Identification of novel small-molecule inhibitors targeting menin-MLL interaction, repurposing the antidiarrheal loperamide

Article abstract of DOI:10.1039/c6ob01248e

Leukemia with a mixed lineage leukemia (MLL) rearrangement, which harbors a variety of MLL fusion proteins, has a poor prognosis despite the latest improved treatment options. Menin has been reported to be a required cofactor for the leukemogenic activity of MLL fusion proteins. Thus, the disruption of the protein-protein interactions between menin and MLL represents a very promising strategy for curing MLL leukemia. Making use of menin-MLL inhibitors with a shape-based scaffold hopping approach, we have discovered that the antidiarrheal loperamide displays previously unreported mild inhibition for the menin-MLL interaction (IC₅₀ = 69 ± 3 μM). In an effort to repurpose this drug, a series of chemical modification analyses was performed, and three of the loperamide-based analogues, DC-YM21, DC-YM25 and DC-YM26 displayed better activities with IC₅₀ values of 0.83 ± 0.13 μM, 0.69 ± 0.07 μM and 0.66 ± 0.05 μM, respectively. Further treatment with DC-YM21 demonstrated potent and selective blockage of proliferation and induction of both cell cycle arrest and differentiation of leukemia cells harboring MLL translocations, which confirmed the specific mechanism of action. In conclusion, molecules of a novel scaffold targeting menin-MLL interactions were reported and they may serve as new potential therapeutic agents for MLL leukemia.

Full text of DOI:10.1039/c6ob01248e

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Identification of novel small-molecule inhibitors targeting menin-
MLL interaction, repurposing the antidiarrheals loperamide†
Received 00th January 20xx,
Accepted 00th January 20xx
Liyan Yue,‡a,b Juanjuan Du,‡c Fei Ye,‡d Zhifeng Chen,e Lianchun Li,f Fulin Lian,a,b Bidong Zhang,a,b
Yuanyuan Zhang,a,b,* Hualiang Jiang,a,b Kaixian Chen,a,e Yuanchao Li,c Bing Zhou,c Yaxi Yang, c,* and
Cheng Luoa,e,
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Leukemia with mixed lineage leukemia (MLL) rearrangement, which harbors a variety of MLL fusion proteins, has a poor
prognosis despite the latest improved treatment options. Menin has been reported to be a required cofactor for the
leukemogenic activity of MLL fusion proteins. Thus, the disruption of the protein-protein interactions between menin and
MLL represents a very promising strategy for curing MLL leukemia. Making use of menin-MLL inhibitors with a shape-based
scaffold hopping approach, we have discovered that the antidiarrheals loperamide displays, previously unreported, mild
inhibition for the menin-MLL interaction (IC50 = 69 ± 3 µM). In an effort to repurpose this drug, a serials of chemical
modification analysis were performed. And three of the loperamide-based analogues, DC_YM21, DC_YM25 and DC_YM26
displayed better activities with IC50 of 0.83 ± 0.13 µM, 0.69 ± 0.07 µM and 0.66 ± 0.05 µM respectively. Further treatment
with DC_YM21 demonstrated potent and selective blockage of proliferation and induction of both cell cycle arrest and
differentiation of leukemia cells harboring MLL translocations, which confirmed specific mechanism of action. In
conclusion, molecules of a novel scaffold targeting menin-MLL interactions were reported and they may serve as new
potential therapeutic agents for MLL leukemia.
Introduction
Dysregulation in epigenetic modifications is closely related to fundamental roles in many biological processes18-21, and
human cancers1-5. During the last decade, huge efforts have aberrances in these “epigenetic modifications” are closely
been put into drug discovery and development of epigenetic related with the development of human cancers21-24. The
targets6-10, especially DNA methyltransferase11-14 and histone Mixed lineage leukemia 1 (also known as histone-lysine N-
methyltransferase (HMT)15-17
.
Histone modifications play methyltransferase 2A, Trithorax-like protein, Zinc finger
protein HRX, CXXC-type zinc finger protein 7, ALL-1, KMT2A
and MLL1) is a specific H3K4 methyltransferase and it is the
a.State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China.
subunit of the COMPASS (Complex Proteins Associated with
Set125)-like multi-subunit (SET1/MLL) complex. MLL1 gene
encodes a 3,969 amino acid, 500 kDa protein with multiple
domains, of which the SET domain that functions as histone
methyltransferase is C-terminal located. After being cleaved by
Taspse1, the active form MLLN320/C180 consisting of N-terminal
320 kDa (MLLN320) and C-terminal 180 kDa (MLLC180) is
generated26, and the two fragments remain non-covalently
bound in a tight complex In the MLL1 complex. MLL1 protein
interacts with WDR5, RBBP5, ASH2L, and Dpy30 through this C-
b.University of Chinese Academy of Sciences
100049, China
，No.19A Yuquan Road, Beijing
c. Department of Medicinal Chemistry, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China.
d.Zhejiang Sci-Tech University, College of Life Sciences, Hangzhou, China.
e. School of Life Science and Technology, Shanghai Tech University, Shanghai
200031, China.
f. Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization,
Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese
Academy of Sciences, Guilin 541006, China.
