High-affinity small molecular blockers of mixed lineage leukemia 1 (MLL1)-WDR5 interaction inhibit MLL1 complex H3K4 methyltransferase activity

Article abstract of DOI:10.1016/j.ejmech.2016.08.036

MLL1-WDR5 protein-protein interaction is essential for MLL1 H3K4 methyltransferase activity. Targeting MLL1 enzymatic activity to regulate expression level of MLL-dependent genes represents a therapeutic strategy for acute leukemia harboring MLL fusion proteins. Herein we reported a series of biphenyl compounds disturbed MLL1-WDR5 interaction. These compounds effectively inhibited MLL1 histone methyltransferase (HMT) activity in vitro and in MV4-11 cell line. The representative compound 30 (DDO-2084) inhibited proliferation and induced apoptosis of MV4-11 cells through deregulating expression level of Hoxa9 and Meis-1 genes, which emphasized our compounds were on-target. Optimization of compound 30 led to high-affinity inhibitors. Especially, compound 42 (DDO-2117, IC₅₀= 7.6 nM) bearing an amino and a 4-aminobutanamido group was the most potent inhibitor reported to-date, and showed the most potent inhibitory activity (IC₅₀= 0.19 μM) in HMT assay.

Full text of DOI:10.1016/j.ejmech.2016.08.036

European Journal of Medicinal Chemistry 124 (2016) 480e489
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
High-affinity small molecular blockers of mixed lineage leukemia 1
(MLL1)-WDR5 interaction inhibit MLL1 complex H3K4
methyltransferase activity
,
b
Dong-Dong Li a, Wei-Lin Chen a, Zhi-Hui Wang a, Yi-Yue Xie a, Xiao-Li Xu a
,
,
,
,
b
,
, b, *
Zheng-Yu Jiang a b, Xiao-Jin Zhang a c, Qi-Dong You a **, Xiao-Ke Guo a
a State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing,
210009, China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China
c Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing, 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
MLL1-WDR5 protein-protein interaction is essential for MLL1 H3K4 methyltransferase activity. Targeting
MLL1 enzymatic activity to regulate expression level of MLL-dependent genes represents a therapeutic
strategy for acute leukemia harboring MLL fusion proteins. Herein we reported a series of biphenyl
compounds disturbed MLL1-WDR5 interaction. These compounds effectively inhibited MLL1 histone
methyltransferase (HMT) activity in vitro and in MV4-11 cell line. The representative compound 30
(DDO-2084) inhibited proliferation and induced apoptosis of MV4-11 cells through deregulating
expression level of Hoxa9 and Meis-1 genes, which emphasized our compounds were on-target. Opti-
mization of compound 30 led to high-affinity inhibitors. Especially, compound 42 (DDO-2117,
IC50 ¼ 7.6 nM) bearing an amino and a 4-aminobutanamido group was the most potent inhibitor re-
Received 9 May 2016
Received in revised form
22 July 2016
Accepted 17 August 2016
Available online 20 August 2016
Keywords:
MLL1-WDR5 interaction
Histone methyltransferase
Biphenyl inhibitors
Gene expression
Leukemia
ported to-date, and showed the most potent inhibitory activity (IC50 ¼ 0.19
mM) in HMT assay.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
mono-, di-, and trimethylated by histone methyltransferases
(HMTs) [5].
Epigenetic regulation plays a vital role in gene expression as the
changes of DNA sequences and transcription factors. DNA methyl-
ation and post-translational modification of histone are two main
categories of epigenetic modification [1]. Two copies of each his-
tone (H2A, H2B, H3 and H4) wrapped with DNA double helix
constitute the basic units of nucleosome [2]. Modifications of his-
tones include methylation, acetylation, phosphorylation, ubiquiti-
nation, sumoylation and other types of marks [3]. Catalyzed by
protein methyltransferases, methyl groups were transferred from
S-adenosyl-methionine (SAM) to the side chain nitrogen atoms of
lysine and arginine residues of histones [4]. Lysine residues can be
MLL1 was one of the HMTs family, specially catalyzing mono-,
di-, and trimethylation of histone 3 lysine 4 (H3K4) through its
evolutionarily conserved SET domain [6]. H3K4 methylation marks
were recognized by different common structural features such as
the royal superfamily and the PHD-finger superfamily (including
the PHD fingers of BPTF and ING proteins) [7], which was essential
for definitive hematopoiesis by regulating transcription activation
of Hox genes and associated cofactors [8].
Dysregulation of MLL1 catalytic function increased the expres-
sion of Hox genes, which was associated with acute lymphoid
leukemia (ALL) and acute myeloid leukemia (AML) [9]. Chromo-
somal rearrangements associated with MLL1 have been shown to
cause MLL fusion proteins that were observed in kinds of leukemia
[10]. MLL fusion proteins, lacking the SET domain and thus the
H3K4 HMT enzymatic activity, cooperated with wild type MLL1
complex to active MLL1 target genes and lead to leukemogenesis
[11].
* Corresponding author. State Key Laboratory of Natural Medicines and Jiangsu
Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing 210009, China.
** Corresponding author. State Key Laboratory of Natural Medicines and Jiangsu
Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing 210009, China.
MLL1 protein alone had weak histone methyltransferase
E-mail addresses: youqd@cpu.edu.cn (Q.-D. You), kexin95@126.com (X.-K. Guo).
http://dx.doi.org/10.1016/j.ejmech.2016.08.036
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

D.-D. Li et al. / European Journal of Medicinal Chemistry 124 (2016) 480e489
481
activity, but the enzymatic activity massively enhanced upon
complex formation of MLL1 with WDR5, ASH2L, and RBBP5 [12].
Disturbing MLL1-WDR5 protein-protein interaction (PPI) with
peptides or small molecules dramatically inhibited the MLL1 H3K4
HMT activity and thus the expression of Hox and Meis-1 genes
[11e15]. Hence, blocking the interaction of MLL1-WDR5 with in-
hibitors may have a potential for the treatment of leukemia
harboring MLL fusion proteins.
activity in vitro and in MV4-11 cell line. Down-regulating MLL-
target genes expression level and inducing apoptosis of MV4-
11 cells of compound 30 emphasized that our compounds were on-
target. Optimization of compound 30 led to high-affinity inhibitors.
