Structure–Activity Relationship Studies on (R)-PFI-2 Analogues as Inhibitors of Histone Lysine Methyltransferase SETD7

Article abstract of DOI:10.1002/cmdc.201800242

SETD7 is a histone H3K4 lysine methyltransferase involved in human gene regulation. Aberrant expression of SETD7 has been associated with various diseases, including cancer. Therefore, SETD7 is considered a good target for the development of new epigenetic drugs. To date, few selective small-molecule inhibitors have been reported that target SETD7, the most potent being (R)-PFI-2. Herein we report structure–activity relationship studies on (R)-PFI-2 and its analogues. A library of 29 structural analogues of (R)-PFI-2 was synthesized and evaluated for inhibition of recombinantly expressed human SETD7. The key interactions were found to be a salt bridge and a hydrogen bond formed between (R)-PFI-2′s NH₂⁺ group and SETD7′s Asp256 and His252 residue, respectively.

Full text of DOI:10.1002/cmdc.201800242

Accepted Article
Title: Structure-Activity Relationship Studies on (R)-PFI-2 Analogs as
Inhibitors of Histone Lysine Methyltransferase SETD7
Authors: Danny C Lenstra, Eddy Damen, Ruben G. G. Leenders,
Richard H. Blaauw, Floris P. J. T. Rutjes, Anita Wegert, and
Jasmin Mecinovic
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Structure-Activity Relationship Studies on (R)-PFI-2 Analogs as
Inhibitors of Histone Lysine Methyltransferase SETD7
Danny C. Lenstra,[a] Eddy Damen,[b] Ruben G. G. Leenders,[b] Richard H. Blaauw,[c] Floris P. J. T.
Rutjes,[a] Anita Wegert,[b] and Jasmin Mecinović*[a]
[a]
D. C. Lenstra, Prof. Dr. F. P. J. T. Rutjes, Dr. J. Mecinović
Institute for Molecules and Materials, Radboud University
Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
E-mail: j.mecinovic@science.ru.nl
[b]
[c]
Dr. E. Damen, Dr. R. G. G. Leenders, Dr. A. Wegert
Mercachem BV
Kerkenbos 1013, 6546 BB Nijmegen, The Netherlands
Dr. R. H. Blaauw
Chiralix
Toernooiveld 100, 6525 EC Nijmegen, The Netherlands
Supporting information for this article is given via a link at the end of the document.
Abstract: SETD7 is a histone H3K4 lysine methyltransferase involved
in human gene regulation. Aberrant expression of SETD7 has been
associated with various diseases, including cancer, therefore SETD7
is considered as a target for the development of new epigenetic drugs.
To date, few selective small molecule inhibitors have been reported
that target SETD7, the most potent being (R)-PFI-2. Here we report
structure-activity relationship studies on (R)-PFI-2 and its analogs. A
library of 29 structural analogs of (R)-PFI-2 was synthesized and
evaluated for inhibition of recombinantly expressed human SETD7.
The key interactions were found to be a salt-bridge and a hydrogen
bond formed between (R)-PFI-2’s NH2+ and SETD7’s Asp256 and
His252, respectively.
Introduction
Covalent posttranslational modifications on the unstructured N-
terminal histone tails play an important role in regulating the
activity of human genes.[1] These modifications are introduced by
‘writers’ and, among others, include methylation, acetylation,
phosphorylation, citrullination, and ubiquitination.[2] Methylation is
one of the most abundant and well-studied modifications on
histones.[3] The transfer of a methyl group from cosubstrate S-
adenosylmethionine (SAM) to lysine residues is catalyzed by
histone lysine methyltransferases (KMTs).[4] KMTs can catalyze
the addition of up to three methyl groups to the ε-amino group of
lysine residues, affording methyllysine (Kme), dimethyllysine
(Kme2) and trimethyllysine (Kme3).[5] With the exception of
DOT1L, an H3K79 methyltransferase, all KMTs contain a highly
conserved SET (Su(var)3-9, enchanter-of-zeste, thritorax)
domain, which is responsible for their catalytic activity.[6]
Figure 1. a) SETD7-catalyzed methylation of H3K4; b) Crystal structure of
SETD7 (grey) in complex with (R)-PFI-2 (cyan) (PDB ID: 4JLG); c) Structure of
(R)-PFI-2, with sites A, B, and C for SAR exploration.
methyltransferases
1
(DNMT1)[9], TAF10[10]
,
FoxO3[11]
,
,
transcription factors YY1 and Pdx1[12], androgen receptor[13]
ARTD1[14], beta-catenin[15], and tumor suppressor p53[16]. Recent
biomedical studies revealed that SETD7 is involved in multiple
signaling pathways, and its aberrant activity has been linked to
various types of cancer[17] and vascular dysfunction in patients
with type 2 diabetes[18]. The development of SETD7 small
molecule inhibitors as chemical probes for studying their
biological activity in cells is therefore highly desired and may lead
to the development of therapeutic agents for the treatment of
human diseases.[19]
SET domain containing lysine methyltransferase 7 (SETD7,
also known as SET7/9) was originally identified as
a
monomethylase of lysine 4 on histone 3 (H3K4).[7] Unlike many
other methyltransferases, SETD7 only monomethylates H3K4,
but does not catalyze di- or trimethylation of lysine. This
methylation mark (H3K4me) has a functional effect on the
chromatin state resulting in activation of transcription. SETD7 has
also been found to catalyze methylation of a range of non-histone
To date, only a few selective SETD7 inhibitors have been
identified. Clinically approved antihistaminic cyproheptadine was
found to inhibit SETD7-mediated methylation of H3K4 (IC50 of 3.4
proteins, including estrogen receptor
α
(ERα)[8], DNA
1
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Figure 2. a) Representative MALDI-TOF MS data for 200 nM SETD7, 21-mer
H3K4 peptide (10 µM), SAM (16 µM) in 50 mM glycine pH 8.8 containing 2.5%
glycerol and 5% DMSO (v/v) after 1 h at 37 °C; b) 10 µM of (R)-PFI-2
(compound 1); c) 10 µM compound 9; d) 10 µM compound 14; e) 10 µM
compound 27. Y-axis represents relative abundance (Rel. abun.).
Scheme 1. Syntheses of (R)-PFI-2 and its analogs 1-28. Reagents and
conditions: i) Boc2O, and NaOH in H2O/dioxane (1:1 v/v), rt, 20 h; ii) EDCI,
HOBt, DIPEA, and R2NH in DCM, rt, 20 h; iii) TFA/DCM (1:1 v/v), rt, 2-4 h; iv)
Pd2(dba)3, Xantphos, DIPEA, and benzylmercaptan in dioxane, 100 °C, 6 h; v)
Benzyltrimethylammonium chloride, trichloroisocyanuric acid, and Na2CO3 in
H2O/MeCN, 0 °C to rt, 1 h; vi) Et3N in DCM, rt, 4 h. Where applicable, Boc-
protected compounds were deprotected using TFA/DCM (1:1 v/v).
HOBt, and DIPEA. Then the Boc group was removed with TFA in
DCM, followed by a basic work-up to afford the free amine. The
sulfonyl part of the inhibitor was synthesized from various aryl
bromides (Scheme 1). These aryl bromides were first reacted with
benzyl mercaptan in the presence of Pd2(dba)3 and Xantphos to
afford aryl benzyl sulfides, followed by oxidation with
trichloroisocyanuric acid to yield the corresponding sulfonyl
chlorides. The sulfonyl chlorides were then coupled to the free
amines in the presence of triethylamine in DCM to afford the
desired structural analogs of (R)-PFI-2.
Initially, all synthesized compounds were tested for their
inhibitory activity using an established matrix-assisted laser
desorption-ionization time-of-flight mass spectrometry (MALDI-
TOF MS) assay, monitoring the monomethylation of a synthetic
21-mer histone 3 peptide mimic containing a lysine at position
4.[25] In a typical assay, 200 nM of SETD7, 10 µM of H3K4 histone
peptide, 16 µM of SAM and 10 or 100 µM of compounds 1-30
were incubated for 1 hour at 37 °C. For compounds showing
>50% inhibition at 100 µM, IC50s were determined. In cases where
<50% inhibition at 100 µM was observed, IC50s were not
determined and this data is presented as IC50 >100 µM.
Enzymatic activity of these compounds at 100 µM concentration
can be found in Figure S1 in Supporting Information.
Representative MALDI data of selected compounds at 10 µM are
shown in Figure 2. In a control experiment without inhibitor (5%
(v/v) of DMSO) nearly quantitative methylation was observed in
the presence of human SETD7 (m/z = 2269.5) (Figure 2a). Under
standard conditions, but in the presence of 10 µM of (R)-PFI-2
(compound 1), no methylation was observed in the MALDI
spectrum (Figure 2b).
