Discovery of a Potent, Selective, and Cell-Active Dual Inhibitor of Protein Arginine Methyltransferase 4 and Protein Arginine Methyltransferase 6

Article abstract of DOI:10.1021/acs.jmedchem.6b01033

Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs) are invaluable chemical tools for testing biological and therapeutic hypotheses. Based on 4, a fragment-like inhibitor of type I PRMTs, we conducted structure-activity

Full text of DOI:10.1021/acs.jmedchem.6b01033

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Article
Discovery of a Potent, Selective and Cell-active Dual Inhibitor of Protein
Arginine Methyltransferase 4 and Protein Arginine Methyltransferase 6
Yudao Shen, Magdalena M Szewczyk, Mohammad S. Eram, David Smil, H. Ümit Kaniskan,
Renato Ferreira de Freitas, Guillermo Senisterra, Fengling Li, Matthieu Schapira, Peter J.
Brown, Cheryl H. Arrowsmith, Dalia Barsyte-Lovejoy, Jing Liu, Masoud Vedadi, and Jian Jin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01033 • Publication Date (Web): 01 Sep 2016
Downloaded from http://pubs.acs.org on September 2, 2016
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Journal of Medicinal Chemistry
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Discovery of a Potent, Selective and Cell-active Dual Inhibitor of Protein Arginine
Methyltransferase 4 and Protein Arginine Methyltransferase 6
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†,#
‡,#
‡,#
‡
†
‡
Yudao Shen, Magdalena M. Szewczyk, Mohammad S. Eram, David Smil, H. Ümit Kaniskan,
‡
‡
‡
‡,§
Renato Ferreira de Freitas, Guillermo Senisterra, Fengling Li, Matthieu Schapira, Peter J. Brown,
‡
,⊥
‡
,†
,‡,§
,†
Cheryl H. Arrowsmith, Dalia Barsyte-Lovejoy, Jing Liu,* Masoud Vedadi,* and Jian Jin*
†Department of Pharmacological Sciences and Department of Oncological Sciences, Icahn School of
Medicine at Mount Sinai, New York, New York 10029, United States
‡Structural Genomics Consortium, University of Toronto, Toronto, Ontario, M5G 1L7, Canada
§Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, M5S 1A8,
Canada
⊥Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto,
Toronto, Ontario, M5G 2M9, Canada
ABSTRACT: Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs)
are invaluable chemical tools for testing biological and therapeutic hypotheses. Based on 4, a fragment-
like inhibitor of type I PRMTs, we conducted structure–activity relationship (SAR) studies and explored
three regions of this scaffold. The studies led to the discovery of a potent, selective and cell-active dual
inhibitor of PRMT4 and PRMT6, 17 (MS049). As compared to 4, 17 displayed much improved potency
for PRMT4 and PRMT6 in both biochemical and cellular assays. It was selective for PRMT4 and
PRMT6 over other PRMTs and a broad range of other epigenetic modifiers and non-epigenetic targets.
We also developed 46 (MS049N), which was inactive in biochemical and cellular assays, as a negative
control for chemical biology studies. Considering possible overlapping substrate specificity of PRMTs,
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1
7 and 46 are valuable chemical tools for dissecting specific biological functions and dysregulation of
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PRMT4 and PRMT6 in health and disease.
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INTRODUCTION
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Arginine residues contain a characteristic guanidinium moiety, which is protonated and
1
positively charged at physiological conditions. The arginine guanidinium group can interact with other
polar groups through its five potential hydrogen bond donors. Consequently, through stable bidentate
salt bridges and hydrogen bonds, arginine is frequently involved in protein/protein and protein/nucleic
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acid complex formations. Arginine methylation, a major post-translational modification of arginine
4
residues, does not change its cationic charge character. Instead, this modification increases the
bulkiness of the guanidinium moiety and alters the charge distribution, hydrophobicity and hydrogen
5
bond formation potential. As a result, arginine methylation can have dramatic effects on the
interactions of the modified protein with other proteins and nucleic acids, thus its physiological
6
function, including RNA processing, DNA repair, transcriptional activation/repression, signal
7
transduction, cell differentiation, and embryonic development.
8-10
As a common post-translational modification in eukaryotic cells,
arginine methylation is
7
catalyzed by S-5′-adenosyl-L-methionine (SAM)-dependent PRMTs. Currently, nine PRMTs have
9
, 11, 12
been identified and grouped into three categories based on their product specificity.
Type I
PRMTs, including PRMT1-4, PRMT6 and PRMT8, catalyze arginine mono- and asymmetric
dimethylation. Type II PRMTs, including PRMT5 and PRMT9, catalyze arginine mono- and symmetric
1
1
dimethylation. PRMT7, which catalyzes arginine monomethylation only, is the sole type III PRMT.
PRMTs have a vital role in the regulation of the arginine methylation pattern and level of a plethora of
7
different substrates, including both histones and non-histone proteins. Thus, it is not surprising that
7, 11, 12
dysregulation of PRMTs has been linked to a variety of human diseases.
For example, PRMT4
(also known as coactivator-associated arginine methyltransferase 1, CARM1) has a broad range of
1
3
substrates including histones (e.g., H3R17, H3R26), transcriptional factors (e.g., SOX2, SOX9, PAX7,
14-17
18
RUNX1),
chromatin remodeling factor (e.g., BAF155), transcriptional coactivators (e.g., CBP,
1
9-21
22-24
p300, SRC3),
RNA binding proteins (e.g., PABP1, HuR, HuD),
splicing factors (e.g., CA150,
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SAP49, SmB, U1C), T cell-specific factor (e.g., TARPP), RNA polymerase II, and mediator of
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RNA polymerase II transcription (e.g., Med12). Overexpression of PRMT4 has been associated with
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AML, breast, prostate, lung and colorectal cancers. The primary methylation targets of PRMT6
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are histone H3R2 and DNA polymerase β. PRMT6 can also methylate HIV Tat and HMGA1a.
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PRMT6 is overexpressed in melanoma , bladder, lung and prostate cancers. Thus, growing
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evidence suggests PRMT4 and PRMT6 as potential therapeutic targets.
In addition, both PRMT4
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9
and PRMT6 dimethylated H3R42 in vitro and in vivo. Furthermore, PRMT4 and PRMT6 showed
4
0
synergic effect in stimulating estrogen receptor alpha (ERα)-dependent transcription. Therefore,
targeting PRMT4 and PRMT6 simultaneously may provide a beneficial therapeutic strategy.
4
1, 42
Potent, selective and cell-active small-molecule inhibitors of PRMTs
would be invaluable
tools for investigating the biology of these protein targets and testing the disease and therapeutic
hypotheses associated with them. Over the last decade, a number of small-molecule PRMT inhibitors
4
3-60
50
have been reported,
PRMT6 selective inhibitor, 2 (Figure 1A). Based on the PRMT4 and PRMT6 inhibitors,
previously discovered 3 (MS023), a potent, selective and cell-active inhibitor of type I PRMTs (Figure
which include a series of PRMT4 inhibitors represented by compound 1 and a
5
7
50, 57
we
4
7
1
A). Recently, benzylpiperidinylethanamine 4 (Figure 1B) was identified as a fragment-like inhibitor
4
8
of type I PRMTs (IC50 = 0.3–19 µM). The crystal structure of PRMT6 in complex with 4 revealed that
the ethylenediamino moiety was anchored in the substrate binding channel and serves as an arginine
4
8
mimetic (Figure 1B). Here, we report structure–activity relationship (SAR) studies that focus on
exploring three regions of the scaffold represented by compound 4. We describe the design, synthesis,
and biological evaluation of novel compounds, which resulted in the discovery of a potent, selective and
cell-active dual inhibitor of PRMT4 and PRMT6, 17 (MS049), and a negative control, 46 (MS049N).
RESULTS
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Design and Synthesis. To improve potency and selectivity of 4, we investigated the following
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three regions: (1) the right-hand side (RHS) piperidinylethanamine moiety, (2) the middle linker moiety,
and (3) the left-hand side (LHS) aromatic moiety (Figure 1B).
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The piperidinylethanamine portion of 4 contains an ethylenediamino group, which is also
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present in the PRMT4 inhibitor 1, PRMT6 selective inhibitor 2, and type I PRMT selective inhibitor
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47, 48, 50, 57
3. The cocrystal structures
of these inhibitors in complex with PRMT4 or PRMT6 clearly
indicate that the ethylenediamino group forms direct and water-mediated hydrogen bonds (H-bonds)
with the proteins in the arginine binding pockets, thus serving as an arginine mimetic. Previous SAR
studies suggested that the ethylenediamino group is a key contributor to the PRMT4 and PRMT6
47, 48, 50, 57
inhibitory activities.
We therefore designed a relatively small set of compounds (Scheme 1A
and Table 1) to explore the SAR of the piperidinylethanamine region. We focused on investigating
substituents on the terminal amino group, the number of carbons between two nitrogen atoms, the
substitution position at the piperidinyl ring, and the ring size. Synthetic routes for preparing compounds
5−10 are outlined in Scheme 1A. Compounds 5−7 and 9−10 were synthesized via a standard sodium
triacetoxyborohydride mediated reductive amination reaction of commercially available
benzylpiperidines or benzylpyrrolidines with corresponding tert-butyloxycarbonyl (Boc) protected α- or
β-amino aldehydes, followed by the removal of the Boc protecting group under acidic conditions.
Compound 8 was synthesized using a nucleophilic substitution reaction between benzylpiperidine and
Boc protected aminomethylepoxide, followed by a deprotection reaction.