Especially, up to today, compound 42 was the most potent small
molecular inhibitor of the MLL1-WDR5 interaction (IC50 ¼ 7.6 nM,
Kd ¼ 13.6 nM), and showed the most potent inhibitory activity
(IC50 ¼ 0.19
mM) in HMT assay.
The first inhibitor of MLL1-WDR5 interaction was commenced
by the report of Ac-ARA-NH2, truncated from WIN motif of MLL1 as
the minimum peptide, bound to WDR5 with high affinity [13].
Based on Ac-ARA-NH2, linear peptidomimetic inhibitors of MLL1-
WDR5 PPI were defined by modification of the Ala residue [11].
Then cyclic peptidomimetic MM-401 was designed by constraining
conformation of the linear peptide MM-102 (Fig. 1a) [15]. Three
small molecular inhibitors (WDR5-0101-0103) were disclosed by
screening diverse compounds libraries with fluorescence polari-
zation (FP) assay [14], then WDR5-47 (Fig. 1b) was obtained from
the optimization of WDR5-0102 on the basis of the co-crystal
structure of WDR5-0102 and WDR5 protein [16]. A more potent
antagonist OICR-9429 (Fig. 1b) was reported to explore the mech-
anism of p30-dependent transformation and establish the essential
p30 cofactor WDR5 as a therapeutic target in CEBPA-mutant AML
[17,18].
2. Results and discussion
2.1. Synthesis of MLL1-WDR5 interaction inhibitors (see supplying
information)
Compounds 2a-g with N-methylpiperazine moiety modification
were acquired following the synthetic route of WDR5-47 (Scheme
1). 2-Fluoro-5-nitroaniline reacted with 2-chloro-4-fluoro-3-
methylbenzoyl chloride to access intermediate 1. Subsequent
displacement of the fluoro with various amines, giving rise to the
However, even the most potent reported small molecular in-
hibitor, OICR-9429 bound to WDR5 protein with only a moderate
activity (Kd ¼ 93 nM). Here, based on our reported inhibitor W-26
(Fig. 1c) and SAR information [16,18,19], we designed and synthe-
tized a series of biphenyl inhibitors to block MLL1-WDR5 PPI.
Among those compounds, 30 effectively inhibited MLL1 HMT
Scheme 1. Synthesis of compounds 2a-g. Reaction: a. 2-chloro-4-fluoro-3-
methylbenzoyl chloride, pyridine, DCM, r.t., 3 h; b. K2CO3, CH3CN, reflux, 6h (2a) or
amines, DIPEA, DMF, 80 ꢀC, 4 h (2b-2g).
Fig. 1. Peptidomimetic and small molecular Inhibitors of MLL1-WDR5 interaction.

482
D.-D. Li et al. / European Journal of Medicinal Chemistry 124 (2016) 480e489
substituted benzamides 2a-g in high yields.
almost all of these modifications led to inactive compounds
(Table 1). That proved the criticality of the N-methylpiperazine
moiety. Hence, the N-methylpiperazine moiety was retained for
further optimization.
We had previously reported that the introduction of aromatic
ring A (Fig. 2) into 5-position of the structure of N-(2-(4-
methylpiperazin-1-yl) phenyl) benzamide was accepted. The aro-
matic ring occupied the hydrophobic groove of WDR5 surrounded
by side chains of Phe133, Phe149 and Tyr191 [19]. Given this po-
All N-(4-(4-methylpiperazin-1-yl)-[1,10-biphenyl]-3-yl) benza-
mide compounds (Fig. 2) in this article were synthetized from 4-
bromo-1-fluoro-2-nitrobenzene. In Scheme 2, 6a-c were prepared
through Suzuki coupling reaction with different boronic acids. The
fluoro of 6a-c was substituted with N-methylpiperazine generating
7a-c, followed by reducing 7a-c with Tin (II) dichloride dehydrate
to product 8a-c. With nitration of 2-chloro-4-fluoro-3-
methylbenzoic acid 3, 2-chloro-4-fluoro-3-methyl-5-nitrobenzoic
acid 4 was acquired. 8a-c reacted with 2-chloro-4-fluoro-3-
sition was located near to the solvent area and the
p-p stacking
methyl-5-nitrobenzoyl chloride
5
or 4-fluoro-3-nitrobenzoyl
interaction with an electron-rich aromatic ring Tyr191, a hydro-
philic or withdrawing substituent at the 4-position of aromatic ring
A was more suitable. That modifications of amide resulted in
complete loss of activity showed the necessity of the amide for this
structure binding to WDR5 [18,19]. Based on SAR information of
this structure, more potent inhibitors were designed and
synthetized.
chloride to get intermediate benzamides W-27, 16e17 or 26. Sub-
sequently, nitro compounds W-27, 16e17 and 26 were reduced to
yield anilines 18e20 and 27.
Compounds 15, 21 and 28 were synthetized from 2-chloro-4-
fluoro-3-methyl-5-nitrobenzoic acid 4 in Scheme 3. Compound 4
was reduced and acetylated to provide 13. Treatment 11 or 8b with
5-acetamido-2-chloro-4-fluoro-3-methylbenzoyl
generated compounds 15 or 28.15 was reduced to yield target
compound 21.
chloride
14
The benzamide moiety (ring C) of N-(4-(4-methylpiperazin-1-
yl)-[1,10-biphenyl]-3-yl) benzamide was optimized firstly. As re-
ported, the introduction of an electron withdrawing group at the o-
position of fluoro led to an appreciable potency gain (Table 2,
compounds W-27 and 16 versus compound W-21 and W-23 in
reference 19, respectively), which was consistent with that an
electron withdrawing group at the o-position of fluoro of the
benzamide moiety decreased the electron density, thus strength-
ened the amide to Ser91 hydrogen bond interaction. This data
indicated that an electron withdrawing group at the benzamide
moiety was favorable in increasing activity of this structure.
