Exploration of cyanine compounds as selective inhibitors of protein arginine methyltransferases: Synthesis and biological evaluation

Article abstract of DOI:10.1021/jm501452j

Protein arginine methyltransferase 1 (PRMT1) is involved in many biological activities, such as gene transcription, signal transduction, and RNA processing. Overexpression of PRMT1 is related to cardiovascular diseases, kidney diseases, and cancers; therefore, selective PRMT1 inhibitors serve as chemical probes to investigate the biological function of PRMT1 and drug candidates for disease treatment. Our previous work found trimethine cyanine compounds that effectively inhibit PRMT1 activity. In our present study, we systematically investigated the structure-activity relationship of cyanine structures. A pentamethine compound, E-84 (compound 50), showed inhibition on PRMT1 at the micromolar level and 6- to 25-fold selectivity over CARM1, PRMT5, and PRMT8. The cellular activity suggests that compound 50 permeated the cellular membrane, inhibited cellular PRMT1 activity, and blocked leukemia cell proliferation. Additionally, our molecular docking study suggested compound 50 might act by occupying the cofactor binding site, which provided a roadmap to guide further optimization of this lead compound.

Full text of DOI:10.1021/jm501452j

Article
pubs.acs.org/jmc
Exploration of Cyanine Compounds as Selective Inhibitors of Protein
Arginine Methyltransferases: Synthesis and Biological Evaluation
†
‡
§
†
‡
§
,‡
Hao Hu, Eric A. Owens, Hairui Su, Leilei Yan, Andrew Levitz, Xinyang Zhao, Maged Henary,*
,†
and Yujun George Zheng*
†
Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, Georgia 30602, United States
‡
Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for Biotechnology and Drug Design, Georgia State
University, 100 Piedmont Avenue SE, Atlanta, Georgia 30303, United States
§
Department of Biochemistry and Molecular Genetics, UAB Stem Cell Institute, The University of Alabama at Birmingham,
Birmingham, Alabama 35294, United States
*
S Supporting Information
ABSTRACT: Protein arginine methyltransferase 1 (PRMT1)
is involved in many biological activities, such as gene trans-
cription, signal transduction, and RNA processing. Over-
expression of PRMT1 is related to cardiovascular diseases,
kidney diseases, and cancers; therefore, selective PRMT1 in-
hibitors serve as chemical probes to investigate the biological
function of PRMT1 and drug candidates for disease treatment.
Our previous work found trimethine cyanine compounds that
effectively inhibit PRMT1 activity. In our present study, we
systematically investigated the structure−activity relationship
of cyanine structures. A pentamethine compound, E-84 (com-
pound 50), showed inhibition on PRMT1 at the micromolar level and 6- to 25-fold selectivity over CARM1, PRMT5, and
PRMT8. The cellular activity suggests that compound 50 permeated the cellular membrane, inhibited cellular PRMT1 activity,
and blocked leukemia cell proliferation. Additionally, our molecular docking study suggested compound 50 might act by
occupying the cofactor binding site, which provided a roadmap to guide further optimization of this lead compound.
2
7−29
INTRODUCTION
The first class comprises peptide derivatives.
The under-
■
lying rationale is that because the SAM-binding pocket of
PRMTs is highly conserved, it might be difficult to discriminate
Protein arginine methylation is a prevalent posttranslational
modification that is mediated by protein arginine methyl-
4
,7,30,31
1−5
between isozymes with SAM analogs.
Instead, inhibitors
transferases (PRMTs).
During this process the methyl group
containing specific substrate sequences might offer enhanced
selectivity. Though these substrate analogs indeed gained some
extra inhibitory activity, there is no remarkable improvement
for the selectivity. Since some of them act as substrate in-
hibitors or irreversible inhibitors and the molecular size of
peptides are relatively large, these peptide inhibitors are more
like PRMT1 function dissecting tools rather than ideal drug
candidates. The second class inhibitors are organic small
molecules, which are normally obtained from random or target-
based screening, such as AMI-1, stilbamidine, allantodapsone,
A few structurally
related inhibitors, including the diamidine compound DB75
recently reported by our group, are rationally investigated.
On one hand, having diverse structures provided more
opportunities for exploration of potential inhibitors; conversely,
the structural dissimilarity from SAM or substrate makes it hard
to generalize the ideal pharmacophores and predict the activity.
of cofactor S-adenosylmethionine (SAM) is transferred to sub-
strate arginine and SAM is converted to S-adenosylhomocysteine
SAH). PRMTs can be divided into three types (types I, II, and
(
6−8
III) according to the degree and position of methylation. Type
I converts arginine into monomethylarginine (MMA) and further
into asymmetric dimethylarginine (ADMA). Type II produces
MMA and symmetric dimethylarginine (SDMA), while type III
can only generate MMA. Among them, PRMT1 is a type I
methyltransferase that accounts for over 80% arginine methylation
27
28
9
in vivo. PRMT1 plays important roles in many biological pro-
32−35
10
RM65, and SAM derivatives (Figure 1).
cessing, such as gene transcription, signal transduction, RNA
3,11−14
processing, and DNA repair.
Moreover, overexpression of
36−43
1
5−18
PRMT1 is reported to be related to cardiovascular diseases,
1
9−22
kidney disease,
and many kinds of cancers, e.g., prostate can-
2
3
24
25,26
cer, breast cancer, and leukemia.
Therefore, the develop-
ment of PRMT1-selective inhibitors will not only benefit the
investigation of the function and biological roles of PRMT1 but
also provide a therapeutic strategy for these diseases.
So far, much work has been done to develop selective PRMT
inhibitors, which could be structurally divided into two classes.
Received: September 21, 2014
©
XXXX American Chemical Society
A
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Figure 1. Structures of SAM, SAH, and reported PRMT inhibitors including AMI-1, uracenadolates, RM65, stilbamidine, DB75, allantodapsone,
MHI-21, and E-4.
Scheme 1. Synthetic Route for the Preparation of 3,3-Dimethylindolenine Based Unsymmetric Trimethine Cyanines 10−16
For example, some derivatives of AMI-1, dubbed as
uracandolates”, turned out to be a CARM1/PRMT4 activity
enhancer. A culmination of these effects has prevented great
strides to be made toward the development of selective PRMT1
inhibitors.
compounds for PRMT inhibition. Having carefully checked
the previous results, we chose compound E-4 (Figure 1) as an
appealing structure for further exploration because it (1)
showed promising inhibitory activity and (2) contained a less
synthetically complex aromatic ring system which likely
increases the potential for further modification. Herein, we
discuss a systematic structure−activity relationship (SAR) for
E-4-like compounds and identified compound 50 as a potent
inhibitor with selectivity for PRMT1 over CARM1/PRMT4,
PRMT5, and PRMT8. Besides, the cellular activity supported
that 50 can permeate cell membrane, inhibit intracellular
PRMT1 activity, and block leukemia cell proliferation. Also,
ligand−enzyme interactions were studied in detail by molecular
docking to understand the SAR and offer structural clues for
further optimization.
“
4
4
Cyanine dyes possess two nitrogen containing hetero-
cycles that are connected by an electron deficient conjugated
methine bridge that provides an important characteristic of
carbocyaninestheir red-shifted absorption and fluorescence
4
5−47
wavelengths.
Through various synthetic methods, the
absorption and fluorescence of these compounds can be tuned
from 400 to 1000 nm. Cyanine−biomolecule interactions
can be easily quantitated spectrophotometrically through the
relative changes in the absorption or emission spectra.
Our previous effort was put into discovering potential
PRMT1 inhibitors from a set of trimethine cyanines, among
which compound MHI-21 (Figure 1) was shown to possess
good potency with moderate selectivity for PRMT1 over other
RESULTS AND DISCUSSION
■
4
8
PRMTs. Because of the intriguing results and the unique
spectroscopic properties of the cyanine structures, we decided
to finely tune their binding properties and create diversified
1. Chemistry and Synthesis. In order to thoroughly
investigate the structure−activity relationship of the cyanine
PRMT inhibiting agents, we prepared a series of tri- and
B
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Scheme 2. Synthetic Route for the Preparation of 3,3-Dimethylindolenine Based Symmetric Trimethine Cyanine PRMT
Inhibiting Agents 22−27
Scheme 3. Synthetic Route for the Preparation of Benzothiazole-Based Trimethine Cyanines without (34−39) and with
(
40 and 41) meso-Modification
Scheme 4. Synthetic Route for the Preparation of Napthothiazole-Based Trimethine Cyanines without (45−47) and with (48)
meso-Modification
pentamethine cyanines based on previously reported cyanine
inhibitors to augment the potency of inhibitory properties. We
first used an effective route toward the preparation of
asymmetric cyanines maintaining a methyl group on one side
of the inhibitors. The first step in the synthesis requires the
quaternization of the 2,3,3-trimethylindolenine heterocyclic
portion that affords compounds 1−7. In parallel, this hetero-
cycle is methylated and subjected to Vilsmeier formylation
followed by basic hydrolysis to afford the aldehyde 9. This
aldehyde reacts effectively with individual 1−7 to yield the final
asymmetric compounds 10−16 (Scheme 1). Several solvents
including acetic anhydride, acetonitrile, pyridine, and ethanol
were used for the development of the synthetic protocol for the
final compound preparation step, and acetic anhydride was
found to perform the transformation with the highest purity.