µM). The substrate in this study was ERα, and in vivo inhibition
led to blocking of estrogen-dependent breast cancer cell growth
in MCF7 cells.[20] A structure-activity relationship study was
performed, however, none of the analogs proved to be more
potent than cyproheptadine.[21] Several SAM derivatives have
also been reported to inhibit SETD7 methyltransferase activity; for
instance, DAAM-3 (IC50 10 µM)[22] and DC-S239 (IC50 4.59 µM).[23]
Unfortunately, SAM-competitive inhibitors usually have poor
selectivity amongst other methyltransferases due to the highly
conserved SAM binding domain in the methyltransferase family.
The most potent known SETD7 inhibitor (IC50 = 2.0 0.2 nM) is
(R)-PFI-2 (Figure 1b-c), while its enantiomer (S)-PFI-2 proved to
be
a very weak SETD7 inhibitor, highlighting that the
stereochemistry of the amino acid side chain defines its
potency.[24] (R)-PFI-2 is a histone competitive inhibitor, and
exhibits an inhibition potency in vitro and in cellular assays.[24]
Comparative studies demonstrated that (R)-PFI-2 is selective for
SETD7 over 18 other human methyltransferases. Although the
structure of (R)-PFI-2 in complex with SETD7 has been solved
(Figure 1b), no further analogs have been reported. To investigate
which structural elements of (R)-PFI-2 are essential for effective
inhibition of SETD7, we carried out a structure-activity relationship
(SAR) study. We modified three distinctive moieties that have a
potential to crucially contribute to the potency of (R)-PFI-2: i) The
amino acid side chain (Figure 1c, part A); ii) the pyrrolidine amide
(Figure 1c, part B); and iii) the tetrahydroisoquinoline moiety
(Figure 1c, part C).
We further examined the potency of (R)-PFI-2 against
recombinantly expressed human SETD7. Under our assay
conditions, an IC50 value of 138 nM was obtained for (R)-PFI-2
(Figure 3, compound 1), indicating very effective inhibition of
SETD7 methyltransferase activity. Previously reported IC50
values for (R)-PFI-2 were 2.0 and 55 nM, respectively;[24, 25b] the
observed difference with our measured IC50 value is due to
differences in employed assay and assay conditions.
Representative inhibition curves can be found in Figure S2. Two
isomers of 1, with ortho- (compound 2) and para-CF3 (compound
Results and Discussion
We initiated our investigations with the synthesis of (R)-PFI-
2 (1) and its 29 structural analogs (Scheme 1). In general, the
synthesis was adapted from literature,[24] and starts from an
unnatural D-amino acid; typically Boc-protected D-phenylalanine
derivatives were coupled to the requisite amines using EDCI,
2
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10.1002/cmdc.201800242
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CF3
Me
O
Cl
F3C
CF3
O
S
O
S
O
O
O
O
O
S
O
O
O
O
O
S
O
N
N
H
S
O
N
N
H
N
H
HN
N
HN
N
H
H
HN
N
HN
N
HN
N
F
F
F
F
F
1: 138 nM
2: 203 nM
3: 173 nM
4: 178 nM
5: 153 nM
Br
O
O
O
O
S
N
H
O
O
O
S
N
H
S
O
O
O
O
S
N
H
N
O
O
S
N
H
O
H
HN
N
O
HN
HN
HN
N
HN
N
F
HN
N
F
F
F
F
6: 132 nM
7: 266 nM
8: 145 nM
9: 79 µM
10: >100 µM
O
O
S
N
H
O
O
S
N
H
O
O
S
N
H
O
O
S
N
H
O
O
S
N
H
O
O
O
O
O
HN
N
O
HN
N
HN
N
HN
N
HN
N
F
F
F
F
F
11: 290 nM
12: 1.3 µM
13: 1.9 µM
14: 20 µM
15: 2.6 µM
O
O
S
N
H
O
O
S
N
H
O
O
S
N
H
O
O
S
N
H
O
O
S
N
H
O
O
O
O
O
O
HN
N
N
N
N
F
F
F
16: >100 µM
17: >100 µM
18: >100 µM
19: >100 µM
20: >100 µM
O
O
S
N
H
O
O
S
N
H
O
O
S
N
H
O
O
S
N
H
O
O
S
N
H
O
O
O
O
O
S
N
N
HN
N
N
N
N
O
N
H
21: >100 µM
22: >100 µM
23: 532 nM
24: >100 µM
25: >100 µM
O
S
O
O
O
S
N
H
O
O
S
N
H
O
O
S
N
H
NH2
O
O
O
O
HN
H2N
HN
H2N
N
H2N
N
N
N
F
F
O
26: 20 µM
27: 2.7 µM
28: >100 µM
29: >100 µM
30: >100 µM
Figure 3. A library of (R)-PFI-2 (compound 1) and its analogs 2-30, showing the maximum half inhibitory concentrations (IC50s).
3) substituents also displayed excellent inhibitory activity with an
IC50 of 203 and 173 nM, respectively. The potencies of analogs 4-
6 bearing a methyl-, chloro-, or bromo functionality at the meta-
position were evaluated next. All compounds were found to be
excellent inhibitors of SETD7 with IC50s ranging from 132 nM to
178 nM. Also compound 7, which does not contain any substituent
on the phenyl ring, was found to be a good SETD7 inhibitor with
an IC50 of 266 nM. Adding an extra fused ring, as in the
naphthalene-containing compound 8, gave an IC50 of 145 nM,
indicating that there is sufficient space available for additional
functionalization. Notably, removing the entire side chain (as in
glycine-based compound 9) led to rather poor inhibition with an
IC50 of 79 µM, implying that the aromatic ring is crucial for potency.
Next, the effect of modifications in the pyrrolidine amide side
chain was investigated (Figure 1c, part B). The analysis of the
crystal structure of (R)-PFI-2 as cocrystal with SETD7 and SAM
(PDB ID: 4JLG) illustrates that the pyrrolidine part of the inhibitor
is positioned inside the lysine channel pointing towards SAM
(Figure 1b). Based on this structural information, it is clear that
space in this pocket is limited and that a significant increase in the
size of the pyrrolidine moiety would presumably not be tolerated.
Indeed, 1-adamantyl substituted compound 10 did not show any
inhibitory activity against SETD7. No significant difference in IC50
3
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Figure 4. Docking studies on various (R)-PFI-2 analogs (cyan) with SETD7 (cartoon, grey) with SAM (grey). SETD7 residues that form key interactions are
shown in purple: a) Redocked structure of (R)-PFI-2 (compound 1), RMSD = 0.638; b) docking of compound 8, containing a naphthalene side chain; c)
docking of compound 12, containing a piperidine amide functionality; d) docking of carbon analog 24; e) docking of benzylamine-type compound 27; f)
superimposition of structures shown in Figures 4a-e, with SETD7 surface visualization (grey).
was observed when the 5-membered ring of the pyrrolidine amide
(compound 7, 266 nM) was replaced by a 4-membered ring: an
IC50 of 290 nM was obtained for azetidine derived analog 11. By
slightly increasing the ring size to piperidine or morpholine amide,
as in analogs 12 and 13, IC50s of 1.3 and 1.9 µM were observed,
respectively. This result shows that a slight increase in ring size
from a 5- to 6-membered already leads to an ~5-fold drop in
potency (compound 7 vs. 12). Replacement of the 5-membered
ring with acyclic diethylamine derivative 14, resulted in a higher
degree of rotational freedom, which could lead to a steric clash
with SETD7’s pocket; this proposition is in line with a surprisingly
higher IC50 value of 20 µM. An almost 10-fold lower IC50 of 2.6 µM
was obtained by decreasing the size and rotational freedom in
dimethylamine derivative 15. Removal of the entire pyrrolidine
amide side chain (as in compound 16), resulted in the formation
of an achiral compound. This significant structural change caused
that 16 is no longer an inhibitor of SETD7 within limits of detection.
Finally, the importance of the tetrahydroisoquinoline rings
was investigated (Figure 1c, part C). The simplest analogs 17-19,
consisting of a phenyl ring, either unsubstituted or with fluorine on
the meta- or para-position, did not significantly inhibit SETD7
methyltransferase activity, indicating the essential role of the
observed in the presence of 100 µM of compound 24, an analog
of 7 that has the positively charged quaternary ammonium group
(under physiological conditions) replaced by a neutral methylene
group of equal size (Figure S1). This result demonstrates that the
+
positively charged NH2 of (R)-PFI-2’s is involved in two
interactions with SETD7, namely as a salt-bridge with Asp252and
a hydrogen bond with His252. Notably, when this nitrogen was
translocated one position, as in tetrahydroquinoline 25, again no
inhibition was observed, whereas for compound 26, only a poor
inhibition with an IC50 value of 20 µM was seen. Based on our
examination of compounds 23-26, it is clearly evident that the
most important contributor to the excellent potency of (R)-PFI-2 is
the nitrogen-containing tetrahydroisoquinoline functionality.
