BRD4 Structure-Activity Relationships of Dual PLK1 Kinase/BRD4 Bromodomain Inhibitor BI-2536

Article abstract of DOI:10.1021/acsmedchemlett.5b00084

A focused library of analogues of the dual PLK1 kinase/BRD4 bromodomain inhibitor BI-2536 was prepared and then analyzed for BRD4 and PLK1 inhibitory activities. Particularly, replacement of the cyclopentyl group with a 3-bromobenzyl moiety afforded the most potent BRD4 inhibitor of the series (39j) with a K_i = 8.7 nM, which was equipotent against PLK1. The superior affinity of 39j over the parental compound to BRD4 possibly derives from improved interactions with the WPF shelf. Meanwhile, substitution of the pyrimidine NH with an oxygen atom reversed the PLK1/BRD4 selectivity to convert BI-2536 into a BRD4-selective inhibitor, likely owing to the loss of a critical hydrogen bond in PLK1. We believe further fine-tuning will furnish a BRD4 "magic bullet" or an even more potent PLK1/BRD4 dual inhibitor toward the expansion and improved efficacy of the chemotherapy arsenal.

Full text of DOI:10.1021/acsmedchemlett.5b00084

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Letter
BRD4 Structure–Activity Relationships of Dual
PLK1 Kinase–BRD4 Bromodomain Inhibitor BI-2536
Lijia Chen, Jeremy L. Yap, Makoto Yoshioka, Maryanna E Lanning, Rachel N
Fountain, Jacob A Scheenstra, Mithun Raje, Jeffrey W Strovel, and Steven Fletcher
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00084 • Publication Date (Web): 18 May 2015
Downloaded from http://pubs.acs.org on May 26, 2015
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BRD4 Structure–Activity Relationships of Dual PLK1 Kinase/BRD4 Bro-
modomain Inhibitor BI-2536
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Lijia Chen,† Jeremy L. Yap,† Makoto Yoshioka,∆ Maryanna E. Lanning,† Rachel N. Fountain,†
Mithun Raje,† Jacob A. Scheenstra,† Jeffrey W. Strovel∆ and Steven Fletcher*†$
†Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N. Pine St., Baltimore, MD
21201; ∆ConverGene, 22, Firstfield Road, Gaithersburg, MD 20878, USA; $University of Maryland Greenebaum Can-
cer Center, 22 S. Green St., Baltimore, MD 21201, USA.
KEYWORDS bromodomain; BRD; BET; protein–protein interaction; kinase; BI-2536
ABSTRACT: A focused library of analogues of the dual PLK1 kinase/BRD4 bromodomain inhibitor BI-2536 was prepared
and then analyzed for BRD4 and PLK1 inhibitory activities. Particularly, replacement of the cyclopentyl group with a 3-
bromobenzyl moiety afforded the most potent BRD4 inhibitor of the series (39j) with a Ki = 8.7 nM, which was equipotent
against PLK1. The superior affinity of 39j to BRD4 possibly derives from improved interactions with the WPF shelf.
Meanwhile, substitution of the pyrimidine NH with an oxygen atom reversed the PLK1/BRD4 selectivity to convert BI-
2536 into a BRD4-selective inhibitor, likely owing to the loss of a critical hydrogen bond in PLK1. Most compounds inhib-
ited growth of the acute myeloid leukemia cell line MV4-11 with sub-micromolar GI50 values. We believe further fine-
tuning will furnish a BRD4 “magic bullet” or an even more potent PLK1/BRD4 dual inhibitor towards the expansion and
improved efficacy of the chemotherapy arsenal.