Asp107 of WDR5 played a significant role in driving the binding
of MLL1 to WDR5. Mutating Asp107 to Ala greatly impaired this
binding [20]. Hence, an amino was introduced into the benzamide
(ring C) to explore the interaction with side chain of Asp107. As
anticipated, all compounds (Table 2, compounds 18, 19, and 30,
IC50 ¼ 47.9, 18.2, and 88.7 nM, respectively) achieved great gain in
potency compared with those without amino (W-21, W-23 and W-
26, IC50 ¼ 465.7, 103.9, and 206.4 nM, respectively). As the inter-
action of the pyridone of compound OICR-9429 to Asp107, this
series of compounds with a key amino were seen a direct and a
water mediated hydrogen bonds to Asp107 in the docking model
[17] (Fig. 3), which may account for their great increase in potency.
To further verify the critical hydrogen bond interaction of the
amino to Asp107, the amino was occluded with an acetyl (Table 2,
compounds 15, 21 and 28). As expected, the inhibition activity of all
compounds dramatically decreased. The additional acetyl may
impede the hydrogen bond interaction with Asp107. Moreover,
considering the groove occupied by benzamide was narrow, the
increasing steric hindrance of acetyl may result in the potency loss.
That hinted an exposed amino was suitable for the interaction with
Asp107 and the narrow pocket. These results revealed that the
amino of the benzamide was essential for compounds to enhance
activity through forming hydrogen bonds with Asp107.
The synthetic route of compounds 22e25 and 29e44 was listed
in Scheme 4. 4-Bromo-1-fluoro-2-nitrobenzene was used as start-
ing material through the alkylation with methyl piperazine to form
9 and compound 9 was reduced to produce 10. Compound 10
reacted with 4-nitrophenylboronic acid through Suzuki coupling
reaction catalyzed by Dichlorobis(triphenylphosphine)palladiu-
m(II) to give compound 11. Compounds 22, 23 and 29 were pre-
pared from compound 11 and respective substituted nitrobenzoyl
chlorides. Compounds 24, 25 and 30 were obtained by reduction of
compounds 22, 23 and 29 in the following step. Compound 30 was
acylated with different acids catalyzed by Reagent Castros to afford
compounds 31e36, 37Be42B and 43e44. Then the t-butylox-
ycarboryl group was removed from compounds 37Be42B with TFA
to provide target compounds 37e42.
2.2. Identification of amino compounds as MLL1-WDR5 PPI potent
inhibitors
2.2.1. Binding affinity evaluated with FP assay and isothermal
titration calorimetry (ITC)
In previous report, the protonated N-methylpiperazine moiety
played a critical role in binding to WDR5 protein through a key
water-mediated hydrogen bond with Cys261 [16]. The Arg3765 of
MLL win peptide and MLL peptidomimetic was sandwiched be-
tween two aromatic rings from Phe133 and Phe263 [11]. To explore
different substitutes in this part and involve in another p-p stack-
ing interaction with Phe263, a series of acyclic chains (2a-c) were
append to the core phenyl ring and the methyl of N-methylpiper-
azine was substituted with aromatic rings (2d-g). Unfortunately,
The hydrophobic substituents of benzamide were critical for
potency improvement [16]. When the methyl, chloro and (or) flu-
oro groups (Table 3) were removed from corresponding com-
pounds only keeping nitro, their activities in blocking MLL1-WDR5
interaction were decreased (22, 23 and 26, IC50 ¼ 1.1, 0.3 and
0.2 mM, respectively), and compounds with a single amino (24, 25
and 27) even showed loss in potency. That directly proved the
criticality of hydrophobic groups in benzamide and may indirectly
indicate that an electron withdrawing group strengthened the
hydrogen bond interaction between amide and Ser91 (22 versus 24,
23 versus 25, and 26 versus 27). But in compounds 19, 20 and 30,
the methyl and chloro substituents of benzamide maintained
interaction with the surrounding hydrophobic side chain of Ala47,
Ala65 and Leu321 (Fig. 3) and kept activity. That further proved the
Fig. 2. Structure of N-(4-(4-methylpiperazin-1-yl)-[1,10-biphenyl]-3-yl)benzamide
compounds.

D.-D. Li et al. / European Journal of Medicinal Chemistry 124 (2016) 480e489
483
Scheme 2. Synthesis of compounds 16e20 and 26e27. Reaction: a. HNO3, H2SO4, r.t., 4 h; b. SOCl2, reflux, 6 h; c. boronic acids, Pd(PPh3)2Cl2, Cs2CO3, dioxane, reflux, 20 h; d. N-
methyl piperazine, DIPEA, DMF, 80 ꢀC, 2 h; e. SnCl2.2H2O, ethyl acetate, reflux, 4 h; f. 5, pyridine, DCM, r.t., 4 h; g. SnCl2.2H2O, ethyl acetate, reflux, 5 h; h. 4-fluoro-3-nitrobenzoyl
chloride, pyridine, DCM, r.t., 4 h; i. SnCl2.2H2O, ethyl acetate, reflux, 6 h.
Scheme 3. Synthesis of compounds 15, 21 and 28. Reaction: a. Pd/C, H2, CH3OH, r.t., 10 h; b. CH3COCl, DMF, pyridine, r.t., 4 h; c. SOCl2, reflux, 6 h; d. 11 or 8b, DCM, pyridine, r.t., 2 h;
e. SnCl2.2H2O, ethyl acetate, reflux, 6 h.
criticality of hydrophobic groups in benzamide, and proper hy-
drophobic substitutes should be explored in future study.
surrounded by side chains of Phe133, Phe149 and Tyr191 and
formed another stacking interaction with Tyr191.
p-p
Docking study was applied to explore the binding model of
compound 30 (Fig. 3a). Compound 30 recapitulated the interaction
of WDR5-47 to WDR5 protein including the hydrogen bonds be-
tween amide and the side chains of Cys261 and Ser91, the water
mediated hydrogen bond between protonate N-methylpiperazine
Overall, the additional direct and indirect hydrogen bonds with
Asp107, along with the occupation of the hydrophobic pocket by
the methyl, chloro and fluoro groups led to greater binding affinity
of compounds with amino (18e20, and 30). Direct binding of
compound 30 to WDR5 protein was assessed by ITC experiments.