Having a benzylamine moiety instead of the cyclic structure, as in
compound 27, a moderately active compound is obtained (IC50
=
2.7 µM). With compound 28, a formation of a salt-bridge is no
longer possible after changing the free amine to an amide
functionality, and consequently no inhibition was observed. The
two individual fragments of which the (R)-PFI-2 scaffold consists
also showed no inhibitory activity (Figure 3, compounds 29 and
30).
Docking studies were then carried out to obtain more
tetrahydroisoquinoline scaffold of (R)-PFI-2 in inhibition of SETD7. detailed information about the potential binding modes of various
IC50 values of >100 µM were observed for several heteroatom-
substituted analogs 20-22, containing for instance a planar or
nonplanar heterocyclic system. To investigate the importance of
the fluorine substituent, we synthesized analog 23, which has an
IC50 value of 532 nM; this is approximately a two-fold increase
with respect to fluorine-containing compound 7 (IC50 = 266 nM).
Approximately 10% inhibition of methyltransferase activity was
(R)-PFI-2 analogs (Figures 4a-f). Initially, to confirm the correct
docking mode, (R)-PFI-2 was successfully redocked into the
structure of SETD7 (RMSD = 0.638 Å, Figure 4a). The ligand
interaction diagram for SETD7–(R)-PFI-2 is shown in Figure S3.
Compound 8 was docked into SETD7 and the binding mode of 8
was found to be similar to that of (R)-PFI-2 (Figure 4b). In the
docked structure of 12, the piperidine group occupies the narrow
4
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Docking studies.
lysine-binding channel; although it is somewhat larger than (R)-
PFI-2’s pyrrolidine group, 12 does not noticeably clash with the
protein (Figure 4c). Finally, two structures with the
tetrahydroisoquinoline core were docked. Despite being a very
poor inhibitor of SETD7 (10% inhibition at 100 µM, Figure S1),
docking of the carbon analog 24 was successful and the apparent
binding mode seems similar to that of (R)-PFI-2, in spite of the
absence of interactions with Asp256 and His252 (Figure 4d). In
contrast, the benzylamine-derived compound 27 does have a
potential to form a salt-bridge with Asp256 and a hydrogen bond
with His252 under physiological conditions, and docking of this
molecule into SETD7 confirmed the existence of the energetically
favorable salt-bridge and hydrogen bond (Figure 4e).
The crystal structure of SETD7 in complex with (R)-PFI-2 and S-
adenosylmethionine (SAM)[24] was downloaded from the Protein Data
Bank (PDB ID: 4JLG). The protein-ligand complex (chain A) was prepared
for docking using the QuickPrep wizard in Molecular Operating
Environment (MOE).[26] All compounds to be docked were prepared using
the default settings of the Wash wizard as implemented in MOE. Molecular
docking was performed using Molegro Virtual Docker v6.0.[27] The active
binding site region was defined as a spherical region, which encompasses
all protein within 10.0 Å of bound (R)-PFI-2. Both the MolDock Score
[GRID] and PLANTS Score [GRID] with a grid resolution of 0.2 Å were
evaluated to score and rank the 5 best docking poses for each compound.
MolDock SE was selected as search algorithm using its default settings.
All figures we prepared with PyMOL visualization software.[28]
Characterization of final compounds.
Conclusions
Compound 1 (25 mg, 75% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3) δ 7.51 – 7.46 (m, 1H), 7.40 – 7.35 (m, 2H), 7.33 – 7.31 (m,
2H), 7.19 (d, J = 8.7, 1H), 4.19 (t, J = 7.5, 1H), 4.03 (bs, 2H), 3.22 – 3.06
(m, 5H), 2.99 (d, J = 7.5, 2H), 2.86 – 2.75 (m, 2H), 2.59 – 2.47 (m, 1H),
1.76 – 1.52 (m, 4H), 1.25 (bs, 1H); 13C NMR (101 MHz, CDCl3) δ 168.3,
160.0, 157.5, 139.0, 138.9, 138.8, 138.7, 136.7, 133.0, 133.0, 131.3, 131.0,
130.7, 130.3, 129.0, 128.5, 128.3, 126.1, 126.1, 126.0, 126.0, 125.2, 124.1,
124.0, 124.0, 123.9, 123.4, 123.3, 122.5, 111.0, 110.8, 110.0, 55.8, 46.2,
45.8, 42.6, 42.1, 42.1, 39.9, 28.8, 28.8, 25.7, 23.8; 19F NMR (377 MHz,
A library of 29 (R)-PFI-2 analogs was synthesized and evaluated
as SETD7 inhibitors with the aim to determine which structural
features contribute to the excellent potency of (R)-PFI-2. Our
results demonstrate that small perturbations in the pyrrolidine
amide side chain, i.e. piperidine amide 12 or dimethylamide 15
were tolerated relatively well, whereas substituents with more
rotational freedom (e.g. diethylamide 14) or with increased size
(e.g. adamantane 10) were not tolerated. Substitutions on the
aromatic side chain led to highly potent (R)-PFI-2 analogs. The
key interactions between SETD7 and (R)-PFI-2 were identified to
be a salt-bridge and a hydrogen bond that are formed between
SETD7’s Asp256/His252 and the tetrahydroisoquinoline’s NH2+ of
(R)-PFI-2. Removal or repositioning of this ammonium group
resulted in analogs that were inactive (compounds 24 and 25) or
only poorly active (compound 26), whereas a replacement of the
tetrahydroisoquinoline moiety by benzylamine (as in compound
27) resulted in an ~10 times less potent inhibitor. It is envisioned
that this study will importantly contribute to the growing field of
epigenetic drug discovery.
CDCl3) δ -62.6, -116.9; MS (ESI) m/z 500.0 [M+H]+; HRMS (ESI) found
21
m/z 500.16332 [M+H]+, calcd for C23H26F4N3O3S+ m/z 500.16310; [α]D
=
-73.2° (dichloromethane).
Compound 2 (31 mg, 83% isolated yield, white solid): 1H NMR (400 MHz,
CDCl3) δ 7.53 – 7.48 (m, 2H), 7.33 (bs, 1H), 7.28 – 7.24 (m, 3H), 7.20 (dd,
J = 1.7, 8.7 Hz, 1H), 4.20 (t, J = 7.3 Hz, 1H), 4.02 (s, 2H), 3.24 – 3.06 (m,
5H), 2.99 (d, J = 7.3 Hz, 2H), 2.88 – 2.70 (m, 2H), 2.67 – 2.56 (m, 1H),
1.79 – 1.52 (m, 4H), 1.26 (bs, 1H); 13C NMR (101 MHz, CDCl3): δ 168.1,
160.0, 157.5, 139.8, 139.8, 139.8, 139.8, 139.0, 138.9, 138.8, 138.7, 129.9,
129.7, 129.4, 128.9, 128.7, 125.4, 125.3, 125.3, 125.3, 123.4, 123.4, 122.7,
111.0, 110.7, 55.7, 46.2, 45.8, 42.8, 42.4, 42.3, 39.9, 29.0, 29.0, 25.7,
23.9; 19F NMR (377 MHz, CDCl3): δ -62.53, -117.11; MS (ESI) m/z 500.1
[M+H]+; HRMS (ESI) found m/z 500.16353 [M+H]+, calcd for
C
23H26F4N3O3S+ m/z 500.16310; [α]D21 = -91.3° (dichloromethane).
Experimental Section
Compound 3 (35 mg, 91% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3) δ 7.58 (d, J = 7.7 Hz, 1H), 7.43 – 7.28 (m, 4H), 7.18 – 7.13
(m, 1H), 4.30 (t, J = 7.7 Hz, 1H), 4.02 (s, 2H), 3.23 (t, J = 6.3 Hz, 3H), 3.18
– 3.00 (m, 4H), 2.84 – 2.73 (m, 2H), 2.68 – 2.61 (m, 1H), 1.81 – 1.57 (m,
4H), 1.24 (bs, 1H); 13C NMR (101 MHz, CDCl3) δ 168.7, 160.0, 157.5,
139.0, 138.9, 138.7, 138.7, 134.1, 133.3, 133.2, 131.6, 129.0, 128.7, 128.6,
128.5, 127.3, 126.0, 125.9, 125.8, 123.4, 123.4, 123.1, 111.0, 110.8, 55.2,
46.0, 45.9, 42.8, 42.4, 42.4, 36.7, 30.9, 29.0, 25.8, 23.9; 19F NMR (377
MHz, CDCl3) δ -62.6, -117.1; MS (ESI) m/z 500.1 [M+H]+; HRMS (ESI)
found m/z 500.16319 [M+H]+, calcd for C23H26F4N3O3S+ m/z 500.16310;
[α]D21 = -74.0° (dichloromethane).