†Electronic
Supplementary
Information
(ESI)
available.
See
DOI: 10.1039/x0xx00000x
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terminal fragment27, 28, playing an essential role in early fusion proteins are dependent on the interaction with menin
development and hematopoiesis. Rearrangement of MLL1 for their oncogenic activity42, and imply that the menin-MLL
gene at chromosome locus 11q23 occurs frequently in patients interaction represents an attractive target for molecular
with de novo acute myeloid (AML) and acute lymphoblastic therapy.
(ALL) leukemia, and has a poor prognosis with only a ~35% Although the interaction between menin and the wild-type
overall 5-year survival rate despite improved treatment MLL protein is reported to be indispensable for Hox genes
options
like
allogeneic
hematopoietic
stem
cell expression in early hematopoietic progenitors to maintain self-
transplantation28, 29. The rearrangement enables MLL1 protein renewal abilities48, recent studies demonstrate that menin is
to fuse its N-terminus to one of partners to yield a collection of not a requisite cofactor for MLL1 during normal hematopoiesis
chimeric fusion proteins. These fusion proteins showed and drugs that target menin-MLL1 is safe and selective for
abnormal functions, upregulating expression of genes such as MLL-rearranged leukemia49, 50. Using a competition-based
HOXA9 and MEIS1, resulting in enhanced proliferation and fluorescence polarization assay with the fluorescein-labelled
30,
blockage of hematopoietic differentiation
31. Over 100 MBM1 peptide and menin protein, Grembecka and Cierpicki
translocations have been reported and more than 70 different discovered the first-generation of small-molecule menin-MLL
partner proteins have been identified32. Among these interaction inhibitors, which belong to the thienopyrimidine
partners, MLL1 is predominantly fused to nine of them, AF4 class43. The optimized MI-2 inhibits the menin-MBM1
(accounts for translocations (t4;11), AF9 (t9;11), ENL (t11;19), interaction with IC50 = 446 nM43. A more potent analog MI-2-2
AF10 (t10;11), AF6 (t6;11), ELL (t11;19), AF1P, AF17, and (IC50 = 46 nM) was obtained after structural studies and further
SEPT6, accounting for almost 90% of MLL rearrangements29. medicinal chemistry optimizations, which fits into the central
The fusion partners are often believed to convert the cavity on menin in the MBM1 pocket by mimicking the key
truncated MLL to a potent transcriptional activator26,33-35
.
interactions of the peptide with menin44. Based on MI-2-2
, MI-
Menin is a 610 amino acid, 67 kDa protein encoded by the 463 (IC50 = 15.3 nM) and MI-503 (IC50 = 14.7 nM) were recently
multiple endocrine neoplasia 1 (Men1) gene36. It functions as a developed to be two lead inhibitors and have favorable drug-
tumor suppressor in the similarly-named familial syndrome, an like properties48. Furthermore, chemical optimization for
inherited disorder that causes tumors in the endocrine glands better activities and drug-like properties have been thoroughly
and the duodenum37. Menin binds directly to the N-terminus discussed recently51, 52, with MI-538 (IC50 = 21±1.4 nM) to be
of WT MLL1/2 and all MLL1 fusion proteins. It simultaneously the most potent inhibitor for in vitro studies51, 52 . The second
interacts with the N-terminus of MLL and the IBD domain of class of menin-MLL interaction inhibitors are based on the
the chromatin associated protein LEDGF (lens epithelium hydroxylmethylpiperidine
scaffold.
