Synthesis and evaluation of protein arginine N-methyltransferase inhibitors designed to simultaneously occupy both substrate binding sites

Article abstract of DOI:10.1039/c4ob01734j

The protein arginine N-methyltransferases (PRMTs) are a family of enzymes that function by specifically transferring a methyl group from the cofactor S-adenosyl-l-methionine (AdoMet) to the guanidine group of arginine residues in target proteins. The most notable is the PRMT-mediated methylation of arginine residues that are present in histone proteins which can lead to chromatin remodelling and influence gene transcription. A growing body of evidence now implicates dysregulated PRMT activity in a number of diseases including various forms of cancer. The development of PRMT inhibitors may therefore hold potential as a means of developing new therapeutics. We here report the synthesis and evaluation of a series of small molecule PRMT inhibitors designed to simultaneously occupy the binding sites of both the guanidino substrate and AdoMet cofactor. Potent inhibition and surprising selectivity were observed when testing these compounds against a panel of methyltransferases.

Full text of DOI:10.1039/c4ob01734j

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Synthesis and evaluation of protein arginine
N-methyltransferase inhibitors designed to
simultaneously occupy both substrate
binding sites†
Cite this: DOI: 10.1039/c4ob01734j
Matthijs van Haren, Linda Quarles van Ufford, Ed E. Moret and Nathaniel I. Martin*
The protein arginine N-methyltransferases (PRMTs) are a family of enzymes that function by specifically
transferring a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to the guanidine group
of arginine residues in target proteins. The most notable is the PRMT-mediated methylation of arginine
residues that are present in histone proteins which can lead to chromatin remodelling and influence gene
transcription. A growing body of evidence now implicates dysregulated PRMT activity in a number of dis-
eases including various forms of cancer. The development of PRMT inhibitors may therefore hold poten-
tial as a means of developing new therapeutics. We here report the synthesis and evaluation of a series of
small molecule PRMT inhibitors designed to simultaneously occupy the binding sites of both the guani-
dino substrate and AdoMet cofactor. Potent inhibition and surprising selectivity were observed when
testing these compounds against a panel of methyltransferases.
Received 13th August 2014,
Accepted 23rd October 2014
DOI: 10.1039/c4ob01734j
www.rsc.org/obc
Introduction
The methylation of arginine side chains is an abundant post-
translational modification encountered across a range of
organisms from bacteria and fungi to plants and mammals.
Approximately 2% of the arginine residues in total protein
extracts from rat liver nuclei are methylated1,2 and similar
levels of arginine methylation appear to be present in the
human proteome.3,4 Arginine methylation is performed by
a dedicated family of enzymes known as the protein arginine
N-methyltransferases (PRMTs). PRMTs employ a bisubstrate
mechanism using the methyl-group donor S-adenosyl meth-
ionine (AdoMet) to methylate substrate proteins/peptides with
the concomitant formation of the by-product S-adenosyl
homocysteine (AdoHcy) (Fig. 1). The first methylation step
catalyzed by PRMTs yields monomethyl-arginine (MMA), an
intermediate that is typically further methylated to form asym-
metric dimethyl-arginine (aDMA) or symmetric dimethyl-
arginine (sDMA).5
Fig. 1 The PRMT catalyzed methylation of arginine residues to generate
monomethyl arginine (MMA), asymmetric dimethyl arginine (aDMA), and
symmetric dimethyl arginine (sDMA).
PRMT-mediated arginine methylation is intimately involved
in a range of cellular functions, including transcriptional regu-
lation, RNA processing, signal transduction and DNA repair.5,6
While dysregulated arginine methylation is implicated in a
number of pathogenic conditions including cardiovascular
disease and inflammation, it is in the areas of epigenetics and
Medicinal Chemistry & Chemical Biology Group, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
E-mail: n.i.martin@uu.nl
†Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
for all new compounds and analytical RP-HPLC traces for the final compounds
1–6, and dose–response curves used for IC50 determination for 1–6. See
DOI: 10.1039/c4ob01734j
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the inhibition of PRMT activity may be a viable approach to
developing novel anticancer therapeutics.7,15
Among the eleven known human PRMTs, the vast majority
of arginine methylation (>85%) is attributable to PRMT1
which exhibits a wide substrate specificity, preferentially
methylating arginine residues flanked by one or more gly-
cines.5,16 By comparison, the other PRMTs have more narrowly
defined substrate tolerances.5,16 While the PRMTs vary signifi-
cantly in size and sequence, a conserved core of amino acids
comprising the catalytic site is strictly maintained.5,16 The
published crystal structures of PRMT1, 3, 4, and 5 exhibit
virtually identical folds and also point to a specific set of active
site residues as being key for catalysis (Fig. 2).17–21 Of particu-
lar note is the “double-E loop” motif common to all PRMTs,
containing the Glu144 and Glu153 residues (PRMT1 number-
ing), known to be critical for substrate recognition.22 As
illustrated in Fig. 2, the PRMTs operate via a bisubstrate mech-
anism wherein the guanidine group of an arginine-containing
peptide substrate is precisely orientated (via a hydrogen-bond
network with Glu144 and Glu153) in proximity to the electro-
philic methylsulfonium group of AdoMet leading to an
“SN2-like” substitution reaction.
Fig. 2 Proposed PRMT “SN2-like” bisubstrate mechanism with key
active site residues shown (PRMT1 numbering scheme). AdoMet is pre-
sented in blue and the arginine residue of a target peptide is presented
in red.
With these conserved active site features in mind we set out
to design a series of novel inhibitors aimed at specifically
exploiting both the adenosine binding domain and “double-E
loop” unique to the PRMTs (compounds 1–6, Fig. 3A). Com-
pounds 1–6 all contain the adenosine group of the AdoMet
cofactor which is further connected to a guanidine moiety via
a variable linker. This design strategy was envisioned to impart
selective binding towards PRMTs relative to other AdoMet-
dependent methyltransferases. Specifically it was expected
that important interactions with key residues conserved in
the PRMT active site, such as Glu144 and Glu153, would be
maintained (Fig. 2). Of note in the structures of the proposed
inhibitors is the omission of the amino acid moiety that is
present in the AdoMet cofactor. This omission was made
based upon the findings of Thompson and coworkers who
oncology that the PRMTs have received most attention. The
aberrant modification of histones is now an established contri-
buting factor in cancer development and there is mounting
evidence that suggests PRMT activity is important in cancer
progression.7–9 Many PRMTs are overexpressed in breast
cancer10 and PRMT4 (also known as CARM1 for coactivator-
associated arginine methyltransferase 1) is associated with
prostate and colorectal cancers.11–13 In addition, PRMT5 levels
are enhanced in various human lymphoid cancer cells, includ-
ing transformed chronic lymphocytic leukemia cell lines.14 Of
particular note are recent studies in which genetic knock-
downs of either PRMT1 or PRMT6 were shown to suppress the
growth of several cancer cell lines.9 These results suggest that
Fig. 3 (A) Bisubstrate PRMT inhibitors 1–6 prepared in the present study and (B) previously described methyltransferase inhibitors containing
the adenosine moiety.