The X-ray crystal structure of PRMT6−compound 4−S-5′-adenosyl-L-homocysteine (SAH, the
cofactor product) complex showed that the middle linker and LHS phenyl moiety of compound 4 make
4
8
hydrophobic interactions with PRMT6. However, it was not clear what was required for these
hydrophobic interactions and whether additional interactions could be made. We therefore explored
these two regions to potentially increase H-bond formations and/or polar interactions. For the middle
linker, we explored different linker lengths (from no linker to a three-atom linker) as well as different
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linker types including various heteroatoms such as O, S, and NH (Scheme 1B and Table 2). In addition,
we investigated linkers containing a hydroxyl methylene, alkyl ether, alkyl amine, ester, or amide
functional group (Scheme 1B and Table 2). Synthetic routes for preparing 11−23 are outlined in Scheme
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B. Compounds 11−18 and 20−23 were synthesized utilizing reductive amination reactions of
commercially available substituted piperidines with N-Boc (methylamino)acetaldehyde followed by the
removal of the protecting group. Compound 19 was prepared by using a 6-step synthetic sequence. First,
reductive amination of aniline with Boc protected piperidine-4-carboxaldehyde provided Boc protected
piperidinylmethylaniline, the aniline nitrogen of which was subsequently protected with a trifluoroactyl
group. Selective removal of the Boc protecting group produced intermediate I1, which underwent
another reductive amination reaction. Sequential removal of the protecting groups afforded the desired
compound 19.
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To investigate SAR of the LHS phenyl moiety, we selected compound 17 as the starting point
because compound 17 was identified as the most potent dual inhibitor of PRMT4 and PRMT6 from our
SAR studies on the middle linker region (see Table 2 and discussion below). We designed compounds
2
4−45 to explore a number of electron donating and withdrawing groups with different steric bulkiness
at the 2-, 3- or 4-postion at the phenyl ring (Scheme 1C and Table 3). These compounds were
synthesized using a 4-step parallel synthetic sequence. Nucleophilic substitution reactions of Boc
protected 4-hydroxypiperidine with various substituted benzyl chlorides or bromides and the subsequent
deprotection afforded 4-benzyloxypiperidine intermediates I2, which were then converted to the desired
compounds 24−45 via reduction amination with N-Boc (methylamino)acetaldehyde, followed by the
removal of the Boc protecting group.
All of the synthesized compounds were evaluated in human PRMT4 and PRMT6 biochemical
assays, which measure the potency of test compounds at inhibiting the transfer of the methyl group from
3
H-SAM to the peptide substrates. IC50 values of the compounds are summarized in Tables 1−3.
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SAR of the RHS Piperidinylethanamine Moiety. The X-ray crystal structure of 4 in the
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complex with PRMT6 and SAH revealed that two nitrogen atoms of the piperidinylethanamine moiety
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formed direct and water mediated H-bonds with Glu155, Glu164, and His317 (Figure 1B). Consistent
with this observation, installation of a small substituent on the terminal primary amino group of 4,
which does not disturb these H-bond interactions, was well tolerated, while changing the distance
between these two nitrogen atoms, which interrupts these interactions, greatly reduced the potency for
both PRMT4 and PRMT6. Specifically, switching the terminal primary amino group (4) to a secondary
methylamino group provided 5, which displayed similar potency (PRMT4, IC50 = 890 ± 140 nM;
PRMT6, IC50 = 170 ± 39 nM) as compound 4 (Table 1). On the other hand, a bulkier ethylamino group
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(
6) decreased the potency for PRMT4 and PRMT6 by 3- to 4-fold, compared with 4. Increasing the
distance between those two nitrogen atoms using 1,3-diaminopropyl group (7) totally abolished the
inhibitory activity against both enzymes. Interestingly, adding a free hydroxyl group to the 1,3-diamino-
propyl group (8) partially recovered the potency for PRMT4 (IC50 = 1,220 ± 140 nM) and PRMT6 (IC50
=
3,040 ± 270 nM), presumably due to extra H-bond interaction(s). These results confirm that the
ethylenediamino group is a preferred moiety for interacting with the arginine binding site. We also
explored the piperidinyl region. Switching the 4-benzyl group at the piperidinyl ring to 3-position (9)
increased potency for PRMT4 (IC50 = 120 ± 28 nM) while maintaining potency for PRMT6 (IC50 = 290
±
30 nM). Replacement of the 6-membered piperidinyl ring with the 5-membered pyrrolidinyl ring (10)
resulted in high potency for both PRMT4 (IC50 = 80 ± 28 nM) and PRMT6 (IC50 = 70 ± 6 nM).
Compound 10 could be a valuable lead for future SAR studies. Because compounds 9 and 10 contain a
chiral center and would require enantiomeric resolution to obtain pure enantiomers, we chose 5, which
is achiral, as the starting point to study SAR of the middle linker for the simplicity reason.
SAR of the Middle Linker. We next investigated the middle linker region by exploring various
linker lengths and introducing various heteroatoms and functional groups. We postulated that changing
the linker length could direct the phenyl group to different locations within the binding site, thus
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affecting inhibitor−protein interactions. Furthermore, heteroatoms such as O and NH and various
functional groups could increase polar interactions or H-bond formations, and in turn improve potency
for PRMT4 and PRMT6. Without the methylene linker, 11 displayed 7-fold and 55-fold potency loss
against PRMT4 and PRMT6, respectively, compared with 5 (Table 2). Among analogs with a one-atom
linker, thioether (13) and hydroxymethine (15) linkers slightly impaired the potency, while amino linker
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(14) showed similar potency for PRMT4 (IC50 = 616 ± 70 nM) and PRMT6 (IC50 = 186 ± 23 nM) as
compound 5. Interestingly, with an ether linker, 12 displayed higher potency (~6-fold) for PRMT4 (IC50
= 149 ± 24 nM) and similar potency for PRMT6 (IC50 = 90 ± 16 nM) as compared to 5. Except an
amide-linker containing compound (22), the synthesized compounds with a two-atom linker, such as
ethylene (16), ethers (17 and 18), methyleneamine (19), ester (20) and amide (21), displayed higher
potency for PRMT4, resulting in inhibitors with almost equal potency for PRMT4 and PRMT6. Among
all derivatives, 17 was the most potent dual inhibitor of PRMT4 (IC50 = 34 ± 10 nM) and PRMT6 (IC50
=
43 ± 7 nM). Compared with 5, 17 was significantly more potent (~26-fold) for PRMT4 and had
moderately improved potency (~4-fold) for PRMT6. The compound with a three-atom ester-linker (23),
however, was less potent than compound 18, which has a two-atom ether-linker, and compound 20,
which has a two-atom ester-linker. Overall, a linker with two atoms between the phenyl and piperidinyl
groups is preferred and the methyleneoxy linker in compound 17 yields the highest potency for PRMT4
and PRMT6. Thus, compound 17 was used as the starting scaffold to investigate the LHS phenyl
moiety.
SAR of the LHS Phenyl Moiety. To examine the effect of substitution at the phenyl moiety of
1
3
7, a number of electron-withdrawing groups, such as F, Cl, Br and CF , and electron donating groups,
such as phenyl, were installed at the 2-, 3- or 4-position of the phenyl ring. SAR results of these
substituted phenyl compounds (24−38) showed that both electron-deficient and electron-rich groups
were well tolerated (Table 3). With the exception of 33, 4-substituted phenyl groups (26, 29, 32 and 38)
slightly improved (by about 2-fold) potency for PRMT4, compared with 17. We therefore explored a
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few additional substituents (39−45) at this position. Similar to the substituents in 24−38, these
additional substituents (in 39−45) were also well tolerated. In general, substitution at the phenyl ring did
not affect PRMT4 and PRMT6 potency drastically. Several compounds (e.g., 30, 38, 45) exhibited
about a 2-fold potency improvement over 17 and could serve as leads for future SAR studies. Because
compound 17 has the simplest structure, we selected it for full characterization in the following studies.
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Characterization of 17 in Selectivity, Biophysical and Mechanism of Action Studies. We
evaluated selectivity of compound 17 and its two close analogs (25 and 37) against other PRMTs (Table
4
). We were pleased to find none of these compounds showed inhibition against type II (PRMT5) and
type III (PRMT7) PRMTs. In addition, all three compounds were more potent against PRMT4 and
PRMT6 than other type I PRMTs. For example, 17 and 25 had good selectivity over PRMT8 (>30-fold)
and excellent selectivity over PRMT1 and PRMT3 (>300-fold). To further assess selectivity of 17, we
tested it against 25 protein lysine methyltransferases (PKMTs), DNA and RNA methyltransferases
(DNMTs/RNMTs), 3 histone lysine demethylases (KDMs), and 9 methyllysine and methylarginine
reader proteins. We found that 17 did not inhibit any of the methyltransferases at 50 µM (Figure 2) or
demethylases (Supporting Figure S1) at 10 µM, and did not display appreciable binding to any of the
methyllysine/methylarginine reader proteins using the DSF assay at 200 µM (Supporting Table S1).
Moreover, we also evaluated the selectivity of 17 over a broad range of non-epigenetic targets in two
61
selectivity profiling panels: CEREP and PDSP . The CEREP platforms cover 100 GPCRs, ion
channels, transporters, and kinases. Compound 17 showed no appreciable inhibition (no more than 50%
inhibition at 10,000 nM) in this panel (Supporting Table S2). The PDSP selectivity panel consists of 44
GPCRs. Compound 17 showed no more than 50% inhibition at 10,000 nM against 41 targets and >50%
inhibition at 10,000 nM against 3 targets in the panel. K
for each of the 3 interacting targets was subsequently performed. Compound 17 had K
7 nM, and 574 nM for sigma1, histamine H3, and sigma2, respectively (Supporting Table S3).
i
determinations in the radioligand binding assay
i
values of 64 nM,
8
However, it is not clear whether 17 has activity in sigma1, histamine H3 and sigma2 functional assays.
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Overall, 17 demonstrated excellent selectivity over a broad range of epigenetic and non-epigenetic
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
targets.