The
HTS
hit,
derived growth factor). As a component of both MLL1 and MLS001171971 showed an activity of IC50 = 5.8 µM, while
MLL2 complex, LEDGF is essential for co-localization of menin modified compounds ML227 and ML399 (Molecular Libraries
and wild type MLL or MLL fusions to target genes38, 39. In this Probe Production Centers Network) displayed better activities
way, menin acts as a co-factor in leukemic transformation. with IC50 values of 883 nM and 90 nM respectively28. A similar
Several studies have demonstrated that menin is required for compound of hydroxyl and aminomethylpiperidine structure
the normal expression of MLL target genes40, 41. A great deal of was found in the same library, and medicinal chemistry
effort has been put into biochemical and structural studies of optimization led to compound MIV-6R (IC50 = 56 nM), which
39,
identifying the menin-MLL interaction motif32,
42-44. Two demonstrated strong and selective effects in MLL leukemia
short motifs in the N-terminus of MLL with high binding affinity cells53, representing the most potent inhibitor of this class.
to menin have been identified45. The menin binding motif 1 Recently, two amino-glycoside antibiotics were reported to
(MBM1) encompasses MLL residues 4-15, binding to menin target the menin-MLL interface54. In addition, peptidomimetics
with high affinity (Kd = 53 nM). The menin binding motif MCP-1 strongly binds to menin with the Ki value of 4.7 nM by
2(MBM2) involves residues 23-40, presenting a lower affinity mimicking MBM155.
(Kd = 1.4 µM)45. Efficient proliferation of myeloid cells Virtual screening methods including scaffold hopping, docking
transformed with oncogenic MLL-AF9 depends on the and pharmacophore as well as other computational methods
existence of menin and loss of menin relieved the have been proved to be useful and efficient approaches for
differentiation block of MLL-transformed leukemic blasts. novel drug discovery12, 14, 16, 56-63. Based on the hypothesis that
Mutations within the N-terminus of MLL fusion proteins that compounds sharing high structural similarities may exhibit
block association with menin showed no activity in inducing similar
bioactivities64,
shape-based
three-dimensional
leukemia in contrast to the intact protein. Additionally, structure superposition has been proved to be a powerful tool
expression of MLL amino terminal peptides, which can disrupt for computational scaffold hopping, providing a desirable
MLL-menin interaction, results in reduction of menin binding approach for discovering novel compounds in a number of
with endogenous MLL as well as growth inhibition of MLL-AF9 cases of ligand-based virtual screening65, 66. In this study,
transformed cells46, 47. These findings demonstrate that MLL scaffold hopping calculations were conducted making use of
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the complex crystal structures of human menin with bound
inhibitor MI-2-2 and MIV-6R respectively (PDB ID: 4GQ4 and
68
4OG8) utilizing SHAFTS67,
. Considering the drug-like
properties of candidates, an in-house library comprising ~1600
compounds was built for 3D similarity searching based on the
existing drugs. As a result, loperamide, an antidiarrheal drug
used in acute and chronic diarrhea as well as in ileostomy, was
identified to inhibit the menin-MLL interaction. Although
loperamide showed mild inhibition to menin-MBM1 complex
(IC50 = 69 ± 3 µM), chemical optimizations led to three
improved molecules DC_YM21, DC_YM25 and DC_YM26 with
IC50 values of 0.83 ± 0.13 µM, 0.69 ± 0.07 µM and 0.66 ± 0.05
µM respectively. These compounds displayed inhibitory
activities against menin-MLL in fluorescence polarization
assays and cell viability assays like the reported inhibitor MI-2-
Figure 1. Seeking loperamide out for targeting menin-MLL interaction. a) The
flowchart of scaffold hopping. b) FP result of loperamide. c-d) CPMG and STD NMR
experiments demonstrate the binding of loperamide to menin.
2
(Supplementary
information)
.
Some
biophysical
experiments including differential scanning fluorimetry (DSF),
surface plasmon resonance (SPR) and competition saturation
transferred difference (STD) were performed to validate the
specific binding of inhibitors to menin at the MBM1 binding
site. The results demonstrated that the molecules we
obtained from loperamide scaffold can disrupt the menin-MLL
interaction both in vitro and in vivo. Further treatment with
DC_YM21 caused growth inhibition, cell cycle arrest,
differentiation induction and down-regulation of MLL fusion
target genes expression, representing the specific action of
menin-MLL inhibitors. Structure-activity relationship of a series
of inhibitors was also discussed, providing some clues for
further menin-MLL inhibitor design and development. In short,
molecules of a novel scaffold targeting menin-MLL interactions
To understand the molecular details of how loperamide
interacts with menin-MLL interface, molecular docking
simulations were performed using Glide 5.569, 70. The results
revealed that loperamide roughly occupies the binding sites of
MBM1 peptides (Figure 2a-b), indicating that several regions
of loperamide can be modified to make this compound fit into
the binding pocket in a better way (Figure 2c). Using
loperamide as a hit compound, chemical optimizations were
performed to improve potencies (see below).