Cancer remains one of the most challenging pathologies
of our time owing to its manifestation through the aber-
rant regulation of multiple signaling pathways; for exam-
ple, the upregulation of the anti-apoptotic Bcl-2 proteins
and the dysregulation of various kinases.1 A conventional
strategy to block cancer cell growth is to inhibit at least
one of the proteins involved in these pathways. However,
whilst target-selective inhibitors may function well in
vitro, the upregulation of compensatory signaling path-
ways often compromises their efficacies in cells. Rational-
ly designed polypharmacology, in which a single synthetic
agent is fashioned to recognize several key biological tar-
gets, carries the promise to deliver drugs that are more
efficacious in cells, as well as overcome the drawbacks
associated with multi-drug regimens.2 Imatinib, like most
of the other FDA-approved kinase inhibitors, owes much
of its success to targeting multiple kinases involved in
tumor development and progression.3 Given the efficacy
of imatinib, a notion that is gaining much traction is that
the next generation of anti-cancer agents should go one
stage further and target multiple protein targets of differ-
ent families that are involved in tumorigenesis.4
(1) is also a potent inhibitor of BRD4, which was con-
firmed through an Alpha Screen (IC50 = 25 nM),9 isother-
mal titration calorimetry (Kd = 37 nM),10 and co-crystal
structures.9,10 The BRD proteins recognize ε-N-acetylated
lysines of histones and have been dubbed epigenetic
“readers”.11 The bromo and extra-terminal domain (BET)
proteins of the BRD family (BRD2, BRD3, BRD4 and
BRDT) have recently emerged as druggable targets for the
development of new anti-cancer agents owing to their
roles in the transcriptional regulation of genes involved in
tumour development (e.g. c-MYC) and survival (e.g.
BCL2).12-14 Particularly, since the BET proteins regulate the
transcription of c-Myc, their inhibition provides an alter-
native and indirect strategy to counter tumorigenesis,
which is urgently needed given the difficulties associated
through targeting c-Myc with synthetic agents.15–21
Herein, we describe a focused structure–activity relation-
ship (SAR) study of BI-2536 (1) against BRD4. Our find-
ings hint at possible strategies to enhance the dual inhibi-
tory activity of BI-2536 as well as to render the ligand
more selective for BRD4 over PLK1. The ability to fine
tune a ligand in this way may help in the expansion of
personalized medicines, and, more generally, may assist
in the delineation of biochemical pathways targeted by
other drugs with polypharmacological profiles.
Polo-like kinase 1 (PLK1) and bromodomain 4 (BRD4) are
both drivers in acute myeloid leukemia (AML).5,6 They are
also intricately involved in mitosis.7,8 Their dual inhibition
by a single drug molecule might provide the platform for
a new strategy to treat AML as well as various other can-
cers. Recently, the laboratories of Knapp and Schönbrunn
independently discovered that the PLK1 inhibitor BI-2536
The crystal structure of BI-2536 bound to the first bromo-
domain of BRD4, BRD4(1), is shown in Figure 1 (PDB ID:
40GI11). The methylated amide of BI-2536 functions as a
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format and colored by atom type. BI-2536 is shown in stick
format and colored by atom type (green = carbon).
mimetic of ε-N-acetylated lysine wherein the carbonyl
forms a water-mediated hydrogen bond with the side
chain amide of N140 while the methyl group is directed
into a hydrophobic sub-pocket formed from F83, M132
and C136. The aniline NH and one of the pyrimidine ni-
trogen atoms bind the backbone amide of Q85 also
through an intermediary water molecule. The ethyl group
1
2
3
4
5
6
7
8
Accordingly, we prepared a focused library of analogues
of BI-2536 by adapting a previously described synthesis of
BI-2536.22 Briefly, the appropriate amino acids 2–5 were
first esterified and then N-alkylated by reductive amina-
tion to deliver compounds 6–11. Regioselective nucleo-
philic aromatic substitutions (SNAr) of 2,6-dichloro-3-
nitropyrimidine furnished tertiary anilines 12–17, wherein
the nitro group directed ortho attack of amines 6–11
through an intramolecular, hydrogen-bonded, six-
membered transition state.23,24 The dihydropteridinone
scaffold was next constructed by reductive heterocycliza-
tions of 12–17 into 18–23, respectively, with iron powder in
hot AcOH. Introduction of the R3 group was achieved by
deprotonation of the anilide NH followed by quenching
with various alkyl iodides and bromides to yield the 2-
chloropyrimidines 24–33. Meanwhile, to complete the
synthesis of BI-2536 and its analogues, the requisite ani-
lines were prepared as in Scheme 2. Briefly, various 4-
nitrobenzoic acids (34a-g) were activated by HBTU and
then coupled to 4-amino-1-methylpiperidine to deliver
amides 35a-g, which were subsequently reduced with tin
(II) chloride (SnCl2) to yield anilines 36a-g. Phenol 38 was
prepared by the condensation of vanillic acid (37) with 4-
amino-1-methylpiperidine. Finally, HCl-assisted SNAr of
the 2-chloropyrimidines 24–33 with anilines 36a-g and
phenol 38 furnished the lead compound BI-2536 (1) and
its analogues 39a–q, whose structures are given in full in
Tables 1 and 2.