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showed that mutation of the active site Arg54 residue that inter-
acts with the AdoMet carboxyl group has very little impact on
PRMT catalysis.22 Our group has previously reported the prepa-
ration of various peptide-based PRMT inhibitors bearing
modified arginine residues in which the guanidine moiety is
substituted in such a way as to convert a PRMT substrate
peptide into an inhibitor.23–26 In the present study we shift our
focus to the preparation of small molecule inhibitors designed
to simultaneously occupy the binding sites of both the guani-
dine unit of the peptide substrate and the adenosine moiety of
the AdoMet cofactor.
Compounds that contain the adenosine moiety have also
been previously developed as effective inhibitors against other
methyltransferases (Fig. 3B). Most notable are the recently
disclosed inhibitors developed by Epizyme against DOT1L,
a lysine methyltransferase implicated in mixed-lineage leuke-
mia. One of Epizyme’s early lead compounds, designated as
Scheme 1 Synthesis of inhibitor 1. Reagents and conditions: (a)
acetone cyanohydrin, PPh3, DEAD, THF, 90%; (b) PtO2, AcOH, H2,
quant.; (c) N,N’-di-Boc-N’’-triflylguanidine, NEt3, CH2Cl2, 75%; (d) TFA–
CH2Cl2 (1 : 1) followed by TFA–CH2Cl2–H2O (1 : 1 : 0.3), 89%.
EPZ004777, is an extremely potent (IC50 = 0.4 nM) and selec- Mitsunobu reaction employing PPh3, DEAD, and acetone
tive DOT1L inhibitor capable of killing leukemia cells in vitro cyanohydrin to yield the known nitrile 8.28 Subsequent
and shows significant extension of survival in a mouse leuke- reduction to the amine followed by treatment with N,N′-bis
mia model.27 In another example, the Diederich group suc- (tert-butoxycarbonyl)-N″-triflylguanidine (Goodman’s reagent)
cessfully developed potent bisubstrate inhibitors of catechol- provided the protected guanidine species 10. Global deprotec-
O-methyltransferase (COMT), an enzyme that has been pro- tion was achieved by treating 10 with 50% TFA in CH2Cl2 to
posed as a target in the development of anti-Parkinson’s remove the Boc groups after which a small volume of water
disease treatments.28 In addition, within the PRMT field itself, was added to the reaction mixture leading to the deprotection
the groups of Dowden and Ward recently described a series of of the isopropylidene group to yield compound 1. This two-
bisubstrate inhibitors with IC50 values in the low micromolar step procedure was found to be necessary to ensure a clean
range.29,30 While somewhat similar to the PRMT inhibitors we and complete deprotection, while avoiding degradation of the
here describe, the compounds prepared by Dowden and Ward desired product.
differ notably in the length of the spacer connecting the
The synthesis of inhibitors 2 and 3 (Scheme 2) began with a
adenosine and guanidine units. When considering the PRMT one pot IBX oxidation of 7 to yield the intermediate aldehyde
transition state model illustrated in Fig. 2, a spacer consisting which was directly converted to alkene 11 via a Wittig reaction
of three atoms appears to most closely approximate the posi- with triphenylcarbethoxymethylenephosphorane. Reduction of
tioning of the adenosine and guanidine groups relative to each 11 with DIBAL yielded an alcohol which was transformed into
other. In the compound series that Dowden and Ward evalu- intermediate 13 by a Mitsunobu reaction employing PPh3,
ated, the spacer length used to link the adenosine and guani- DEAD, and phthalimide.28 Removal of the phthalimide group
dine units ranged from five to seven atoms. In contrast, when with methylamine provided amino compound 14 which was in
designing inhibitors 1–6 we were specifically interested in turn converted into protected guanidine 15 by treatment with
examining the impact of shorter spacers consisting of two to Goodman’s reagent. Hydrogenation of 15 followed by deprotec-
four atoms with the aim of more accurately mimicking the tion using the procedure described above for 1 yielded com-
PRMT transition state geometry. As shown in Fig. 3A inhibitors pound 2 while compound 3 was obtained by the direct
2 and 3 both contain three-carbon spacers while the spacer in deprotection of 15.
compound 1 is one atom shorter. Also prepared were inhibi-
The preparation of thioether-linked inhibitor 4 (Scheme 3)
tors 4–6 which contain four atom spacers each including a began with the conversion of 7 to thioacetate intermediate 16
different heteroatom. The activities of all 6 inhibitors were via the Mitsunobu reaction with PPh3, DIAD, and thioacetic
then evaluated against a panel of three PRMTs including acid. A one-pot deacetylation and alkylation procedure was
PMRT1, 4 and 6 as well as G9a, a well-characterized lysine then applied to convert 16 into the phthalimide 17. Phtha-
methyltransferase.
limide removal using methylamine followed by treatment with
Goodman’s reagent yielded protected guanidine 19. Deprotec-
tion as described above yielded inhibitor 4. The synthesis of
oxyether-linked inhibitor 5 (Scheme 4) began with benzoyl pro-
tection of the adenosine unit in 7 to give intermediate 20.
Results and discussion
The synthetic routes used in the preparation of PRMT inhibi- Alkylation with chloroethylamine then provided direct access
tors 1–6 all started from the commercially available 2′,3′-O-iso- to amine 21 after which treatment with Goodman’s reagent
propylideneadenosine building block 7. The synthesis of 1 generated the protected guanidine 22. Removal of the Boc and
(Scheme 1) began with the one carbon homologation of 7 via a isopropylidene groups as described above followed by
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Scheme 3 Synthesis of inhibitor 4. Reagents and conditions: (a) thioa-
cetic acid, PPh3, DIAD, THF, 85%; (b) 2-bromoethyl-phthalimide,
NaOCH3, MeOH, 49%; (c) methylamine, EtOH, 89%; (d) N,N’-di-Boc-N’’-
triflylguanidine, NEt3, CH2Cl2, 92%; (e) TFA–CH2Cl2 (1 : 1) followed by
TFA–CH2Cl2–H2O (1 : 1 : 0.3), 74%.
Scheme 2 Synthesis of inhibitors 2 and 3. Reagents and conditions:
(a) IBX, Ph3PvCHCO2Et, DMSO, 79%; (b) DIBAL-H, hexane, CH2Cl2, 78%;
(c) phthalimide, PPh3, DEAD, THF, 83%; (d) MeNH2, EtOH, 94%; (e) N,N’-
di-Boc-N’’-triflylguanidine, NEt3, CH2Cl2, 92%; (f) 10% Pd/C, H2, EtOH,
47%; (g) TFA–CH2Cl2 (1 : 1) followed by TFA–CH2Cl2–H2O (1 : 1 : 0.3),
61% for 2 and 70% for 3.
treatment with ammonium hydroxide to remove the benzoyl
group yielded inhibitor 5.