Symmetric PRMT-inhibiting cyanines previously synthesized
were moderately effective without heterocyclic modification. To
potentially increase inhibitory efficacy, we have incorporated
halogens on the phenyl ring of such inhibitors. The synthetic
route began with Fischer indole synthesis and methylation
which afforded the indolenine heterocyclic derivatives 6 and
different positions of halogen incorporation. Since benzothia-
zoles have been reported to bind the minor groove of duplex
49−51
DNA,
we incorporated a steric restraint toward preventing
this interaction. Reacting benzothiazole salts 28−33 with
triethyl orthoacetate afforded the cyanine compounds featuring
a methyl group in the central position of the polymethine
bridge (Scheme 3). The delocalized cation allows this central
methyl group to be acidic; reacting at this position in the
presence of pyridine with a corresponding aldehyde affords the
central modification of compound 41. Similar methyl-
incorporating chemistry cannot be applied to the dimethy-
lindolenine core because of steric interactions with the dimethyl
groups. Identical reaction conditions were applied to the
naphthothiazole heterocyclic compound shown in Scheme 4.
In order to draw conclusions about the length of the poly-
methine bridge, we elongated the structure one vinylene unit
(
−CHCH−), comparing trimethine compound 26 to
pentamethine compound 50. The route to 50 began with the
formation of brominated salt 6. Separately, reacting muco-
bromic acid with a warm ethanolic solution of aniline afforded
the polymethine linking precursor 49. These two intermediates
reacted under basic conditions in acetic anhydride to yield 50
(Scheme 5).
1
7−21. These quaternary salts were then allowed to react with
triethyl orthoformate, which yielded the symmetric compounds
2
2−27 (Scheme 2).
Similar chemistry was applied to the preparation of anal-
In addition, using similar synthetic methodology, we pre-
ogous benzothiazole-based cyanine compounds 34−39 with
pared the pentamethine analogs shown in Scheme 6 for direct
C
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Scheme 5. Synthetic Route for the Preparation of Brominated Pentamethine Cyanine Dye 50 with an N-Butyl Substituent
Scheme 6. Synthetic Route for the Preparation of
Benz[e]indolenine Pentamethine Cyanine Dyes 51−54
exhibited no inhibition even at 90 μM, indicating the double-
headed structure is essential for maintaining activity. As ex-
pected, the activities increased along with the structural
symmetry. This can be seen by comparing 10 (9% at 30 μM)
vs E-14 (42% at 30 μM), 11 (32% at 30 μM) vs E-6 (48% at
3
0 μM), and 12 (35% at 10 μM) vs E-4 (41% at 10 μM).
Interestingly, the structural symmetry seems not as important
as the “tail” hydrophobicity, as can be seen in that compound
12 (35% at 10 μM) with a bulky phenylpropyl tail is a better
inhibitor than E-6 and E-14 with shorter tails.
Then further modifications were introduced on both the
head” and “tail” of the dimer structures. The addition of either
negative or positive charged groups on the end of the butyl
“tail” adversely affected the inhibitory activity. For example, at
0 μM, charged compounds 13 (2%) and 16 (N.I.) showed
weaker or no activity compared with compounds 11 (8%)
containing a neural butyl tail. As to the modification on the
polymethine length comparison to our previously published
trimethine compounds.
“
2
. Inhibitory Activity of the New Cyanine Compounds
52
for PRMT1 and SAR Analysis. Scintillation proximity assay
1
was used to test inhibitory activity of each new compound
on PRMT1 inhibition. Briefly, inhibitor was incubated with
3
[
H]-labeled SAM (S-adenosylmethionine) and H4(1−20)-
“
nude (unsubstituted) head”, the incorporation of 5-sulfonate
BTN peptide (a biotinylated 20-amino-acid peptide from H4
histone N-terminus) before initiating the reaction by adding
recombinant His6x-PRMT1. The inhibition ratio was used to
assess the potency of each inhibitor against PRMT1 activity
group abolished activity by comparing 13 and 14 (N.I.).
However, 5-bromination brought the inhibition rate up to 36%
of 15 from 8% of 11, which is reasonable because halogen
substituents predominately prevail in drug development as a
hydrophobic group, Lewis acid, or forming “halogen bond” in
(
Tables 13). It should be noted that all the enzyme
reactions in this work were controlled under initial rate
condition, where the peptide methylation was linear with
time.
53,54
ligand−protein interactions.
On the basis of the SAR mentioned above, as well as the fact
that many PRMT1 inhibitors are reported to contain symmetric
The compound represented by E-4 can be considered as a
dimer”: a polymethine spacer connecting two identical
monomers”. Each “monomer” can be further divided into
3
2,34,35,38,39
“
“
structures,
we prepared symmetric 5,5′-dichloro- and
5
,5′-dibromoindolium cyanines (compounds 22−27). Again, as
an aromatic ring (head) and an N-substituted side chain (tail).
the “tail” became longer and bulkier, as seen from 22 through
24 and from 25 through 27, the potency was enhanced. Com-
pared to the prototypes, all halogenated compounds gained
extra activity. For example, 10 μM phenylpropyl derivatives 24
and 27 even displayed about 100% inhibition on PRMT1,
compared with E-4. It seems bromine played a slightly (if any)
better role than chlorine on potency enhancement.
4
8
The IC50 values were reported for indolium-headed E-14,
E-6, and E-4 and benz[e]indolium-headed E-5, E-18, and E-8
(
for structures, see Table 1). Since the potency can vary under
different assay conditions, we began by retesting the PRMT1
inhibition for these compounds at 10 μM under the same
experimental condition to make sure the data are comparable
with each other. In consistence with our previous results,
we found that when the “head” or “tail” became bulkier or
more hydrophobic, the activity was improved. For example,
the bigger-headed E-5 (27% at 10 μM), E-18 (91% at 10 μM),
and E-8 (100% at 10 μM) showed superior activities
compared with E-14 (4% at 10 μM), E-6 (no inhibition,
N.I., at 10 μM), and E-4 (41% at 10 μM), respectively. Also,
E-4 and E-8, which both have a phenylpropyl group as the
SAR for Thiazolium-Based Cyanines (Series 2). The second
series of compounds (Series 2, see Table 2) were obtained by
replacement of the indolium-based cyanines with a benzothia-
zole ring containing a sulfur atom. It turned out this subtle
change was efficacious for the benzothiazoliums, especially for
those with low potency. For example, compounds 35 and 37
showed inhibition up to 79% and 88% at the concentration of
0 μM, in contrast to the corresponding indolium species E-6
N.I.) and compound 25 (11%), respectively. Besides, the
activity was retained when the bromine atom on C-6 (38, 93%)
was moved to C-5 (39, 91%).
The success of the 3,3-dimethyl-to-sulfur alteration strategy,
coupled with the “bulkier head, better inhibitor” SAR, prompted
us to generate naphthothiazoliums 45−47. As expected, these
compounds indeed turned out to be satisfactory inhibitors to
PRMT1, though they failed to exhibit further improvement on
1
(
“
tail”, showed better inhibition than those with the same
“
heads” but shorter “tails” (E-14 and E-6, and E-5 and E-18,
respectively).
SAR for Indolium-Based Cyanines (Series 1). The initial
investigation was focused on the indolium derivatives (series 1,
see Table 1). To elucidate the impact of symmetry on activity,
general “monomers” (1−3) and simple asymmetrical indolium
cyanines (10−16), where modifications were introduced into
just one head, were synthesized. It turned out compounds 1−3
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Table 1. Inhibition and IC50 for Indolium-Related Compounds
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Table 1. continued
a
b
c
Cmpd# = compound number. sd = standard deviation. N.I. = no inhibition.
activity comparing to the benzothiazoliums 34−35 and the
benzoindoliums E-5, E-18, and E-8.
Meanwhile, it is worth noting that the halogen substitutions
(69%) demonstrated that longer chain could result in better
activity, perhaps because the gained flexibility renders the
molecule a better binding conformation or the longer distance
between two “heads” is more favored by the binding pocket.
Besides, the nonchlorinated compound 51 (89%) has better
activity than 52. Further development of 51 and 52 by
changing “tail” to phenylpropyl group yielded 53 and 54,
respectively. The better activity of 54 (96%) than 53 (70%)
indicates that the effect of spacer chlorination might not be
uniform, likely because compounds with different “tails” have
slight differences regarding to the binding poses. On the basis
of IC50 values which are in range of less than 3-fold, halogen-
substitution on the central methine carbon subtly affected the
activity of these cyanine compounds in PRMT1 inhibition.