Inhibition studies.
For determination of maximum half inhibitory concentrations (IC50s), a
MALDI-TOF MS based assay monitoring methylation of a 21-mer histone
3 peptide mimic containing a lysine at position 4 was used. Briefly, SETD7
(200 nM final concentration) was preincubated with compound (100%
DMSO stock solution at appropriate concentration) for 5 minutes at 37 °C
in 18 µL of 50 mM glycine pH 8.8 containing 2.5% glycerol as assay buffer.
Then, 2 µL of SAM (16 µM final concentration) and 21-mer H3K4 peptide
(10 µM final concentration) premixture was added to initiate the enzymatic
reaction, thereby obtaining a final reaction volume of 20 µL containing 5%
DMSO (v/v). The reaction was left to incubate for an additional 1 hour at
37 °C after which it was quenched by the addition of 20 µL of methanol.
For MALDI-TOF MS analysis, a 2 µL aliquot from the quenched reaction
mixture was mixed with 8 µL of saturated HCCA matrix (α-cyano-4-
Compound 4 (47 mg, 69% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3): δ 7.34 – 7.31 (m, 1H), 7.20 (dd, J = 1.8, 8.8 Hz, 1H), 7.11 (t,
J = 7.9 Hz, 1H), 7.03 – 6.98 (m, 1H), 6.94 – 6.88 (m, 2H), 4.16 (dd, J = 6.7,
8.5 Hz, 1H), 4.03 (bs, 2H), 3.22 – 3.01 (m, 5H), 2.94 – 2.73 (m, 4H), 2.49
– 2.38 (m, 1H), 2.27 (s, 3H), 1.71 – 1.45 (m, 4H), 1.26 (bs, 1H); 13C NMR
(101 MHz, CDCl3) δ 168.7, 160.0, 157.5, 139.1, 139.1, 138.7, 138.6, 138.0,
135.5, 130.1, 128.4, 128.3, 128.2, 127.8, 126.4, 123.4, 123.4, 111.1, 110.8,
56.2, 46.1, 45.7, 42.7, 42.2, 42.2, 40.4, 28.9, 25.7, 23.9, 21.3; 19F NMR
(377 MHz, CDCl3) δ –117.2; MS (ESI) m/z 446.1 [M+H]+; HRMS (ESI)
found m/z 446.19004 [M+H]+, calcd for C23H29FN3O3S+ m/z 446.19006;
[α]D21 = -91.1° (dichloromethane).
hydroxycinnamic acid, Sigma-Aldrich). From this mixture
1 µL was
deposited on the MALDI plate for crystallization. Methyltransferase activity
was calculated by taking the peak area of monomethylation state
(including all isotopes and adducts), and is expressed relative to the
reaction in which no compound was present (control experiment in which
just 5% DMSO was present). The calculated activities were plotted against
the corresponding compound concentrations and fitted to the Hill-equation
using Origin Pro. Each experiment was performed in triplicate.
5
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10.1002/cmdc.201800242
ChemMedChem
FULL PAPER
Compound 5 (14 mg, 81% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3) δ 7.32 – 7.29 (bs, 1H), 7.22 – 7.14 (m, 3H), 7.08 – 7.00 (m,
2H), 4.19 – 4.13 (t, J = 7.4 Hz, 1H), 4.02 (s, 2H), 3.28 – 3.09 (m, 5H), 2.91
(d, J = 7.4 Hz, 2H), 2.85 – 2.76 (q, J = 6.1 Hz, 2H), 2.66 – 2.56 (m, 1H),
1.79 – 1.58 (m, 4H), 1.26 (bs, 1H); 13C NMR (126 MHz, CDCl3) δ 168.4,
159.8, 157.8, 138.8, 138.8, 138.8, 137.7, 134.2, 129.7, 129.4, 128.6, 128.5,
127.7, 127.3, 123.4, 123.4, 111.0, 110.8, 55.9, 46.2, 45.9, 42.8, 42.3, 42.3,
39.9, 28.9, 28.9, 25.8, 23.9; 19F NMR (377 MHz, CDCl3) δ -117.0;
[M+H]+; HRMS (ESI) found m/z 512.23804 [M+H]+, calcd for
C28H35FN3O3S+ m/z 512.23831; [α]D21 = -19.1° (dichloromethane).
Compound 11 (39 mg, 81% isolated yield, white solid): 1H NMR (400 MHz,
CDCl3) δ 7.35 (bs, J = 1.6 Hz, 1H), 7.30 – 7.22 (m, 4H), 7.15 – 7.10 (m,
2H), 4.05 (s, 2H), 3.90 (dd, J = 9.0, 6.1 Hz, 1H), 3.82 – 3.64 (m, 3H), 3.13
(t, J = 5.9 Hz, 2H), 3.07 (td, J = 8.9, 6.2 Hz, 1H), 2.95 – 2.76 (m, 4H), 2.08
– 1.99 (m, 1H), 1.97 – 1.83 (m, 1H), 1.30 – 1.22 (m, 1H); 13C NMR (101
MHz, CDCl3) δ 169.2, 160.1, 157.6, 139.1, 139.0, 138.9, 138.8, 135.7,
129.4, 128.7, 128.5, 128.5, 127.2, 123.4, 123.3, 111.0, 110.8, 53.7, 49.7,
47.6, 42.8, 42.4, 42.3, 40.2, 29.0, 28.9, 15.2; 19F NMR (377 MHz, CDCl3)
MS (ESI) m/z 466.1 [M+H]+; HRMS (ESI) found m/z 466.13652 [M+H]+,
21
calcd for C22H26ClFN3O3S+ m/z 466.13674; [α]D
=
-88.9°
(dichloromethane).
δ -117.2; MS (ESI) m/z 418.2 [M+H]+; HRMS (ESI) found m/z 418.16006
Compound 6 (18 mg, 72% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3) δ 7.35 – 7.29 (m, 2H), 7.24 – 7.16 (m, 2H), 7.14 – 7.06 (m,
2H), 4.16 (t, J = 7.5 Hz, 1H), 4.05 (s, 2H), 3.28 – 3.10 (m, 5H), 2.88 (d, J =
7.5 Hz, 2H), 2.85 – 2.78 (m, 2H), 2.66 – 2.55 (m, 1H), 1.79 – 1.58 (m, 4H)
1.26 (bs, 1H); 13C NMR (126 MHz, CDCl3) δ 168.4, 159.7, 157.8, 138.9,
138.9, 138.7, 138.6, 138.0, 132.3, 130.2, 130.0, 128.8, 128.3, 128.2, 128.2,
123.3, 123.3, 122.4, 111.0, 110.8, 55.9, 46.2, 45.9, 42.7, 42.2, 42.2, 39.8,
39.7, 29.7, 29.3, 28.7, 25.8, 23.9; 19F NMR (377 MHz, CDCl3) δ -117.1;
[M+H]+, calcd for C21H25FN3O3S+ m/z 418.15965; [α]D
= -87.0°
21
(dichloromethane).
Compound 12 (47 mg, 96% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3) δ 7.34 – 7.31 (m, 1H), 7.28 – 7.18 (m, 4H), 7.16 – 7.09 (m,
2H), 4.41 (dd, J = 7.8, 6.6 Hz, 1H), 4.02 (s, 2H), 3.36 – 3.26 (m, 1H), 3.26
– 3.17 (m, 1H), 3.10 (t, J = 5.9 Hz, 2H), 3.04 – 2.72 (m, 6H), 1.51 – 1.07
(m, 6H), 0.99 – 0.80 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 168.3, 160.0,
157.6, 138.9, 138.8, 138.8, 138.7, 135.5, 129.6, 128.6, 128.5, 128.5, 127.2,
123.5, 123.5, 111.2, 111.0, 53.6, 46.3, 43.1, 42.8, 42.4, 42.3, 40.9, 29.0,
MS (ESI) m/z 511.9 [M+H]+; HRMS (ESI) found m/z 510.08574 [M+H]+,
21
calcd for C22H26BrFN3O3S+ m/z 510.08523; [α]D
=
-50.8°
29.0, 25.6, 25.1, 24.0; 19F NMR (377 MHz, CDCl3)
δ -117.2;
(dichloromethane).
MS (ESI) m/z 446.1 [M+H]+; HRMS (ESI) found m/z 446.19111 [M+H]+,
calcd for C23H29FN3O3S+ m/z 446.19136; [α]D21 = -92.0° (dichloromethane).