With low Kd value of 30 (Kd ¼ 202.0 nM), this series of compounds
were confirmed to directly bind to WDR5 protein (Fig. 4). Consid-
ering the physicochemical property of these compounds, com-
pound 30 (IC50 ¼ 88.7 nM) was selected to verify the on-target
biological activities in MV4-11 cell line and for further optimization.
and Cys261, the
p-p stacking interaction with Phe133, and the
hydrophobic interaction with the pocket formed by Ala65, Ala47,
and Leu321. But the additional amino formed a direct and an in-
direct hydrogen bonds interaction with Asp107 of WDR5 protein.
Aromatic ring A occupied the hydrophobic groove of WDR5,

484
D.-D. Li et al. / European Journal of Medicinal Chemistry 124 (2016) 480e489
Scheme 4. Synthesis of compounds 22e25 and 29e44. Reaction: a. N-methyl piperazine, DIPEA, DMF, 80 ꢀC, 4 h; b. SnCl2.H2O, ethyl acetate, reflux, 6 h; c. 4-Nitrophenylboronic
acid, Pd(PPh3)2Cl2, Cs2CO3, dioxane, reflux, 20 h; d. 5, pyridine, DCM, r.t., 4 h; e. SnCl2.H2O, ethyl acetate, reflux, 6 h; f. acids, BOP, TEA, DMF, r.t., 12 h. g. 37Be42B, TFA, DCM, r.t., 2 h;
h. 4-fluoro-3-nitrobenzoyl chloride or 3-nitrobenzoyl chloride, pyridine, DCM, r.t., 4 h; i. SnCl2.H2O, ethyl acetate, reflux, 6 h.
Table 1
Table 2
Activity of compounds with N-methylpiperazine modification disturbing the inter-
Activity of compounds with R1 and R2 modification disturbing the interaction of
action of MLL1 probe-WDR5.
MLL1 probe-WDR5.
Cpd.
R
IC50/mM (FP)
2a
>20
Cpd.
R1
R2
IC50/nM (FP)
W-21
W-23
W-26
W-27
15
16
17
18
19
20
21
28
29
30
MM-102
ePh
eH
eH
eH
465.7 24.6
103.9 11.9
206.1 26.4
207.2 34.7
>20 (mM)
77.1 6.1
92.5 7.7
47.9 3.8
18.2 0.3
70.0 5.5
2b
2c
>20
4-Pyridyl
4eNH2ePh
ePh
4eNO2ePh
4-Pyridyl
4eCOOCH3ePh
ePh
4ePyridyl
4eCOOCH3ePh
4eNH2ePh
4-Pyridyl
4eNO2ePh
4eNH2ePh
e
>20
eNO2
eNHCOCH3
eNO2
eNO2
eNH2
eNH2
eNH2
eNHCOCH3
eNHCOCH3
eNO2
eNH2
e
2d
2e
2f
>20
>20
>20
>20
3186 101
1363 226
285.3 8.8
88.7 4.9
2g
1.7 0.4 (2.4 1.7 reported)
WDR5-47
MM-102
338.2 31.7 (nM)
e
1.7 0.4 (nM) (2.4 1.7 nM reported)

D.-D. Li et al. / European Journal of Medicinal Chemistry 124 (2016) 480e489
485
should inhibit the catalytic activity of MLL complex in vitro and in
leukemia cells carrying MLL fusion proteins.
To explore the inhibition activity for MLL complex methyl-
transferase in vitro, compound 30 was evaluated in a recombinant
MLL complex (MLL1, WDR5, RbBP5, and ASH2L proteins) Alpha
Screen assays in vitro. The reported peptidomimetic MM-102 (87%
inhibition at 30
control. With an amino at the benzamide (compared with W-26,
20% inhibition at 30 M), compound 30 improved inhibition ac-
tivity of MLL1-WDR5 interaction and MLL1 HMT activity (Fig. 5b,
75% inhibition at 30 M).
mM, IC50 ¼ 0.75
mM) was selected as a positive
m
mM, IC50 ¼ 1.65
m
After characterizing the inhibition of MLL1 HMT activity in vitro,
MV4-11 cells harboring MLL-AF4 fusion protein were applied to
evaluate the inhibition activity of HMT. As well as in vitro study, the
reported peptidomimetic MM-102 was selected as a positive con-
trol. Compound 30 was able to reduce MLL1-dependent H3K4me1
at 5
with 10
H3K4me2 was reduced concentration-dependently when treated
with a range of concentrations of 30 for 7 days, and 5 M of 30 was
M of MM-102 in inhibiting H3K4me2 in
mM, while no obvious differences in H3K4me1 was observed
mM of MM-102. Consistent with the inhibition in vitro,
m
at the same level with 10
western blotting (Fig. 6).
m
Taken together, compound 30 was originally designed to block
the interaction of MLL1-WDR5, effectively and potently inhibiting
the MLL1 H3K4 methyltransferase activity in recombinant MLL
complex Alpha Screen assays and in MV4-11 cell line.
2.2.3. Compound 30 reduced the expression level of Hoxa9 and
Meis-1 genes
H3K4me markers were essential for the activation of MLL1
target Hox genes and cofactor Mesi-1 gene [23,24], which were
correlated with the methyltransferase activity of MLL complex [25].
MLL1-WDR5 interaction was required for the integrity of MLL1
complex, and therefore, its HMT activity. To assess whether com-
pounds disturbing MLL1-WDR5 interaction affect the expression
level of Hoxa9 and Meis-1 genes, RT-PCR experiments were per-
formed in MV4-11 cell line harboring MLL-AF4 fusion protein.