We next confirmed binding of 17 to PRMT4 using isothermal titration calorimetry (ITC) in the
presence of SAH with a K
d
value of 100 nM (n = 2) (Figure 3A). In the case of PRMT6 binding was
value of 87 ± 35 nM (Figure 3B). Binding of 17 to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
confirmed by ITC in the presence of SAM with a K
d
PRMT4 and PRMT6 was confirmed by another orthogonal method, differential scanning fluorimetry
62
(
m
DSF). In DSF experiments, an increase in melting temperature (ΔT ) of PRMT4 (3.3 °C) and
PRMT6 (11.3 °C) was observed upon binding of 17 at 200 µM (Figure 3C and 3D).
To assess mechanism of action (MOA) of 17, we evaluated the effect of SAM and peptide
concentrations on IC50 values of 17 against PRMT4 and PRMT6. As illustrated in Figures 3E and 3F,
increasing the cofactor SAM or peptide substrate concentrations had no effects on IC50 values of 17 for
either PRMT4 or PRMT6, suggesting that this inhibitor is noncompetitive with both the cofactor SAM
and peptide substrate. This is not surprising as similar noncompetitive inhibition phenomena were
4
7, 48, 57
observed for arginine binding-site occupying PRMT inhibitors, such as compounds 2, 3 and 4.
Considering its similar chemical structure to 4, compound 17 most likely occupies the substrate
arginine) binding site of PRMT4 and PRMT6. It is likely that the binding affinity of the peptide
substrate mainly derived from the interactions outside of the arginine-binding site. Thus 17 can’t
(
4
7, 48, 63, 64
compete off the peptide substrate.
Another plausible interpretation is that the binding of 17
4
7
induces major protein conformational changes and traditional enzymatic kinetic may not apply.
Cell-based Studies. We first assessed the effect of 17 on inhibition of ectopically expressed
PRMT6 in HEK293 cells. Overexpression of the wild type (WT) PRMT6 but not catalytic mutant
(V86/D88A) robustly increased the asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a)
3
2
level (Figure 4A, first two columns). Compound 17 (20 h exposure) reduced the H3R2me2a mark in
HEK293 cells in a concentration dependent manner (IC50 = 0.97 ± 0.05 µM (n = 3)) (Figure 4A). The
effect of the 8 µM 17 treatment matched with that of the catalytically inactive mutant (Figure 4A).
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2
8
Recently, Med12 was identified as a non-histone substrate of PRMT4. Compound 17 treatment (72 h
exposure) inhibited endogenous PRMT4 methyltransferase activity in a concentration dependent manner
resulting in reduced levels of cellular asymmetric arginine dimethylation of Med12 (Med12-Rme2a,
IC50 = 1.4 ± 0.1 µM (n = 3)) in HEK293 cells (Figure 4B). To further confirm the cellular assay results,
we designed and synthesized a negative control compound, 46, by replacing the methyl amino group
with the methyl ether group on the RHS of 17 (Figure 5 and Supporting Scheme 1). This single atom
switch (from nitrogen to oxygen) completely abolished inhibitory activity against PRMT4 and PRMT6
in vitro (Figure 5). As expected, 46 did not decrease cellular levels of H3R2me2a or Med12me2a at
concentrations up to 30 µM (Supporting Figure S2A and S2B). It is worth noting that in our experiments
further characterizing compound 17, we observed that 17 unexpectedly reduced asymmetric
dimethylation of histone H4 arginine 3 (H4R3me2a) in HEK293 cells while compound 46 did not
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Supporting Figure S2C). It was shown previously that PRMT1 is the major isoform responsible for
6
5
methylation of H4R3, however PRMT6 is also able to methylate H4R3 in vitro and in cells when
6
6
overexpressed. It is unclear what contributes to the inconsistency between the biochemical and cellular
assay results, which will be a subject of future investigations. Lastly, to evaluate cell toxicity of 17 and
46, we investigated effects of 17 and 46 on growth inhibition of HEK293 cells over 4 days (Supporting
Figure S3). Both compounds did not display significant cell toxicity at up to 50 µM within the time of
the treatment.
CONCLUSIONS
We conducted SAR studies and explored three regions of the fragment-like inhibitor of PRMTs,
4
. By optimizing the three regions of this hit, we discovered compound 17 as a potent, selective and
cell-active dual inhibitor of PRMT4 and PRMT6. In biochemical assays, 17 was highly potent and
selective for PRMT4 and PRMT6 over a broad range of epigenetic modifiers, including other PRMTs,
PKMTs, DNMTs, KDMs, and methyllysine/methylarginine reader proteins, and non-epigenetic targets,
including GPCRs, ion channels, transporters, and kinases. In cellular assays, 17 potently inhibited the
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methyltransferase activity of PRMT4 and PRMT6 and reduced levels of Med12me2a and H3R2me2a in
HEK293 cells. We also discovered 46, a close analog of 17, which was inactive in biochemical and
cellular assays, as a negative control for chemical biology studies. Both 17 and 46 were not toxic and
did not affect the growth of HEK293 cells. Therefore, 17 and 46 are valuable chemical tools for the
biomedical community to test biological and therapeutic hypotheses concerning PRMT4 and PRMT6 in
cellular systems.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Chemistry General Procedures. HPLC spectra for all compounds were acquired using an Agilent 1200
Series system with DAD detector. Chromatography was performed on a 2.1×150 mm Zorbax 300SB-
C
18 5 µm column with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1%
formic acid as solvent B at a flow rate of 0.4 mL/min. The gradient program was as follows: 1% B (0−1
min), 1−99% B (1−4 min), and 99% B (4−8 min). High-resolution mass spectra (HRMS) data were
acquired in positive ion mode using an Agilent G1969A API-TOF with an electrospray ionization (ESI)
source. Nuclear Magnetic Resonance (NMR) spectra were acquired on eith a Bruker DRX-600
1
13
spectrometer (600 MHz H, 150 MHz C) or a Bruker Avance-III 500 MHz spectrometer (500 MHz
1
13
H, 125 MHz C). Chemical shifts are reported in ppm (δ). Preparative HPLC was performed on
Agilent Prep 1200 series with UV detector set to 254 nm. Samples were injected into a Phenomenex
Luna 75 x 30 mm, 5 µm, C18 column at room temperature. The flow rate was 40 mL/min. A linear
2
gradient was used with 10% (or 50%) of MeOH (A) in H O (with 0.1 % TFA) (B) to 100% of MeOH
(A). HPLC was used to establish the purity of target compounds. All final compounds had > 95% purity
using the HPLC methods described above. Because compound 20 was not very stable when treated with
HCl, the free base form was used in biological assays. All of the rest of compounds were tested in
biological assays using their HCl salt forms.
General procedure A for reductive amination (step a). To a solution of the amine (1 equiv.) in
CH
2
Cl
2
(0.13 M solution) was added aldehyde (1.1 equiv.). The resulting solution was stirred at room
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Journal of Medicinal Chemistry
temperature for 15 mins prior to the addition of sodium triacetoxyborohydride (1.4 equiv.), after which
the solution was stirred for an additional 16 h. The reaction was then quenched with aqueous sodium
bicarbonate solution and extracted with EtOAc (3 x). The combined organic extracts were dried with
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
anhydrous Na
silica gel column chromatography to afford the Boc protected amine. (Note: When the amine starting
material is in the HCl salt form, Et N (1.1 equiv.) was added to the reaction before aldehyde addition.)
2 4
SO , filtered, and concentrated under reduced pressure. The residue was then purified by
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
General procedure B for Boc deprotection (step b). To a solution of the Boc protected amine (1
equiv.) in CH Cl (0.20 M solution) was added HCl/dioxane (4 M solution, 10 equiv.). The resulting
2
2
solution was stirred at room temperature for 2 h prior to removal of all solvents under reduced pressure,
and dissolution of the residue in water. The aqueous solution was frozen, and lyophilized for 16 h to
afford the hydrochloride salt. Alternatively, HCl/MeOH (3 M solution, 10 equiv.) was used to remove
the Boc protecting group at rt for 12 h.
General procedure C for benzyl ether formation (step g). To a solution of tert-butyl 4-
hydroxypiperidine-1-carboxylate (1 equiv.) in DMF (0.13 M solution) was added NaH (60% dispersion
in mineral oil, 2 equiv.) in portions at 0 °C. After 15 min, a solution of benzyl bromide or chloride (2
equiv.) in DMF (1 M solution) was added dropwise. The resulting mixture was slowly warmed to room
temperature and stirred for overnight, before being quenched with water. The reaction was extracted
2 2 2 4
with CH Cl (3 x). The combined organic extracts were dried with anhydrous Na SO , filtered, and
concentrated under reduced pressure. The resulting residue was purified by silica gel column
chromatography to afford the desired benzyl ether.
1
General procedure D for converting compound 17 and 24−46 to free base form. The H NMR of
the HCl salt contains different sets of signals, presumably due to different salt forms. A small portion of
the HCl salt was converted to free base by washing with aqueous saturated NaHCO
3
and extraction with
EtOAc. The combined organic layers were dried over Na SO and concentrated. The resulting residue
2
4
was used directly to obtain NMR spectra. Note: The HPLC-MS spectra were identical for the HCl salt
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form and free base form. In addition, the HCl salt form and free-base form of compound 17 showed
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
identical results in the biochemical assay.
2-(4-Benzylpiperidin-1-yl)-N-methylethan-1-amine (5). The title compound was prepared according
1
to the general procedure A and B (HCl/dioxane) (68% over two steps). H NMR (500 MHz, DMSO-d
6
):
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
δ 10.90 (br s, 1H), 10.18 (br s, 2H), 7.30 – 7.24 (m, 2H), 7.20 – 7.13 (m, 3H), 3.74 – 3.65 (m, 1H), 3.53
– 3.45 (m, 1H), 2.81 – 2.76 (m, 2H), 2.53 – 2.47 (m, 2H), 2.30 (t, J = 6.5 Hz, 2H), 2.26 (s, 3H), 1.85 –
13
1
.75 (m, 2H), 1.54 – 1.48 (m, 2H), 1.48 – 1.41 (m, 1H), 1.22 – 1.10 (m, 2H). C NMR (125 MHz,
DMSO-d
6
): δ 139.9, 129.4 (2), 128.8 (2), 126.5, 52.7, 52.2 (2), 42.7, 41.9, 35.3, 32.7, 29.1 (2). HRMS
+
(
ESI-TOF) m/z: [M+H] calcd for C15
H
25
N
2
, 233.2018; found: 233.2019.