Structural Optimizations of Loperamide Scaffold
were reported and they may serve as new potential A series of derivatives were obtained by both similarity search
therapeutic agents for MLL leukemia.
and chemical synthesis (Table 1) to get a deeper
understanding of structure activity relationship (SAR). After
the validation of FP assay, we found that the quarternization
of piperidin (R1 region in Figure 2c) or the substitution of
piperidinring to piperazine totally abolished potency of the
Results and discussion
Identifying Loperamide as a Mild menin-MLL Interface Inhibitor
compounds (DC_YM1, DC_YM7, Table 1). Introduction of a
Since MI-2-2 and MIV-6R are representative compounds of
double bond into the piperidin group, which yield 4-(4-(4-
chlorophenyl)-5,6-dihydropyridin-1(2H)-yl)-N,N-dimethyl-2,2-
diphenylbutanamide (DC_YM6), led to a 4-fold increase in
activity (IC50 = 18 ± 2 µM, Table 1 and Supplementary Figure 2).
On the basis of these data, we concluded that 1,2,3,6-
tetrahydropyridine is the preferred group in the linker region.
Next, the dimethylacetamide group (R2 region in Figure 2c) at
the head region was replaced with different acylamino groups
to explore its role in activities. The inhibitory activity of 4-(4-(4-
chlorophenyl)-5,6-dihydropyridin-1(2H)-yl)-N-methyl-2,2-
diphenylbutanamide (DC_YM2), with hydrogen atom replacing
one methyl group, was slightly weaker versus loperamide
(IC50= 79 ± 4 µM, Table 1). Removal of two methyl groups
two classes of menin-MLL inhibitors, they were used to query
against the in-house database with SHAFTS (Figure 1a)
67, 68. A
set of 12 compounds were selected out by 3D similarity
calculation for primary validation by fluorescence polarization
(FP) assay with the FITC-labelled MLL-derived peptide MBM1.
The results of FP assays indicated that loperamide, an
antidiarrheal drug used for acute and chronic diarrhea as well
as ileostomy, showed mild inhibition for menin-MBM1
complex (IC50 = 69 ± 3 µM, Figure 1b). NMR Carr-Purcell-
Meiboom-Gill (CPMG) and saturation transfer difference (STD)
experiments provided evident evidence of the direct binding
between loperamide and menin (Figure 1c-d) which makes us
to conclude that loperamide binds directly to menin and
disrupts the interactions of menin-MBM1 complex.
resulted in decrease in activity with IC50 > 200 µM (DC_YM3
,
Table 1) implying that alkyl groups at R2 position is important
for inhibitory activity. Introduction of the preferred group
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1,2,3,6-tetrahydropyridine at R1 position as well as the bulkier due to steric clash (DC_YM18- DC_YM20, Table 2). Since there
ethyl group at R2 position, yielded about 4-fold increase in is a hydrogen bond formed between the nitrile group of MIV-
activity (IC50 = 16 ± 2 µM, DC_YM8
,
Table 1). We found that -6R and the indole nitrogen of W341 on menin53, we explored
substitution of the dimethylacetamide group with ethanamine, weather introduction of hydrogen-bond acceptors at R3 region
which includes an alkyl hydrophobic group, 4-(4-(4- can improve activities. Consequently, the inhibitory activity of
chlorophenyl)-5,6-dihydropyridin-1(2H)-yl)-2,2-diphenylbutan- N,N-dimethyl-4-(4-(4-(methylsulfon-amido)-phenyl)-5,6-
1-amine (DC_YM9), also shows inhibition of the menin-MLL dihydropyridin-1(2H)-yl)-2,2-diphenylbutanamide (DC_YM13),
interactions (IC50 = 26 ± 3 µM, Table 1). However, the with methane sulfonamide replacing the chlorine atom, was
replacement of acylamino groups with hydrophilic group nitrile increased by about 3-fold (IC50 = 5.9 ± 0.4 µM, DC_YM13
or hydroxyl totally abolished the activity of inhibitors (DC_YM4 Table ). Halogen bonding, specific intermolecular
DC_YM5 Table 1), validating the essential role of hydrophobic interaction between a halogen atom and an electron-rich
substituents at R2 position in compounds. partner (O, N, S, or ), is a distinct class of hydrogen-bond-like
As shown in Figure 2a-b, there are very few interactions interactions45. Therefore, introducing a fluorine atom also lead
between menin and the tail group region of loperamide (R3 to an improvement in activity (IC50 = 7.6 ± 2.1 µM, DC_YM12
,
,
2
a
,
π
,
region in Figure 2c), which gives us space for optimization. Table 2). As for DC_YM14-DC_YM16, the inhibitory activities
Replacement of chlorine atom with methoxy group in the tail were degraded in different degrees, probably due to formation
region resulted in a modest ~1.5-fold increase in IC50 value of intramolecular hydrogen-bonds in these compounds. The
(IC50 = 13 ± 2 µM, DC_YM10, Table 2), implying that extension binding pocket in menin that interacts with the tail region of
of tail group with hydrophobic substituents can improve inhibitors is negatively charged (Figure 2a) and replacement of
inhibitory activity. Introduction of hydrophilic group nitrile R3 region with the negative-charged carboxyl completely
slightly decreased the inhibitory activity, validating the abolished the interaction between menin and the compound
importance of hydrophobic substituents (IC50 = 22 ± 4 µM,
(DC_YM17, Table 2). These results demonstrated that the
DC_YM11 Table 2). However, introducing phenyl rings which binding affinity could be improved through introduction of
,
adopt a larger size reduced the inhibitory activity completely
proper hydrophobic substituents and hydrogen-bond
acceptors at the tail region of inhibitors.