3. Specificity Profiling. One of our goals is to obtain
selective inhibitors for PRMT1 over other PRMT isoforms. So
several potent inhibitors were picked out and further evaluated
against different PRMTs. First, IC50 values against PRMT1 for
those with inhibition higher than 90% at 10 μM were deter-
mined to identify accurate potencies. The fractional activities
were plotted with the concentrations and fitted to the Hill
equation. Generally speaking, the result was well matched with
that from the previous screening. Unexpectedly, the most
potent inhibitor is the benzoindolium E-8 (0.61 μM), which is
about 5-fold more potent than its naphthothiazolium counter-
part 47 (3.05 μM).
on the aromatic ring enhanced the activity by comparing 37
(
88%) vs 34 (46%), 38 (93%) vs 35 (79%), and bulkier “tails”
in these thiazolium-based compounds exhibited the better
activities (comparing 34−36 or 45−47). These trends are the
same as in series 1, indicating these two series of compounds
probably have the same binding mode to PRMT1.
Modifications of the Spacers of Indolium Derivatives
(
Series 3). So far, the thiazolium-based structures (series 2)
seem to be more promising for further study than the
dimethylindolium derivatives (series 1). Nevertheless, some
benzothiazolium cyanines have also been reported to bind
49−51
the minor groove of dsDNA.
On the contrary, the
dimethylindolium lost the double strand DNA (dsDNA)
binding ability probably because of steric hindrance in
5
5,56
ligand−DNA interaction caused by the dimethyl moiety.
From this perspective, we assumed that the steric hindrance
on spacer of the thiazole-containing cyanine would also impair
the interaction with dsDNA. Therefore, methyl and styryl
groups were introduced onto the meso-carbon in the trimethine
spacer. For benzothiazoliums, substitution at the central
methine carbon with methyl group (40, 77%) retained the
activity of the parent molecule (35, 79%), but substitution with
more bulky group styrene (41, 48%) decreased the potency. As
for the naphthothiazoliums, methylation at the central methine
carbon enhanced the potency, as indicated by comparing
For each enzyme assay for the other PRMT members, the
3
concentrations of [ H]SAM and the substrate were set around
45 (40%) and 48 (90%). These results held promise for
respective K values so that the resulting IC values of the
m
50
developing the thiazole-based cyanine structures into PRMT-
specific inhibitors rather than dsDNA binders.
same compound can be compared between different PRMTs.
As shown in Figure 2 (and Table S1), indolium-based com-
pounds E-8 and 24 were very potent for blocking the activities
of all the tested PRMTs. E-8 only showed very minor specificity
for PRMT1 (0.61 μM) over PRMT5 (1.4 μM) and PRMT8
(1.74 μM), while compound 24 possesses no selectivity. The
thiazolium compound 39 exhibited moderate (3- to 4-fold)
PRMT1 (2.03 μM) selectivity over CARM1 (7.23 μM) and
PRMT5 (8.36 μM). Compared to it, compound 47 showed
even lower (about 2-fold) selectivity to the same set of enzymes
On the other hand, because the dimethylindolium-based
structure has the innate property to reject binding with dsDNA,
it was subjected to further modifications including elongation of
spacer and addition of halogen atom on meso-carbon (Table 3).
Since it was easy to tell how modifications affected the potency
for those with moderate inhibitory activity, compound 26
(
48%) and E-5 (27%) were chosen as “parent” compounds.
The elevated inhibition ratio of compounds 50 (96%) and 52
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Table 2. Inhibition and IC50 for Thiazolium-Based Compounds
a
b
Cmpd# = compound number. sd = standard deviation.
5
7,59,62
(3.05 μM for PRMT1, 5.8 μM for CARM1, and 7.04 μM for
other than reflecting the true Ki.
Overall, there was a
PRMT5), but its inhibitory activity on PRMT1 is more than
general observation that the compounds with high n exhibited
a poor degree of selectivity. Compound 50, on the other hand,
H
9-fold higher than on PRMT8 (28.4 μM). As for the longer-
spacer compound 50, it displays more than 6-, 10-, and 25-fold
selectivity for PRMT1 (3.38 μM) over CARM1 (21.5 μM),
PRMT5 (35.4 μM), and PRMT8 (84.9 μM), respectively.
It is worthy to note that all the compounds but 50 showed
steep slopes for the concentration−potency curves, with Hill
has n approximate to unity for all the PRMTs, meaning it is
H
most likely that this compound exerted inhibition through
specific ligand−enzyme binding rather than by promiscuous
interaction.
4. Compound 50 Binding with PRMT1. Because some
PRMT inhibitors, such as NS-1 and TBBD (ellagic acid), act by
binding with the substrate peptide instead of the enzyme, we
coefficients (n ) apparently higher than 1 (Figure 2, Table S2,
H
57
Figure S1). There could be several explanations for it. The
43,63
first one could be that the compounds interacted with PRMT1
also tried to detect the binding preference of compound 50.
58
59
dimer or oligomer in a positive cooperative way, which
means the binding of one ligand could enhance the affinity of
the nearby site for the second ligand such that the IC50 curve
We harnessed the fluorescence property of compound 50 and
found its fluorescence intensity increased about 6-fold upon
binding to PRMT1 (Figure S3). The binding affinity is 2.3 μM,
became steeper (so n larger than 1). The second possibility is
which is in the close range of IC value for PRMT1 inhibition.
H
50
that compound aggregates are the active form interacting with
This validates that there is a direct interaction between com-
pound 50 and PRMT1.
enzymes. This is possible considering the inhibition via
6
0,61
aggregation is common in the early stage of drug discovery
and cyanine dyes have a tendency to aggregate as π-stacked
5. Docking. To better understand the obtained SAR, it is
intriguing to investigate the interaction at the molecular
level. Therefore, molecular simulations were used to begin
speculating about the molecular recognition between 50 and
PRMT1. The reported crystal structures (PDB codes 1ORI,
5
1
structures. This behavior is undesirable because it usually
causes nonspecific binding and thus low selectivity. The third
reason is that the compounds are tight binding inhibitors,
58
which makes K much lower than the enzyme concentration. In
1OR8, and 1ORH) by Zhang and Cheng provided substantial
information on cofactor and substrate binding sites of rat
d
this case, the IC50 only relates to the enzyme concentration
G
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Table 3. Inhibition and IC50 for Compounds with Modified Spacers
a
b
Cmpd# = compound number. sd = standard deviation.
PRMT1 (rPRMT1). Nevertheless, the crystals were obtained at
nonphysiological condition (pH 4.7), so they were considered
inappropriate for virtual screening study.
In the following docking study, a spherical area that covered
both SAM and arginine binding pockets was chosen as the
binding site (Figure S2) and the conformers ranking top 10 for
the -CDOCKER_ENERGY values were generated. It turned
out that there was no significant difference for these 10 con-
formers regarding the orientations (Figure 3C; the pocket
surface was rendered according to hydrophobicity), which
suggested 50 could fit the pocket very well. Conformer 1 (with
the highest -CDOCKER_ENERGY value) was selected and
superimposed with SAH (Figure 3A), which was maintained at
the same orientation as in the crystal structure (PDB code
1OR8). As shown in Figure 3A, the binding site can be divided
into three parts: a deeply buried pocket (BP), an exterior
surface cavity (ESC), and a narrow channel connecting the two
areas. The molecule of 50 spanned BP and ESC: (1) half of the
molecule occupied the BP which comprised the site housing
the adenosyl group of SAH and entrance of substrate arginine
to the pocket; (2) the other half protruded out to the ESC area;
(3) the pentamethine spacer bound to the channel. An analysis
of the volume and hydrophobicity distribution of the pocket
shed light on the underlying molecular basis for the
summarized SAR: (1) Both the BP and ESC showed medium
to high hydrophobicity with the highest areas located near the
two distal bromines of compound 50. This was consistent with
the experimental phenomenon that higher hydrophobicity of
32−34,40,41
Recently, we built a homology-modeled human PRMT1
(
named HM-hPRMT1) from which the interaction between
41
an inhibitor DB75 and hPRMT1 was reveal. On the basis of
the HM-hPRMT1 structure, we attempted to perform a
molecular docking on 50 using the implemented docking
64
tool, CDORKER, of Discovery Studio. Unfortunately, this initial
trial failed. In the rPRMT1 crystal structure, the first 1−40 residues
at N-terminal, including a conserved helix αX that contributes the
recognition of SAM among PRMTs, were not resolved because of
the disordered structure, indicating there was high flexibility of this
sequence. In addition, several independent studies on the crystal
65
66,67
structures of rat PRMT3, mouse CARM1,
and Trypanosoma
68
brucei PRMT6 demonstrated the corresponding segments also
had conformation alteration upon the binding of cofactor (SAM
and SAH). On the basis of these facts, we postulated that the
N-terminal acted as a “lid” of the pocket and could be adjusted to
house ligands of different sizes. The failure of our first trial was
probably because modeled SAM binding sites were too small to
accommodate compound 50. Therefore, we attempted to take
the “lid” off the pocket by deleting the residues 1−40 in the
HM-hPRMT1 (the resulting structure named PRMT1_αX(−))
to get an enlarged binding pocket.
H
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58
several negatively charged grooves on the surface of PRMT1.