Treating MV4-11 cells with compound 30 resulted in a strong
and concentration dependent reduction in the expression level of
Hoxa9 and Meis-1 genes as compared to the DMSO control (Fig. 7).
Approximately 50% and 80% reduction in both genes expression
Fig. 3. Docking study of compounds 30 (a) and 41 (b) to WDR5 protein (PDB code:
4IA9). The carbon atoms of compounds 30, 41 and WDR5 residues were colored white,
light green, and purple, respectively. Hydrogen bonds were represented as blue dashed
lines and
p-p stacking interaction were plotted in orange lines. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)
Table 3
Activity of compounds removing the chloro, methyl and (or) fluoro disturbing the
interaction of MLL1 probe-WDR5.
were observed with 2.5 and 10 mM of compound 30, respectively.
These results suggested that inhibiting MLL1-WDR5 interaction
with our compounds was an efficient strategy to regulate expres-
sion levels of MLL-fusion protein dependent genes, emphasizing
on-target effects for these compounds and validating their specific
mechanism of action.
2.2.4. Compounds 30 selectively inhibited proliferation of leukemia
cells harboring MLL fusion proteins by inducing apoptosis
Down-regulation or suppression the expression level of Hoxa9
and Meis-1 genes selectively inhibited growth of acute leukemia
cell lines carrying MLL fusion protein [26]. Compound 30 was
evaluated the inhibition of cells growth harboring with or without
Cpd.
R1
R2
IC50/nM (FP)
22
23
24
25
26
27
4eNO2ePh
4eNO2ePh
4eNH2ePh
4eNH2ePh
4ePyridyl
4ePyridyl
4eFe3eNO2
3eNO2
4eFe3eNH2
3eNH2
4eFe3eNO2
4eFe3eNH2
1093 50
285.1 40
MLL fusion protein, and MM-102 (IC50 ¼ 20.7
mM) was selected as
M) strongly inhibited
13.6 1.0 (
>20 ( M)
197.6 24.5
5.8 1.3 ( M)
mM)
positive control. Compound 30 (IC50 ¼ 17.7
m
m
cell proliferation in MV4-11 cells. When treating K562 and THP-
1 cell lines carrying no MLL fusion protein with compounds 30, no
inhibition was observed (89.1%, 84.2% activity, respectively) at the
m
concentration of 35 mM.
2.2.2. Compound 30 inhibited MLL complex HTM activity in vitro
and in MV4-11 cells
MLL1-WDR5 interaction was critical for the integrity of MLL1
complex, and thus the H3K4 methyltransferase activity [20e22].
Our compounds, which were designed to disturb this interaction,
To further evaluate the anti-proliferation activity of our com-
pounds, apoptosis induction experiment was carried out in MV4-11
leukemia cell line. As illustrated in Fig. 8, compound 30 effectively
induced apoptosis of MV4-11 cells in a concentration-dependent
manner and about 65% cells underwent death with treatment of

486
D.-D. Li et al. / European Journal of Medicinal Chemistry 124 (2016) 480e489
Fig. 4. Binding affinity of compounds 30, 41 and 42 evaluated with ITC. Upper panels show heat of binding plotted versus time. Lower panels show fit to a single-site binding model
to the binding isotherms. Kd (S.D.) derived from the fit is indicated.
Fig. 6. Western blot analyses for the inhibition of HMT activity after treatment of MV4-
11 cells with DMSO and 0.625
MM-102 was selected as positive control. H3K4me1 and H3K4me2 were determined
using -actin as loading control.
mM, 1.25 mM, 2.5 mM, 5.0 mM, and 10 mM compound 30.
b
Fig. 5. (a) Competitive binding curves of compounds 30, 41 and 42 determined using
FP assay. 10mer-Thr-FAM was chosen as the fluorescent probe. (b) Inhibition of HMT
activity of reconstituted MLL1 core complex as measured with Alpha Screen assays,
and MM-102 was selected as positive control. Values are shown as mean SD (n ¼ 3).
30 at 10 mM for 72h. These data proved our compounds effectively
and specially inhibit proliferation of leukemia cells carrying MLL
fusion protein through inducing cells apoptosis.
The on-target effects of compound 30 were verified, but 30
bound to WDR5 protein with moderate activity (IC50 ¼ 88.7 nM).
Thus, 30 was selected as a candidate compound for further
optimization.
2.3. Further modification of compound 30
Fig. 7. Inhibition of Hoxa9 and Meis-1 genes expression in MV4-11 cells after treatment
with DMSO and 0.625 M, 1.25 M, 2.5 M and 10 M compound 30 for 7 days
m
m
m
m
assessed by RT-PCR. *p < 0.05, **p < 0.01, statistically significant difference from the
nontreated blank control group.
2.3.1. Binding affinity evaluation of compounds 31e44
Retaining the 5-amino-2-chloro-4-fluoro-3-methylbenzamide

D.-D. Li et al. / European Journal of Medicinal Chemistry 124 (2016) 480e489
487
disturbing MLL1-WDR5 interaction (34 versus 37, 35 versus 38, 36
versus 39, and 32 versus 41). When the amino was occluded with a
t-butyloxy carbonyl (compounds 37B, 38B, 39B, 40B and 42B), a
slight decrease in potency was observed. That implied a hydro-
phobic group was not suitable for the solvent area of WDR5 protein
and proved the vital effect of an exposed amino. The length of the
linker between the amino and amide may have effect on the po-
tency. Compounds 41 (IC50 ¼ 8.5 nM) and 42 (IC50 ¼ 7.6 nM), with a
three carbon linker, were the most two potent inhibitors in block-
ing the MLL1-WDR5 interaction. Docking study (Fig. 3b) showed
that the binding model of compound 41 was same as compound 30,
but the substituent group of aromatic A located in the solvent area.