is projected into
a small hydrophobic sub-pocket
(V87/L92/L94/Y97), while the cyclopentyl moiety and the
N-methylpiperidine point to the solvent. Close inspection
of the binding site indicates that replacement of the am-
ide methyl group with a slightly larger ethyl or isopropyl
might enhance binding affinity to BRD4; large groups,
such as benzyl, conversely, are not expected to be ac-
commodated here. Introduction of a kink into the cyclo-
pentyl group might allow it to interact better with the
WPF shelf (W81, P82, F83). Finally, elaboration of the 3-
methoxy group is predicted to improve binding affinity
through interactions with L92 and/or L94. One of the
pyrimidine nitrogens and the aniline NH form critical
hydrogen bonds with C133 in PLK1 (PDB ID = 2RKU). As
suggested by Knapp et al., substitution of this NH with
NMe will result in the loss of a hydrogen bond donor and
a concomitant reduction in PLK1 binding affinity.10 At the
same time, this is expected to have limited impact on the
inhibition of BRD4 since this group points to the solvent.
Thus, towards the optimization of the BRD4 inhibitory
activity of BI-2536, the methyl, ethyl and cyclopentyl moi-
eties were varied, as was the 3-methoxy group. Substitu-
tion of the aniline NH with an oxygen atom should render
this fragment incapable of functioning as a hydrogen
bond donor, and was, therefore, predicted to furnish
BRD4 selectivity. The aminopiperidine moiety was main-
9
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Table 1 shows the derivatives with variations of substitu-
ents on the dihydropteridinone ring along with associated
BRD4 and PLK1 inhibitory activities. The data is presented
as Ki values, which were obtained with BROMOscan® and
KINOMEscan®, DiscoveRx‘s proprietary ligand binding
competition assays that measure interactions between
test compounds and bromodomains or kinases, respec-
tively. We obtained similar binding data for the parent
compound BI-2536 to that reported previously for
BRD49,10 and PLK1.25 It is unsurprising that BI-2536 is
>250-fold more selective for PLK1 given the extensive op-
timization that led to its discovery.25 Replacement of the
CONMe group with a CONH group reduced its activity by
almost 150-fold, supporting its functional role as a mimet-
ic of ε-N-acetylated lysine moieties. Interestingly, alt-
hough the binding pocket that accommodates this methyl
group appears large enough to allow occupancy by slight-
ly larger groups such as ethyl and isopropyl, all groups
bigger than methyl resulted in weaker inhibitors. For ex-
ample, the Ki values for compounds with R3 = Me, Et, iPr,
Bn were 56, 470, 4500 and >10,000 nM, respectively. Alt-
hough still potent inhibitors, increasing the size of the R3
group was not tolerated by PLK1, either.
N
O
N
N
O
N
N
H
N
N
H
OMe
BI-2536 (1)
tained to assist with compound solubility.