The binding mode of loperamide revealed that menin-
loperamide interactions are mostly mediated by the
hydrophobic contacts between protein and the head group
region of compound (R4 region in Figure 2c). However, two
benzene rings cannot fit into the F9 and P10 hydrophobic
pockets very well (Figure 2a). Thus, one of the phenyl rings at
the head region of loperamide was substituted with different
groups to explore ways of improving its potency. As a result,
cycloalkanes or chain hydrocarbons substituents both led to
significant improvement of the inhibitory activity. Cyclopentyl
was found to be the optimal group at this position while
introducing a 2-fluoro or 3-fluoro to another phenyl ring led to
decrease in activities (DC_YM21-DC_YM23, Table 3). Among
these compounds, 4-(4-(4-chlorophenyl)-5,6-dihydropyridin-
1(2H)-yl)-2-cyclopentyl-2-phenylbutan-1-amine
exhibited
about 30-fold increase in activity versus DC_YM9 (IC50 = 0.83 ±
0.13 μM, DC_YM21, Table 3). Molecular docking simulations
were performed to investigate the binding mode between
DC_YM21 and menin. As shown in Figure 3a, the phenyl and
cyclopentyl rings in the head group of DC_YM21 overlaps with
side chains of F9 and P10 in MLL, thus closely mimicking the
interactions between menin and MLL. As guided by
modifications on the tail group region (Table 2), the chlorine
atom was replaced with fluorine atom and methane
sulfonamide group resulting in increased inhibitory activities
Figure 2. Binding mode of loperamide to menin cavity. a) Molecular modeling of
loperamide/menin complex. The crystal structure of MIV-6R binding at active
site in menin is also shown for comparison (PDB ID: 4GO8). Loperamideis colored
in magenta, and MIV-6R is colored in blue. b) Schematic diagram showing
interactions between loperamide and menin. Residues involved in the
hydrophobic interactions are shown as starbursts. Molecular graphics figures
were prepared with LigPlot+ program71
. c) Four regions in loperamide to be
modified by chemistry optimization.starbursts.
against the menin-MLL interactions (DC_YM25-DC_YM26,
Table 3). Molecular modeling implies that the improved
affinity is probably contributed by hydrogen bonds and
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Table 1. Structures and IC50 values for loperamide and its derivatives
Compound
Structure
Ki (µM)
IC50 (µM)
O
O
O
O
N
Loperamide
15 ± 1
69 ± 3
N
OH
Cl
Cl
Cl
N
O
DC_YM1
DC_YM2
DC_YM3
DC_YM4
DC_YM5
DC_YM6
DC_YM7
DC_YM8
DC_YM9
-
N.I.
79 ± 4
> 200
N.I.
N
OH
N
18 ± 1
HN
OH
N
-
NH2
OH
Cl
Cl
Cl
Cl
Cl
N
N
C
-
OH
N
H
O
-
N.I.
OH
O
N
4.1 ± 0.5
18 ± 2
N
N
N
O
N
-
N.I.