Direct binding of compounds 41 and 42 to WDR5 protein were
also assessed by ITC experiments. As the FP results, the affinity of
optimized compounds 41 and 42 (Fig. 4, Kd ¼ 7.5, 13.6 nM,
respectively) were more 15-times improved than compound 30
(Kd ¼ 202.0 nM). With the highest binding affinities to WDR5,
compounds 41 and 42 also showed the most potent inhibitory ac-
tivity in HMT assay with IC50 of 0.30 and 0.19 mM (Fig. 5b) in vitro.
In conclusion, modification of the 4-position of aromatic ring A
with an amino tag and a linker with appropriate length, high-
affinity inhibitors 41 (Kd ¼ 7.5 nM) and 42 (Kd ¼ 13.6 nM) were
defined. These two compounds were the most potent small mo-
lecular inhibitors in blocking MLL1-WDR5 interaction reported to-
date.
2.3.2. Compounds 31e44 selectively inhibited proliferation of
leukemia cells
Compounds 31e44 optimized from compound 30 were evalu-
ated their anti-proliferation activity of leukemia cell lines. As
shown in Table 5, almost all compounds showed stronger inhibition
in MV4-11 cells growth than compound 30. Compounds (32, 34, 35,
36, 37B, 38B, 39B and 42B) with a hydrophobic tag were more
efficacious in growth inhibitory than those with a hydrophilic one
(41, 37, 38, 39, 37, 38, 39 and 42). The polarity and cell membrane
permeability of compounds may account for the difference in the
activity. What's more, selective inhibition for leukemia cells
harboring with or without MLL fusion proteins was clear when
treating MV4-11, K562 and THP-1 cell lines with compounds 41 and
42 (Table 6). These data indicated that compounds optimized from
30 improved the anti-proliferation activity and specialty for leu-
kemia cells carrying MLL fusion protein.
Fig. 8. Induction of apoptosis by DMSO and 2.5 mM, 5.0 mM, and 10 mM compound 30
in MV4-11 cells harboring MLL-AF4 protein. Concentration-dependent effects of
compound 30 on apoptosis, analyzed by annexin V/propidium iodide (PI) staining.
*p < 0.05, **p < 0.01, ***p < 0.001, statistically significant difference from the non-
treated blank control group.
3. Conclusion
WDR5 was a critical protein for the integrity of MLL complex
and MLL1 HMT activity [12,27,28]. Disturbing the interaction of
MLL1-WDR5 with small molecular inhibitors represents a validated
and attractive therapeutic strategy in acute leukemia with trans-
locations of MLL genes [11,15,25,29].
of this structure, further modification was carried out at the 4-
position of aromatic ring A that located in solvent area of WDR5
protein. From the docking model (Fig. 3a), the aromatic ring A was
partially solvent exposed. But modification of this position did
realize great gain in potency. The SAR study of aromatic ring A
indicated that substitutes at 3-position resulted in twisting
conformation of benzamide as well as the twist angle between the
In the present study, we designed and synthetized a series of
potent compounds based on the SAR information and reported
compound. A key amino was introduced into benzamide, which
played a vital role in binding to WDR5 protein through a direct and
an indirect hydrogen bonds with Asp107. With the additional
amino, the binding affinity to WDR5 was great improved, as well as
inhibition activity of MLL1 HMT in recombinant MLL complex
(MLL1, WDR5, RbBP5, and ASH2L proteins) Alpha Screen assays in
vitro. The representative compound 30 (DDO-2084) verified the
inhibition activity of MLL1-dependent H3K4 methylation using
western blotting experiment in MV4-11 leukemia cells. Down-
regulation of expression level of Hoxa9 and Meis-1 genes, selec-
tive and effective inhibition of proliferation of leukemia cells
harboring MLL1 fusion proteins and induction of apoptosis by
disturbing MLL1-WDR5 interaction of 30 emphasized that our
aromatic ring A and core phenyl ring B [19]. To maintain the
p-p
stacking interaction between the core phenyl ring B and Phe133,
and to engage in an additional stacking interaction with Tyr191,
optimization of aromatic ring A was selected at 4-position.
As shown in Table 4, almost all compounds substituted at 4-
postion kept affinity to WDR5, except compounds 32, 33 and 37B
(IC50 ¼ 340.8, 421.9 and 1553 nM, respectively). The large hydro-
phobic groups of these compounds were located near to the solvent
area of WDR5 protein, which may cause the potency loss. From the
results, an aliphatic group seemed to be more suitable than an ar-
omatic one (compounds 33) in the 4-position of aromatic ring A.
What's more, a hydrophilic tag substituent increased the activity of

488
D.-D. Li et al. / European Journal of Medicinal Chemistry 124 (2016) 480e489
Table 4
Activity of compounds optimized from 30 disturbing the interaction of MLL1 probe-WDR5.
Cpd.
R
IC50/nM (FP)
Cpd.
R
IC50/nM (FP)
31
32
eNHCOCH3
70.1 4.5
340.8 31.0
39
39B
eNHCOCH2CH2NH2
eNHCOCH2CH2NHBoc
50.2 5.3
102.1 14.8
33
34
35
eNHCOPh
eNHCOCH2CH3
eNHCOCH(CH3)2
421.9 24.8
81.5 3.4
95.6 12.9
40
40B
41
eNHCOCH(i-Pro)NH2
eNHCOCH(i-Pro)NHBoc
30.1 2.6
113.4 10.3
8.5 0.7
36
37
37B
38
eNHCOCH2CH2CH3
eNHCOCH2NH2
eNHCOCH2NHBoc
eNHCOCH(CH3)NH2
76.0 1.9
62.5 4.8
1553 122.3
15.0 2.1
42
42B
43
eNHCO(CH2)3NH2
eNHCO(CH2)3NHBoc
eNHCOCH2CH(CH3)2
7.6 0.1
80.4 16.9
104.6 4.6
75.8 4.3
44
38B
eNHCOCH(CH3)NHBoc
77.9 3.8
MM-102
e
1.7 0.4
Table 5
which may stimulate druggable compounds on this target. And our
studies will pave the way toward further optimization of this
structure into chemical probes for in vivo studies in MLL leukemia
models and for potential therapeutic applications.