These grooves on the PRMT1 surface were considered as
potential docking sites for the positively charged substrate
segments, which usually are glycine- and arginine-rich (GAR)
70,71
sequences.
Though currently there is still no direct
evidence that shows the small molecule inhibitors act by
occupying peptide binding pocket for PRMTs, the situation is
encouraging in the area of discovery of protein lysine
73
72
transferases (PKMTs) inhibitors: AZ-505 and BIX-01294
75
73
74
related compounds (e.g., UNC0224, UNC0638, and E72 )
have all been shown to interact with the peptide binding groove
according to crystal structures, and the backbone amino groups
in these molecules are involved in electrostatic or hydrogen-
bond interaction. Form this point of view, the positively
charged cyanine compounds might also act by targeting the
peptide binding groove on PRMT1.
41
6
. Cellular Activity. In one of our previous reports, we
showed that DB75, another PRMT1-specific inhibitor, has cell
membrane permeable capability and blocks proliferation of
leukemia cells. In order to determine whether compound 50 is
also an intracellular PRMT1 inhibitor, we applied different
concentrations of 50 to several leukemia cell cultures and
then measured cellular proliferation by viability assays. We
found that 50 was a powerful agent that significantly inhibited
leukemia cell growth at 100 nM in Meg01 and MOLM13 cells
and 200 nM in HEL cells (Figure 4A−C). HEL cells are more
resistant to E84 than Meg01 and MOLM13 cells (Figure 4 F)
possibly because of HEL cells’ genetic background: the con-
stitutively active JAK2 mutation makes cells less dependent on
PRMT1-mediated signaling pathways. These results were
consistent with what we have observed previously with DB75.
At both 24 and 48 h of incubation, 50 showed a nanomolar
level requirement for 50% inhibition (Figure 4F), indicating 50
can more efficiently target PRMT1 in cells than that of DB75.
Cell pellets harvested from 50 supplemented culture showed a
blue color (unpublished data), meaning 50 can permeate cell
membrane and can be used as an intracellular PRMT1 stain.
Treatment with 50 on leukemia cells led to significant loss of
total protein arginine methylation (Figure 4G), demonstrating
that 50 inhibited intracellular PRMT1 activity. All data above
indicated that 50 permeated the cell membrane, inhibited
intracellular PRMT1 activity, and blocked leukemia cell
proliferation.
Figure 2. IC50 and n of compounds 24, E-8, 39, 47, and 50 against
H
PRMTs. Note that only compound 50 (E-84) shows satisfactory
selectivity for PRMT1 as well as Hill coefficient around 1 against all
PRMTs.
“heads” and “tails” resulted in better activities. (2) The BP
seemed to fit one of the “head−tail” units of the compound
very well, meaning the ligand can be fully contacted with this
part. In contrast, the interaction between the molecule and ESC
is much looser because of the larger volume of ESC, indicating
the compound substituent in ESC can be replaced with a larger
group to result in better spatial complementation in a future
study. (3) The channel bridging BP and ESC was so narrow
that even the bromine on spacer shifted slightly toward the BP
to avoid the collision with pocket wall. This explained the poor
activity of compound 41 in which there is a very bulky styryl
group attached to the spacer.
A detailed inspection on the ligand−enzyme interaction
revealed some hydrophobic, charge−charge, and hydrogen
bond forces between the skeleton of 50 and side chains of
surrounding residues (Figure 3B). The cation is delocalized
across the nitrogen atoms of 50 at physiological pH and can
involve electrostatic interactions and/or hydrogen bond,
indicating their essential role in lowering the binding energy
(
thus increasing the binding affinity). Because a molecule may
CONCLUSION
bind with protein with more than one orientation, it is more
necessary to identify which residues make universal interactions
with these poses and thus are essential for the ligand binding.
Hence, the first 10 generated conformers of compound 50 were
analyzed globally (Figure 3D). As for the residues, C101, E129,
and M155 formed noncovalent bonds with all 10 conformers
and K127 and E153 with no less than eight conformers. It is
noteworthy that C101, E129, M155 are conserved residues
■
We reported herein a set of cyanine compounds as PRMT
inhibitors. Among them, a pentamethine compound E-84
(compound 50) showed inhibition on PRMT1 with the
potency at low micromolar level as well as specificity for
PRMT1 over CARM1, PRMT5, and PRMT8 ranging from
6- to 25-fold. The following cellular activity studies showed that
E-84 permeated cell plasma membrane, decreased intracellular
arginine methylation, and blocked leukemia cell proliferation.
Besides, the structure−activity relationship for the cyanine
compounds was systematically investigated with these features:
(1) the two nitrogen-containing “head” is necessary for
maintaining the activity; (2) increasing hydrophobicity
generally leads to better inhibition; (3) a sulfur atom in posi-
tion 3 of indolium group results in equal or better activity than
dimethylmethine; (4) pentamethine as the spacer seems better
than trimethine; (5) no larger than bromine can be tolerated on
the meso-carbon of the spacer. Molecular docking study
suggested E-84 might act by partially occupying the cofactor
58
among PRMT family that contribute to the SAM binding and
E153 was considered as a very essential residue for positioning
the substrate arginine in a proper orientation to approach the
+
58,69
methyl group (S -Me) in SAM.
The docking study
suggested that the binding of compound 50 inhibited the
activity of PRMT1 by impeding the correct positioning of both
cofactor and substrate.
Nonetheless, it should be kept in mind that the above
conclusion is just deduced on the basis of the docking results
and we could not exclude the possibility that the compound can
occupy the substrate peptide binding site. Actually, there are
I
DOI: 10.1021/jm501452j
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Docking result of compound 50. (A) Binding pocket for compound 50. The hydrophobic surface is rendered as brown and hydrophilic
surface as blue. Conformer 1 of 50 (yellow) and SAH (green, retaining the same orientation as in crystal structure 1OR8) are shown in stick mode.
The backbone of PRMT1_αX(−) is shown as ribbon. (B) Noncovalent bond interactions between the conformer 1 and residues. Conformer 1
(
yellow) and the involved residues (cyanine) are shown in stick mode. Dash lines represent the interactions: hydrophobic interaction is colored as
light purple, electrostatic force as brown, and hydrogen bond (H-bond) as green. (C) Overlapping of 10 conformers of 50 in the binding pocket with
conformer 1 rendered as yellow and others as dark gray. Note there is no significant difference between the poses with regard to the spatial
arrangement. (D) Histogram for the noncovalent bonds between 10 conformers and PRMT1_αX(−). Blue columns represent all the favorable
interactions including hydrophobic interaction, electrostatic force, and H-bond. The colors of the columns in the other three histograms are the same
as the corresponding interactions in (B).
binding site as well as blocking the proximity of cofactor and
substrate arginine.
2. Synthesis of Cyanine Compounds. All chemical reactions
were maintained under a positive pressure of nitrogen unless otherwise
stated. The reaction progress was monitored using silica gel 60 F254
thin layer chromatography plates (Merck EMD Millipore, Darmstadt,
Germany). Open column chromatography was utilized for the
purification of all final compounds using 60−200 μm, 60A classic
column silica gel (Dynamic Adsorbents, Norcross, GA). The nuclear
magnetic resonance spectra were obtained using high quality Kontes
NMR tubes (Kimble Chase, Vineland, NJ) rated to 500 MHz and were
recorded on a Bruker Avance 400 MHz spectrometer interfaced to a
PC using Topspin 3.1. High-resolution accurate mass spectra were
obtained at the Georgia State University Mass Spectrometry Facility
using a Waters Q-TOF micro (ESI-Q-TOF) mass spectrometer or
utilizing a Waters Micromass LCT TOF ES+ Premier mass spec-
trometer. Liquid chromatography utilized a Waters 2487 single
EXPERIMENTAL SECTION
■
1
. Materials. The reagents for the synthesis of the final PRMT
inhibiting agents were obtained commercially from Acros Organics or
Matrix Scientific. Fmoc-protected amino acids and resins were purchased
from NovaBioChem or ChemPep, and 2-(6-chloro-1H-benzotriazole-1-yl)-
1
,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) was pur-
3
chased from ChemPep. [ H]SAM (15 or 18 Ci/mmol). Streptavidin-
coated PVT SPA beads were purchased from PerkinElmer. Isopropyl
β-D-1-thiogalactopyranoside (IPTG) was obtained from RPI Corp.
Other chemical reagents were purchased from Fisher, Sigma, and
VWR. H3.3 protein was purchased from New England Biolabs.