Compound 7 (13 mg, 64% isolated yield, white solid): 1H NMR (500 MHz,
CDCl3) δ 7.33 (bs, 1H), 7.25 – 7.18 (m, 4H), 7.14 – 7.09 (m, 2H), 4.16 (dd,
J = 8.9, 6.2 Hz, 1H), 4.02 (bs, 2H), 3.17 – 2.87 (m, 7H), 2.85 – 2.73 (m,
2H), 2.44 – 2.39 (m, 1H), 1.67 – 1.56 (m, 3H), 1.52 – 1.45 (m, 1H) 1.25
(bs, 1H); 13C NMR (126 MHz, CDCl3) δ 168.8, 160.1, 158.1, 139.2, 139.2,
139.1, 135.9, 129.8, 129.1, 128.9, 128.8, 127.5, 123.8, 123.8, 111.4, 111.2,
56.5, 46.4, 46.0, 43.2, 42.8, 42.7, 40.9, 29.4, 29.3, 26.0, 24.2. 19F NMR
(377 MHz, CDCl3) δ -117.3; MS (ESI) m/z 432.1 [M+H]+; HRMS (ESI)
found m/z 454.15748 [M+Na]+, calcd for C22H26FN3O3SNa+ m/z
545.15766; [α]D21 = -67.1° (dichloromethane).
Compound 13 (37 mg, 91% isolated yield, slightly yellow solid): 1H NMR
(400 MHz, CDCl3) δ 7.34 (s, J = 7.8 Hz, 1H), 7.30 – 7.20 (m, 4H), 7.16 –
7.09 (m, 2H), 4.39 (dd, J = 9.4, 5.8 Hz, 1H), 4.03 (s, 2H), 3.47 – 3.28 (m,
3H), 3.28 – 3.07 (m, 4H), 3.04 – 2.64 (m, 7H), 1.25 (bs, 1H); 13C NMR (126
MHz, CDCl3) δ 169.0, 159.8, 157.9, 139.0, 139.0, 138.9, 138.8, 135.4,
129.6, 129.0, 128.8, 128.7, 127.5, 123.4, 123.4, 111.1, 110.9, 66.2, 65.6,
53.3, 45.7, 42.8, 42.4, 42.4, 42.2, 41.0, 29.1, 29.0; 19F NMR (377 MHz,
CDCl3) δ -117.3; MS (ESI) m/z 448.1 [M+H]+; HRMS (ESI) found m/z
21
448.17145 [M+H]+, calcd for C22H27FN3O4S+ m/z 448.17063; [α]D = -
Compound 8 (25 mg, 85% isolated yield, off-white solid): 1H NMR (500
MHz, CDCl3) δ 7.81 – 7.76 (m, 1H), 7.73 – 7.68 (m, 2H), 7.54 (bs, 1H),
7.48 – 7.42 (m, 2H), 7.25 – 7.21 (m, 2H), 7.16 (dd, J = 8.7, 1.7 Hz, 1H),
4.30 (t, J = 7.5 Hz, 1H), 3.93 – 3.86 (m, 2H), 3.26 – 3.14 (m, 3H), 3.07 (dd,
J = 7.5, 1.8 Hz, 2H), 3.01 (t, J = 5.9 Hz, 2H), 2.71 – 2.55 (m, 3H), 1.70 –
1.50 (m, 3H), 1.45 – 1.38 (m, 1H), 1.25 (bs, 1H); 13C NMR (126 MHz,
CDCl3) δ 168.8, 159.6, 157.6, 138.9, 138.9, 138.7, 138.6, 133.3, 133.2,
132.3, 128.5, 128.4, 128.2, 128.0, 127.6, 127.5, 127.4, 126.2, 125.8, 123.2,
123.2, 110.9, 110.7, 56.2, 46.1, 45.8, 42.7, 42.2, 42.2, 40.4, 28.7, 28.7,
25.7, 23.9; 19F NMR (377 MHz, CDCl3) δ -117.4; MS (ESI) m/z 482.1
[M+H]+; HRMS (ESI) found m/z 482.19176 [M+H]+, calcd for
C26H29FN3O3S+ m/z 482.19136; [α]D21 = -97.1° (dichloromethane).
86.2° (dichloromethane).
Compound 14 (41 mg, 85% isolated yield, white solid): 1H NMR (400 MHz,
CDCl3) δ 7.34 – 7.30 (m, 1H), 7.26 – 7.19 (m, 4H), 7.15 – 7.09 (m, 2H),
4.34 (dd, J = 7.7, 6.5 Hz, 1H), 4.01 (s, 2H), 3.31 – 3.20 (m, 1H), 3.13 –
3.00 (m, 3H), 2.99 – 2.82 (m, 4H), 2.78 (q, J = 5.2 Hz, 2H), 1.25 (bs, 1H),
0.92 (t, J = 7.1 Hz, 3H), 0.84 (t, J = 7.1 Hz, 3H); 13C NMR (126 MHz, CDCl3)
δ 169.5, 159.8, 157.9, 139.3, 139.2, 138.8, 138.7, 135.6, 129.5, 128.6,
128.5, 128.5, 127.2, 123.3, 123.3, 111.1, 110.9, 54.1, 42.8, 42.3, 42.3,
41.5, 40.9, 40.6, 29.0, 28.9, 13.7, 12.6; 19F NMR (377 MHz, CDCl3) δ -
117.3; MS (ESI) m/z 434.1 [M+H]+; HRMS (ESI) found m/z 434.19037
[M+H]+, calcd for C22H29FN3O3S+ m/z 434.19082; [α]D
21
= -64.5°
(dichloromethane).
Compound 9 (18 mg, 75% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3) δ 7.43 (bs, 1H), 7.34 (dd, J = 8.7, 1.7 Hz, 1H), 4.05 (s, 2H),
3.67 (s, 2H), 3.43 (t, J = 6.8 Hz, 2H), 3.29 (t, J = 6.8 Hz, 2H), 3.13 (t, J =
5.9 Hz, 2H), 2.85 (t, J = 5.9 Hz, 2H), 2.01 – 1.92 (m, 2H), 1.90 – 1.81 (m,
2H), 1.35 (t, J = 7.3 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 164.7, 160.3,
157.8, 139.1, 139.1, 137.8, 137.8, 129.0, 128.8, 123.5, 123.5, 111.3, 111.0,
46.2, 45.9, 45.4, 44.3, 42.8, 42.5, 42.4, k 29.0, 25.8, 24.1; 19F NMR (377
MHz, CDCl3) δ -117.0; MS (ESI) m/z 342.0 [M+H]+; HRMS (ESI) found m/z
342.12923 [M+H]+, calcd for C15H22N3O3S+ m/z 342.12876.
Compound 15 (38 mg, 95% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3) δ 7.31 (bs, 1H), 7.26 – 7.18 (m, 4H), 7.13 – 7.08 (m, 2H),
4.42 (dd, J = 7.9, 6.8 Hz, 1H), 4.03 (s, 2H), 3.11 (t, J = 5.9 Hz, 2H), 2.91
(d, J = 8.4 Hz, 2H), 2.80 (q, J = 5.7 Hz, 2H), 2.67 (s, 3H), 2.48 (s, 3H), 1.24
(bs, 1H); 13C NMR (101 MHz, CDCl3) δ 170.3, 160.0, 157.6, 139.0, 139.0,
138.8, 138.7, 135.6, 129.3, 128.6, 128.5, 128.4, 127.2, 123.3, 123.3, 111.0,
110.7, 54.0, 42.8, 42.3, 42.3, 40.5, 36.5, 35.5, 28.9, 28.9; 19F NMR (377
MHz, CDCl3) δ -117.3; MS (ESI) m/z 406.1 [M+H]+; HRMS (ESI) found m/z
406.16060 [M+H]+, calcd for C20H25FN3O3S+ m/z 406.16006; [α]D = -
21
Compound 10 (42 mg, 77% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3) δ 7.29 – 7.26 (m, 1H), 7.25 – 7.19 (m, 3H), 7.13 (dd, J = 8.6,
1.8 Hz, 1H), 7.09 – 7.02 (m, 2H), 5.45 (bs, 1H), 4.03 (s, 2H), 3.77 (t, J =
7.0 Hz, 1H), 3.12 (t, J = 5.9 Hz, 2H), 2.95 (qd, J = 13.8, 7.0 Hz, 2H), 2.83
– 2.76 (m, 2H), 2.04 – 1.97 (m, 3H), 1.80 – 1.71 (m, 6H), 1.67 – 1.55 (m,
6H), 1.26 (bs, 1H); 13C NMR (101 MHz, CDCl3) δ 168.5, 160.2, 157.7,
138.9, 138.8, 138.2, 138.1, 135.8, 129.3, 128.8, 128.7, 128.7, 128.5, 127.2,
123.5, 123.5, 111.1, 110.9, 58.6, 52.1, 42.7, 42.3, 42.3, 41.1, 39.4, 36.2,
29.3, 28.8, 28.8; 19F NMR (377 MHz, CDCl3) δ -116.6; MS (ESI) m/z 512.1
91.1° (dichloromethane).