Figure 1. Crystal structure of BI-2536 bound to BRD4(1) (PDB
ID = 4OGI).10 Key side chains of BRD4(1) are shown in stick
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NO2
R2
R1
R1
N
1
2
3
4
a, b
c
R2
H2N
CO2H
N
H
CO2Me
Cl
N
N
R1
CO2Me
5
6
7
8
: R1= (R)-Bn, R2=Cp
12: R1= (R)-Et, R2= Cp
13: R1= H, R2= Cp
14: R1= (S)-Et, R2= Cp
2
3
: R1= (R)-Et
: R1= H
6
7
8
: R1= (R)-Et, R2= Cp
: R1= H, R2= Cp
: R1= (S)-Et, R2= Cp
15: R1= (R)-Bn, R2=Cp
16: R1= (R)-Et, R2= iBu
17: R1= (R)-Et,
9
10: R1= (R)-Et, R2= iBu
11: R1= (R)-Et,
4: R1= (S)-Et
: R1= (R)-Bn
R2= 3-Br-C6H4CH2
R2= 3-Br-C6H4CH2
5
9
R3
10
11
12
13
14
15
16
17
18
19
20
21
22
H
N
O
e
d
N
O
N
N
Cl
N
N
R2
R1
Cl
N
N
R2
R1
24: R1= (R)-Et, R2= Cp, R3= Me
25: R1= H, R2= Cp, R3= Me
26: R1= (S)-Et, R2= Cp, R3= Me
27: R1= (R)-Bn, R2= Cp, R3= Me
28: R1= (R)-Et, R2= iBu, R3= Me
29: R1= (R)-Et,
R2= 3-Br-C6H4CH2, R3= Me
30: R1= (R)-Et, R2= Cp, R3= Et
31: R1= (R)-Et, R2= Cp, R3= iPr
32: R1= (R)-Et, R2= Cp, R3= iBu
33: R1= (R)-Et, R2= Cp, R3= Bn
18: R1= (R)-Et, R2= Cp
19: R1= H, R2= Cp
20: R1= (S)-Et, R2= Cp
21: R1= (R)-Bn, R2=Cp
22: R1= (R)-Et, R2= iBu
23: R1= (R)-Et,
R2= 3-Br-C6H4CH2
Scheme 1. (a) SOCl2, MeOH, 0 °C to reflux, 16 h; (b) cyclopentanone, isobutyraldehyde or 3-bromobenzaldehyde, NaBH(OAc)3,
NaOAc, CH2ClCH2Cl, RT, 16 h; (c) 2,4-dichloro-5-nitropyrimidine, K2CO3, acetone, RT, 16 h; (d) Fe, AcOH, 70 °C, 1 h, 100 °C, 4 – 5
h; (e) alkyl iodide or benzyl bromide, NaH, DMF, 0 °C to RT, 3 h.
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R3
N
NO2
N
O
N
O
a
c
O
N
H
N
R4
N
H
X
N
N
R2
R1
R4
R4
NY2
R4
O
OH
34g
a
b
c
d
e
= 3-OMe
= H
= 2-OMe
= 3-F
= 3-OiBu
f = 3-OBn
= 3-OCp
35a
36a
-
35g (Y = O)
36g (Y = H)
34a
-
1
,
39a-39p
b
-
OH
N
O
g
N
O
d
c
OMe
N
N
O
N
H
N
O
OMe
OH
N
H
N
OMe
O
OH
38
37
39q
Scheme 2. (a) 4-amino-1-methylpiperidine, HBTU, Et3N, DMF, RT, 16 h. (b) SnCl2•2H2O, EtOAc/ethanol, 10:3, 500C, 16 h; (c) 24–
33, c. HCl, EtOH/dioxane/H2O, 1:1:1, 100 °C, 24 – 48 h; (d) 4-amino-1-methylpiperidine, EDCI•HCl, HOBt•H2O, Et3N, CH3CN, RT,
16 h.
In the co-crystal structure of BRD4(1)–BI-2536, the ethyl
group at the R1 position points into the pocket formed
by V87, L92, L94 and Y97. Changing the stereochemistry
from R to S did not affect the binding affinity to BRD4
(compare 1 with 39g) but resulted in a two-fold drop in
potency against PLK1. With respect to BRD4 inhibition,
the S-Et group can point into a pocket on the other side
of V87/L92/L94/Y97 formed by P82, F83 and I146, and
this accounts for the subtle effect of the chirality
change. Deleting the ethyl group afforded a reduction in
BRD4 inhibitory activity of 26-fold (39f: Ki = 1400 nM),
whilst replacing it with a bulky R-benzyl group (com-
pound 39h) completely abolished binding. Together,
these data indicate that a small hydrophobic group is
optimal here, consistent with the crystal structure. In
PLK1 (PDB ID = 2RKU), the R-Et group is surrounded by
L59, C67, A80, K82 and L130. Similar to BRD4, this
pocket is largely tolerant of the chirality change but
cannot accommodate a larger benzyl group.