J
DOI: 10.1021/jm501452j
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 4. Compound 50 inhibited proliferation of leukemia cell lines via blocking of PRMT1 activity. (A−F) Compound 50 inhibited leukemic cell
growth. Serial diluted 50 was added to the Meg01 (A), MOLM13 (B), and HEL (C) cell cultures, and each day the cell growth was measured by
viability assay. As a control, the cells were treated with the same volume of DMSO as that added in 2 μM 50 treated samples. (D) Drug sensitivity
curves of all cell lines after 24 h of treatment of 50. (E) Drug sensitivity curves of all cell lines after 48 h of treatment of 50. (F) Calculated IC50
concentration of 50 for all cell lines. (G) Arginine methylation level of 50-treated leukemic cells. Meg01, MOLM13, and HEL cells were cultured
with the presence of 50 or DMSO. Extract was harvested after 24 h of treatment, and samples were resolved by SDS−PAGE. Arginine methylation
status was detected by using anti-methyl-R antibodies.
wavelength absorption detector with wavelengths set between 640 and
00 nm depending on the particular photophysical properties. The
column used in LC was a Waters Delta-Pak 5 μm, 100A 3.9 mm ×
50 mm reversed phase C18 column. Evaporative light scattering
detection analyzes trace impurities that cannot be observed by
alternative methods; a SEDEX 75 ELSD was utilized in tandem with
liquid chromatography to confirm purity. Integrals for the ELSD and
absorption peaks were utilized to confirm >95% purity for all final
compounds that were tested in PRMT inhibition assays.
The synthetic procedures for 1−9 and 17−21 have been previously
reported by our laboratory and were utilized in the subsequent
reactions without purification or modification.
Synthesis of Halogenated Trimethine Cyanines with
Indolenine Heterocycle (22−27). Appropriate heterocyclic salt
was added to an oven dried round-bottom flask which was filled with
nitrogen. Acetic anhydride (5 mL) was added to the round-bottom
flask, and the solution was heated to 90 °C. Triethyl orthoformate
column fractions were combined and concentrated to afford the pure
products.
5-Bromo-2-((E)-3-((E)-5-bromo-1-ethyl-3,3-dimethylindolin-2-
7
1
ylidene)prop-1-en-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium Iodide
1
(22). H NMR (400 MHz, DMSO-d ) δ 1.295 (d, J = 6.4 Hz, 6H),
6
1.696 (s, 12H), 4.153 (d, J = 6.4 Hz, 4H), 6.559 (d, J = 13.2 Hz, 2H),
1
3
7
.515 (s, 4H), 7.835 (s, 2H), 8.316 (t, J = 13.2 Hz, 1H); C NMR
(
100 MHz, DMSO-d ) δ 12.67, 27.64, 49.59, 103.39, 113.31, 123.58,
6
1
29.06, 130.10, 140.88, 143.28, 173.91. 67% yield; mp >260 °C.
-Butyl-2-((E)-3-((E)-1-butyl-5-chloro-3,3-dimethylindolin-2-
1
ylidene)prop-1-en-1-yl)-5-chloro-3,3-dimethyl-3H-indol-1-ium Io-
1
dide (23). H NMR (400 MHz, DMSO-d ) δ 0.932 (t, J = 6.4 Hz,
6
6
H), 1.410 (d, J = 6.8 Hz, 4H), 1.697 (s, 16H), 4.131 (s, 4H), 6.597
(
d, J = 13.2 Hz, 2H), 7.505 (s, 4H), 7.827 (s, 2H), 8.321 (t, J = 13.2
13
Hz, 1H); C NMR (100 MHz, DMSO-d ) δ 14.28, 19.98, 27.74,
6
2
1
9.64, 44.43, 49.58, 103.61, 113.59, 123.51, 129.03, 130.09, 141.32,
43.19, 150.52, 174.31. 54% yield; mp >260 °C.
(
3
3 mol equiv ) was added via syringe to the stirring solution. After
0 min, the reaction turned bright-pink and the reaction progress was
monitored using UV−vis spectrophotometry and TLC, eluting with
% methanol in DCM. After consumption of the starting materials, the
5
-Chloro-2-((E)-3-((E)-5-chloro-3,3-dimethyl-1-(3-phenylpropyl)-
indolin-2-ylidene)prop-1-en-1-yl)-3,3-dimethyl-1-(3-phenylpropyl)-
1
3
H-indol-1-ium Bromide (24). H NMR (400 MHz, DMSO-d ) δ
5
6
1
.681 (s, 12H), 2.036 (s, 4H), 2.766 (s, 4H), 4.179 (s, 4H), 6.424 (d,
reaction mixture was allowed to cool and the solution was triturated
with ether, resulting in the precipitation of the product. The com-
pounds were isolated using silica gel chromatography with gradient
elution from 10% hexanes in DCM to 5% methanol in DCM. The
J = 12.4 Hz, 2H), 7.204 (m, 10H), 7.466 (d, J = 8.0 Hz, 4H), 7.818 (s,
2H), 8.293 (t, J = 13.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d
) δ
6
27.70, 29.15, 32.52, 44.31, 49.62, 103.54, 113.49, 123.52, 126.54,
K
DOI: 10.1021/jm501452j
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
5
28.72, 128.86, 129.02, 130.14, 141.29, 141.36, 143.20, 150.42, 174.35;
(q, J = 6.8 Hz, 4H), 6.62 (d, J = 12.4 Hz, 2H), 7.74 (m, 4H), 7.82 (t,
9% yield; mp >260 °C.
J = 12.8 Hz, 1H), 8.29 (s, 2H); 13C NMR (100 MHz, DMSO-d ) δ
6
5
-Bromo-2-((E)-3-((E)-5-bromo-1-ethyl-3,3-dimethylindolin-2-
13.01, 42.20, 115.48, 117.61, 125.97, 127.93, 131.38; 46% yield.
ylidene)prop-1-en-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium Iodide
6-Bromo-2-((1E,3Z)-3-(6-bromo-3-butylbenzo[d]thiazol-2(3H)-
1
(
25). H NMR (400 MHz, DMSO-d ) δ 1.308 (t, J = 7.2 Hz, 6H),
ylidene)prop-1-en-1-yl)-3-butylbenzo[d]thiazol-3-ium Iodide(38).
6
1
1
7
8
2
1
.693 (s, 12H), 4.148 (m, J = 7.2 Hz, 4H), 6.565 (d, J = 13.2 Hz, 2H),
.446 (d, J = 8.4 Hz, 2H), 7.634 (d, J = 8.4 Hz, 2H), 7.948 (s, 2H),
H NMR (400 MHz, DMSO-d ) δ 0.93 (t, J = 7.2 Hz, 6H), 1.42
6
(
m, 4H), 1.69 (m, 4H), 4.31 (t, J = 7.2 Hz, 4H), 6.62 (d, J = 12.8 Hz,
.316 (t, J = 13.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d ) δ 12.67,
13
6
2H), 7.77 (m, 5H), 8.28 (s, 2H); C NMR (100 MHz, DMSO-d ) δ
6
7.64, 49.58, 103.42, 113.75, 118.11, 126.32, 131.91, 141.30, 143.58,
50.59, 173.76; 65% yield; mp >260 °C.
1
1
5
4.15, 19.83, 29.85, 46.80, 99.84, 115.73, 117.63, 125.91, 127.77,
+
31.34, 141.20, 147.52, 165.24; HRMS calcd for C H Br N S m/z
25
27
2
2 2
5
-Bromo-2-((E)-3-((E)-5-bromo-1-butyl-3,3-dimethylindolin-2-
76.9977, obsd m/z 576.9983; 76% yield; mp >260 °C.
ylidene)prop-1-en-1-yl)-1-butyl-3,3-dimethyl-3H-indol-1-ium Iodide
5
-Bromo-2-((1E,3Z)-3-(5-bromo-3-butylbenzo[d]thiazol-2(3H)-
1
(
1
6
26). H NMR (400 MHz, DMSO-d ) δ 0.934 (t, J = 7.2 Hz, 6H),
.410 (q, J = 7.2 Hz, 4H), 1.696 (s, 16H), 4.121 (t, J = 7.2 Hz, 4H),
.570 (d, J = 13.6 Hz, 2H), 7.455 (d, J = 8.4 Hz, 2H), 7.635 (d, J = 8.4
6
ylidene)prop-1-en-1-yl)-3-butylbenzo[d]thiazol-3-ium Iodide (39).
1
H NMR (400 MHz, DMSO-d ) δ 0.94 (t, J = 7.2 Hz, 6H), 1.45
6
(m, 4H), 1.68 (m, 4H), 4.33 (t, J = 7.2 Hz, 4H), 6.63 (d, J = 12.8 Hz,
13
Hz, 2H), 7.945 (s, 2H), 8.320 (t, J = 13.2 Hz, 2H). C NMR (100
MHz, DMSO-d ) δ 14.28, 19.98, 27.74, 29.62, 44.41, 49.59, 103.61,
1
2
2
8
1
1
5
H), 7.60 (d, J = 8.4 Hz, 2H), 7.79 (t, J = 12.8 Hz, 1H), 7.95 (d, J =
6
13
.4 Hz, 2H), 8.08 (s, 2H); C NMR (100 MHz, DMSO-d ) δ 0.58,
6
14.02, 118.13, 126.26, 131.88, 141.75, 143.49, 174.19; 73% yield, mp
4.20, 19.78, 29.91, 46.70, 100.02, 116.78, 121.59, 125.06, 125.10,
65−268 °C.
+
28.43, 143.14, 147.65; HRMS calcd for C H Br N S m/z
2
5
27
2
2 2
5
-Bromo-2-((E)-3-((E)-5-bromo-3,3-dimethyl-1-(3-phenylpropyl)-
76.9977, obsd m/z 576.9983; 77% yield; mp >260 °C.