The preparation of inhibitor 6 bearing an amine spacer
started with chlorination of 7 followed by conversion to azide
27 (Scheme 5). After hydrogenation to generate amine 28,
reductive amination with the known aldehyde 25 (prepared Scheme 4 Synthesis of inhibitor 5. Reagents and conditions:
(a) TMS-Cl, BzCl, pyridine, 77%; (b) chloroethylamine, NaH, DMF, 32%;
(c) N,N’-di-Boc-N’’-triflylguanidine, NEt3, CH2Cl2, 87%; (d) TFA–CH2Cl2
(1 : 1) followed by TFA–CH2Cl2–H2O (1 : 1 : 0.3); (e) NH4OH, MeOH, 79%
over 2 steps.
from ethanolamine) yielded the Cbz protected intermediate
29. Following Boc protection and Cbz group removal, treat-
ment with Goodman’s reagent yielded protected guanidine 31
after which global deprotection led to compound 6. For
compounds 1–6 final purifications were performed using pre-
Compounds 1–6 were evaluated as inhibitors of PRMT1, 4,
and 6 as these three PRMTs are responsible for the majority of
arginine methylation in vivo and also display good in vitro
parative RP-HPLC followed by concentration of pure fractions
and lyophilisation to yield each compound as an amorphous
solid.
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Scheme 5 Synthesis of inhibitor 6. Reagents and conditions: (a) Cbz-Cl, NEt3, CH2Cl2, 63%; (b) sulfur trioxide-pyridine complex, DIPEA, DMSO,
CH2Cl2, 38%; (c) thionyl chloride, HMPA, 77%; (d) NaN3, DMF, 90%; (e) 10% Pd/C, H2, MeOH, quant.; (f) 25, Na(OAc)3BH, DCE, 44%; (g) Boc2O, NEt3,
CH2Cl2, 50%; (h) 10% Pd/C, H2, MeOH; (i) N,N’-di-Boc-N’’-triflylguanidine, NEt3, CH2Cl2, 97% over 2 steps; ( j) TFA–CH2Cl2 (1 : 1) followed by
TFA–CH2Cl2–H2O (1 : 1 : 0.3), 74%.
activity. In addition, the lysine methyltransferase G9a was also expected to most closely approximate the spacing between the
included to assess the inhibitor selectivity against other two substrates in the PRMT transition state. In addition, owing
methyltransferase families. Enzyme activity was measured to the presence of the double bond in its alkyl spacer, com-
using an established chemiluminescence-based assay wherein pound 3 is more rigid and as such could be expected to be a
a substrate peptide derived from either the histone H4 or H3 more potent PRMT inhibitor than compound 2. The results of
tail is treated with AdoMet and the methyltransferase of inter- the inhibition studies however revealed compound 3 to be a
est. After incubation, a primary antibody is then added to slightly weaker inhibitor of both PRMT1 and 6 compared to
specifically bind the methylated arginine or lysine residue fol- compound 2 containing a fully saturated spacer. In general,
lowed by washing and treatment with a horseradish peroxidase compound 2 was shown to be a more active, albeit less selec-
(HRP) conjugated secondary antibody. In the final step, an tive, inhibitor of the three PRMTs tested. In contrast, com-
HRP substrate is added to generate a chemiluminescent signal pounds
1 and 3 were particularly potent and selective
that is measured using a standard microplate reader. Inclusion inhibitors of PRMT4 with IC50 values of 120 nM and 150 nM
of potential inhibitors in the first step of this process in turn respectively. The near 100-fold selectivity of both 1 and 3 for
allows for the convenient detection and quantitation of PRMT4 vs. PRMT1 is of particular note in light of previously
enzyme inhibition. Compounds 1–6 were initially screened reported findings with structurally similar PRMT inhibitors.
against each enzyme at a threshold concentration of 50 μM. Specifically, Dowden and Ward found that compounds con-
The known methyltransferase inhibitor S-adenosyl-L-homo- taining longer (five- to seven-atom long) spacers between the
cysteine (AdoHcy), a by-product of the AdoMet cofactor result- adenosine and guanidine groups were generally more active
ing from methylation, was also included as a reference against PRMT1 and displayed little or no inhibition of
compound. In cases where no appreciable inhibition was PRMT4.29,30 By comparison, the selectivity exhibited by com-
observed at 50 μM further inhibition studies were not per-
formed. Conversely, in cases where >50% inhibition was
observed at the threshold inhibitor concentration of 50 μM,
complete IC50 curves were generated (Table 1).
Table 1 Inhibition constantsa for compounds 1–6 against PRMT1, 4, 6
and PKMT G9a
As shown in Table 1, compounds 1–3 bearing the shorter
PRMT1
PRMT4
PRMT6
G9a
two- and three-atom spacers between the adenosine and guani-
dine groups generally displayed potent inhibition of the
PRMTs tested while having no measurable effect on the activity
of the lysine methyltransferase G9a. In contrast, compounds
4–6 which contain longer four-atom spacers were found for the
most part to be devoid of activity at the threshold concen-
tration tested. These findings would appear to be in line with
the transition state model used to illustrate PRMT catalysis
(Fig. 2). As described above in the design strategy section, com-
pounds 2 and 3, containing the 3-carbon linkers, were
AdoHcy
6.21 0.56
11.09 2.77
1.30 0.38
16.96 3.73
>50
>50
>50
0.67 0.19
0.12 0.02
0.56 0.25
0.15 0.05
>50
>50
>50
0.20 0.25
20.23 8.67
0.72 0.33
5.15 1.27
>50
>50
3.20 3.93
16.64 6.43
>50
>50
>50
3.18 2.67
1
2
3
4
5
6
>50
>50
a IC50 values (μM) based on the best fit of dose–response curves
generated from at least seven unique inhibitor concentrations (each
concentration measured in duplicate). Curve fitting performed with
GraphPad Prism 5.0.
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pounds 1 and 3 in the present study suggests that by incorpor-
ating shorter spacers preferential inhibition of PRMT4 vs.
PRMT1 can be achieved. Also worth mentioning among the
generally inactive compounds 4–6 (bearing longer four-atom
spacers) are two notable and somewhat surprising exceptions.
As shown in Table 1, thioether linked analogue 4 showed good
inhibition of G9a (IC50 3.18 μM) while the amino-linked
species 6 was specific for PRMT6 (IC50 3.20 μM).