Whilst modifying the R1 and R3 groups exhibited only
detrimental impacts on BRD4 binding, changing the R2
group from cyclopentyl to 3-bromobenzyl increased the
BRD4 binding affinity by 7-fold (39j: Ki = 8.7 nM) alt-
hough a bulkier isobutyl group (39i) was not tolerated.
The potent binding affinity of 39j places it amongst the
most active BRD4 inhibitors known,14 and might be ex-
plained by hydrophobic interactions with the WPF shelf.
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Notably, compound 39j bound PLK1 25-fold weaker than
selectivity, and further optimization here might furnish
a selective, sub-nanomolar BRD4 inhibitor.
1
2
3
BI-2536, such that affinities for BRD4 and PLK1 had be-
come effectively equipotent. Taken together, these data
indicate that the R2 group appears to be a dictator of
4
5
Table 1. Structure–activity relationships of the R1, R2, R3, R4 and X groups. Cp = cyclopentyl.
6
R3
N
7
8
9
N
O
O
N
N
H
2
X
N
N
R2
R1
10
11
12
13
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R4
3
Compound
R1
R2
R3
R4
X
BRD4
Ki (nM)a
PLK1
Ki (nM)a
BI-2536
39a
39b
39c
(R)-Et
(R)-Et
(R)-Et
(R)-Et
(R)-Et
(R)-Et
H
Cp
Cp
Cp
Cp
Cp
Cp
Cp
Cp
Cp
iBu
Me
H
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
H
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
56 ± 9
7950 ± 1550
470 ± 10
4500 ± 300
>10000
>10000
1400 ± 400
54 ± 4
0.22 ± 0.01
ND
Et
0.91 ± 0.13
ND
iPr
iBu
Bn
39d
39e
39f
ND
ND
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
ND
39g
39h
39i
(S)-Et
(R)-Bn
(R)-Et
(R)-Et
(R)-Et
(R)-Et
(R)-Et
(R)-Et
(R)-Et
(R)-Et
(R)-Et
0.42 ± 0.04
4.20 ± 0.99
6.95 ± 1.63
5.80 ± 0.99
ND
>10000
970 ± 30
8.7 ± 1.3
60 ± 7
39j
3-Br-C6H4CH2
39k
39l
Cp
Cp
Cp
Cp
Cp
Cp
Cp
2-OMe
3-F
37 ± 5
ND
39m
39n
39o
39p
39q
46 ± 2
ND
3-OiBu
3-OBn
3-OCp
3-OMe
29 ± 9
ND
100 ± 0
27 ± 8
ND
0.84 ± 0.01
240 ± 14
99 ± 11
aData represent the mean and standard deviation of two independent experiments.
Shifting the 3-methoxy group to the 2-position (com-
pound 39l) did not have a significant impact on the bind-
ing to BRD4. Moreover, its deletion or replacement with a
fluorine atom also had little effect. Substitution of the 3-
methoxy group with bulkier isobutoxy (39n) and cyclo-
pentyloxy (39p) moieties afforded a 2-fold enhancement
in binding affinity to BRD4, which may be due to im-
proved contacts with L92 and/or L94, with a concomitant
4-fold reduction in binding affinity to PLK1, as predicted.10
Akin to our findings with the R2 group, careful modifica-
tion of the R4 group might provide further improvements
to BRD4 inhibitory activity. One of the most striking ob-
servations of our SAR work is seen when comparing the in
vitro data for BI-2536 with 39q wherein substitution of
the pyrimidine NH with an isosteric O afforded more
than a 1000-fold drop in activity against PLK1, yet only
resulted in a 2-fold reduction in activity against BRD4.
This result is consistent with the NH engaging in a critical
hinge interaction with C133 as a hydrogen bond donor in
the active site of PLK1, an interaction that is lost upon its
replacement with an oxygen atom; this group points to
the solvent in BRD4 and so a limited effect on binding
affinity was observed. The dramatic impact on binding
profiles indicates that the isosteric identity here is a key
determinant of ligand specificity, and may be exploited
towards achieving BRD4 selectivity. Indeed, the data for
39q implies that the PLK1 selective inhibitor BI-2536 has
been transformed into a BRD4 selective inhibitor.