Synthesis of Benzothiazole cyanine with meso-Methyl
indolin-2-ylidene)prop-1-en-1-yl)-3,3-dimethyl-1-(3-phenylpropyl)-
1
3
1
4
H-indol-1-ium Bromide (27). H NMR (400 MHz, DMSO-d ) δ
6
Moiety 40. Butylated benzothiazole salt (1 mol equiv) was added
to a round-bottom flask, and triethyl orthoacetate (10 mol equiv) was
added followed by freshly distilled pyridine (7 mL). The resulting
mixture was refluxed under heavy stirring for 4 h. The bright pink
solution was allowed to cool. Ether (100 mL) was added to the round-
bottom flask, and it was allowed to stand for 2 h. The resulting pink
crystals were filtered and dissolved in DCM. The pink solution was
washed with water, sat. solution of sodium bicarbonate, and brine. The
organic layer was combined and concentrated to afford the meso-
methylated benzothiazole carbocayanine PRMT binding agent 40.
.694 (s, 12H), 2.038 (q, J = 7.2 Hz, 4H), 2.772 (t, J = 8.0 Hz, 4H),
.181 (t, J = 7.2 Hz, 4H), 6.442 (d, J = 13.6 Hz, 2H), 7.192 (d, J = 8.8
Hz, 4H), 7.243 (m, 6H), 7.399 (d, J = 8.4 Hz, 2H), 7.618 (d, J = 7.6
Hz, 2H), 7.939 (s, 2H), 8.302 (t, J = 13.2, 1H); 13C NMR (100 MHz,
DMSO-d ) δ 27.71, 29.16, 44.28, 49.61, 103.58, 113.93, 118.17,
1
7
6
26.26, 126.54, 126.59, 128.73, 128.86, 141.36, 141.71, 143.49, 174.20;
8% yield; mp >260 °C.
Compounds 28−33 were prepared according to literature
procedure without modification and were used without purification
in the proceeding steps.
3
-Butyl-2-((1E,3Z)-3-(3-butylbenzo[d]thiazol-2(3H)-ylidene)-2-
Synthesis of Benzothiazole Trimethine Cyanines 34−39.
Benzothiazole heterocyclic salt (1 mol equiv) bearing alkyl groups on
the heterocyclic nitrogen was added to a round-bottom flask followed
by acetic anhydride (5 mL). This mixture was heated to 90 °C, and
triethyl orthoformate (3 mol equiv) was added to the reaction mixture.
After 15 min, the reaction solution turned deep purple indicating the
progress on the reaction. After the reaction was completed as indicated
by TLC analyses, the contents were allowed to cool and diethyl ether
was added. The flask was cooled on an ice bath for 1 h, and crystals
began to develop. These crystals were filtered and washed with ether
and hexanes. The pure compounds were isolated using column
chromatography.
1
methylprop-1-en-1-yl)benzo[d]thiazol-3-ium Iodide (40). H NMR
400 MHz, MeOD-d ) δ 1.080 (t, J = 7.2 Hz, 6H), 1.607 (m, 4H),
(
4
1
6
7
.934 (p, J = 7.2 Hz, 4H), 2.698 (s. 3H), 4.495 (t, J = 7.2 Hz, 4H),
.553 (s, 2H), 7.463 (t, J = 7.6 Hz, 2H), 7.650 (t, J = 7.6 Hz, 2H),
1
3
.730 (d, J = 8.4 Hz, 2H), 7.926 (d, J = 8.0, Hz, 2H); C NMR (100
MHz DMSO-d ) δ 14.24, 19.93, 29.60, 46.67, 14.05, 123.38, 123.44,
6
1
25.32, 125.86, 128.79, 128.85, 140.41, 160.49; 74% yield; mp >260 °C.
Synthesis of Styryl-Modified Cyanine 41. Precursor 40 (1 mol
equiv) was added to a round-bottom flask followed by benzaldehyde
12 mol equiv) followed by ethanol (5 mL) and piperidine (5 mol
(
equiv). The resulting mixture was heated to reflux for 12 h and was
followed by a shift in the absorption spectrum and TLC. The pink
colored starting solution gradually turned deep purple resulting in the
formation of the product. After the starting material was consumed,
the reaction mixture was concentrated and extracted in DCM. The
organic layer was washed with sat. sodium bicarbonate and brine, dried
over sodium sulfate, and concentrated. The residue was purified with
open column chromatography, eluting with 3% methanol in DCM to
obtain the pure compound with a meso-styryl group.
3
-Ethyl-2-((1E,3Z)-3-(3-ethylbenzo[d]thiazol-2(3H)-ylidene)prop-
1
1
-en-1-yl)benzo[d]thiazol-3-ium Iodide (34). H NMR (400
MHz,DMSO-d ) δ 1.35 (t, J = 6.8 Hz, 6H), 4.39 (d, J = 6.8 Hz,
6
4H), 6.63 (d, J = 13.2 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.58 (t, J = 8.0
13
Hz, 2H), 7.80 (m, 3H), 8.01 (d, J = 7.6 Hz, 2H); C NMR (100
MHz, DMSO-d ) δ 13.08, 21.53, 41.94, 99.10, 113.84, 123.59, 125.67,
1
6
28.59, 141.27, 147.13, 164.64; 72% yield; mp >260 °C.
3
-Butyl-2-((1E,3Z)-3-(3-butylbenzo[d]thiazol-2(3H)-ylidene)prop-
1
1
-en-1-yl)benzo[d]thiazol-3-ium Iodide (35). H NMR (400 MHz,
3-Butyl-2-((1E,3E)-2-((Z)-(3-butylbenzo[d]thiazol-2(3H)-ylidene)-
methyl)-4-phenylbuta-1,3-dien-1-yl)benzo[d]thiazol-3-ium Iodide
DMSO-d ) δ 0.94 (t, = 7.2 Hz, 6H), 1.43 (m, 4H), 1.72 (m, 4H), 4.34
6
1
(
41). H NMR (400 MHz, CDCl ) δ 1.013 (s, 6H), 1.260 (s, 4H),
(
7
t, J = 7.2 Hz, 4H), 6.62 (d, J = 12.8 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H),
3
.58 (t, J = 7.6 Hz, 2H), 7.78 (m, 3H), 8.00 (d, J = 7.6 Hz, 2H); 13
1.660 (s, 4H), 1.933 (s, 4H), 4.631 (s, 4H), 6.983 (s, 2H), 7.696−
C
7
2
1
.258 (m, 15H); 13C NMR (100 MHz, DMSO-d ) δ 14.15, 19.90,
NMR (100 MHz, DMSO-d ) δ 13.70, 19.41, 29.44, 46.08, 98.78,
1
yield; mp >260 °C.
6
6
9.96, 46.73, 110.22, 113.82, 123.25, 125.43, 126.40, 128.45, 128.69,
29.58, 130.15, 136.32, 140.63, 140.70, 161.82.
13.64, 123.06, 125.22, 128.08, 139.48, 141.26, 146.68, 164.62; 66%
3
-Ethyl-2-methylnaphtho[2,1-d]thiazol-3-ium Iodide (42). 1H
3
-(3-Phenylpropyl)-2-((1E,3Z)-3-(3-(3-phenylpropyl)benzo[d]-
thiazol-2(3H)-ylidene)prop-1-en-1-yl)benzo[d]thiazol-3-ium Bro-
mide (36). H NMR (400 MHz, DMSO-d ) δ2.09 (t, J = 7.2 Hz,
NMR (400 MHz, DMSO-d ) δ 1.51 (t, J = 7.2 Hz, 3H), 3.30 (s,
6
1
3H), 4.88 (q, J = 7.2 Hz, 2H), 7.87 (m, 2H), 8.30 (d, J = 8.0 Hz, 1H),
8.43 (m, 3H); 75% yield.
6
4
7
H), 2.79 (m, 4H), 4.38 (t, J = 7.2 Hz, 4H), 6.43 (d, J = 12.0 Hz, 2H),
.26 (m, 12H),7.56 (t, J = 6.8 Hz, 2H), 7.69 (d, J = 7.2 Hz, 2H), 7.76
3-Butyl-2-methylnaphtho[2,1-d]thiazol-3-ium Iodide (43). 1H
13
(
t, J = 12.8 Hz, 1H), 7.99 (d, J = 7.2 Hz, 2H); C NMR (100 MHz,
NMR (400 MHz, DMSO-d ) δ 0.95 (t, J = 7.2 Hz, 3H), 1.49 (q,
6
DMSO-d ) δ 28.57, 31.78, 45.82, 113.37, 122.89, 125.09, 125.94,
J = 7.2 Hz, 2H), 1.884 (m, 2H), 3.31 (s, 3H), 4.83 (t, J = 7.2 Hz, 2H),
7.86 (m, 2H), 8.30 (d, J = 7.6 Hz, 1 H), 8.41 (m, 3H); 90% yield.
2-Methyl-3-(3-phenylpropyl)naphtho[2,1-d]thiazol-3-ium Bro-
mide (44). Compound 44 was used without purification.