The binding modes of compounds 1–3 were next evaluated
via docking studies. Ideally, a comparison of these compounds
docked into the active sites of PRMT1, 4, and 6 might be
expected to provide insight into the selectivity observed in the
inhibition assays. This, however, is not possible given that the
published crystal structure for PRMT1 (from rat, 1OR8.pdb17)
is not suitable for docking studies as has been previously
noted by others.30–32 Specifically, the PRMT1 structure was
obtained at pH 4.7 far from the value of 8.0–8.5 required for
the enzyme activity resulting in a structure with a disordered
N-terminus. In addition, the recently reported structure of
PRMT6 (from the parasite Trypanosoma brucei, 4LWP.pdf33) is
unfortunately not applicable for docking analysis as it also
contains a disordered N-terminus. By comparison, the crystal
structure of human PRMT4 obtained by Bertrand and co-
workers is well suited to docking studies.20 Compounds 1–3
were thus docked into the PRMT4 active site (PDB code
2Y1W20) revealing a number of key interactions. As illustrated
in Fig. 4 the hydrogen bond network around the adenine ring
is well conserved for each compound. Due to its shorter
length, the linker in compound 1 is fully extended so as to
allow the guanidine group to hydrogen bond with Glu258 and
Met260. This extended conformation however leads to loss of a
hydrogen bonding interaction between Ser217 and the fura-
nose ring. Compounds 2 and 3 which contain longer three-
atom linkers are able to hydrogen bond with Glu258 and
Met260 without the loss of the interaction with Ser217. In
addition, both 2 and 3 benefit from H-bonding interactions
between their guanidine units and Met163 and Met269
respectively. It is interesting to note that in each of the
docking analyses performed, hydrogen bonding interactions
between the guanidine moieties of 1–3 and Glu258 were found
but none were detected with Glu267. As described above
(Fig. 2), the “double-E loop” is conserved across all PRMTs and
is critical for PRMT catalysis (in the case of PRMT4 Glu258
and Glu267 correspond to these two residues). The observation
that the guanidine groups in compounds 1–3 do not appear to
simultaneously interact with both of these glutamate side
chains suggests that the compounds may be optimized to
further enhance binding.
Fig. 4 Proposed hydrogen bond network after docking and minimiz-
ation of compounds 1–3 in the PRMT4 active site (aliphatic hydrogens
omitted for clarity). (A) Compound 1; (B) compound 2; (C) compound 3.
The potent PRMT4 inhibition displayed by compounds 1–3
places them amongst the most active PRMT4 inhibitors
reported to date. Only the pyrazole-based compounds reported
by researchers at both MethylGene and Bristol-Myers exhibit
more potent PRMT4 inhibition, however they lack activity in
cell-based assays.34,35 The groups of Bedford and Mai also
recently generated novel PRMT4 inhibitors which, while gener-
ally less active, showed very good PRMT4 selectivity versus
other methyltransferases and in some cases exhibited activity
in cellular models of disease.36 As a preliminary measure of
cellular activity, we evaluated the effects of compounds 1–3 on
cell proliferation using a standard MTT assay with Caco-2
colon cancer cells and MCF-7 breast cancer cells. These investi-
gations revealed that compounds 1–3 have no significant effect
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on the viability of these cell lines at concentrations up to staining; ninhydrin staining. Flash chromatography was per-
100 µM. This may be due to the inability of the highly charged formed using Merck type 60, 230–400 mesh silica gel. The
compounds to enter the cells and suggests that inhibitors final compounds 1–6 were purified by preparative scale
containing less polar analogues of the guanidine moiety RP-HPLC using a Reprosil-Pur C18 column (10 µm, 250 ×
(i.e. amidine or N-hydroxy guanidine) may be more effective.
22 mm) eluted with a water–methanol gradient moving from
0% to 50% MeOH (0.1% TFA) over 60 minutes at a flow-rate of
1.4 mL min−1 with UV detection at 214 nm. Purity was con-
firmed to be ≥95% by analytical RP-HPLC using a Reprosil-Pur
C18 column (5 µm, 250 × 4.6 mm) eluted with a water–metha-
Conclusion
We here describe the design and synthesis of a series of PRMT nol gradient moving from 0% to 50% MeOH (0.1% TFA) over
inhibitors designed to simultaneously occupy both the 40 minutes at a flow rate of 0.7 mL min−1 with UV detection at
AdoMet and peptide substrate binding sites. Given that the 214 nm and 254 nm.
active site architectures of all human PRMTs are highly con-
served, the selectivity observed amongst the inhibitors pre-
Instrumentation for compound characterization
pared is rather surprising. Of particular note is the potent 1H NMR spectra were recorded at 300 MHz or 400 MHz with
PRMT4 inhibition achieved by those inhibitors with shorter chemical shifts reported in parts per million (ppm) downfield
two- and three-atom linkers connecting the adenosine and relative to tetramethylsilane (TMS) or H2O (δ 4.79). 1H NMR
guanidine moieties. By comparison, analogues containing data are reported in the following order: multiplicity (s,
four-atom linkers were generally inactive. Compound 4 bearing singlet; d, doublet; t, triplet; q, quartet and m, multiplet),
a four-atom thioether linkage was, however, found to effec- coupling constant (J) in hertz (Hz) and the number of protons.
tively inhibit the lysine methyltransferase G9a and none of the When appropriate, the multiplicity is preceded by br, indicat-
PRMTs tested. In addition, compound 6 with a four-atom ing that the signal was broad. 13C NMR spectra were recorded
linker comprising of a secondary amine was found to inhibit at 75.5 MHz with chemical shifts reported relative to CDCl3
PRMT6 exclusively. Docking studies were also performed with (δ 77.16). The 13C NMR spectra of compounds 1–6 (as their
inhibitors 1–3 and PRMT4 and indicate the possible modes of TFA salts) were recorded in D2O and therefore referenced to
binding that support the strong inhibition observed with these the TFA quartet at 116.6. High-resolution mass spectrometry
three compounds. These docking studies also suggest possibi- (HRMS) analysis was performed using an ESI instrument.
lities for further improving the inhibitor affinity by introducing
Synthetic procedures and compound characterization
hydrogen-bonding substituents on the guanidine moiety to
allow for simultaneous interactions with both Glu258 and
2-(2-((3aR,4R,6R,6aR)-6-(6-Amino-9H-purin-9-yl)-2,2-dimethyl-
Glu267. To this end, future investigations will involve the tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)ethyl)-N,N′-di-Boc-guanidine
incorporation of N-hydroxy and N-amino substituted guani- (10). To a suspension of amine 928 (570 mg, 1.5 mmol) in
dines26,37 into the inhibitor structures here described. Aside CH2Cl2 (2 mL), a solution of N,N′-bis(tert-butoxycarbonyl)-
from the possibility of enhancing inhibition, such modifi- N″-triflylguanidine (392 mg, 1.0 mmol) and NEt3 (416 µL,
cations may also serve to improve the cell permeability of 3.0 mmol) in CH2Cl2 (3 mL) was added. The mixture was
these inhibitors. The results of these ongoing studies will be stirred overnight at room temperature and diluted with CH2Cl2
reported in due course.