N
O
N
3.5 ± 0.5
5.8 ± 0.8
16 ± 2
27 ± 4
Cl
N
NH2
Cl
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Table 2. Structures of loperamide-based analogues modified in the tail group region
O
N
N
r
A
Compound
Structure
Ki (µM)
IC50 (µM)
H
OC
DC_YM10
2.7 ± 0.4
4.7 ± 0.9
1.6 ± 0.5
1.2 ± 0.1
39 ± 3
6.5 ± 0.7
21 ± 1
-
13 ± 2
3
N
C
DC_YM11
DC_YM12
DC_YM13
DC_YM14
DC_YM15
DC_YM16
DC_YM17
DC_YM18
DC_YM19
22 ± 4
7.6 ± 2.1
5.9 ± 0.4
175 ± 13
30 ± 3
94 ± 5
N.I.
F
O
S
O
NH
O
N
H
O
NH2
S
O
O
NH2
H
COO
l
C
-
N.I.
-
N.I.
N
DC_YM20
-
N.I.
O
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Table 3. Structures and activities of analogues with substitution of alkyl group in the head region
Ki(µM)
Compound
Structure
IC50 (µM)
N
DC_YM21
0.08 ± 0.03
0.83 ± 0.13
NH2
Cl
F
N
DC_YM22
DC_YM23
0.36 ± 0.05
0.59 ± 0.15
2.1 ± 0.2
3.2 ± 0.7
NH2
Cl
F
N
N
N
N
NH2
NH2
NH2
Cl
DC_YM24
DC_YM25
1.4 ± 0.3
6.9 ± 1.2
Cl
0.05 ± 0.02
0.69 ± 0.07
F
DC_YM26
DC_YM27
0.04 ± 0.01
0.88 ± 0.24
0.66 ± 0.05
NH2
O
N
S
O
H
N
O
S O
4.4 ± 1.1
HN
NH2
N.I. refers to no inhibitory activity
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DC_YM25 and DC_YM26
(Table 3). To determine the binding
affinity of DC_YM21 with menin, the melting temperature (Tm
value) of menin protein was first measured with a differential
scanning fluorimetry assay. The presence of DC_YM21 at 10
µM and 20 µM both led to significant increases in Tm values
(ΔTm = 2.42 ± 0.25℃ and 3.40 ± 0.21℃ respectively, Figure 4a),
indicating the direct interactions between the compound and
menin in the dose-dependent manner. In contrast, the
analogue of DC_YM21
in FP assay (Table 1), caused no shift in the differential
scanning fluorimetry assay Figure 4a). The equilibrium
, DC_YM3, which displayed poor activity
(
dissociation constant value of DC_YM21 and menin was also
determined to be 3.07 ± 0.21 µM (Figure 4b) by a Surface
Plasmon Resonance (SPR) experiment.
Figure 3. Binding mode of potent inhibitors. a) Molecular modeling of
DC_YM21/menin complex. The crystal structure of MLL peptide binding at active site in
menin is also shown for comparison (PDB code 4GQ6). DC_YM21 is colored in magenta,
and MLL peptide is colored in yellow. b) Schematic diagram showing interactions
between DC_YM26 and menin. Residues involved in the hydrophobic interactions are
shown as starbursts, and hydrogen-bonding interactions are denoted by dotted green
In order to confirm that DC_YM21 binds directly to menin and
competes with MLL, competition saturation transfer difference
(STD) NMR experiments were also performed using the MBM1
peptide. A significant and dose-dependent decrease in the STD
effect of DC_YM21 was detected upon adding the MBM1 at
the final concentration of 10 µM, 50 µM, and 100 µM (Figure
4c), indicating that DC_YM21 binds menin at the same site of
MBM1 binding.
lines. Molecular graphics figures were prepared with LigPlot+ program72
.
Disrupting the Menin and MLL Fusion Protein Interaction in Cells
by DC_YM21
To test the capacity of potent compounds in disrupting the
menin and MLL fusion protein interaction in cells, co-
immunoprecipitation experiments were performed in HEK293
cells transfected with flag-MLLN. Menin was expressed
endogenously in
a
high level and could be co-
immunoprecipitated with Flag-MLLN in the DMSO treated
sample (Figure 5a). After treatment with DC_YM21 in the
indicated concentration for 12 h, less menin protein was
detected in co-immunoprecipitation samples (Figure 5a),
demonstrating the effective disrupting of menin-MLL fusion
protein interactions in human cells.
Figure 4. DC_YM21 binds to menin and disrupt menin-MLL interface in vitro. a)
Changes in thermodynamic stability of menin upon binding of DC_YM21 and DC_YM3.
b) Surface Plasmon Resonance (SPR) experiment of DC_YM21 binding to menin. c)
Competition with MBM1 peptide in NMR STD experiments containing DC_YM21 (200
μM) and menin (5 µM).