Synthesis of Napthathiazole-Based Trimethine Cyanines.
Corresponding alkylated heterocycle was added to an oven-dried and
6
+
1
27.91, 128.05, 128.25, 140.51, 141.07; HRMS calcd for C H N S
35 33 2 2
m/z 545.2080, obsd m/z 545.2061; 67% yield; mp 220−221 °C.
6
-Bromo-2-((1E,3Z)-3-(6-bromo-3-ethylbenzo[d]thiazol-2(3H)-
ylidene)prop-1-en-1-yl)-3-ethylbenzo[d]thiazol-3-ium Iodide (37).
1
H NMR (400 MHz, DMSO-d ) δ 1.32 (t, J = 6.8 Hz, 6H), 4.36
6
L
DOI: 10.1021/jm501452j
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
nitrogen cooled round-bottom flask followed by acetic anhydride
Hz, 6H), 1.962 (s, 12H), 4.297 (t, J = 8 Hz, 4H), 6.37 (d, J = 12 Hz,
2H), 6.637 (t, J = 12 Hz, 1H), 7.51 (t, J = 8 Hz, 2H), 6.68 (t, J = 8 Hz,
2H), 7.74 (d, J = 8 Hz, 2H), 8.08 (t, J = 8 Hz, 4H), 8.25 (d, J = 8 Hz,
(
8 mL) and triethyl orthoformate (3 mol equiv). The reaction mixture
was heated at 90 °C, and the reaction was monitored using UV−vis
and TLC. After the starting materials were consumed, the reaction
mixture was poured into diethyl ether under heavy stirring resulting in
the precipitation of the compound. The crystals were filtered and
isolated using column chromatography to obtain the final binding
agents.
2H), 8.46 (t, J = 12 Hz, 2H); 13C NMR (100 MHz, DMSO-d ) δ
6
11.96, 26.17, 48.04, 50.19, 102.05, 110.90, 121.58, 124.20, 125.07,
127.11, 127.19, 129.39, 129.83, 130.76, 132.72, 138.77, 152.54, 172.71;
+
HRMS calcd for [C H N ] 511.3113, found 511.3098; 79% yield;
37
39
2
mp 266−268 °C.
3
-Ethyl-2-((1E,3Z)-3-(3-ethylnaphtho[2,1-d]thiazol-2(3H)-
2-((1E,3Z,5E)-3-Chloro-5-(3-ethyl-1,1-dimethyl-1H-benzo[e]indol-
1
ylidene)prop-1-en-1-yl)naphtho[2,1-d]thiazol-3-ium Iodide (45). H
2(3H)-ylidene)penta-1,3-dien-1-yl)-3-ethyl-1,1-dimethyl-1H-benzo-
1
NMR (400 MHz, DMSO-d ) δ 1.40 (t, J = 6.4 Hz, 6H), 4.47 (q, J =
[e]indol-3-ium Iodide (52). H NMR (400 MHz, DMSO-d ) δ 1.39
6
6
6
.0 Hz, 4H), 6.67 (d, J = 12.4 Hz, 2H), 7.55 (t, J = 7.6 Hz, 2H), 7.93
(s, 6H), 2.00 (s, 12H), 4.37 (s, 4H), 6.38 (d, J = 16 Hz, 2H), 7.55 (t,
2H), 7.73 (t, 2H), 7.84 (d, J = 8 Hz, 2H), 8.12 (m, 4H), 8.27 (d, J = 4
13
(
m, 5H), 8.06 (d, J = 7.2 Hz, 2H), 8.14 (d, J = 7.6 Hz, 2H); C NMR
(
100 MHz, DMSO-d ) δ 13.47, 42.43, 99.21, 113.18, 121.40, 123.52,
Hz, 2H), 8.65 (d, J = 16 Hz, 2H); 13C NMR (100 MHz, DMSO-d ) δ
6
6
1
26.75, 127.00, 129.07, 129.59, 129.93, 130.64, 139.39, 145.86, 163.44;
11.81, 25.80, 50.72, 98.52, 111.19, 121.60, 121.71, 124.67, 126.96,
127.37, 129.47, 130.01, 131.15, 133.41, 138.56, 146.10, 174.17; HRMS
+
HRMS calcd for C H N S m/z 465.1454, obsd m/z 465.1455; 70%
yield; mp >260 °C.
29
25
2 2
+
calcd for [C H N Cl] 545.2724, found 545.2717; 68% yield; mp
37
38
2
3
-Butyl-2-((1E,3Z)-3-(3-butylnaphtho[2,1-d]thiazol-2(3H)-
231−233 °C.
1
ylidene)prop-1-en-1-yl)naphtho[2,1-d]thiazol-3-ium Iodide (46). H
NMR (400 MHz, DMSO-d ) δ 0.96 (t, J = 7.2 Hz, 6H), 1.47 (m, 4H),
2-((1E,3E,5E)-5-(1,1-Dimethyl-3-(3-phenylpropyl)-1H-benzo[e]-
indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-3-(3-phenyl-
6
1
1
(
8
.78 (m, 4H), 4.45 (t, J = 7.2 Hz, 4H), 6.69 (d, J = 12.8 Hz, 2H), 7.61
t, J = 8.0 Hz, 2H), 7.74 (t, J = 8.0 Hz, 2H), 7.93 (m, 5H), 8.10 (d, J =
.0 Hz, 2H), 8.16 (d, J = 9.2 Hz, 2H); 13C NMR (100 MHz, DMSO-
d6) δ 14.22, 19.88, 30.28, 47.00, 99.44, 113.48, 121.30, 123.59, 126.71,
27.11, 129.15, 129.65, 129.88, 130.65, 139.86, 145.96, 163.86; HRMS
propyl)-1H-benzo[e]indol-3-ium Iodide (53). H NMR (400 MHz,
DMSO-d ) δ 1.95 (s, 12H), 2.05 (t, 8.4 Hz, 4H), 2.78 (t, J = 8.0 Hz,
6
4H), 4.29 (t, J = 6.8 Hz, 4H), 6.27 (d, J = 13.6, 2H), 6.55 (t, J = 12.4
Hz, 1H), 7.31−7.17 (m, 10H), 7.49 (t, J = 11.2 Hz, 2H), 7.72−7.64
1
(m, 4H), 8.07−8.03 (m, 4H), 8.24 (d, J = 8.4, 2H), 8.49 (t, J = 13.2,
+
2
13
calcd for C H N S m/z 521.2080, obsd m/z 521.2075; 60% yield;
2H); C NMR (100 MHz, DMSO-d ) δ 27.24, 29.53, 32.55, 43.74,
33
33
2
6
mp >260 °C.
51.19, 103.40, 112.01, 119.44, 122.59, 125.21, 125.54, 128.05, 128.19,
128. 73, 128.90, 130.38, 130.75, 131.74, 133.63, 140.13, 141.49,
3
-(3-Phenylpropyl)-2-((1E,3Z)-3-(3-(3-phenylpropyl)naphtho[2,1-
+
d]thiazol-2(3H)-ylidene)prop-1-en-1-yl)naphtho[2,1-d]thiazol-3-
ium Iodide (47). H NMR (400 MHz, DMSO-d ) δ 2.15 (m, 4H),
153.41, 174.13; HRMS calcd for [C H N ] 691.4052, found
51
51
2
1
6
691.4028; 33% yield; mp 95−98 °C.
2
.82 (m, 4H), 4.515 (m, 4H), 6.52 (d, J = 12.4 Hz, 2H), 7.21 (m, 2H),
.28 (m, 7H), 7.64 (t, J = 7.6 Hz, 2H), 7.76 (t, J = 7.6 Hz, 2H), 7.92
2
-((1E,3Z,5E)-3-Chloro-5-(1,1-dimethyl-3-(3-phenylpropyl)-1H-
7
(
(
benzo[e]indol-2(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-3-(3-
m, 3H), 8.00 (d, J = 8.0 Hz, 2H), 8.15 (t, J = 9.6 Hz, 4H); 13C NMR
1
phenylpropyl)-1H-benzo[e]indol-3-ium Bromide (54). H NMR (400
100 MHz, DMSO-d ) δ 29.60, 32.34, 46.82, 113.39, 121.42, 123.60,
MHz, DMSO-d ) δ 1.98 (s, 12H), 2.04 (t, J = 4.0 Hz, 4H), 2.82 (t, J =
6
6
1
26.59, 126.74,127.16, 128.70, 128.90, 129.19, 129.69, 129.88, 130.69,
4.0 Hz, 4H), 4.31 (t, J = 8.0 Hz, 4H), 6.235 (d, J = 16.0 Hz, 2H),
7.23−7.36 (m, 10H), 7.57 (t, J = 8.0 Hz, 2H), 7.72 (t, J = 8.0 Hz, 2H),
7.82 (d, J = 8.0 Hz, 2H), 8.12 (t, J = 8.0 Hz, 4H), 8.28 (d, J = 8.0 Hz,
+
139.86, 141.18, 145.94; HRMS calcd for C H N S m/z 645.2393,
43
37
2 2
obsd m/z 645.2375; 50% yield; mp 231−233 °C.