(15 mL). The organic layer was washed with 1 N KHSO4
(10 mL), saturated NaHCO3 (10 mL) and brine (10 mL), dried
over Na2SO4 and filtered. Evaporation in vacuo and column
chromatography (SiO2: EtOAc–MeOH (98 : 2)) afforded 10
(422 mg, 75%) as a white powder. Rf 0.41 (95 : 5 EtOAc–MeOH).
1H NMR (300 MHz, CDCl3): δ 8.41 (br m, 1H), 8.30 (s, 1H), 7.87
Experimental
Reagents and general methods
All reagents employed were of American Chemical Society (s, 1H), 6.15 (br s, 2H), 6.02 (s 1H), 5.44 (d, J = 6.3 Hz, 1H),
(ACS) grade or finer and were used without further purification 4.97–4.87 (m, 1H), 4.23 (q, J = 6.6 Hz, 1H), 3.67–3.56 (m, 1H),
unless otherwise stated. Compounds 8,28 9,28 11–14,28 16,38 3.43–3.33 (m, 1H), 2.01 (q, J = 6.2 Hz, 2H), 1.57 (s, 3H), 1.45 (s,
20,39 24,40 25,41 26,42 27,43 28,44 and 29–3045 were synthesized 9H), 1.36 (s, 9H), 1.34 (s, 3H). 13C NMR (75 MHz, CDCl3):
according to previously described procedures. All known com- δ 163.5, 155.9, 155.7, 153.1, 152.8, 149.3, 120.3, 114.8, 90.1,
pounds prepared had NMR spectra, mass spectra, and optical 85.6, 84.0, 83.9, 82.8, 79.1, 38.1, 32.1, 28.3, 27.9, 27.2, 25.4.
rotation values consistent with the assigned structures. All HRMS (ESI): calculated for C25H38N8O7 [M + Na]+ 585.2761,
reactions and fractions from column chromatography were found 585.2773.
monitored by thin layer chromatography (TLC) using
plates with a UV fluorescent indicator (normal SiO2, Merck 60 tetrahydrofuran-2-yl)ethyl)guanidine
2-(2-((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxy-
(1). Compound 10
F254). One or more of the following methods were used (56.5 mg, 0.1 mmol) is dissolved in a 1 : 1 mixture of TFA and
for visualization: UV absorption by fluorescence quenching; CH2Cl2 (10 mL) and stirred for 2 hours at room temperature.
phosphomolybdic acid : ceric sulfate : sulfuric acid : H2O H2O (1.5 mL) is added, and the mixture is stirred for
(10 g : 1.25 g : 12 mL : 238 mL) staining; KMnO4 staining; PPh3 30 minutes at room temperature. Evaporation in vacuo, purifi-
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cation by preparative HPLC and freeze-drying of the pure frac- δ 157.1, 150.3, 148.4, 144.9, 142.9, 130.1, 128.1, 119.2, 89.0,
tions afforded the final compound 1 (38.8 mg, 89%) as a white 84.5, 73.9, 73.8, 42.0. HRMS (ESI): calculated for C13H18N8O3
1
powder. H NMR (400 MHz, D2O): δ 8.46 (s, 1H), 8.45 (s, 1H), [M + H]+ 335.1580, found 335.1576.
6.12 (d, J = 4.7 Hz, 1H), 4.87 (t, J = 5.0 Hz, 1H), 4.35 (t, J =
2-(2-((((3aS,4S,6R,6aR)-6-(6-Amino-9H-purin-9-yl)-2,2-dimethyl-
5.1 Hz, 1H), 4.27–4.19 (m, 1H), 3.37 (t, J = 6.6 Hz, 2H), tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)thio)-ethyl)isoindoline-
2.17–2.06 (m, 1H). 13C NMR (75 MHz, D2O): δ 157.0, 150.2, 1,3-dione (17). To a solution of thioacetate 1638 (500 mg,
148.5, 144.7, 143.3, 119.3, 89.2, 82.6, 73.9, 73.5, 38.4, 31.9. 1.37 mmol) in MeOH (30 mL), 2-bromoethylphthalimide
HRMS (ESI): calculated for C12H18N8O3 [M + H]+ 323.1580, (536 mg, 2.10 mmol) was added and the mixture was cooled to
found 323.1578.
−20 °C under a N2 atmosphere. Sodium methoxide (165 mg,
2-((E)-3-((3aR,4R,6R,6aR)-6-(6-Amino-9H-purin-9-yl)-2,2-dimethyl- 3.06 mmol) was added and the mixture was stirred for
tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)allyl)-N,N′-di-Boc-guanidine 90 minutes at −20 °C and for 72 hours at room temperature.
(15). To a suspension of amine 1428 (332 mg, 1.0 mmol) in The solvent was evaporated in vacuo and extracted from water–
CH2Cl2 (2 mL), a solution of N,N′-bis(tert-butoxycarbonyl)-N″- chloroform (3 × 30 mL). The organic layers were combined,
triflylguanidine (392 mg, 1.0 mmol) and NEt3 (139 µL, dried over Na2SO4 and filtered. Evaporation in vacuo and
1.0 mmol) in CH2Cl2 (2 mL) was added. The mixture was column chromatography (SiO2: CH2Cl2–MeOH (9 : 1)) afforded
stirred until completion followed by TLC (EtOAc–MeOH (9 : 1)). compound 17 (332 mg, 49%) as a white foam. Rf 0.61 (9 : 1
The mixture was diluted with CH2Cl2 (6 mL) and the organic CH2Cl2–MeOH). 1H NMR (300 MHz, CDCl3): δ 8.37 (s, 1H),
layer was washed with 1 N KHSO4 (10 mL), saturated NaHCO3 7.95 (s, 1H), 7.88–7.79 (m, 2H), 7.66–7.75 (m, 2H), 6.09 (d, J =
(10 mL) and brine (10 mL), dried over Na2SO4 and filtered. 2.1 Hz, 1H), 5.98 (br s, 2H), 5.49 (dd, J = 6.3, 2.1 Hz, 1H), 5.08
Evaporation in vacuo and column chromatography (SiO2: (dd, J = 6.3, 3.3 Hz, 1H), 4.41 (td, J = 6.7, 3.3 Hz, 1H), 3.82 (t,
EtOAc–MeOH (98 : 2)) afforded compound 15 (526 mg, 92%) as J = 6.9 Hz, 2H), 2.92 (dd, J = 6.8, 2.7 Hz, 2H), 2.83 (t, J = 6.9 Hz,
a
white powder. Rf 0.43 (9 : 1 EtOAc–MeOH); 1H NMR 2H), 1.61 (s, 3H), 1.39 (s, 3H). 13C NMR (75 MHz, CDCl3):
(300 MHz, CDCl3): δ 8.36 (s, 1H), 8.34 (t, J = 5.1 Hz, 1H), 7.89 δ 168.0, 155.9, 153.2, 149.1, 134.0, 131.9, 123.3, 120.2, 114.5,
(s, 1H), 6.09 (d, J = 2.1 Hz, 1H), 6.01 (br s, 2H), 5.79–5.76 (m, 90.7, 86.7, 84.1, 83.7, 36.8, 33.7, 30.4, 27.1, 25.3. HRMS (ESI):
2H), 5.50 (dd, J = 6.3, 2.1 Hz, 1H), 5.01 (dd, J = 6.3, 3.6 Hz, 1H), calculated for C23H24N6O5S [M
4.68 (t, J = 4.2 Hz, 1H), 4.01–3.97 (m, 2H), 1.63 (s, 3H), 1.49 497.1662.