Compound 16 (31 mg, 81% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3) δ 7.34 (bs, 1H), 7.30 – 7.19 (m, 4H), 7.12 – 7.07 (m, 2H),
4.04 (s, 1H), 3.23 (t, J = 6.9 Hz, 1H), 3.11 (t, J = 5.9 Hz, 1H), 2.86 – 2.75
(m, 4H), 2.06 (bs, 1H), 1.25 (bs, 1H); 13C NMR (101 MHz, CDCl3) δ 160.3,
157.8, 138.9, 138.9, 138.7, 138.6, 137.6, 128.8, 128.8, 128.7, 128.7, 128.5,
126.9, 123.3, 123.3, 111.1, 110.8, 44.2, 42.7, 42.3, 42.3, 35.8, 28.9, 28.9;
19F NMR (377 MHz, CDCl3) δ -116.9. MS (ESI) m/z 335.1 [M+H]+; HRMS
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800242
ChemMedChem
FULL PAPER
(ESI) found m/z 335.12305 [M+H]+, calcd for C17H20FN2O2S+ m/z
335.12295.
Compound 23 (56 mg, 99% isolated yield, off-white solid): 1H NMR (400
MHz, CDCl3) δ 7.56 – 7.48 (m, 2H), 7.26 – 7.16 (m, 3H), 7.15 – 7.04 (m,
3H), 4.13 (dd, J = 8.9, 6.2 Hz, 1H), 4.07 (s, 2H), 3.21 – 3.06 (m, 3H), 3.06
– 2.71 (m, 7H), 2.40 – 2.26 (m, 1H), 1.64 – 1.48 (m, 3H), 1.50 – 1.36 (m,
1H), 1.23 (bs, 1H); 13C NMR (101 MHz, CDCl3) δ 168.7, 140.1, 137.8,
135.8, 135.6, 129.4, 128.4, 128.0, 127.1, 126.8, 124.5, 56.0, 47.8, 45.9,
45.6, 43.2, 40.5, 28.8, 25.7, 23.8; MS (ESI) m/z 414.1 [M+H]+; HRMS (ESI)
found m/z 414.18487 [M+H]+, calcd for C22H28N3O3S+ m/z 414.18514.
[α]D21 = -96.9° (dichloromethane).
Compound 17 (88 mg, 88%, white solid): 1H NMR (500 MHz, CDCl3) δ
7.84 – 7.80 (m, 2H), 7.56 – 7.51 (m, 1H), 7.49 – 7.43 (m, 2H), 7.27 – 7.19
(m, 3H), 7.17 – 7.12 (m, 2H), 5.95 – 5.90 (d, J = 9.5 Hz, 1H), 4.17 – 4.05
(td, J = 9.5, 5.9 Hz, 1H), 3.14 – 3.06 (m, 1H), 3.03 – 2.89 (m, 3H), 2.87 –
2.80 (m, 1H), 2.32 – 2.24 (m, 1H), 1.57 – 1.36 (m, 4H); 13C NMR (126 MHz,
CDCl3) δ 168.4, 139.9, 135.7, 132.6, 129.4, 128.8, 128.4, 127.2, 127.2,
56.0, 45.9, 45.5, 40.7, 25.6, 23.7; MS (ESI) m/z 359.0 [M+H]+; HRMS
(ESI) found m/z 381.12487 [M+Na]+, calcd for C19H22N2O3SNa+ m/z
381.12433; [α]D21 = -125.8° (dichloromethane).
Compound 24 (153 mg, 86% isolated yield, colorless oil): 1H NMR (400
MHz, CDCl3) δ 7.51 – 7.45 (m, 2H), 7.25 – 7.17 (m, 3H), 7.15 – 7.08 (m,
3H), 5.77 (d, J = 9.6 Hz, 1H), 4.08 (td, J = 9.6, 5.9 Hz, 1H), 3.14 – 3.03 (m,
1H), 3.02 – 2.85 (m, 3H), 2.85 – 2.66 (m, 5H), 2.31 – 2.19 (m, 1H), 1.78 (p,
J = 3.3 Hz, 4H), 1.58 – 1.32 (m, 4H); 13C NMR (126 MHz, CDCl3) δ 168.6,
142.6, 137.9, 136.7, 135.8, 129.4, 129.4, 128.4, 127.8, 127.1, 124.2, 55.9,
45.8, 45.4, 40.7, 29.5, 29.3, 25.6, 23.8, 22.7, 22.7; MS (ESI) m/z 413.1
[M+H]+; HRMS (ESI) found m/z 435.17130 [M+Na]+, calcd for
Compound 18 (94 mg, 94% isolated yield, white solid): 1H NMR (500 MHz,
CDCl3) δ 7.62 (dt, J = 8.0, 1.7, 1.0 Hz, 1H), 7.51 (dt, J = 8.0, 2.5, 1.7 Hz,
1H), 7.45 (td, J = 8.0, 5.2 Hz, 1H), 7.28 – 7.20 (m, 4H), 7.17 – 7.13 (m,
2H), 6.27 (d, J = 9.7 Hz, 1H), 4.20 (ddd, J = 9.7, 8.4, 6.7 Hz, 1H), 3.23 –
3.11 (m, 1H), 3.07 – 2.94 (m, 4H), 2.45 – 2.36 (m, 1H), 1.68 – 1.42 (m,
4H); 13C NMR (126 MHz, CDCl3) δ 168.3, 163.1, 161.1, 142.4, 142.3,
135.6, 130.7, 130.7, 129.4, 128.5, 127.2, 123.0, 122.9, 119.7, 119.6, 114.5,
114.3, 56.2, 46.0, 45.7, 40.5, 25.7, 23.8; 19F NMR (377 MHz, CDCl3) δ -
109.8; MS (ESI) m/z 376.9 [M+H]+; HRMS (ESI) found m/z 399.11732
[M+Na]+, calcd for C19H21FN2O3SNa+ m/z 399.11546; Optical rotation
[α]D21 = -116.4° (dichloromethane).
C
23H28O3N2SNa+ m/z 435.17183. [α]D21 = -92.7° (dichloromethane).
Compound 25 (32 mg, 79% isolated yield, slightly yellow oil): 1H NMR (400
MHz, CDCl3); δ 7.30 – 7.23 (m, 2H), 7.19 – 7.09 (m, 3H), 7.09 – 7.02 (m,
2H), 6.27 (d, J = 8.5 Hz, 1H), 5.49 (d, J = 9.8 Hz, 1H), 3.94 (td, J = 9.8, 5.7
Hz, 1H), 3.25 (dd, J = 6.1, 5.0 Hz, 2H), 3.06 – 2.87 (m, 3H), 2.87 – 2.51
(m, 4H), 2.20 – 2.06 (m, 1H), 1.85 – 1.75 (m, 2H), 1.51 – 1.38 (m, 3H),
1.35 – 1.23 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 168.1, 147.4, 135.0,
128.4, 127.9, 127.3, 126.0, 125.9, 124.1, 119.0, 111.2, 54.8, 44.8, 44.5,
40.5, 39.7, 25.9, 24.6, 22.8, 20.1; MS (ESI) m/z 414.0 [M+H]+; HRMS
(ESI) found m/z 436.16680 [M+Na]+, calcd for C22H27N3O3SNa+ m/z
436.16708; [α]D21 = -134.3° (dichloromethane).
Compound 19 (78 mg, 78% isolated yield, white solid): 1H NMR (500 MHz,
CDCl3) δ 7.83 – 7.77 (m, 2H), 7.27 – 7.18 (m, 3H), 7.15 – 7.06 (m, 4H),
6.15 (d, J = 9.8 Hz, 1H), 4.19 – 4.09 (m, 1H), 3.18 – 3.08 (m, 1H), 3.05 –
2.89 (m, 4H), 2.44 – 2.35 (m, 1H), 1.63 – 1.51 (m, 3H), 1.50 – 1.41 (m,
1H); 13C NMR (126 MHz, CDCl3) δ 168.6, 166.0, 163.9, 136.2, 136.2,
135.6, 130.0, 129.9, 129.4, 128.5, 127.2, 116.0, 115.8, 56.0, 46.0, 45.7,
40.4, 25.6, 23.8; 19F NMR (377 MHz, CDCl3) δ -105.4; MS (ESI) m/z 377.1
[M+H]+; HRMS (ESI) found m/z 399.11607 [M+Na]+, calcd for
C19H21FN2O3SNa+ m/z 399.11546; [α]D21 = -96.7° (dichloromethane).