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may account for the imperfect correlation between the in
1
vitro and cell data.
2
3
Table 3. MV4-11 cell viability exposed to BI-2536 analogues.
4
5
6
Compound
MV4-11,
GI50 (ꢀM)a
Compound
MV4-11,
GI50 (ꢀM)a
7
8
9
BI-2536
(+)-JQ-1
GSK-461364
39a
0.0152
0.08
39h
39i
1.87
0.04
10
11
12
13
14
15
16
17
0.679
0.219
0.392
2.67
39j
0.675
0.003
0.015
0.004
0.154
0.356
39k
39l
39b
39c
39m
39n
39o
39p
Figure 2. A high scoring, GOLD-predicted docking mode of
compound 39j in BRD4(1). Compound 39j shown in stick
format and colored by atom type (green = carbon).
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39d
2.53
39e
7.28
To gain a better appreciation of how our most potent
compound 39j likely binds BRD4(1), we employed molec-
ular modeling with GOLD. Figure 2 shows a GOLD high
scoring docking pose of the most potent BRD4 inhibitor
compound 39j bound to BRD4. Comparing to Figure 1,
compound 39j and BI-2536 bind to BRD4 in a very similar
pattern, and form largely identical interactions with
BRD4. Structurally, the only difference between these two
molecules is the replacement of the cyclopentyl group in
BI-2536 with a 3-bromobenzyl moiety. In BI-2536, the
cyclopentyl group does not appear to be involved in any
notable interactions with BRD4 as it is directed out into
the solvent. In contrast, the installation of the benzyl CH2
in 39j provides the extra flexibility to reach out to the
WPF shell (W81, P82, and F83), possibly accounting for
the 7-fold improvement in activity. Furthermore, the
bromine atom potentially forms a hydrophobic interac-
tion with I146, which might also explain the greater bind-
ing affinity for 39j. Therefore, future investigation into
the R2 position with flexible and hydrophobic arylmethyl
groups may lead to more potent BRD4 inhibitors.
39f
2.19
0.240
1.34
39g
39q
0.019
aCompound concentration that elicits 50% growth inhibition
of MV4-11 cells.
In summary, building on the initial discovery by Knapp
and Schönbrunn, an SAR campaign of the PLK1 inhibitor
BI-2536 has revealed opportunities for its continued op-
timization as a dual inhibitor of PLK1 and BRD4, as well
as a selective inhibitor of the latter. The impacts of struc-
tural modifications were largely in agreement with pre-
dictions made based on the PDB of the BRD4(1)–BI-2536
co-crystal structure. We believe that combining further
modifications to the R2 and R4 groups, as well as switch-
ing the pyrimidine NH to O, could provide selective, sub-
nanomolar BRD4 inhibitors. At the same time, we antici-
pate it will be feasible to enhance BRD4 inhibitory activi-
ty yet maintain dual PLK1/BRD4 inhibitory activity
through careful selection of the R4 side chain whilst re-
taining, most critically, the pyrimidine NH group, as well
as the cyclopentyl group and the R-stereochemistry of the
ethyl group. More generally, it is considered that drugs
with polypharmacological profiles may likewise be opti-
mized for inhibitory activities against specific deconvo-
luted targets, which may assist in a better understanding
of the biochemical pathways targeted within cells.
Compounds were also analyzed in a CellTiter-Blue® cell
viability assay with the MV4-11 cell line, which has an
acute myeloid leukemia cell line that expresses high levels
of BRD4 and PLK1. As positive controls, the BRD4-
selective inhibitor (+)-JQ-1 and the PLK1-selective inhibi-
tor GSK-461364 were used. The growth inhibition (GI50)
data is shown in Table 2, which represents the concentra-
tion of small-molecule that elicits 50% growth inhibition
of the MV4-11 cells. The overall trend of in vitro data is
mirrored by the cell data, supporting the conclusion that
cell death is through inhibition of BRD4 and PLK1. There
are a few anomalous results that require discussion. The
cell data for 39j and 39p are less potent, while 39i is more
potent than would be anticipated given their in vitro data.