2
-(4-Benzylpiperidin-1-yl)-N-ethylethan-1-amine (6). The title compound was prepared according to
1
the general procedure A and B (HCl/MeOH) (64% over two steps). H NMR (600 MHz, CD
3
OD): δ
7.29 (t, J = 7.5 Hz, 2H), 7.23 – 7.13 (m, 3H), 3.66 (d, J = 12.2 Hz, 2H), 3.55 – 3.48 (m, 2H), 3.46 (t, J =
6
3
2
.6 Hz, 2H), 3.15 (q, J = 7.2 Hz, 2H), 3.07 – 2.96 (m, 2H), 2.62 (d, J = 6.8 Hz, 2H), 1.96 – 1.84 (m,
+
H), 1.74 – 1.59 (m, 2H), 1.36 (t, J = 7.3 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H] calcd for C16
H
27
N
2
,
47.2174; found: 247.2176.
3
-(4-Benzylpiperidin-1-yl)-N-methylpropan-1-amine (7). The title compound was prepared according
1
to the general procedure A and B (HCl/MeOH) (68% over two steps). H NMR (600MHz, CD
3
OD): δ
7
.29 (t, J = 7.6 Hz, 2H), 7.23 – 7.14 (m, 3H), 3.59 (d, J = 12.2 Hz, 2H), 3.20 (t, J = 8.1 Hz, 2H), 3.11 (t,
J = 7.8 Hz, 2H), 2.97 (t, J = 12.8 Hz, 2H), 2.74 (s, 3H), 2.62 (d, J = 6.6 Hz, 2H), 2.24 – 2.11 (m, 2H),
+
1.98 – 1.82 (m, 3H), 1.66 – 1.51 (m, 2H). HRMS (ESI-TOF) m/z: [M+H] calcd for C16
H
27
N
2
,
2
47.2174; found: 247.2165.
1
-(4-Benzylpiperidin-1-yl)-3-(methylamino)propan-2-ol (8). To a solution of 4-benzylpiperidine
(0.053 g, 0.3 mmol) in i-PrOH (5 mL) was added tert-butyl methyl(oxiran-2-ylmethyl)carbamate (0.067
g, 0.36 mmol). The reaction solution was heated at 80 °C for 16 h before being concentrated and passed
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through a short silica gel plug. After concentration under reduced pressure, the Boc protecting group
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
was remove using the general procedure B (HCl/MeOH) (53% over two steps). H NMR (600 MHz,
3
CD OD): δ 7.29 (t, J = 7.6 Hz, 2H), 7.23 – 7.17 (m, 3H), 4.47 – 4.39 (m, 1H), 3.72 – 3.65 (m, 1H), 3.64
–
1
3.59 (m, 1H), 3.24 – 3.13 (m, 3H), 3.10 – 2.93 (m, 3H), 2.75 (s, 3H), 2.62 (d, J = 6.8 Hz, 2H), 1.96 –
+
.84 (m, 3H), 1.70 – 1.53 (m, 2H). HRMS (ESI-TOF) m/z: [M+H] calcd for C16
H
27
N
2
O, 263.2123;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
found: 263.2122.
2
-(3-Benzylpiperidin-1-yl)-N-methylethan-1-amine ((±)-9). The title compound was prepared
1
according to the general procedure A and B (HCl/dioxane) (77% over two steps). H NMR (500 MHz,
DMSO-d
H), 3.74 – 3.65 (dm, 1H), 3.57 – 3.40 (m, 6H), 2.90 – 2.80 (m, 1H), 2.80 – 2.70 (m, 1H), 2.65 – 2.45
(dm, 1H), 2.57 (s, 3H), 2.25 – 2.15 (m, 1H), 1.85 – 1.75 (m, 2H), 1.70 – 1.61 (m, 1H), 1.20 – 1.06 (m,
6
): δ 10.90 (br s, 1H), 9.53 (br s, 2H), 7.35 – 7.29 (m, 2H), 7.26 – 7.20 (m, 1H), 7.20 – 7.16 (m,
2
1
3
1
H). C NMR (125 MHz, DMSO-d
6
): δ 138.9, 129.5 (2), 128.9 (2), 126.8, 57.2, 53.6, 52.4, 42.6, 39.1
+
(
hidden by solvent), 35.6, 32.7, 27.8, 22.5. HRMS (ESI-TOF) m/z: [M+H] calcd for C15
H
25
N
2
,
233.2018; found: 233.2017.
2
-(3-Benzylpyrrolidin-1-yl)-N-methylethan-1-amine ((±)-10). The title compound was prepared
1
according to the general procedure A and B (HCl/dioxane) (80% over two steps). H NMR (500 MHz,
DMSO-d
6
): δ 11.40 (br s, 1H), 9.42 (br s, 2H), 7.35 – 7.29 (m, 2H), 7.28 – 7.20 (m, 3H), 3.75 – 3.65 (m,
1
–
H), 3.65-3.55 (m, 1H), 3.55-3.45 (m, 3H), 3.35 – 3.10 (m, 3H), 2.90 – 2.66 (m, 3H), 2.58 (s, 3H), 2.20
1
3
1.95 (m, 1H), 1.85 – 1.55 (m, 1H). C NMR (125 MHz, DMSO-d
6
): δ 140.1, 129.1 (2), 129.0 (2),
+
126.8, 60.7, 58.1, 52.9, 44.1, 44.0, 39.1, 38.4, 32.7. HRMS (ESI-TOF) m/z: [M+H] calcd for C14
H
23
N
2
,
2
19.1861; found: 219.1862.
N-Methyl-2-(4-phenylpiperidin-1-yl)ethan-1-amine (11). The title compound was prepared according
1
to the general procedure A and B (HCl/dioxane) (72% over two steps). H NMR (500 MHz, DMSO-d
6
):
δ 10.99 (br s, 1H), 9.45 (br s, 2H), 7.38 – 7.32 (m, 2H), 7.28 – 7.22 (m, 3H), 3.73 – 3.65 (m, 2H), 3.50 –
3
.52 (m, 4H), 3.20 – 3.09 (m, 2H), 2.90 – 2.81 (m, 1H), 2.62 (s, 3H), 2.16 – 2.06 (m, 2H), 2.04 – 1.96
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1
3
(
m, 2H). C NMR (125 MHz, DMSO-d
6
): δ 144.6, 129.1 (2), 127.1, 127.0 (2), 53.0, 52.2 (2), 42.8,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
3
8.9, 32.8, 30.2 (2). HRMS (ESI-TOF) m/z: [M+H] calcd for C14H N , 219.1861; found: 219.1859.
23 2
N-Methyl-2-(4-phenoxypiperidin-1-yl)ethan-1-amine (12). The title compound was prepared
1
according to the general procedure A and B (HCl/dioxane) (60% over two steps). H NMR (500 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
DMSO-d ): δ 11.15 (br s, 1H), 9.52 (s, 2H), 7.35 – 7.28 (m, 2H), 7.08 – 6.93 (m, 3H), 3.70 – 3.63 (m,
2H), 3.54 – 3.43 (m, 5H), 3.27 – 3.11 (m, 2H), 2.60 (s, 3H), 2.30 – 2.15 (m, 2H), 2.11 – 1.92 (m, 2H).
13
C NMR (125 MHz, DMSO-d
6
): δ 157.1, 130.1 (2), 121.8, 116.8 (2), 70.5, 51.1, 48.0 (2), 42.7, 32.7,
+
2
8.6, 26.7. HRMS (ESI-TOF) m/z: [M+H] calcd for C14
H
23
N
2
O, 235.1810; found: 235.1811.
N-Methyl-2-(4-(phenylthio)piperidin-1-yl)ethan-1-amine (13). The title compound was prepared
1
according to the general procedure A and B (HCl/dioxane) (81% over two steps). H NMR (500 MHz,
6
DMSO-d ): δ 11.26 – 10.94 (br s, 1H), 9.49 (s, 2H), 7.46 – 7.42 (m, 2H), 7.40 – 7.35 (m, 2H), 7.32 –
7.28 (m, 1H), 3.64 – 3.55 (m, 2H), 3.55 – 3.40 (m, 5H), 3.15 – 3.00 (m, 2H), 2.58 (s, 3H), 2.14 – 2.04
13
(
m, 2H), 1.98 – 1.82 (m, 2H). C NMR (125 MHz, DMSO-d
6
): δ 133.4, 131.6 (2), 129.7 (2), 127.6,
+
5
2.4 (2), 51.9, 42.6, 40.0 (hidden by solvent), 32.6, 29.7 (2). HRMS (ESI-TOF) m/z: [M+H] calcd for
14 23 2
C H N S, 251.1582; found: 251.1577.
1
-(2-(Methylamino)ethyl)-N-phenylpiperidin-4-amine (14). The title compound was prepared
1
according to the general procedure A and B (HCl/dioxane) (78% over two steps). H NMR (500 MHz,
DMSO-d
6
): δ 11.00 – 10.80 (br s, 1H), 9.52 (s, 2H), 7.44 – 7.27 (m, 2H), 7.26 – 7.03 (m, 3H), 4.95 (br
s, 1H), 3.70 – 3.63 (m, 2H), 3.52 – 3.45 (m, 1H), 3.45 – 3.35 (m, 4H), 3.15 – 3.04 (m, 2H), 2.59 (t, J =
1
3
5.0 Hz, 3H), 2.22 – 2.10 (m, 2H), 2.08 – 1.94 (m, 2H). C NMR (125 MHz, DMSO-d
6
): δ 137.7, 130.2
+
(
2), 126.5, 121.6 (2), 66.8, 51.9, 51.3 (2), 42.7, 32.8, 26.8 (2). HRMS (ESI-TOF) m/z: [M+H] calcd for
14 24 3
C H N , 234.1970; found: 234.1968.
