same order of
magnitude as their predecessors, and they exhibited improved
clearance, volume of distribution and longer half lives.
8. Osborne, T.; Roska, R. L.; Rajski, S. R.; Thompson, P. R. J. Am. Chem. Soc. 2008,
130, 4574.
9. (a) Allan, M.; Manku, S.; Therrien, E.; Nguyen, N.; Styhler, S.; Robert, M.-F.;
Goulet, A. C.; Petschner, A. J.; Rahil, G.; Robert Macleod, A.; Déziel, R.;
Besterman, J. M.; Nguyen, H.; Wahhab, A. Bioorg. Med. Chem. Lett. 2009, 19,
1218; (b) Purandare, A. V.; Chen, Z., International Patent WO 06/069155 A2,
2006.; (c) 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; (d) Huynh, T.; Chen, Z.; Pang, S.; Geng, J.; Bandiera, T.; Bindi, S.;
Vianello, P.; Roletto, F.; Thieffine, S.; Galvani, A.; Vaccaro, W.; Poss, M. A.;
Trainor, G. L.; Lorenzi, M. V.; Gottardis, M.; Jayaraman, L.; Purandare, A. V.
Bioorg Med. Chem. Lett. 2009, 19, 2924.
Table 4
PK profile in rat of selected compounds
Compd N. AUC (PO)b
M h/(mg/Kg)
N. AUC (IV)b
Cl (IV)
V
ss(IV) t1/2(IV) %F
l
lM h/(mg/Kg) (L/h)/Kg L/Kg
h
2ba
12
23d
23m
24
0.01
0.07
0.03
0.03
0.03
0.04
2.1
1.8
1.3
1.0
86
33.6
1.4
0.1
0.2
0.6
0.07
2.1
0.5
1.4
1.8
—
4
2
3
3
c
1.2
1.2
2.3
2.2
10. Zacharie, B.; Lagraoui, M.; Dimarco, M.; Penney, C. L.; Gagnon, L. J. Med. Chem.
1999, 42, 2046.
11. (a) All experimental details can be found in MethylGene patent application,
Wahhab, A.; Therrien, E.; Allan, M.; Manku, S. International Patent WO 08/
104077 A1, 2008; (b) The CARM1 enzyme (N-terminal His-tagged,
recombinant mouse CARM1, expressed in Sf9 cells) was purchased from
Millipore (cat# is 14-575, Lot # is DAM1473541). Histone H3 (Sigma–Aldrich)
a
b
c
Values obtained in our laboratories.
N. AUC is the normalized AUC values.
The compound is cleared extremely rapidly. An accurate value could not be
calculated due to the very low N. AUC (IV).

6732
E. Therrien et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6725–6732
(Amersham Pharmacia Biotech) as
was used as the substrate, and the methylation was monitored using tritiated
a
methyl donor. The reactions were
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
performed at 30 °C for a total of 15 min, using enzyme (PRMT1), substrate
(histone H4), and co-factor (SAM) in the absence and presence of compound.
Assay protocol: 2
96-well plate. PRMT1 enzyme was diluted in a Tris–HCl (pH 9) buffer (to a final
enzyme concentration of 0.2 M) and 10 l of the cold enzyme solution was
immediately added to the compound and allowed to pre-incubate for 10 min at
room temperature. A mixture of SAM (20% [3H] SAM) and histone H4 (10 l)
was then added and incubated for 15 min at 30 °C for a final concentration of
3.5 M histone and 0.2 M SAM (0.033 mCi/ml). The reaction was stopped by
adding 20 l of SAH for a final concentration of 60 M and, 10 l of the
ll of the diluted compound was added to a U-Bottom of a PP
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 8 ll
ll of the diluted compound was
l
l
l
l
of the cold enzyme solution was immediately added to the compound and
allowed to pre-incubate for 10 min at room temperature. A mixture of SAM
l
(20% [3H] SAM) and histone H3 (10
15 min at 30 °C for a final concentration of 0.0125
(0.033 mCi/ml). The reaction was stopped by adding 20
concentration of 60 M and, 10 l of the quenched reaction is spotted onto P30
l
l) was then added and incubated for
g/ l histone and 2 M SAM
l of SAH for a final
l
l
l
l
l
l
l
l
l
quenched reaction is spotted onto P30 Filtermat paper. The 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 (Wallac, filtermat A 1450-
421) and the radioactivity was read using a Wallac Microbeta counter (CCPM).
The SET9 enzyme (N-terminal His-tagged, human SET9 recombinant protein,
expressed in E. coli) was purchased from upstate (Cat # 14-469, Lot # 32194).
Histone H3 (purchased from upstate) was used as the substrate, and the
methylation was monitored using tritiated S-Adenosyl-Methionine (SAM)
l
l
Filtermat paper. The 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).
12. Yue, W. W.; Hassler, M.; Roe, S. M.; Thompson-Vale, V.; Pearl, L. H. EMBO J.
2007, 26, 4402.
13. (a) Corbeil, C. R.; Moitessier, N. J. Chem. Inf. Model. 2009, 49(4), 997; (b) Corbeil,
C. R.; Englebienne, P.; Moitessier, N. J. Chem. Inf. Model. 2007, 47(2), 435; (c)
http://www.fitted.ca.; (d) We are confident of the proposed binding mode of
compound 12, however we are unable to predict the activity of inhibitors
based on subtle structural changes.
14. To test the specificity of our inhibitors for CARM1 versus other arginine or
lysine methyltransferases, compound 12 was tested against the PRMT1
arginine and the Set7/9 lysine methyltransferases and was found to be
(Amersham Pharmacia Biotech) as a methyl donor. The reactions were
performed at 37 °C for a total of 45 min, using enzyme (SET9), substrate
(histone H3), and co-factor (SAM) in the absence and presence of compound.
Assay protocol: 2
96-well plate. SET9 enzyme was diluted in a Tris–HCl (pH 9) buffer (to a final
enzyme concentration of 0.0005 g/ l) and 8 l of the cold enzyme solution
ll of the diluted compound was added to a U-Bottom of a PP
l
l
l
was immediately added to the compound and allowed to pre-incubate for
10 min at room temperature. A mixture of SAM (20% [3H] SAM) and histone H3
significantly less active against both enzymes (IC50 >100
enzyme (N-terminal GST-tagged, human PRMT1 recombinant protein,
expressed in E. coli) was purchased from upstate (Cat 14-474, Lot
25336). Histone H4 (recombinant protein expressed in E. coli, Cat # 14-412, Lot
23215, purchased from upstate) was used as the substrate, and the
methylation was monitored using tritiated S-Adenosyl-Methionine (SAM)
l
M). The PRMT1
(10
concentration of 0.06
reaction was stopped by adding 100 l
Flashplate (Perkin Elmer basic flashplate). The flashplate was washed twice
with 10% TCA. The dry plate was read using a Wallac Microbeta counter
(CCPM).
l
l) was then added and incubated for 45 min at 37 °C for
g/ histone and 0.2 SAM (0.011 mCi/ml). The
l of water and transferred to a White
a final
l
ll
lM
#
#
#