Inhibition activity of compounds inhibited growth of MV4-11 cells harboring MLL-
AF4 fusion protein.
Cpd.
IC50
/mM (MV:4e11)
Cpd.
IC50/mM (MV:4e11)
30
31
32
33
34
35
36
37
17.7 2.3
12.5 1.3
3.8 0.5
5.4 0.2
6.5 0.2
4.5 0.2
2.3 0.1
13.2 1.2
4.1 0.5
11.1 0.1
4.3 0.1
39
39B
40
40B
41
42
42B
43
44
25.1 11.1
4.6 0.1
2.8 0.1
3.8 0.1
9.2 0.9
7.4 1.4
4.8 0.4
3.9 0.2
4.8 0.2
20.7 1.5
Conflicts of interest
The authors declare no other conflicts of interest.
Acknowledgments
37B
38
38B
This work was supported by the project 81230078 (key pro-
gram), 81502915 (Youth Foundation) and 81573346 of National
Natural Science Foundation of China, Project Program of State Key
Laboratory of Natural Medicines, China Pharmaceutical University
MM-102
(No.
SKLNMZZCX201611),
2013ZX09402102-001-005
and
Table 6
2014ZX09507002-005-015 of the National Major Science and
Technology Project of China (Innovation and Development of New
Drugs), Specialized Research Fund for the Doctoral Program of
Higher Education (SRFDP, 20130096110002), Project of the Priority
Academic Program Development of Jiangsu Higher Education
Institutions.
Selective inhibition of growth of leukemia cell lines with or without MLL fusion
proteins.
Cpd.
IC50
(MV:4e11)
/
m
M
activity@35
M (K562)
activity@35
(THP-1)
mM
m
30
41
42
MM-
17.7 2.3
9.2 0.9
7.4 1.4
89.1%
113.7%
84.2%
84.2%
84.6%
86.8%
NDa
20.7 1.5 (25
m
M reported) 37.8 1.4 mM (IC50)
Appendix A. Supplementary data
102
a
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.08.036.
ND ¼ not determined.
compounds were on-target.
References
Modification of 30 with a 4-aminobutanamido group afforded to
compounds 42 (DDO-2117). Especially, 42 was the most potent
inhibitor with IC50 of 7.6 nM, and Kd of 13.6 nM reported to-date,
and showed the most potent inhibitory activity in HMT assay
[1] B.E. Bernstein, A. Meissner, E.S. Lander, The mammalian epigenome, Cell. 128
(2007) 669e681.
[2] S. Khorasanizadeh, The nucleosome: from genomic organization to genomic
regulation, Cell. 116 (2004) 259e272.
with IC50 of 0.19
m
M in vitro. It was the breakthrough achieving in
[3] K.A. Gelato, W. Fischle, Role of histone modifications in defining chromatin
structure and function, Biol. Chem. 389 (2008) 353e363.
[4] M. Chopra, S.K. Bohlander, Disturbing the histone code in leukemia:
MLL1-WDR5 PPI small molecular blockers in low nanomolar range,

D.-D. Li et al. / European Journal of Medicinal Chemistry 124 (2016) 480e489
489
translocations and mutations affecting histone methyl transferases, Cancer
Genet. 208 (2015) 192e205.
[5] C. Martin, Y. Zhang, The diverse functions of histone lysine methylation, Nat.
Rev. Mol. Cell Biol. 6 (2005) 838e849.
[6] Y.L. Dou, T.A. Milne, A.J. Tackett, E.R. Smith, A. Fukuda, J. Wysocka, C.D. Allis,
B.T. Chait, J.L. Hess, R.G. Roeder, Physical association and coordinate function
of the H3K4 methyltransferase MLL1 and the H4K16 acetyltransferase MOF,
Cell. 121 (2005) 873e885.
[7] A.J. Ruthenburg, C.D. Allis, J. Wysocka, Methylation of lysine 4 on histone H3:
intricacy of writing and reading a single epigenetic mark, Mol. Cell. 25 (2007)
15e30.
A. Skucha, S. Vittori, E. Kuznetsova, D. Smil, D. Barsyte-Lovejoy, F. Li, G. Poda,
M. Schapira, H. Wu, A. Dong, G. Senisterra, A. Stukalov, K.V. Huber,
A. Schonegger, R. Marcellus, M. Bilban, C. Bock, P.J. Brown, J. Zuber,
K.L. Bennett, R. Al-Awar, R. Delwel, C. Nerlov, C.H. Arrowsmith, G. Superti-
Furga, Pharmacological targeting of the Wdr5-MLL interaction in C/EBPalpha
N-terminal leukemia, Nat. Chem. Biol. 11 (2015) 571e578.
[18] M. Getlik, D. Smil, C. Zepeda-Velazquez, Y. Bolshan, G. Poda, H. Wu, A. Dong,
E. Kuznetsova, R. Marcellus, G. Senisterra, L. Dombrovski, T. Hajian, T. Kiyota,
M. Schapira, C.H. Arrowsmith, P.J. Brown, M. Vedadi, R. Al-Awar, Structure-
based optimization of a small molecule antagonist of the interaction between
WD repeat-containing protein
5 (WDR5) and mixed-lineage leukemia 1
[8] M.G. Guenther, R.G. Jenner, B. Chevalier, T. Nakamura, C.M. Croce, E. Canaani,
R.A. Young, Global and Hox-specific roles for the MLL1 methyltransferase,
Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 8603e8608.
[9] B. Argiropoulos, R.K. Humphries, Hox genes in hematopoiesis and leukemo-
genesis, Oncogene 26 (2007) 6766e6776.
(MLL1), J. Med. Chem. 59 (2016) 2478e2496.
[19] D.D. Li, W.L. Chen, X.L. Xu, F. Jiang, L. Wang, Y.Y. Xie, X.J. Zhang, X.K. Guo,
Q.D. You, H.P. Sun, Structure-based design and synthesis of small molecular
inhibitors disturbing the interaction of MLL1-WDR5, Eur. J. Med. Chem. 118
(2016) 1e8.