(
1-(2-(Methylamino)ethyl)piperidin-4-yl)(phenyl)methanol (15). The title compound was prepared
1
according to the general procedure A and B (HCl/dioxane) (85% over two steps). H NMR (500 MHz,
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Journal of Medicinal Chemistry
DMSO-d
6
): δ 10.46 (br s, 1H), 9.39 (br s, 2H), 7.36 – 7.29 (m, 4H), 7.29 – 7.24 (m, 1H), 5.45 (br s, 1H),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
.30 (br s, 1H), 3.74 – 3.65 (dm, 1H), 3.62 – 3.54 (m, 2H), 3.54 – 3.44 (m, 2H), 2.95 – 2.80 (m, 2H),
1
3
2.58 (s, 3H), 1.98 – 1.90 (m, 1H), 1.80 – 1.70 (m, 1H), 1.70 – 1.57 (m, 2H), 1.46 – 1.37 (m, 1H). C
NMR (125 MHz, DMSO-d
6
): δ 144.4, 128.5 (2), 127.5, 126.9 (2), 66.8, 52.8, 52.6, 52.2, 42.8, 41.0,
+
3
2.8, 26.2, 25.3. HRMS (ESI-TOF) m/z: [M+H] calcd for C15
H
25
N
2
O, 249.1967; found: 249.1966.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N-Methyl-2-(4-phenethylpiperidin-1-yl)ethan-1-amine (16). The title compound was prepared
1
according to the general procedure A and B (HCl/dioxane) (69% over two steps). H NMR (500 MHz,
DMSO-d
6
): δ 10.77 (br s, 1H), 9.56 (br s, 2H), 7.30 – 7.25 (m, 2H), 7.24 – 7.14 (m, 3H), 3.75 – 3.63 (m,
2
H), 3.63 – 3.43 (m, 7H), 3.00 – 2.90 (m, 2H), 2.51 (s, 3H), 1.93 – 1.86 (m, 2H), 1.60 – 1.45 (m, 4H).
13
C NMR (125 MHz, DMSO-d
6
): δ 142.4, 128.8 (2), 128.7 (2), 126.2, 52.8 (2), 52.1, 42.7, 37.6, 32.8,
+
32.7, 32.5, 29.2 (2). HRMS (ESI-TOF) m/z: [M+H] calcd for C16
H
27
N
2
, 247.2174; found: 247.2172.
2-(4-(Benzyloxy)piperidin-1-yl)-N-methylethan-1-amine (17). The title compound was prepared
according to the general procedure A (with Et N) and B (HCl/MeOH) (64% over two steps). A small
3
portion of the HCl salt was converted to the free base form using the general procedure D to obtain the
1
NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.39 – 7.30 (m, 4H), 7.29 – 7.22 (m, 1H), 4.55 (s, 2H),
3
.54 – 3.44 (m, 1H), 2.84 – 2.74 (m, 2H), 2.71 (t, J = 6.8 Hz, 2H), 2.50 (t, J = 6.8 Hz, 2H), 2.41 (s, 3H),
1
3
2.28 – 2.16 (m, 2H), 2.00 – 1.89 (m, 2H), 1.71 – 1.59 (m, 2H); C NMR (150 MHz, CD
3
OD): δ 140.2,
+
1
29.3, 128.8, 128.6, 75.4, 70.9, 58.2, 52.4, 49.3, 36.1, 32.0. HRMS (ESI-TOF) m/z: [M+H] calcd for
15 25 2
C H N O, 249.1967; found: 249.1970.
N-Methyl-2-(4-(phenoxymethyl)piperidin-1-yl)ethan-1-amine (18). The title compound was prepared
1
according to the general procedure A and B (HCl/MeOH) (67% over two steps). H NMR (600 MHz,
3
CD OD): δ 7.26 (t, J = 7.8 Hz, 2H), 6.96 – 6.88 (m, 3H), 3.91 (d, J = 5.6 Hz, 2H), 3.75 (d, J = 12.0 Hz,
2H), 3.59 – 3.47 (m, 4H), 3.14 (t, J = 12.5 Hz, 2H), 2.81 (s, 3H), 2.21 – 2.10 (m, 3H), 1.90 – 1.77 (m,
+
2
H). HRMS (ESI-TOF) m/z: [M+H] calcd for C15
H
25
N
2
O, 249.1967; found: 249.1969.
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Journal of Medicinal Chemistry
Page 18 of 54
N-((1-(2-(Methylamino)ethyl)piperidin-4-yl)methyl)aniline (19). To a solution of aniline (0.027 mL,
.3 mmol) in CH Cl (5 mL) was added tert-butyl 4-formylpiperidine-1-carboxylate (0.64 g, 0.3 mmol).
The resulting solution was stirred for 30 min before being treated with sodium triacetoxyborohydride
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
2
2
(
0.095 g, 0.45 mmol) for 16 h. The reaction was quenched with aqueous saturated NaHCO
3
2
and the
SO and
product was extracted with EtOAc (3 x). The combined organic layers were dried over Na
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
concentrated. The resulting residue was passed through a short silica gel plug to get the crude desired
product. The resulting intermediate (0.087 g, 0.3 mmol) was dissolved in CH Cl (5 mL). To the
2
2
solution was added triethylamine (0.084 mL, 0.6 mmol) followed by the addition of trifluoroacetic
anhydride (0.054 mL, 0.36 mmol) at 0 °C. The reaction was allowed to warm to rt and stirred for 16 h at
rt. The reaction was quenched with aqueous saturated NaHCO
3
2
and the product was extracted with
SO and concentrated. The resulting
EtOAc (3 x). The combined organic layers were dried over Na
4
residue was purified by silica gel column to provide tert-butyl 4-((2,2,2-trifluoro-N-
1
phenylacetamido)methyl)piperidine-1-carboxylate (0.098 g, 85% over 2 steps). H NMR (600 MHz,
CD
H), 2.86 – 2.58 (m, 2H), 1.83 – 1.72 (m, 1H), 1.68 (d, J = 13.3 Hz, 2H), 1.45 (s, 9H), 1.22 – 1.10 (m,
2H). To a solution of tert-butyl 4-((2,2,2-trifluoro-N-phenylacetamido)methyl)piperidine-1-carboxylate
0.077g, 0.2 mmol) in CH Cl (2 mL) was added trifluoroacetic acid (1 mL). The reaction was stirred for
h at rt before being concentrated under reduce pressure. After the resulting residue was dissolved in
CH Cl (5 mL), tert-butyl methyl(2-oxoethyl)carbamate (0.035 g, 0.2 mmol) was added. The solution
was stirred for 30 mins, before triethylamine (0.041 mL, 0.3 mmol) and sodium triacetoxyborohydride
0.064 g, 0.3 mmol) were added. The reaction was stirred for 16 h at rt. The reaction was quenched with
aqueous saturated NaHCO and the product was extracted with EtOAc (3 x). The combined organic
layers were dried over Na SO and concentrated. The resulting residue was passed through a short silica
3
OD): δ 7.54 – 7.42 (m, 3H), 7.36 (d, J = 7.4 Hz, 2H), 4.05 (d, J = 13.4 Hz, 2H), 3.71 (d, J = 7.3 Hz,
2
(
2
2
2
2
2
(
3
2
4
gel plug. After evaporation of solvents, the resulting residue was dissolved in methanol (2.5 mL) and
aqueous NaOH (5 N, 0.5 mL). The reaction mixture was heated at 50 °C for 2 h before being diluted
with EtOAc and water. The mixture was extracted with EtOAc (3 x). The combined organic layers were
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Journal of Medicinal Chemistry
dried over Na
2
SO
4
and concentrated. The residue was purified by silica gel column to provide tert-butyl
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
methyl(2-(4-((phenylamino)methyl)piperidin-1-yl)ethyl)carbamate. A solution of tert-butyl methyl(2-
(4-((phenylamino)methyl)piperidin-1-yl)ethyl)carbamate in 3 N HCl (2 mL) was stirred for 12 h. After
concentration of the reaction mixture under reduced pressure, the resulting residue was dissolved in
MeOH (0.5 mL). The resulting solution was triturated with ethyl ether to precipitate the desired product
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
as white solid (0.047 g, 66% over 4 steps). H NMR (600 MHz, D
2
O): δ 7.43 (t, J = 7.7 Hz, 2H), 7.37 (t,
J = 7.4 Hz, 1H), 7.30 (d, J = 7.9 Hz, 2H), 3.65 – 3.47 (br s, 2H), 3.40 (s, 4H), 3.31 (d, J = 6.6 Hz, 2H),
3.09 – 2.88 (m, 2H), 2.67 (s, 3H), 2.09 – 1.93 (m, 3H), 1.62 – 1.44 (m, 2H). HRMS (ESI-TOF) m/z:
+
[
M+H] calcd for C15
H
26
N
3
, 248.2127; found: 248.2130.
1
-(2-(Methylamino)ethyl)piperidin-4-yl benzoate (20). The title compound was prepared according to
the general procedure A and B (HCl/MeOH). The product was further purified by prep-HPLC. After
evaporation of solvents, the residue was basified with aqueous saturated NaHCO and extracted with
CH Cl (3x). The combined organic layers were dried over Na SO and concentrated to provide desired
3
2
2
2
4
product as a free base (65% over two steps). Note: Conversion the free base to its HCl salt was
1
attempted but led to slow decomposition. H NMR (600 MHz, CD
3
OD): δ 8.06 – 7.99 (m, 2H), 7.66 –
7
.57 (m, 1H), 7.48 (t, J = 7.8 Hz, 2H), 5.10 – 4.99 (m, 1H), 2.93 (t, J = 6.3 Hz, 2H), 2.84 – 2.74 (m,
2H), 2.61 (t, J = 6.3 Hz, 2H), 2.57 (s, 3H), 2.50 – 2.41 (m, 2H), 2.09 – 2.00 (m, 2H), 1.93 – 1.82 (m,
+
2
H). HRMS (ESI-TOF) m/z: [M+H] calcd for C15
H
23
N
2
O
2
, 263.1760; found: 263.1761.