2H), 8.58 (d, J = 12.0 Hz, 2H); 13C NMR (100 MHz, DMSO-d ) δ
Synthesis of meso-Methylated Napthothiazole Compound
8. 3-Ethyl-2-((1E,3Z)-3-(3-ethylnaphtho[2,1-d]thiazol-2(3H)-yli-
6
4
2
1
1
7
7.65, 29.77, 33.39, 44.93, 52.44, 100.59, 112.99, 123.47, 123.60,
26.49, 127.42, 128.68, 129.17, 129.55, 129.80, 131.22, 131.73, 132.90,
35.16, 140.62, 141.89, 147.75, 176.31; HRMS calcd for C H N Cl
dene)-2-methylprop-1-en-1-yl)naphtho[2,1-d]thiazol-3-ium Iodide
1
(48). H NMR (400 MHz, DMSO-d ) δ 1.51 (t, J = 6.8 Hz, 6H),
6
5
1
50
2
2
.74 (s, 3H), 4.67 (q, J = 6.8 Hz, 4H), 6.72 (m, 2H), 7.66 (t, J = 7.6
25.3663, found 725.3663; 47% yield; mp 165−168 °C.
. Peptide Synthesis. Peptides were synthesized using standard
solid phase peptide synthesis protocols, purified on C-18 RP-HPLC,
Hz, 2H), 7.78 (t, J = 7.6 Hz, 2H), 8.07 (m, 2H), 8.11 (d, J = 8.4 Hz,
3
2H), 8.16 (d, J = 8.0 Hz, 2H), 8.23 (d, J = 9.2 Hz, 2 H).
Pentamethine precursor 49 has been previously described by our
43,76,77
and confirmed with MALDI-MS as described before.
The se-
lab and was used as described without any additional modifications or
purification.
quence of the NH -terminal 20 aa peptide of histone H4, H4(1−20),
56
2
is Ac-SGRGKGGKGLGKGGAKRHRK. The sequence of K20-
biotinated H4(1−20), H4(1−20)_BTN, is Ac-SGRGKGG-
KGLGKGGAKRHRK(biotin). The biotin is connected to the side
chain amino group. The sequence of the glycine- and arginine-rich
peptide, R4, is Ac-GGRGGFGGRGGKGGRGGFGGRGGFG. Under-
lined R letters are the arginines that are methylated in the assay.
Synthesis of Pentamethine Cyanine Binding Agents 50−54.
Corresponding heterocyclic derivative (1 mol equiv) was added to a
round-bottom flask followed by the pentamethine precursor (2 mol
equiv). This mixture was dissolved in acetic anhydride (5 mL), and
sodium acetate was added (4 mol equiv). The solution was stirred and
heated to 60 °C for 2−4 h or until TLC indicated the complete
reaction of starting materials. The reaction mixture was allowed to
cool, and diethyl ether was added to the round-bottom flask. The
obtained crystals were dissolved in DCM and gravity-filtered to
remove unreacted sodium acetate from the final compound. The liquid
was concentrated and purified on column chromatography, eluting
with 2−5% methanol in DCM.
4. Protein Expression and Purification. PRMT1, -3, -6, and -8
are His6x-tagged proteins, and PRMT4 (usually also called CARM1)
is a GST-tagged protein. The expression and purification have been
41−43,48,77−79
described in previous work.
Generally speaking, the
plasmid was transformed into E. coli BL21(DE3) using heat shock
method and the expression of protein was induced with IPTG. Next,
bacteria were precipitated and harvested by centrifugation, followed by
cell lysis in cell disruptor. Then His6x-tagged protein was purified on
Ni-NTA beads and GST-tagged protein on glutathione agarose beads.
Protein concentration was determined with Bradford assay.
5
-Bromo-2-((1E,3Z)-3-bromo-5-((E)-5-bromo-1-butyl-3,3-dime-
thylindolin-2-ylidene)penta-1,3-dien-1-yl)-1-butyl-3,3-dimethyl-3H-
indol-1-ium Iodide (50). H NMR (400 MHz, DMSO-d ) δ 0.936 (t,
J = 7.2 Hz, 6H), 1.377 (q, J = 7.2 Hz, 4H), 1.727 (s, 16H), 4.178 (bs,
4
1
6
5
. Single-Dose Inhibition Study with PRMT1 Enzyme.
H), 6.340 (d, J = 13.2 Hz, 2H), 7.498 (d, J = 8.4 Hz, 2H), 7.630 (d,
52
13
Scintillation proximity assay (SPA) was performed in a 96-well
plate at room temperature (about 25 °C). The reaction buffer con-
tained 50 mM HEPES (pH 8.0), 1 mM EDTA, 50 mM NaCl, and
J = 8.4 Hz, 2H), 8.031 (s, 2H), 8.466 (d, J = 13.2 Hz, 2H); C NMR
100 MHz, DMSO-d ) δ 14.12, 19.93, 26.96, 29.38, 44.31, 50.10,
(
1
1
6
00.67, 114.20, 118.52, 123.23, 126.44, 131.76, 141.63, 144.25, 148.19,
0
.5 mM dithiothreitol (DTT). In the tested group, 6 μL of compound
74.47; 61% yield; mp 260−262 °C.
3
(10, 30, or 90 μM) was incubated with 18 μL mixture of [ H]-labeled
3
-Ethyl-2-((1E,3E,5E)-5-(3-ethyl-1,1-dimethyl-1H-benzo[e]indol-
2
3
(3H)-ylidene)penta-1,3-dien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-
SAM (0.5 μM) and His6x-PRMT1 (0.02 μM) for 5 min before
initiating the reaction with the addition of 6 μL of H4(1−20)_BTN
1
-ium Iodide (51). H NMR (400 MHz, DMSO-d ) δ 1.333 (t, J = 8
6
M
DOI: 10.1021/jm501452j
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
peptide (1 μM). After 8 min, the reaction was quenched with 30 μL of
isopropanol and then mixed with 10 μL of streptavidin-coated SPA
beads (20 mg/mL). The products were determined by Microbeta2
scintillation counter. Positive control was carried out under the same
conditions with 6 μL of water replacing the tested compound and
Ten poses of compound 50 with highest −CDOCKER_ENERGY
were generated. Then the crystal structure of PRMT1 (PDB code
1OR8) together with the cocrystallized SAH was superimposed with
PRMT1_αX(−) docked with conformer 1 of compound 50 to do the
comparison. Other parameters were used with the default setting.
3
negative control only contained PRMT1 and [ H]SAM. Inhibition
ratio is calculated by the following equation: inhibition % = ((P − A)/
ASSOCIATED CONTENT
Supporting Information
Additional experimental and computational data. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
(
P − N)) × 100, in which A is the remaining activity of tested group,
*
S
P is positive control, and N is negative control.
. IC50 and n Determination. To measure the IC and n , serial
6
H
50
H
concentrations of the tested compound were tested against varied
PRMTs. SPA assay was performed for PRMT1, PRMT5, and PRMT8
in the same way as the single-dose inhibition study with H4(1−20)
AUTHOR INFORMATION
Corresponding Authors
M.H.: phone, 404-413-5566; fax, 404-413-5505; e-mail,
mhenary1@gsu.edu.
Y.G.Z.: phone, 706-542-0277; fax, 706-542-5358; e-mail,
■
*
_
BTN as substrate. For the enzymes with R4 peptide (PRMT3 and
PRMT6) or H3.3 protein (CARM1) as substrate, P81 filter paper
41−43,48
binding assay
was used. In this assay, the reaction was carried
out as in SPA assay and quenched by applying 20 μL of mixture on
P81 filter paper disks. Then the disks were successively subjected to
*
yzheng@uga.edu.
air-drying and wash (50 mM NaHCO buffer, pH 9). The redried
3
disks were submerged in liquid scintillation cocktail, and the
Microbeta2 counter was used to quantify the products. The readout
was plotted against concentration and then fitted with IC equation
Notes
The authors declare no competing financial interest.
50
n
H
modified with the Hill equation: A = (1/(1 + ([I]/IC ) )), in which
50
ACKNOWLEDGMENTS
■
nH is the Hill coefficient, A is the remaining activity of tested group,
This work was supported by NIH Grant R01GM086717
(Y.G.Z.) and a Brains and Behavior grant (M.H.). We thank the
members of Zheng group for PRMT protein expression and
histone peptide synthesis. E.A.O was supported through a
Graduate Fellowship through the Center for Diagnostics and
Therapeutics.
and [I] is the compound concentration. For each reaction, the con-
3
centrations of [ H]SAM and the substrate were set around the
respective K values (which were predetermined).
m
7
. Fluorescence Titration. The fluorescence intensity was
measured using a Fluoro-Max 4 fluorimeter (Horiba Jobin Yvon). A
25 μL mixture containing 0.6 μM compound 50 and 30 μM PRMT1
1
was added into a fluorescence cuvette (Hellma 105.253-QS) and
incubated for 2 min. Then an increasing amount of compound 50
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