Compound 26 (62 mg, 95% isolated yield, off-white solid): 1H NMR (500
MHz, CDCl3) δ 7.53 (dd, J = 8.0, 2.0 Hz, 1H), 7.46 (bs, 1H), 7.26 – 7.18
(m, 3H), 7.17 – 7.09 (m, 3H), 4.12 (dd, J = 9.2, 6.0 Hz, 1H), 4.02 (q, J =
16.2 Hz, 2H), 3.20 – 3.08 (m, 3H), 3.06 – 2.88 (m, 4H), 2.83 (t, J = 6.0,
2H), 2.38 – 2.29 (m, 1H), 1.64 – 1.35 (m, 4H); 13C NMR (126 MHz, CDCl3)
δ 168.6, 140.2, 137.3, 136.5, 135.7, 129.7, 129.4, 128.4, 127.1, 125.2,
124.7, 56.0, 47.9, 45.9, 45.5, 43.2, 40.6, 29.2, 25.6, 23.8;
MS (ESI) m/z 414.1 [M+H]+; HRMS (ESI) found m/z 414.18545 [M+H]+,
calcd for C22H28N3O3S+ m/z 414.18514; [α]D21 = -107.9° (dichloromethane).
Compound 20 (88 mg, 99% isolated yield, white solid): 1H NMR (500 MHz,
CDCl3) δ 7.32 (d, J = 2.3 Hz, 1H), 7.30 – 7.19 (m, 4H), 7.17 – 7.11 (m, 2H),
6.89 (d, J = 8.5 Hz, 1H), 5.81 (d, J = 9.8 Hz, 1H), 4.33 – 4.21 (m, 4H), 4.13
(td, J = 5.9, 9.55 Hz, 1H), 3.18 – 3.03 (m, 2H), 3.01 – 2.89 (m, 3H), 2.38 –
2.29 (m, 1H), 1.67 – 1.53 (m, 3H), 1.50 – 1.39 (m, 1H); 13C NMR (126 MHz,
CDCl3) δ 168.6, 147.2, 143.2, 135.8, 132.5, 129.4, 128.4, 127.1, 120.9,
117.5, 116.8, 64.5, 64.1, 56.0, 45.9, 45.6, 40.6, 25.7, 23.8;
Compound 27 (35 mg, 87% isolated yield, slightly yellow solid): 1H NMR
(400 MHz, CDCl3) δ 7.56 (dd, J = 8.0, 1.8 Hz, 1H), 7.51 – 7.46 (m, 1H),
7.42 (dd, J = 9.4, 1.8 Hz, 1H), 7.26 – 7.17 (m, 3H), 7.16 – 7.08 (m, 2H),
4.19 (dd, J = 8.5, 6.6 Hz, 1H), 3.94 (s, 2H), 3.14 (dd, J = 13.0, 6.1 Hz, 1H),
3.10 – 2.99 (m, 2H), 3.00 – 2.84 (m, 2H), 2.50 – 2.36 (m, 1H), 1.71 – 1.54
(m, 3H), 1.54 – 1.41 (m, 1H); 13C NMR (126 MHz, CDCl3) δ 168.4, 161.0,
159.0, 140.5, 140.5, 135.6, 135.2, 135.1, 129.5, 129.5, 129.4, 128.5, 127.2,
123.1, 123.0, 114.3, 114.1, 56.2, 46.1, 45.7, 40.5, 40.0, 39.9, 25.7, 23.8;
19F NMR (377 MHz, CDCl3) δ -116.5; MS (ESI) m/z 406.1 [M+H]+; HRMS
(ESI) found m/z 406.16057 [M+H]+, calcd for C20H25FN3O3S+ m/z
406.16006; [α]D21 = -82.2° (dichloromethane).
MS (ESI) m/z 417.0 [M+H]+; HRMS (ESI) found m/z 439.13019 [M+Na]+,
21
calcd for C21H24N2O5SNa+ m/z 439.13036. [α]D
=
-106.4°
(dichloromethane).
Compound 21 (87 mg, 98% isolated yield, white solid): 1H NMR (500 MHz,
CDCl3) δ 9.20 (s, 1H), 8.44 (dd, J = 0.6, 1.90 Hz, 1H), 8.18 (dd, J = 0.6,
8.6 Hz, 1H), 7.92 (dd, J = 1.9, 8.6 Hz, 1H), 7.23 – 7.14 (m, 3H), 7.14 – 7.09
(m, 2H), 6.21 (bs, 1H), 4.19 (dd, J = 6.4, 8.7 Hz, 1H), 3.10 – 2.93 (m, 3H),
2.93 – 2.77 (m, 2H), 2.42 – 2.33 (m, 1H), 1.51 – 1.36 (m, 3H), 1.35 – 1.24
(m, 1H); 13C NMR (126 MHz, CDCl3) δ 168.4, 157.9, 155.4, 137.4, 135.6,
133.8, 129.4, 128.4, 127.2, 125.0, 123.9, 122.0, 56.2, 45.9, 45.6, 40.5,
25.6, 23.7; MS (ESI) m/z 416.1 [M+H]+; HRMS (ESI) found m/z 438.09165
Compound 28 (69 mg, 75% isolated yield, white solid): 1H NMR (500 MHz,
DMSO-d6) δ 8.39 (s, 1H), 8.14 (s, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.71 (d, J
= 8.5 Hz, 2H), 7.58 (s, 1H), 7.28 – 7.09 (m, 4H), 4.10 (t, J = 7.6 Hz, 1H),
3.24 – 3.15 (m, 1H), 2.92 (dt, J = 6.9, 11.5 Hz, 1H), 2.88 – 2.66 (m, 4H),
1.71 – 1.60 (m, 1H), 1.52 - 1.45 (m, 3H); 13C NMR (126 MHz, DMSO-d6)
δ 168.0, 167.1, 143.5, 137.8, 137.0, 129.7, 128.6, 128.3, 127.1, 126.7,
56.1, 46.0, 45.6, 38.6, 25.2, 23.8; MS (ESI) m/z 402.0 [M+H]+; HRMS
(ESI) found m/z 424.13147 [M+Na]+, calcd for C20H23N3O4SNa+ m/z
424.13070; [α]D21 = -91.7° (dichloromethane).
21
[M+Na]+, calcd for C20H21N3O3S2Na+ m/z 438.09220. [α]D = -107.9°
(dichloromethane).
Compound 22 (89 mg, 97% isolated yield, white solid): 1H NMR (500 MHz,
CDCl3) δ 7.62 – 7.55 (m, 2H), 7.25 – 7.17 (m, 3H), 7.15 – 7.10 (m, 2H),
6.74 (d, J = 8.3 Hz, 1H), 5.91 (d, J = 9.9 Hz, 1H), 4.63 (t, J = 8.8 Hz, 2H),
4.16 – 4.06 (m, 1H), 3.29 – 3.09 (m, 3H), 3.05 – 2.88 (m, 4H), 2.38 – 2.30
(m, 1H), 1.60 – 1.50 (m, 3H), 1.47 – 1.40 (m, 1H); 13C NMR (126 MHz,
CDCl3) δ 168.8, 163.7, 135.8, 131.5, 129.4, 128.7, 128.4, 128.2, 127.1,
124.6, 108.8, 72.3, 55.9, 45.9, 45.6, 40.5, 29.0, 25.6, 23.8;
Compound 29 (20 mg, 99% isolated yield, TFA salt, white solid): 1H NMR
(500 MHz, CD3OD) δ 7.67 (d, J = 1.5 Hz, 1H), 7.57 (dd, J = 9.2, 1.6 Hz,
1H), 4.47 (s, 2H), 3.57 (t, J = 6.3 Hz, 2H), 3.23 (t, J = 6.3 Hz, 2H); 13C NMR
(126 MHz, CD3OD) δ 160.0, 158.0, 145.1, 145.0, 135.4, 135.3, 122.0,
122.0, 120.3, 120.1, 110.9, 110.7, 40.7, 39.3, 39.2, 24.5, 24.5; 19F NMR
MS (ESI) m/z 401.0 [M+H]+; HRMS (ESI) found m/z 423.13553 [M+Na]+,
21
calcd for C21H24N2O4SNa+ m/z 423.13553. [α]D
=
-124.2°
(dichloromethane).
7
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10.1002/cmdc.201800242
ChemMedChem
FULL PAPER
(377 MHz, MeOD) δ -77.0 (CF3COO-), -118.1; MS (ESI) m/z 231.1 [M+H]+;
HRMS (ESI) found m/z 231.06035 [M+H]+, calcd for C9H12FN2O2S+ m/z
231.05922.
[19] a) F. Paneni, S. Costantino, R. Battista, L. Castello, G. Capretti,
S. Chiandotto, G. Scavone, A. Villano, D. Pitocco, G. Lanza, M.
Volpe, T. F. Lüscher, F. Cosentino, Circ. Cardiovasc. Genet. 2015,
8, 150; b) H. U. Kaniskan, M. L. Martini, J. Jin, Chem. Rev. 2017;
c) H. Ü. Kaniskan, K. D. Konze, J. Jin, J. Med. Chem. 2015, 58,
1596-1629; d) H. Ü. Kaniskan, J. Jin, Curr. Opin. Chem. Biol.