+
H]+ 497.1607, found
(s, 18H), 1.40 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 163.4, 155.9,
9-((3aR,4R,6S,6aS)-6-(((2-Aminoethyl)thio)methyl)-2,2-dimethyl-
155.6, 153.2, 153.1, 149.4, 129.7, 129.1, 120.2, 114.6, 90.3, 87.3, tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine (18). For
84.6, 84.2, 83.2, 79.4, 41.9, 28.3, 28.0, 27.1, 25.4. HRMS (ESI): deprotection of the phthalimide, compound 17 (271 mg,
calculated for C26H38N8O7 [M + H]+ 575.2942, found 575.2921.
0.55 mmol) was dissolved in a solution of methylamine in
2-(2-((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxy- EtOH (33%, 9 mL) and stirred at room temperature for
tetrahydrofuran-2-yl)propyl)guanidine (2). To a solution of 16 hours. The solvent was evaporated in vacuo and the residue
compound 15 (250 mg, 0.44 mmol) in EtOH (10 mL), a suspen- was redissolved in CHCl3 (20 mL). The organic layer was
sion of Pd/C (10%, 200 mg, excess) in EtOH (5 mL) was added. extracted with 10% aqueous acetic acid (25 mL). The aqueous
The mixture was hydrogenated (5 bar H2) for 72 hours. The layer was washed with CHCl3 (3 × 20 mL), the pH was adjusted
catalyst was removed by filtration over celite and the solvent to >12 using 2 N NaOH and then was extracted with CHCl3 (4 ×
was evaporated in vacuo. Purification by column chromato- 20 mL). The final organic layers were dried over Na2SO4 and fil-
graphy (SiO2: EtOAc–MeOH (98 : 2)) afforded the saturated tered and the solvents were evaporated in vacuo to afford free
intermediate (118 mg, 47%) as a white powder, which was sub- amine 18 (177 mg, 89%). Rf 0.18 (9 : 1 CH2Cl2–MeOH).
jected to the deprotection and purification procedure as 1H NMR (300 MHz, CDCl3) δ 8.26 (s, 1H), 7.88 (s, 1H), 6.67 (s,
described for compound 1 to afford the final compound 2 2H), 6.03 (d, J = 2.1 Hz, 1H), 5.47 (dd, J = 6.4, 2.1 Hz, 1H), 5.01
(39 mg, 61%) as a white powder. 1H NMR (400 MHz, D2O): (dd, J = 6.4, 3.1 Hz, 1H), 4.32 (td, J = 6.9, 3.1 Hz, 1H), 2.85–2.61
δ 8.46 (s, 1H), 8.45 (s, 1H), 6.12 (d, J = 4.9 Hz, 1H), 4.88–4.81 (m, 4H), 2.53 (t, J = 6.3 Hz, 2H), 1.54 (s, 3H), 1.52 (br s, 2H),
(m, 1H), 4.31 (t, J = 5.1 Hz, 1H), 4.22–4.14 (m, 1H), 3.24 (t, J = 1.33 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 156.1, 153.1, 149.1,
6.7 Hz, 2H), 1.99–1.53 (m, 4H). 13C NMR (75 MHz, D2O): 120.2, 114.4, 90.8, 86.9, 84.0, 83.8, 41.0, 36.7, 34.0, 27.1, 25.3.
δ 157.0, 150.6, 148.6, 145.3, 142.8, 119.2, 88.6, 84.7, 74.0, 73.4, HRMS (ESI): calculated for C15H22N6O3S [M + H]+ 367.1552,
41.0, 29.9, 24.6. HRMS (ESI): calculated for C13H20N8O3 found 367.1535.
[M + H]+ 337.1737, found 337.1733.
2-(2-((((3aS,4S,6R,6aR)-6-(6-Amino-9H-purin-9-yl)-2,2-dimethyl-
2-((E)-3-((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxy- tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-thio)ethyl)-N,N′-di-
tetrahydrofuran-2-yl)allyl)guanidine (3). Starting from com- Boc-guanidine (19). Following the procedure as described for
pound 15 (100 mg, 0.17 mmol), the deprotection and compound 15, amine 18 (160 mg, 0.44 mmol) was guanidiny-
purification procedure as described for compound 1 afforded lated. After purification by column chromatography (SiO2:
the final compound 3 (41 mg, 70%) as a white powder. CH2Cl2–MeOH (9 : 1)), compound 19 was obtained as a clear
1H NMR (400 MHz, D2O): δ 8.45 (m, 2H), 6.17 (d, J = 4.0 Hz, oil (245 mg, 92%). Rf 0.63 (9 : 1 CH2Cl2–MeOH). 1H NMR
1H), 5.97–5.91 (m, 2H), 4.86–4.81 (m, 1H), 4.65 (br s, 1H), 4.39 (300 MHz, CDCl3) δ 8.50 (t, J = 5.3 Hz, 1H), 8.25 (s, 1H), 7.90 (s,
(t, J = 5.2 Hz, 1H), 3.91 (s, 2H). 13C NMR (75 MHz, D2O): 1H), 6.54 (s, 2H), 6.03 (d, J = 1.8 Hz, 1H), 5.44 (dd, J = 6.4,
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1.8 Hz, 1H), 5.00 (dd, J = 6.3, 3.1 Hz, 1H), 4.32 (td, J = 6.7, 92.0, 86.1, 85.0, 83.2, 81.7, 79.4, 71.1, 69.4, 40.0, 28.2, 28.0,
3.1 Hz, 1H), 3.50 (q, J = 6.2 Hz, 2H), 2.79 (m, 2H), 2.64 (t, J = 27.1, 25.3. HRMS (ESI): calculated for C33H44N8O9 [M + Na]+
6.4 Hz, 2H), 1.54 (s, 3H), 1.40 (s, 18H), 1.32 (s, 3H). 13C NMR 719.3129, found 719.3180.
(75 MHz, CDCl3): δ 163.4, 156.0, 155.9, 153.0, 149.1, 120.1,
114.4, 90.6, 86.5, 84.0, 83.6, 83.1, 79.2, 39.8, 33.9, 31.6, 28.2, tetrahydrofuran-2-yl)methoxy)ethyl)guanidine
28.0, 27.0, 25.3. HRMS (ESI): calculated for C26H40N8O7S from protected guanidine 22 (56 mg, 0.08 mmol), the depro-
[M + Na]+ 631.2638, found 631.2685.