Growth Inhibition and Cell Cycle Arrest in Leukemia Cells
Inhibitors targeting the menin-MLL fusion protein interaction
are supposed to cause cell growth inhibition and induce cell
cycle arrest in cells with MLL fusions43, 44, 47. Several leukemia
cells were chosen and treated with potent inhibitors to assess
the effect of growth inhibition.
hydrophobic interactions between the tail group of DC_YM26
and the binding pocket which is mainly formed by M322, E326,
W341, and E363 (Figure 3b). Para-substitution of the R4 region
is vital for inhibitory activity and meta-methane sulfonamide
replacement decreased activity probably due to steric clashes,
Treatment with DC_YM21 after 7 days effectively blocked the
proliferation of MV4;11 and KOPN-8 cells, with half-maximal
growth inhibitory concentration (GI50) values of 1.67±0.51 µM
as compound DC_YM27 was much less active than DC_YM26
.
Overall, the structure-activity relationships (SAR) of menin-MLL
inhibitors shed light on further chemical modification in order
to improve inhibitory activity.
and 2.08±0.42 µM respectively，whereas minimal effect on
proliferation was observed in leukemia cells without MLL
translocation (Figure 5b). In contrast, DC_YM3 showed a much
weaker effect on proliferation of MV4;11 cells (Figure 5c).
Besides, impacts of several other potent molecules as well as
the reported inhibitor MI-2-2 were measured in the MV4;11
Inhibition of menin-MLL Interface in vitro
Taken both in vitro and in vivo behaviors into consideration,
DC_YM21 was believed to be one of the most potent inhibitors
against menin-MLL interface. DC_YM21 disrupted the menin-
MBM1 complex with nanomolar activity (IC50 = 0.83±0.13 μM)
in the FP competition assay, which is only slight weaker than
8 | J. Name., 2012, 00, 1-3
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Figure 5. Menin-MLL inhibitor DC_YM21 displays significant biological activities. a) HEK293 cells were transfected with Flag-MLL1-167 and co-immunoprecipitation experiment was
performed after treatment with inhibitors for 12 h. b) AlamarBlue cell viability assay of human leukemia cells with or without MLL-fusion translocations after 7 days’ treatment
with DC_YM21. The experiment was performed three times. c) AlamarBlue cell viability assay of MV4;11 cells after 3 days’ treatment with DC_YM21 and DC_YM3. The experiment
was performed three times. The experiment was performed three times. d) Effect of DC_YM21 on cell cycle progression measured by FACS in MV4;11 cells after 2 days’ treatment.
e) Quantification of CD11b expression in MV4;11 cells treated for 7 days with DC_YM21. Date represent the mean values for quad-ruplicates ±SD. f) Wright-Giemsa-stained
cytospins for MV4;11 cells after 10 days’ treatment. The experiment was performed four times. g) Expression of the Hox genes and MEIS1, DLX2 genes by quantitative Real-Time
PCR in MV4;11 cells treated for 3 days with DC_YM21 referenced to DMSO-treated samples. Data represent the mean values for duplicates ± SD.
Upregulating of HOX cluster genes and HOX cofactor MEIS1 is
cells (Supplementary Table2), indicating the same level symbolic in human MLL leukemia cells30, 31. MV4;11 cells were
activity of these compounds in cellular responses causing. The treated with DC_YM21 at various concentrations and the Real-
effect of DC_YM21 on cell cycle progression was measured by Time PCR experiment was performed to monitor several MLL-
FACS analysis in MV4;11 cells (Figure 5d). DC_YM21 caused an fusion protein target genes. Particularly, homebox genes
increase in the number of cells in the G0/G1 phase at the HOXA10 and MEIS1, as well as one of the downstream targets
concentration of 15 µM by 2 days, and a substantial decrease DXL248 were profoundly decreased upon treatment with
in the S phase was observed. Thus, these results validated the DC_YM21 compared with the DMSO control sample (Figure
selective killing of MLL leukemia cells by the menin-MLL 5g). HOXA9 also showed a mild down-regulation effect.
inhibitors DC_YM21
.