2017, 39, 100-108.
[20] Y. Takemoto, A. Ito, H. Niwa, M. Okamura, T. Fujiwara, T. Hirano,
N. Handa, T. Umehara, T. Sonoda, K. Ogawa, M. Tariq, N.
Nishino, S. Dan, H. Kagechika, T. Yamori, S. Yokoyama, M.
Yoshida, J. Med. Chem. 2016, 59, 3650-3660.
Compound 30 (943 mg, 80% isolated yield, yellow solid): 1H NMR (400
MHz, CDCl3) δ 7.32 – 7.26 (m, 2H), 7.25 – 7.18 (m, 3H), 3.71 (t, J = 7.2
Hz, 1H), 3.52 – 3.43 (m, 1H), 3.42 – 3.29 (m, 2H), 2.95 (dd, J = 13.1, 7.2
Hz, 1H), 2.84 – 2.75 (m, 2H), 1.86 – 1.60 (m, 6H). 13C NMR (126 MHz,
CDCl3) δ 173.1, 137.9, 129.3, 128.4, 126.7, 55.2, 46.0, 45.8, 43.0, 25.9,
24.0. MS (ESI) m/z 219.0 [M+H]+; HRMS (ESI) found m/z 241.13149
[M+Na]+, calcd for C13H18N2ONa+ m/z 241.13168.
[21] T. Fujiwara, K. Ohira, K. Urushibara, A. Ito, M. Yoshida, M. Kanai,
A. Tanatani, H. Kagechika, T. Hirano, Bioorg. Med. Chem. 2016,
24, 4318-4323.
[22] H. Niwa, N. Handa, Y. Tomabechi, K. Honda, M. Toyama, N.
Ohsawa, M. Shirouzu, H. Kagechika, T. Hirano, T. Umehara, S.
Yokoyama, Acta Crystallogr. D Biol. Crystallogr. 2013, 69, 595-
602.
Acknowledgements
We thank the Netherlands Organization for Scientific Research
(NWO, NCI-TA grant 731.015.202) for funding.
[23] F. Meng, S. Cheng, H. Ding, S. Liu, Y. Liu, K. Zhu, S. Chen, J. Lu,
Y. Xie, L. Li, R. Liu, Z. Shi, Y. Zhou, Y. C. Liu, M. Zheng, H. Jiang,
W. Lu, H. Liu, C. Luo, J. Med. Chem. 2015, 58, 8166-8181.
[24] D. Barsyte-Lovejoy, F. Li, M. J. Oudhoff, J. H. Tatlock, A. Dong,
H. Zeng, H. Wu, S. A. Freeman, M. Schapira, G. A. Senisterra, E.
Kuznetsova, R. Marcellus, A. Allali-Hassani, S. Kennedy, J. P.
Lambert, A. L. Couzens, A. Aman, A. C. Gingras, R. Al-Awar, P.
V. Fish, B. S. Gerstenberger, L. Roberts, C. L. Benn, R. L. Grimley,
M. J. Braam, F. M. Rossi, M. Sudol, P. J. Brown, M. E. Bunnage,
D. R. Owen, C. Zaph, M. Vedadi, C. H. Arrowsmith, Proc. Natl.
Acad. Sci. USA 2014, 111, 12853-12858.
[25] a) D. C. Lenstra, A. H. K. Al Temimi, J. Mecinović, Bioorg. Med.
Chem. Lett. 2018, 28, 1234-1238; b) K. Guitot, T. Drujon, F.
Burlina, S. Sagan, S. Beaupierre, O. Pamlard, R. H. Dodd, C.
Guillou, G. Bolbach, E. Sachon, D. Guianvarc'h, Anal. Bioanal.
Chem. 2017, 409, 3767-3777.
Keywords: Histone lysine methyltransferase • (R)-PFI-2 •
SETD7 • Epigenetics • Structure-Activity Relationship
Conflict of interest
The authors declare no conflict of interest.
References
[1] A. J. Bannister, T. Kouzarides, Cell Res. 2011, 21, 381-395.
[2] K. A. Gelato, W. Fischle, Biol. Chem. 2008, 389, 353-363.
[3] E. L. Greer, Y. Shi, Nat. Rev. Genet. 2012, 13, 343-357.
[4] C. Martin, Y. Zhang, Nat. Rev. Mol. Cell Biol. 2005, 6, 838-849.
[5] K. Hyun, J. Jeon, K. Park, J. Kim, Exp. Mol. Med. 2017, 49, e324.
[6] M. Wong, P. Polly, T. Liu, Am. J. Cancer. Res. 2015, 5, 2823-2837.
[7] K. Nishioka, S. Chuikov, K. Sarma, H. Erdjument-Bromage, C. D.
Allis, P. Tempst, D. Reinberg, Genes Dev. 2002, 16, 479-489.
[8] K. Subramanian, D. Jia, P. Kapoor-Vazirani, D. R. Powell, R. E.
Collins, D. Sharma, J. Peng, X. Cheng, P. M. Vertino, Mol. Cell
2008, 30, 336-347.
[26] Chemical Computing Group Molecular Operating Environment
(MOE), https://www.chemcomp.com/.
[27]
Qiagen
https://www.qiagenbioinformatics.com/.
[28] Schrödinger PyMOL, https://pymol.org/.
Molegro
Virtual
Docker
v6.0,
[9] P. O. Esteve, H. G. Chin, J. Benner, G. R. Feehery, M.
Samaranayake, G. A. Horwitz, S. E. Jacobsen, S. Pradhan, Proc.
Natl. Acad. Sci. USA 2009, 106, 5076-5081.
[10] A. Kouskouti, E. Scheer, A. Staub, L. Tora, I. Talianidis, Mol. Cell
2004, 14, 175-182.
[11] D. R. Calnan, A. E. Webb, J. L. White, T. R. Stowe, T. Goswami,
X. Shi, A. Espejo, M. T. Bedford, O. Gozani, S. P. Gygi, A. Brunet,
Aging 2012, 4, 462-479.
[12] W. J. Zhang, X. N. Wu, T. T. Shi, H. T. Xu, J. Yi, H. F. Shen, M.
F. Huang, X. Y. Shu, F. F. Wang, B. L. Peng, R. Q. Xiao, W. W.
Gao, J. C. Ding, W. Liu, Sci. Rep. 2016, 6, 21718.
[13] L. Gaughan, J. Stockley, N. Wang, S. R. McCracken, A.
Treumann, K. Armstrong, F. Shaheen, K. Watt, I. J. McEwan, C.
Wang, R. G. Pestell, C. N. Robson, Nucleic Acids Res. 2011, 39,
1266-1279.
[14] I. Kassner, A. Andersson, M. Fey, M. Tomas, E. Ferrando-May,
M. O. Hottiger, Open Biol. 2013, 3, 120173.
[15] C. Shen, D. Wang, X. Liu, B. Gu, Y. Du, F. Z. Wei, L. L. Cao, B.
Song, X. Lu, Q. Yang, Q. Zhu, T. Hou, M. Li, L. Wang, H. Wang,
Y. Zhao, Y. Yang, W. G. Zhu, FASEB J. 2015, 29, 4313-4323.
[16] J. K. Kurash, H. Lei, Q. Shen, W. L. Marston, B. W. Granda, H.
Fan, D. Wall, E. Li, F. Gaudet, Mol. Cell 2008, 29, 392-400.
[17] a) J. Zhou, M. Xie, Y. Shi, B. H. Luo, G. H. Gong, J. N. Li, J. P.
Wang, W. J. Zhao, Y. Zi, X. Y. Wu, J. F. Wen, Oncol. Rep. 2015,
34, 111-120; b) Y. Zhang, J. Liu, J. Lin, L. Zhou, Y. Song, B. Wei,
X. Luo, Z. Chen, Y. Chen, J. Xiong, X. Xu, L. Ding, Q. Ye,
Oncotarget 2016, 7, 9859-9875.
[18] F. Paneni, S. Costantino, R. Battista, L. Castello, G. Capretti, S.
Chiandotto, G. Scavone, A. Villano, D. Pitocco, G. Lanza, M.
Volpe, T. F. Luscher, F. Cosentino, Circ. Cardiovasc. Genet. 2015,
8, 150-158.
8
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Entry for the Table of Contents
This work describes structure-activity relationship studies on (R)-PFI-2, the most potent inhibitor of biomedically important histone
lysine methyltransferase SETD7. Synthesis and evaluation of 29 structural analogs of (R)-PFI-2 revealed key interactions that
essentially contribute to excellent potency.
9
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