N-(1-(2-(Methylamino)ethyl)piperidin-4-yl)benzamide (21). The title compound was prepared
according to the general procedure A and B (HCl/MeOH). The product was further purified by prep-
HPLC. After evaporation of solvents, the residue was basified with aqueous saturated NaHCO
3
and
extracted with CH Cl (3x). The combined organic layers were dried over Na SO and concentrated.
2
2
2
4
The resulting residue was dissolved in 3 mL of MeOH and was treated with 3 N HCl in MeOH (0.5 mL)
1
for 1 h. After concentration, the title compound was obtained as white solid (59% over two steps). H
NMR (600 MHz, CD
3
OD): δ 7.83 (d, J = 7.7 Hz, 2H), 7.59 – 7.50 (m, 1H), 7.47 (t, J = 7.6 Hz, 2H),
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Journal of Medicinal Chemistry
Page 20 of 54
4
.20 (t, J = 12.2 Hz, 1H), 3.79 (d, J = 12.3 Hz, 2H), 3.59 – 3.52 (m, 4H), 3.25 (t, J = 12.8 Hz, 2H), 2.81
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
(s, 3H), 2.30 – 2.24 (m, 2H), 2.17 – 2.02 (m, 2H). HRMS (ESI-TOF) m/z: [M+H] calcd for C15
H
24
N
3
O,
262.1919; found: 262.1920.
1
-(2-(Methylamino)ethyl)-N-phenylpiperidine-4-carboxamide (22). The title compound was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
prepared according to the general procedure A and B (HCl/dioxane) (65% over two steps). H NMR
(500 MHz, DMSO-d ): δ 10.94 (br s, 1H), 10.67 (br s, 1H), 10.22 (br s, 2H), 7.57 (d, J = 8.0 Hz, 2H),
.25 – 7.20 (m, 2H), 6.99 – 6.94 (m, 1H), 3.63 – 3.55 (m, 2H), 3.44 – 3.29 (m, 6H), 3.04 – 2.91 (m, 2H),
6
7
2
1
1
3
6
.68 – 2.58 (m, 1H), 2.45 – 2.42 (m, 1H), 2.10 – 1.89 (m, 4H). C NMR (125 MHz, DMSO-d ): δ
72.2, 139.6, 129.1 (2), 123.7, 119.7 (2), 52.3, 52.0 (2), 42.8, 38.7 (hidden by solvent), 32.8, 26.1 (2).
+
HRMS (ESI-TOF) m/z: [M+H] calcd for C15
H
24
N
3
O, 262.1919; found: 262.1921.
(
1-(2-(Methylamino)ethyl)piperidin-4-yl)methyl benzoate (23). The title compound was prepared
according to the general procedure A and B (HCl/MeOH). The product was further purified by prep-
HPLC. After evaporation of solvents, the residue was basified with aqueous saturated NaHCO and
extracted with CH Cl (3x). The combined organic layers were dried over Na SO and concentrated.
3
2
2
2
4
The resulting residue was dissolved in 3 mL of MeOH and was treated with 3 N HCl in MeOH (0.5 mL)
1
for 1 h. After concentration, the title compound was obtained as white solid (62% over two steps). H
NMR (600 MHz, CD
J = 5.8 Hz, 2H), 3.75 (d, J = 12.1 Hz, 2H), 3.58 – 3.48 (m, 4H), 3.19 – 3.09 (m, 2H), 2.81 (s, 3H), 2.24
2.15 (m, 1H), 2.15 – 2.07 (m, 2H), 1.97 – 1.84 (m, 2H).
3
OD): δ 8.10 – 8.02 (m, 2H), 7.65 – 7.60 (m, 1H), 7.49 (t, J = 7.8 Hz, 1H), 4.28 (d,
–
2-(4-((2-Fluorobenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (24). The title compound was
prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et N) and B
HCl/MeOH) (35% over four steps). A small portion of the HCl salt was converted to the free base form
3
(
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.44 (t, J =
7
.1 Hz, 1H), 7.36 – 7.24 (m, 1H), 7.15 (t, J = 7.4 Hz, 1H), 7.06 (t, 1H), 4.60 (s, 2H), 3.58 – 3.39 (m,
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Journal of Medicinal Chemistry
1
2
H), 2.86 – 2.73 (m, 2H), 2.71 (t, J = 6.8 Hz, 2H), 2.50 (t, J = 6.8 Hz, 2H), 2.41 (s, 3H), 2.30 – 2.16 (m,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
H), 2.06 – 1.84 (m, 2H), 1.77 – 1.58 (m, 2H). MS (ESI) m/z 267.2 [M+H] .
2-(4-((3-Fluorobenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (25). The title compound was
prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et N) and B
HCl/MeOH) (38% over four steps). A small portion of the HCl salt was converted to the free base form
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.39 – 7.24
(m, 1H), 7.14 (d, J = 7.6 Hz, 1H), 7.08 (d, J = 9.8 Hz, 1H), 6.98 (t, J = 8.5 Hz, 1H), 4.55 (s, 2H), 3.55 –
3
1
1
.39 (m, 1H), 2.89 – 2.66 (m, 4H), 2.52 (t, J = 6.7 Hz, 2H), 2.46 (s, 3H), 2.33 – 2.07 (m, 2H), 2.00 –
1
3
.90 (m, 2H), 1.77 – 1.58 (m, 2H). C NMR (150 MHz, CD
3
OD): δ 163.7, 162.1, 141.9, 129.7, 122.7,
+
13.7, 74.2, 68.6, 55.9, 50.8, 47.4, 34.1, 30.6. MS (ESI) m/z 267.2 [M+H]
.
HRMS (ESI-TOF) m/z:
+
[M+H] calcd for C15
H24FN
2
O, 267.1867; found 267.1874.
2-(4-((4-Fluorobenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (26). The title compound was
prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et N) and B
HCl/MeOH) (44% over four steps). A small portion of the HCl salt was converted to the free base form
3
(
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.58 – 7.24
(m, 2H), 7.23 – 6.72 (m, 2H), 4.51 (s, 2H), 3.64 – 3.40 (m, 1H), 2.89 – 2.73 (m, 4H), 2.54 (t, J = 6.6 Hz,
2H), 2.50 (s, 3H), 2.33 – 2.17 (m, 2H), 2.03 – 1.86 (m, 2H), 1.72 – 1.58 (m, 2H). MS (ESI) m/z 267.2
+
[M+H] .
2
-(4-((2-Chlorobenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (27). The title compound was
prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et N) and B
HCl/MeOH) (30% over four steps). A small portion of the HCl salt was converted to the free base form
3
(
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.51 (d, J =
7.1 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.32 – 7.23 (m, 2H), 4.62 (s, 2H), 3.68 – 3.45 (m, 1H), 2.93 –
2
1
.71 (m, 4H), 2.54 (t, J = 6.5 Hz, 2H), 2.49 (s, 3H), 2.33 – 2.21 (m, 2H), 2.04 – 1.91 (m, 2H), 1.77 –
+
.61 (m, 2H). MS (ESI) m/z 283.1 [M+H] .
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Journal of Medicinal Chemistry
Page 22 of 54
2
-(4-((3-Chlorobenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (28). The title compound was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et N) and B
3
(HCl/MeOH) (35% over four steps). A small portion of the HCl salt was converted to the free base form
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.36 (s, 1H),
7
2
1
.33 – 7.28 (m, 1H), 7.28 – 7.23 (m, 2H), 4.53 (s, 2H), 3.53 – 3.43 (m, 1H), 2.85 (t, J = 6.5 Hz, 2H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.82 – 2.74 (m, 2H), 2.54 (t, J = 6.5 Hz, 2H), 2.52 (s, 3H), 2.35 – 2.18 (m, 2H), 2.01 – 1.87 (m, 2H),
+
.74 – 1.58 (m, 2H). MS (ESI) m/z 283.1 [M+H] .
2
-(4-((4-Chlorobenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (29). The title compound was
prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et
3
N) and B
(HCl/MeOH) (44% over four steps). A small portion of the HCl salt was converted to the free base form
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.32 (s, 4H),
4
2
.52 (s, 2H), 3.52 – 3.44 (m, 1H), 2.77 (t, J = 6.5 Hz, 4H), 2.51 (t, J = 6.7 Hz, 2H), 2.46 (s, 3H), 2.30 –
+
.16 (m, 2H), 1.99 – 1.89 (m, 2H), 1.72 – 1.59 (m, 2H). MS (ESI) m/z 283.1 [M+H] .
2
-(4-((2-Bromobenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (30). The title compound was
prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et
3
N) and B
(HCl/MeOH) (27% over four steps). A small portion of the HCl salt was converted to the free base form
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.55 (d, J =
7
3
.9 Hz, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.34 (t, J = 7.3 Hz, 1H), 7.18 (t, J = 8.2 Hz, 1H), 4.58 (s, 2H),
.59 – 3.48 (m, 1H), 2.84 – 2.69 (m, 4H), 2.52 (t, J = 6.7 Hz, 2H), 2.46 (s, 3H), 2.32 – 2.18 (m, 2H),
+
2.04 – 1.88 (m, 2H), 1.78 – 1.60 (m, 2H). MS (ESI) m/z 327.1 [M+H] .
2
-(4-((3-Bromobenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (31). The title compound was
prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et N) and B
3
(HCl/MeOH) (38% over four steps). A small portion of the HCl salt was converted to the free base form
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.51 (s, 1H),
7
.41 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 4.52 (s, 2H), 3.52 – 3.43 (m,
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Journal of Medicinal Chemistry
1
2
H), 2.78 (t, J = 6.6 Hz, 4H), 2.52 (t, J = 6.6 Hz, 2H), 2.47 (s, 3H), 2.30 – 2.18 (m, 2H), 1.99 – 1.90 (m,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
H), 1.72 – 1.60 (m, 2H). MS (ESI) m/z 327.0 [M+H] .