tection procedure as described for compound 1 afforded the
1-(2-(((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxy-
(5). Starting
1-(2-((((2S,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxy- benzoyl-protected intermediate which was subsequently depro-
tetrahydrofuran-2-yl)methyl)thio)ethyl)guanidine (4). Starting tected overnight in a mixture of NH4OH (3 mL) and MeOH
from protected guanidine 19 (140 mg, 0.23 mmol), the depro- (5 mL). Purification by preparative HPLC and freeze-drying as
tection and purification procedure as described for compound described for compound 1 afforded the final compound 5
1
1 afforded the final compound 4 (63 mg, 74%). 1H NMR (22.4 mg, 79%). H NMR (400 MHz, D2O): δ 8.47 (s, 1H), 8.41
(400 MHz, D2O): δ 8.49 (s, 1H), 8.41 (s, 1H), 6.12 (d, J = 4.7 Hz, (s, 1H), 6.17 (d, J = 4.6 Hz, 1H), 4.84–4.80 (m, 1H), 4.48 (t, J =
1H), 4.85 (t, J = 5.0 Hz, 1H), 4.43 (t, J = 5.1 Hz, 1H), 4.36–4.28 5.0 Hz, 1H), 4.40–4.33 (m, 1H), 3.95–3.79 (m, 2H), 3.79–3.65
(m, 1H), 3.37 (t, J = 6.5 Hz, 2H), 3.12–2.95 (m, 2H), 2.78 (t, J = (m, 2H), 3.44–3.32 (m, 2H). 13C NMR (75 MHz, D2O): δ 157.4,
6.5 Hz, 2H). 13C NMR (75 MHz, D2O): δ 157.4, 150.5, 148.8, 150.3, 148.5, 144.9, 142.9, 119.1, 89.0, 84.0, 74.5, 70.6, 70.4,
145.1, 143.5, 119.5, 89.2, 84.5, 74.2, 72.9, 41.2, 34.3, 31.6. 69.4, 41.5. HRMS (ESI): calculated for C13H20N8O4 [M + H]+
HRMS (ESI): calculated for C13H20N8O3S [M + H]+ 369.1457, 353.1686, found 353.1653.
found 369.1465.
2-(2-((((3aR,4R,6R,6aR)-6-(6-Amino-9H-purin-9-yl)-2,2-dimethyl-
N-(9-((3aR,4R,6R,6aR)-6-((2-Aminoethoxy)methyl)-2,2-dimethyl- tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-(Boc)amino)ethyl)-
tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide N,N′-di-Boc-guanidine (31). The Cbz-protected intermediate
(21). To
a
solution of benzoyl-protected adenosine 2039 3045 (148 mg, 0.25 mmol) was deprotected overnight using
(617 mg, 1.5 mmol) in dry DMF (10 mL) under Ar at 0 °C, NaH 10% palladium on activated charcoal (105 mg, excess) in
(dispersion in mineral oil (50%), 720 mg, 15 mmol) was added methanol (6 mL) at room temperature under a H2 atmosphere.
over a period of 1 hour. The mixture was stirred for 1 hour After removal of the catalyst over celite and concentration the
allowing it to warm to room temperature. The mixture was deprotected amine was guanidinylated following the procedure
then cooled to −5 °C and chloroethylamine hydrochloride as described for compound 15. After purification by column
(1.05 g, 9.0 mmol) was added. The mixture was stirred for chromatography (SiO2: CH2Cl2–MeOH (925 : 75)), compound
5.5 hours, allowing it to warm to room temperature, and 31 was obtained as a clear oil (168 mg, 97% over two steps).
quenched with MeOH (6 mL). Evaporation in vacuo and Rf 0.54 (9 : 1 CH2Cl2–MeOH). 1H NMR (300 MHz, CDCl3)
column chromatography with the product absorbed on silica δ 8.40–8.20 (m, 2H), 7.89 (s, 1H), 6.44 (s, 2H), 6.01 (m, 1H),
(SiO2: first CH2Cl2–MeOH (9 : 1), then CH2Cl2–MeOH–NEt3 5.40 (dd, J = 6.3, 1.9 Hz, 1H), 4.94 (m, 1H), 4.51–4.21 (m, 1H),
(9 : 1 : 0.5)) afforded amine 21 (220 mg, 32%) as an off-white 3.80–3.12 (m, 6H), 1.53 (s, 3H), 1.40 (s, 18H), 1.37 (s, 9H), 1.31
powder. Rf 0.32 (9 : 1 : 0.5 CH2Cl2–MeOH–NEt3); 1H NMR (s, 3H). 13C NMR (75 MHz, CDCl3): δ 163.4, 156.3, 155.9, 155.6,
(300 MHz, CDCl3) δ 8.72 (s, 1H), 8.54 (s, 1H), 7.97–7.88 (m, 153.1, 149.2, 120.2, 114.6, 90.5, 85.7, 84.7, 83.8, 83.0, 82.4,
2H), 7.55–7.33 (m, 3H), 6.39 (br s, 2H), 6.19 (d, J = 2.1 Hz, 1H), 80.3, 79.1, 49.0, 47.0, 39.0, 28.3, 28.0, 27.8, 27.2, 25.4. HRMS
5.38 (dd, J = 6.2, 2.1 Hz, 1H), 5.12 (dd, J = 6.2, 2.6 Hz, 1H), (ESI): calculated for C31H49N9O9 [M + Na]+ 714.3551, found
4.44–4.35 (m, 1H), 3.69–3.48 (m, 4H), 3.02–2.86 (m, 2H), 1.55 714.3565.
(s, 3H), 1.34 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 165.6, 152.4,
1-(2-((((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxy-
151.7, 149.2, 142.9, 133.3, 132.7, 128.6, 128.0, 123.4, 114.1, tetrahydrofuran-2-yl)methyl)amino)ethyl)-guanidine (6). Start-
90.8, 86.1, 84.6, 81.2, 71.0, 69.0, 39.8, 27.0, 25.3. HRMS (ESI): ing from protected guanidine 31 (160 mg, 0.23 mmol), the
calculated for C22H26N6O5 [M + H]+ 455.2043, found 455.2002.
deprotection and purification procedure as described for com-
N-(9-((3aR,4R,6R,6aR)-6-((2-(N,N′-Di-Boc-guanidino)-ethoxy)- pound 1 afforded the final compound 6 (61 mg, 74%). 1H
methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]-dioxol-4-yl)-9H- NMR (400 MHz, D2O): δ 8.46–8.41 (m, 2H), 6.16 (d, J = 4.0 Hz,
purin-6-yl)benzamide (22). Following the procedure as 1H), 4.88 (t, J = 4.0 Hz, 1H), 4.54–4.43 (m, 2H), 3.72–3.54 (m,
described for compound 15, amine 21 (220 mg, 0.48 mmol) 4H), 3.39 (t, J = 5.9 Hz, 2H). 13C NMR (75 MHz, D2O): δ 157.4,
was guanidinylated. After purification by column chromato- 150.3, 148.4, 144.7, 143.9, 119.7, 90.2, 79.8, 73.6, 71.8, 49.8,
graphy (SiO2: CH2Cl2–MeOH (98 : 2)), compound 22 was 46.5, 37.8. HRMS (ESI): calculated for C13H21N9O3 [M + H]+
obtained as a white powder (291 mg, 87%). Rf 0.24 (98 : 2 352.1846, found 352.1834.