[10] A. Kohlmann, C. Schoch, M. Dugas, S. Schnittger, W. Hiddemann, W. Kern,
T. Haferlach, New insights into MLL gene rearranged acute leukemias using
gene expression profiling: shared pathways, lineage commitment, and part-
ner genes, Leukemia 19 (2005) 953e964.
[11] H. Karatas, E.C. Townsend, F. Cao, Y. Chen, D. Bernard, L. Liu, M. Lei, Y. Dou,
S. Wang, High-affinity, small-molecule peptidomimetic inhibitors of MLL1/
WDR5 proteineprotein interaction, J. Am. Chem. Soc. 135 (2012) 669e682.
[12] Y.L. Dou, T.A. Milne, A.J. Ruthenburg, S. Lee, J.W. Lee, G.L. Verdine, C.D. Allis,
R.G. Roeder, Regulation of MLL1 H3K4 methyltransferase activity by its core
components, Nature Struct, Mol. Biol. 13 (2006) 713e719.
[13] H. Karatas, E.C. Townsend, D. Bernard, Y. Dou, S. Wang, Analysis of the binding
of mixed lineage leukemia 1 (MLL1) and histone 3 peptides to WD repeat
domain 5 (WDR5) for the design of inhibitors of the MLL1-WDR5 interaction,
J. Med. Chem. 53 (2010) 5179e5185.
[14] G. Senisterra, H. Wu, A. Allali-Hassani, G.A. Wasney, D. Barsyte-Lovejoy,
L. Dombrovski, A. Dong, K.T. Nguyen, D. Smil, Y. Bolshan, T. Hajian, H. He,
A. Seitova, I. Chau, F. Li, G. Poda, J.F. Couture, P.J. Brown, R. Al-Awar,
M. Schapira, C.H. Arrowsmith, M. Vedadi, Small-molecule inhibition of MLL
activity by disruption of its interaction with WDR5, Biochem. J. 449 (2013)
151e159.
[15] F. Cao, E.C. Townsend, H. Karatas, J. Xu, L. Li, S. Lee, L. Liu, Y. Chen, P. Ouillette,
J. Zhu, J.L. Hess, P. Atadja, M. Lei, Z.S. Qin, S. Malek, S. Wang, Y. Dou, Targeting
MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia, Mol. Cell.
53 (2014) 247e261.
[16] Y. Bolshan, M. Getlik, E. Kuznetsova, G.A. Wasney, T. Hajian, G. Poda,
K.T. Nguyen, H. Wu, L. Dombrovski, A. Dong, G. Senisterra, M. Schapira,
C.H. Arrowsmith, P.J. Brown, R. Al-awar, M. Vedadi, D. Smil, Synthesis, opti-
mization, and evaluation of novel small molecules as antagonists of WDR5-
MLL interaction, ACS Med. Chem. Lett. 4 (2013) 353e357.
[20] J.J. Song, R.E. Kingston, WDR5 interacts with mixed lineage leukemia (MLL)
protein via the histone H3-binding pocket, J. Biol. Chem. 283 (2008)
35258e35264.
[21] A. Patel, V.E. Vought, V. Dharmarajan, M.S. Cosgrove, A conserved arginine-
containing motif crucial for the assembly and enzymatic activity of the
mixed lineage leukemia protein-1 core complex,, J. Biol. Chem. 283 (2008)
32162e32175.
[22] A. Patel, V. Dharmarajan, M.S. Cosgrove, Structure of WDR5 bound to mixed
lineage leukemia protein-1 peptide, J. Biol. Chem. 283 (2008) 32158e32161.
[23] T.A. Milne, Y. Dou, M.E. Martin, H.W. Brock, R.G. Roeder, J.L. Hess, MLL asso-
ciates specifically with a subset of transcriptionally active target genes, Proc.
Natl. Acad. Sci. U. S. A. 102 (2005) 14765e14770.
[24] T.A. Milne, S.D. Briggs, H.W. Brock, M.E. Martin, D. Gibbs, C.D. Allis, J.L. Hess,
MLL targets SET domain methyltransferase activity to Hox gene promoters,
Mol. Cell. 10 (2002) 1107e1117.
[25] T.A. Milne, J. Kim, G.G. Wang, S.C. Stadler, V. Basrur, S.J. Whitcomb, Z.X. Wang,
A.J. Ruthenburg, K.S.J. Elenitoba-Johnson, R.G. Roeder, C.D. Allis, Multiple in-
teractions recruit MLL1 and MLL1 fusion proteins to the HOXA9 locus in
leukemogenesis, Mol. Cell. 38 (2010) 853e863.
[26] J. Faber, A.V. Krivtsov, M.C. Stubbs, R. Wright, T.N. Davis, M. van den Heuvel-
Eibrink, C.M. Zwaan, A.L. Kung, S.A. Armstrong, HOXA9 is required for survival
in human MLL-rearranged acute leukemias, Blood 113 (2009) 2375e2385.
[27] Y. Li, J. Han, Y. Zhang, F. Cao, Z. Liu, S. Li, J. Wu, C. Hu, Y. Wang, J. Shuai, J. Chen,
L. Cao, D. Li, P. Shi, C. Tian, J. Zhang, Y. Dou, G. Li, Y. Chen, M. Lei, Structural
basis for activity regulation of MLL family methyltransferases, Nature 530
(2016) 447e452.
[28] Y. Dou, J.L. Hess, Mechanisms of transcriptional regulation by MLL and its
disruption in acute leukemia, Int. J. Hematol. 87 (2008) 10e18.
[29] H. Liu, E.H. Cheng, J.J. Hsieh, MLL fusions: pathways to leukemia, Cancer Biol.
Ther. 8 (2009) 1204e1211.
[17] F. Grebien, M. Vedadi, M. Getlik, R. Giambruno, A. Grover, R. Avellino,