2-(4-((4-Bromobenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (32). The title compound was
prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et N) and B
HCl/MeOH) (30% over four steps). A small portion of the HCl salt was converted to the free base form
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.48 (d, J =
8
.3 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 4.51 (s, 2H), 3.53 – 3.43 (m, 1H), 2.81 (t, J = 6.5 Hz, 4H), 2.53 (t,
J = 6.6 Hz, 2H), 2.48 (s, 3H), 2.30 – 2.17 (m, 2H), 2.04 – 1.88 (m, 2H), 1.72 – 1.60 (m, 2H). MS (ESI)
+
m/z 327.0 [M+H] .
N-Methyl-2-(4-((2-(trifluoromethyl)benzyl)oxy)piperidin-1-yl)ethan-1-amine (33). The title
compound was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with
Et
3
N) and B (HCl/MeOH) (45% over four steps). A small portion of the HCl salt was converted to the
1
free base form using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD):
δ 7.72 (d, J = 7.7 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H),
4
2
.70 (s, 2H), 3.58 – 3.45 (m, 1H), 2.85 – 2.77 (m, 2H), 2.74 (t, J = 6.7 Hz, 2H), 2.51 (t, J = 6.7 Hz, 2H),
.44 (s, 3H), 2.34 – 2.15 (m, 2H), 2.03 – 1.88 (m, 2H), 1.77 – 1.55 (m, 2H). HRMS (ESI-TOF) m/z:
+
[M+H] calcd for C16
H
24
F
3
N
2
O, 317.1835; found 317.1832.
N-Methyl-2-(4-((3-(trifluoromethyl)benzyl)oxy)piperidin-1-yl)ethan-1-amine (34). The title
compound was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with
Et
3
N) and B (HCl/MeOH) (50% over four steps). A small portion of the HCl salt was converted to the
1
free base form using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD):
δ 7.65 (s, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 4.62 (s, 2H),
3.55 – 3.48 (m, 1H), 2.85 – 2.75 (m, 2H), 2.72 (t, J = 6.8 Hz, 2H), 2.50 (t, J = 6.8 Hz, 2H), 2.42 (s, 3H),
+
2
.31 – 2.16 (m, 2H), 2.06 – 1.89 (m, 2H), 1.80 – 1.62 (m, 2H). HRMS (ESI-TOF) m/z: [M+H] calcd for
16 24 3 2
C H F N O, 317.1835; found 317.1834.
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Journal of Medicinal Chemistry
Page 24 of 54
N-Methyl-2-(4-((4-(trifluoromethyl)benzyl)oxy)piperidin-1-yl)ethan-1-amine (35). The title
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with
Et
3
N) and B (HCl/MeOH) (44% over four steps). A small portion of the HCl salt was converted to the
1
free base form using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD):
δ 7.62 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 7.9 Hz, 2H), 4.63 (s, 2H), 3.56 – 3.46 (m, 1H), 2.88 – 2.73 (m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
2
H), 2.54 (t, J = 6.6 Hz, 2H), 2.49 (s, 3H), 2.34 – 2.17 (m, 2H), 2.07 – 1.88 (m, 2H), 1.80 – 1.60 (m,
+
H). HRMS (ESI-TOF) m/z: [M+H] calcd for C16
H
24
F
3
N
2
O, 317.1835; found 317.1841.
2
-(4-([1,1'-Biphenyl]-2-ylmethoxy)piperidin-1-yl)-N-methylethan-1-amine (36). The title compound
N) and B
HCl/MeOH) (45% over four steps). A small portion of the HCl salt was converted to the free base form
was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et
3
(
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.53 – 7.43
(m, 1H), 7.50 – 7.37 (m, 2H), 7.37 – 7.29 (m, 5H), 7.27 – 7.17 (m, 1H), 4.39 (s, 2H), 3.35 – 3.24 (m,
1
H), 2.75 – 2.60 (m, 4H), 2.46 (t, J = 6.7 Hz, 2H), 2.41 (s, 3H), 2.21 – 2.03 (m, 2H), 1.84 – 1.71 (m,
+
2H), 1.59 – 1.45 (m, 2H). MS (ESI) m/z 325.2 [M+H] .
2
-(4-([1,1'-Biphenyl]-3-ylmethoxy)piperidin-1-yl)-N-methylethan-1-amine (37). The title compound
was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et N) and B
3
(HCl/MeOH) (41% over four steps). A small portion of the HCl salt was converted to the free base form
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.66 – 7.54
(m, 3H), 7.52 (d, J = 7.7 Hz, 1H), 7.49 – 7.36 (m, 3H), 7.36 – 7.26 (m, 2H), 4.59 (s, 2H), 3.54 – 3.46
(m, 1H), 2.85 – 2.73 (m, 4H), 2.51 (t, J = 6.6 Hz, 2H), 2.48 (s, 3H), 2.26 – 2.15 (m, 2H), 2.01 – 1.91 (m,
+
2
H), 1.73 – 1.60 (m, 2H). MS (ESI) m/z 325.2 [M+H] .
2
-(4-([1,1'-Biphenyl]-4-ylmethoxy)piperidin-1-yl)-N-methylethan-1-amine (38). The title compound
was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et
3
N) and B
(HCl/MeOH) (40% over four steps). A small portion of the HCl salt was converted to the free base form
1
3
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD OD): δ 7.63 – 7.56
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Page 25 of 54
Journal of Medicinal Chemistry
(
m, 4H), 7.45 – 7.39 (m, 4H), 7.32 (t, J = 7.3 Hz, 1H), 4.59 (s, 2H), 3.57 – 3.47 (m, 1H), 2.87 – 2.70 (m,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
H), 2.52 (t, J = 6.7 Hz, 2H), 2.46 (s, 3H), 2.35 – 2.15 (m, 2H), 2.02 – 1.89 (m, 2H), 1.76 – 1.62 (m,
+
2H). HRMS (ESI-TOF) m/z: [M+H] calcd for C21
H
29
N
2
O, 325.2274; found 325.2269.
N-Methyl-2-(4-((4-(trifluoromethoxy)benzyl)oxy)piperidin-1-yl)ethan-1-amine (39). The title
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compound was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with
Et
3
N) and B (HCl/MeOH) (34% over four steps). A small portion of the HCl salt was converted to the
1
free base form using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD):
δ 7.44 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 4.56 (s, 2H), 3.55 – 3.45 (m, 1H), 2.86 – 2.73 (m,
2
2
H), 2.70 (t, J = 6.8 Hz, 2H), 2.50 (t, J = 6.8 Hz, 2H), 2.41 (s, 3H), 2.31 – 2.17 (m, 2H), 2.06 – 1.90 (m,
+
H), 1.75 – 1.61 (m, 2H). HRMS (ESI-TOF) m/z: [M+H] calcd for C16
H
24
F
3
N
2
O
2
, 333.1784; found
333.1776.
N-Methyl-2-(4-((4-(2,2,2-trifluoroethoxy)benzyl)oxy)piperidin-1-yl)ethan-1-amine (40). The title
compound was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with
Et
3
N) and B (HCl/MeOH) (21% over four steps). A small portion of the HCl salt was converted to the
1
free base form using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD):
δ 7.31 (d, J = 8.2 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 4.56 – 4.44 (m, 4H), 3.49 – 3.43 (m, 1H), 2.84 –
2.72 (m, 4H), 2.52 (t, J = 6.6 Hz, 2H), 2.47 (s, 3H), 2.28 – 2.15 (m, 2H), 1.98 – 1.89 (m, 2H), 1.70 –
+
1
.59 (m, 2H). HRMS (ESI-TOF) m/z: [M+H] calcd for C17
H
26
F
3
N
2
O
2
, 347.1941; found 347.1938.
2
-(4-((4-isobutylbenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (41). The title compound was
N) and B
HCl/MeOH) (20% over four steps). A small portion of the HCl salt was converted to the free base form
prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et
3
(
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.24 (d, J =
7.6 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H), 4.50 (s, 2H), 3.55 – 3.40 (m, 1H), 2.82 – 2.75 (m, 2H), 2.73 (t, J =
6
.7 Hz, 2H), 2.50 (t, J = 6.7 Hz, 2H), 2.46 (d, J = 7.3 Hz, 2H), 2.43 (s, 3H), 2.25 – 2.17 (m, 2H), 1.97 –
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Journal of Medicinal Chemistry
Page 26 of 54
1
.91 (m, 2H), 1.85-1.82 (m, 1H), 1.68 – 1.59 (m, 2H), 0.89 (d, J = 6.6 Hz, 6H). HRMS (ESI-TOF) m/z:
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
[M+H] calcd for C19
H
33
N
2
O, 305.2587; found 305.2580.
2-(4-((4-Cyclohexylbenzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (42). The title compound
was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et N) and B
HCl/MeOH) (10% over four steps). A small portion of the HCl salt was converted to the free base form
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.24 (d, J =
7
6
1
.9 Hz, 2H), 7.17 (d, J = 7.8 Hz, 2H), 4.49 (s, 2H), 3.50 – 3.43 (m, 1H), 2.85 – 2.69 (m, 4H), 2.50 (t, J =
.8 Hz, 2H), 2.44 (s, 3H), 2.29 – 2.12 (m, 2H), 1.97 – 1.89 (m, 2H), 1.89 – 1.78 (m, 4H), 1.78 – 1.71 (m,
+
H), 1.69 – 1.58 (m, 2H), 1.49 – 1.35 (m, 4H), 1.36 – 1.21 (m, 2H). HRMS (ESI-TOF) m/z: [M+H]
35 2
calcd for C21H N O, 331.2744; found 331.2745.