1
CH2Cl2–MeOH). H NMR (300 MHz, CDCl3) δ 9.30 (br s, 1H),
8.73 (s, 1H), 8.44 (t, J = 4.8 Hz, 1H), 8.27 (s, 1H), 8.03–7.92 (m,
Methyltransferase inhibition assays
2H), 7.57–7.36 (m, 3H), 6.22 (d, J = 2.4 Hz, 1H), 5.33 (dd, J = Methyltransferase inhibition assays were performed as pre-
6.1, 2.4 Hz, 1H), 4.99 (dd, J = 6.1, 2.0 Hz, 1H), 4.56–4.48 (m, viously described46 using commercially available chemilumi-
1H), 3.75–3.19 (m, 6H), 1.58 (s, 3H), 1.40 (s, 9H), 1.38 (s, 9H), nescent assay kits for PRMT1, PRMT4 and PRMT6 and G9a
1.35 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 164.8, 163.3, 156.1, (purchased from BPS Bioscience, San Diego, CA, USA). The
153.2, 151.4, 149.4, 133.5, 132.7, 128.7, 127.9, 123.4, 114.0, enzymatic reactions were conducted in duplicate at room
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temperature for 1 hour (20 minutes for PRMT1) in substrate- eagle’s medium) supplemented with 10% FBS and 10 000 U
coated well plates at a final reaction volume of 50 µL contain- per mL penicillin and 10 mg per mL streptomycin at 37 °C
ing the manufacturer’s proprietary assay buffer, AdoMet (at a under a 5% CO2 atmosphere in air. Monolayers were passaged
concentration of 5-times the respective Km value for each on reaching ∼90% confluency with trypsin–EDTA. The MCF-7
enzyme), the methyltransferase enzyme: PRMT1 (80 ng per and Caco-2 cells were plated in duplicate into 96-well plates in
reaction), PRMT4 (200 ng per reaction), PRMT6 (180 ng per media at a concentration of 5000 cells per well. At 40–50% con-
reaction), G9a (160 ng per reaction), and inhibitors 1–6 (in the fluency (48 hours post-seeding) compounds 1–3 were added at
range of concentrations: 0.001–100 µM in water). Before concentrations of 1, 10 and 100 µM respectively to each well
addition of the AdoMet, the enzyme was first incubated with plate (each compound concentration applied to four separate
the inhibitor for 15 minutes at 37 °C. Positive controls were wells per plate). The plates were then incubated for 24 hours
performed in the absence of inhibitors using water to keep the in the presence of the test compounds after which the media
final volume consistent. Blanks and substrate controls were was removed and the cells incubated for 2 hours in fresh
performed in the absence of the enzyme and AdoMet respect- media. The cells were next treated with a MTT solution at a
ively. Following the enzymatic reactions, 100 μL of primary final concentration of 0.1 mg ml−1 in a serum-free medium for
antibody (recognizing the respective immobilized methylated 4 hours at 37 °C/5% CO2. After 4 hours, the plates were centri-
arginine product) was added to each well and the plate was fuged and the MTT solution was removed. DMSO was added to
incubated at room temperature for an additional 1 hour. Then dissolve the blue formazan product and UV absorption was
100 μL of secondary horseradish peroxidase (HRP)-conjugated measured at 550 nm.
antibody was added to each well and the plate was incubated
Docking studies
at room temperature for additional 30 minutes. Finally, 100 μL
of an HRP substrate mixture was added to the wells and the
luminescence was measured directly using a standard micro-
plate reader. In all cases enzyme activity measurements were
performed in duplicate at each of the inhibitor concentrations
evaluated.
Compounds 1–3 were built in YASARA (Version 13.9.848),
based on the S-adenosyl homocysteine structure in complex
with PRMT349 (PDB code 1F3L). After aligning 1F3L to the
structure of PRMT420 (PDB code 2Y1W) using MUSTANG50
compounds 1–3 were in a good starting position for docking to
PRMT4, using the default run_docklocal macro. According to
this macro the ligand and all protein sidechains within 7 Å of
the ligand were energy minimized using the steepest descent
and simulated annealing with the NOVA forcefield,51 followed
by docking with AutoDock Vina.52 Of the best docking pose,
all ligand and protein atoms were again energy minimized
with the steepest descent and simulated annealing with the
NOVA forcefield, to yield the images used in generating Fig. 4.
The luminescence data were analysed using GraphPad
Prism (version 5.0). The luminescence of the positive control
(Lp) in each dataset was defined as 100% activity. This value
was included in the IC50 graphs at a concentration of two log
values below the lowest concentration tested. The lumines-
cence data of the negative controls (Ln) in each dataset were
subtracted from the obtained luminescence data. The percent
activity in the presence of each inhibitor was calculated accord-
ing to the following equation: % activity = (L − Ln)/(Lp − Ln),
where L = the luminescence in the presence of the compound,
Ln = the luminescence in the absence of the enzyme, and Lp =
the luminescence in the absence of the inhibitor. The percent
activity values were plotted as a function of inhibitor concen-
trations and fitted using non-linear regression analysis of the
Sigmoidal dose–response curve generated using the equation
Y = B + (T − B)/1 + 10((Logic50−X)×Hill Slope), where Y = percent
activity, B = minimum percent activity, T = maximum percent
activity, X = the logarithmic concentration of the compound
and Hill Slope = slope factor or Hill coefficient. The IC50 value
was determined by the concentration resulting in a half-
maximal percent activity. The standard errors were reported
using the symmetrical CI function. The IC50 values measured
for AdoHcy which served as a reference compound are similar
to those reported by the supplier of the methyltransferase kits.
Acknowledgements
We thank Javier Sastre Toraño for HRMS analysis. Financial
support provided by Utrecht University and the Netherlands
Organization for Scientific Research (VIDI grant to NIM) is
acknowledged.
Notes and references
1 L. C. Boffa, J. Karn, G. Vidali and V. G. Allfrey, Biochem.
Biophys. Res. Commun., 1977, 74, 969–976.
2 W. K. Paik and S. Kim, in Advances in Enzymology and
Related Areas Molecular Biology, John Wiley & Sons, Inc.,
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