Considering its polypharmacological profile, it is possible
that BI-2536, and its analogues, bind other targets in addi-
tion to BRD4 and PLK1, such as PLK2 and PLK3,25 which
ASSOCIATED CONTENT
Supporting Information. Synthetic procedures and assay
protocols. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* sfletcher@rx.umaryland.edu; 410-706-6361.
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Page 6 of 7
C.; Kung, A. L.; Bradner, J. E.; Mitsiades, C. S. BET
Bromodomain Inhibition as a Therapeutic Strategy to
Target c-Myc. Cell 2011, 146, 904–917.
Author Contributions
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All authors have given approval to the final version of the
manuscript.
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Wyce, A.; Ganji, G.; Smitheman, K. N.; Chung, C.-W.;
Korenchuk, S.; Bai, Y.; Barbash, O.; Le, B.; Craggs, P. D.;
McCabe, M. T.; Kennedy-Wilson, K. M.; Sanchez, L. V;
Gosmini, R. L.; Parr, N.; McHugh, C. F.; Dhanak, D.;
Prinjha, R. K.; Auger, K. R.; Tummino, P. J. BET Inhibition
Silences Expression of MYCN and BCL2 and Induces
Cytotoxicity in Neuroblastoma Tumor Models. PLoS One
2013, 8, e72967.
Brand, M.; Measures, A. M.; Wilson, B. G.; Cortopassi, W.
A.; Alexander, R.; Höss, M.; Hewings, D. S.; Rooney, T. P.;
Paton, R. S.; Conway, S. J. Small Molecule Inhibitors of
Bromodomain–Acetyl-lysine Interactions. ACS Chem. Biol.
2015, 10, 22–39.
Fletcher, S.; Prochownik, E. V. Small-Molecule Inhibitors of
the Myc Oncoprotein. Biochim. Biophys. Acta 2014, 1849,
525–543.
Albihn, A.; Johnsen, J. I.; Henriksson, M. A. MYC in
Oncogenesis and as a Target for Cancer Therapies. Adv.
Cancer Res. 2010, 107, 163–224.
Yap, J. L.; Chauhan, J.; Jung, K.-Y.; Chen, L.; Prochownik, E.
V.; Fletcher, S. Small-Molecule Inhibitors of Dimeric
Transcription Factors: Antagonism of Protein–protein and
protein–DNA Interactions. Medchemcomm 2012, 3, 541.
Chauhan, J.; Wang, H.; Yap, J. L.; Sabato, P. E.; Hu, A.;
Prochownik, E. V; Fletcher, S. Discovery of Methyl 4’-
Methyl-5-(7-nitrobenzo[c][1,2,5]oxadiazol-4-Yl)-[1,1'-
Biphenyl]-3-Carboxylate, an Improved Small-Molecule
Inhibitor of c-Myc-Max Dimerization. ChemMedChem 2014,
9, 2274–2285.
Wang, H.; Chauhan, J.; Hu, A.; Pendleton, K.; Yap, J. L.;
Sabato, P. E.; Jones, J. W.; Perri, M.; Yu, J.; Cione, E.; Kane,
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Max Heterodimerization with Improved Cell-Penetrating
Analogs of the Small Molecule 10074-G5. Oncotarget 2013,
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ACKNOWLEDGMENT
We thank the University of Maryland School of Pharmacy
(start-up funds (SF); fellowship (LC)), ConverGene and the
American Foundation for Pharmaceutical Education (fellow-
ship to JLY) for financial support of this research. Chemietek
is acknowledged for the generous donation of GSK-461364.
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ABBREVIATIONS
BRD4, bromodomain 4; BET, bromodomain and extra-
terminal domain; PLK1, polo-like kinase 1.
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Table of Contents Graphic
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small hydrophobic groups;
Me optimal
BI-2536 (1)
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N
O
N
N
O
N
N
small hydrophobic groups;
chirality of Et group
unimportant
H
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N
N
H
OMe
many substituents tolerated;
3-alkyoxy groups optimal
3-bromobenzylic group
improves activity
replacement with O:
BRD4 selective
(PLK1 activity reduced >1000-fold)
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