N-methyl-2-(4-((4-(tert-pentyl)benzyl)oxy)piperidin-1-yl)ethan-1-amine (43). The title compound
was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with Et N) and B
HCl/MeOH) (11% over four steps). A small portion of the HCl salt was converted to the free base form
3
(
1
using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD): δ 7.31 (d, J =
8
6
.0 Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 4.51 (s, 2H), 3.51 – 3.44 (m, 1H), 2.86 – 2.73 (m, 2H), 2.69 (t, J =
.9 Hz, 2H), 2.49 (t, J = 6.8 Hz, 2H), 2.40 (s, 3H), 2.27 – 2.16 (m, 2H), 1.99 – 1.89 (m, 2H), 1.71 – 1.59
+
(m, 4H), 1.27 (s, 6H), 0.65 (t, J = 7.5 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H] calcd for C20
H35Cl
2
N
2
O,
3
19.2744; found 319.2744.
2
-(4-((4-(2,3-dichlorophenoxy)benzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine (44). The title
compound was prepared in four steps according to the general procedure C, B (HCl/MeOH), A (with
Et
3
N) and B (HCl/MeOH) (25% over four steps). A small portion of the HCl salt was converted to the
1
free base form using the general procedure D to obtain the NMR sample. H NMR (600 MHz, CD
3
OD):
δ 7.40 – 7.30 (m, 3H), 7.26 (t, J = 8.2 Hz, 1H), 6.97 – 6.85 (m, 3H), 4.53 (s, 2H), 3.55 – 3.45 (m, 1H),
2
.85 – 2.76 (m, 2H), 2.72 (t, J = 6.8 Hz, 2H), 2.50 (t, J = 6.8 Hz, 2H), 2.42 (s, 3H), 2.29 – 2.19 (m, 2H),
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Page 27 of 54
Journal of Medicinal Chemistry
+
2
4
2 2 2
.01 – 1.89 (m, 2H), 1.73 – 1.60 (m, 2H). HRMS (ESI-TOF) m/z: [M+H] calcd for C21H27Cl N O ,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
09.1444; found 409.1439.
2-(4-((4-(3,5-dimethyl-1H-pyrazol-1-yl)benzyl)oxy)piperidin-1-yl)-N-methylethan-1-amine
(45).
The title compound was prepared in four steps according to the general procedure C, B (HCl/MeOH), A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(with Et
3
N) and B (HCl/MeOH) (25% over four steps). A small portion of the HCl salt was converted to
1
the free base form using the general procedure D to obtain the NMR sample. H NMR (600 MHz,
3
CD OD): δ 7.50 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 6.06 (s, 1H), 4.62 (s, 2H), 3.57 – 3.47 (m,
1
8
3
H), 2.85 – 2.76 (m, 2H), 2.74 (t, J = 6.8 Hz, 2H), 2.51 (t, J = 6.8 Hz, 2H), 2.43 (s, 2H), 2.30 – 2.18 (m,
+
H), 2.02 – 1.91 (m, 2H), 1.75 – 1.62 (m, 2H). HRMS (ESI-TOF) m/z: [M+H] calcd for C19
H
30
N
4
O,
43.2492; found 343.2490.
4
-(Benzyloxy)-1-(2-methoxyethyl)piperidine (46). To a mixture of 4-(benzyloxy)piperidine
hydrochloride (0.23 g, 1.0 mmol), potassium carbonate (0.28 g, 2.0 mmol), potassium iodide (0.016 g,
.1 mmol) in acetonitrile (10 mL) was added 1-bromo-2-methoxyethane (0.15 g, 1.1 mmol). After being
refluxed for overnight, the reaction mixture was cooled down to rt and quenched with water. The
mixture was extracted with EtOAc (3 x). Combined organic layers were dried over Na SO and
0
2
4
concentrated under reduced pressure. The resulting residue was purified by flash chromatography on
1
silica gel column to provide desired product as yellow oil (20%). H NMR (600 MHz, CD
3
OD): δ 7.69
–
3
7.28 (m, 4H), 7.28 – 7.22 (m, 1H), 4.53 (s, 2H), 3.51 (t, J = 5.6 Hz, 2H), 3.49 – 3.42 (m, 1H), 3.32 (s,
H), 2.88 – 2.74 (m, 2H), 2.55 (t, J = 5.6 Hz, 2H), 2.32 – 2.18 (m, 2H), 1.98 – 1.87 (m, 2H), 1.73 – 1.59
1
3
3
(m, 2H). C NMR (150 MHz, CD OD): δ 138.7, 127.9, 127.3, 127.1, 73.8, 69.7, 69.4, 57.5, 57.1, 51.0,
+
+
3
0.3. MS (ESI) m/z 250.2 [M+H] . HRMS (ESI-TOF) m/z: [M+H] calcd for C15
2
H24NO , 250.1802;
found 250.1799.
PRMT biochemical assays. A radioactivity based scintillation proximity assay (SPA) was used to
assess the effect of test compounds on inhibiting the methyl transfer reaction catalyzed by PRMTs as
4
5
described previously. The full length human PRMT4 (residues 1-608) and PRMT6 (residues 1-375)
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Journal of Medicinal Chemistry
Page 28 of 54
4
8, 49
were expressed and purified as described previously
and were used in all of the biochemical and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
biophysical assays. The tritiated S-adenosyl-L-methionine ( H-SAM) obtained from PerkinElmer Life
Sciences (cat#NET155V001MC; specific activity range 12−18 Ci/mmol) served as the methyl donor.
3
The ( H) methylated biotin labelled peptide would be captured in streptavidin/scintillant-coated
3
scintillation proximity FlashPlates Plus (PerkinElmer Life Sciences) which brings the incorporated H-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
methyl and the scintillant to close proximity resulting in light emission that is quantified by tracing the
radioactivity signal (counts per minute) as measured by a TopCount NXT™ Microplate Scintillation
and Luminescence Counter (PerkinElmer Life Sciences). When necessary, non-tritiated SAM (AK
Scientific, Union City, CA) was used to supplement the reactions. The reaction mixture for the PRMT6
assay (10 µL) contained 20 mM bis-tris-propane (pH 7.5) containing 0.01% Tween-20, 10 mM
dithiothreitol
(DTT),
25
nM
PRMT6,
0.6
µM
H4
1-24
peptide
(
SGRGKGGKGLGKGGAKRHRKVLRDK-Biotin) and 2.3 µM SAM. For PRMT4 assay, the reaction
mixture contained 20 mM bicine (pH 8.5), 0.01% Triton X-100, 25 nM PRMT4, 0.7 µM H3 1-25
peptide (ARTKQTARKSTGGKAPRKQLATKAAGK-Biotin) and 1.9 µM SAM. For the IC50
determination the compounds were serially diluted in the reaction buffer and were added to the reaction
mixture before starting the reactions by addition of the substrate. The reactions were quenched by
addition of equal volume of 7.5 M guanidine hydrochloride and the volume finalized to 120 µL by the
addition of 20 mM Tris-HCl, pH 8.0. After the transfer to the SPA plates (FlashPlate® PLUS;
PerkinElmer Life Sciences), the plates were incubated for at least 3 h before measuring the signals using
a TopCount NXT™ Microplate Scintillation and Luminescence Counter (PerkinElmer Life Sciences).
The IC50 values were determined under balanced conditions at K
m
concentrations of both substrate and
3
cofactor by titration of test compounds in the reaction mixture. The H-SAM can be purchased only at
low concentration (40-50 µM) in acidic solution which need to be diluted at least in 1:10 ratio to prepare
the final reaction mixture (maximum of 5 µM). All IC50 values were determined at K
m
of substrates. For
enzymes that have K values higher than 5 µM for SAM (for example PRMT3) we needed to increase
m
the concentration of SAM by adding unlabeled SAM. Same limitation existed when assessing the
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Page 29 of 54
Journal of Medicinal Chemistry
competition with SAM which saturation concentration of SAM was required. In case of the enzymes
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
m
with apparent SAM K below 5 µM, due to high cost of H-SAM, we still diluted H-SAM with
unlabeled SAM as long as the signal-to-noise was reliable for assays.
Selectivity assays. The methyltransferase selectivity of 17 was assessed at two compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7-70
concentrations of 10 and 50 µM as described previously.
The inhibitory effect of 17 against three
lysine demethylases, KDM1A (LSD1), KDM3A (JMJD1A), and KDM4A (JMJD2A) was assessed
71
using the method described by Yu et. al. The selectivity also was tested against 9 reader proteins
(UHRF1, BRPF1, WDR5, HGDF2, TDRD3, RBBP1, FXR1, EED, and SND1) using DSF and/or DSLS
7
2
based assays as described elsewhere. Selectivity assays of 100 non-epigenetic targets (kinases,
GPCRs, ion channels, and transporters) were conducted by CEREP. The 5HT2B radiolabel binding
assay was performed by the NIMH Psychoactive Drug Screening Program.
Mechanism of action (MOA) studies. To determine the MOA of the 17, the competition of the
compound with SAM and peptide substrate was determined individually. In brief, the peptide
concentration was kept at saturation and compound potency was monitored at different SAM
concentrations (0.5, 1, 4, 8, 12, 16, 20, and 25×K
concentration was kept at saturation and IC50 values were determined at different peptide concentrations
(0.5, 1, 2, 4, 8, 12, 16, and 20×K ).
m
). To test the competition with peptide, the SAM
m
Biophysical assays. Isothermal titration calorimetry (ITC) was performed in a Nano ITC instrument
4
7
(
TA Instruments, USA) following the procedure described previously. The ITC titrations were
performed at 25 °C by using 2 µL injections with a total of 25 injections. For PRMT6, the cell was
loaded with 50 µM PRMT6, 100 µM SAM with final DMSO concentration of 2.5% DMSO in 50 mM
Tris-HCl (pH 8.5) containing 150 mM NaCl. The syringe was loaded with 200 µM of 17, 100 µM SAM
and 2.5% DMSO prepared in the same buffer. For PRMT4, the cell was loaded with 35 µM PRMT4,
1
20 µM SAH and 3% DMSO in phosphate buffered saline. The syringe was loaded with 300 µM 17,
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