Cell Differentiation and Downregulation of MLL Fusion Target
Genes Induced by DC_YM21
Chemistry
A total of 24 loperamide-based analogues were synthesized for optimization
of inhibitory activity against the menin-MLL interaction. Firstly, to identify
the role of amide in loperamide, analogues having a cyano or hydroxyl in
place of the amide group were readily synthesized. As outline in Scheme 1,
the first route employs substitution with commercially available 4-(4-
chlorophenyl)-4-hydroxypiperidine (1) and 4-bromo-2,2-
Having measured the selective growth inhibition effect of
compounds, we further assessed the impact on induction of
differentiation in two MLL leukemia cells MV4;11 and KOPN-8
(Figure 5e-f and Supplementary Figure 3A). After treatment
with DC_YM21, a marked morphology change from primitive
blasts to more mature myeloid cells was observed (Figure 5f).
A significant increase in cells positive for CD11b, a myeloid
differentiation marker, was detected after 7 day’s treatment of
MV4;11 cells (Figure 5e). Together, these results reveal that
disrupting the interaction of menin-MLL fusion protein by
DC_YM21 relieves the differentiation block induced by MLL
fusion proteins in human MLL leukemia cells.
diphenylbutanenitrile (2) yielding DC_YM4 in high yield. Similarly, tosylate (5)
was synthesized in three steps starting from the benzophenone73, followed
by subsequent substitution with 1 to afford the desired analogue DC_YM5.
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Full experimental details can be found within Supporting Information.
Scheme 2 Synthesis of Compound DC_YM6 and DC_YM7. Reagents and conditions: (i)
LDA, 1,1,1-trifluoro-N-phenyl-N-(trifluoromethyl sulfonyl)methane sulfonamide,
-
78~0°C, 3 h, 92%; (ii) 4-chlorobenzeneboronic acid, PdCl2(PPh3)2, NaHCO3, THF, reflux, 2
h, 95%; (iii) hydrogen chloride (gas), EA, r.t., 1 h, 90%; (iv) 9, Na2CO3, MeCN, 80°C, 4 h,
80-82%.
CN
Cl
N
C
N
s
OT
OH
OH
DC YM4
_
Cl
Synthesis of DC_YM8 was performed according to Scheme 3.
N
2
1
H
Acyl chlorination of 4-bromo-2,2-diphenylbutyric acid (11
proceeded efficiently with SOCl2 producing the
)
i
OH
OH
diphenylbutanoyl chloride, which was then subjected to
N
diethylamine to afford N,N-diethyl-substituted amide (12)75.
s
OT
OH
5
Similar approach was employed for synthesis of DC_YM8
which substitution with 4-(4-chlorophenyl)-1,2,3,6-
tetrahydropyridine hydrochloride salt ( ). The process was
,
Cl
DC YM5
_
v
i
8
OH
OH
optimized to provide good overall yield of 68% over three
steps.
iii
O
ii
OH
O
O
Et
N
Et
4
3
r
B
Et
O
H
COO
N
O
Et
i ii
,
Scheme 1 Synthesis of Compound DC YM4 and DC_YM5. Reagents and conditions: (i) 1,
Na2CO3, MeCN, 80°C, 4 h, 80-83%; (ii) LDA, EA, -78℃, 2 h, 95%; (iii) LiAlH4, THF, 0°C,
1 h, 90%; (iv) TsCl, pyridine, 0°C, 4 h, 72%.
r
B
r
B
11
12
Next, we intend to remove the hydroxyl of piperidin ring and
introduce double bond or nitrogen atom to build the
tetrahydropyridine or piperazine scaffold and a strategy with
Suzuki coupling as a key step74 was employed. Starting from
the commercially available Boc-protected piperidone followed
by trifluoromethylsulfonylation, we were able to obtain 4-
chlorophenyl-1,2,3,6-tetra hydropyridine core and subsequent
O
N
iii
N
l
C
D
C
YM8
_
Scheme 3 Synthesis of Compound DC_YM8. Reagents and conditions: (i) 11, SOCl2,
CHCl3, reflux, 4 h; (ii) diethylamine, Na2CO3, PhMe/H2O, 0~5°C, 2 h; (iii) 8, Na2CO3,
MeCN, 80°C, 4 h, 3 steps 68%.
deprotection provided
with an important intermediate
8 as hydrochloride salt. Substitution
9
(commercially available or
prepared according to the previous work75) using Na2CO3 as a
weak base to form DC_YM6 and DC_YM7, respectively,
Scheme 2.
For preparation of DC_YM9, we explored the possibility of
removing carbonyl group on head region. Thus, reduction of
nitrile to primary amine gave us a feasible method. However,
initial screening of various reduction conditions, including
borohydride, LiAlH4 or borane complexs, we failed to get the
desired product due to the inert nitrile in compound 13. Finally,
lewis acid-assisted reduction strategy was accomplished
utilizing fresh preparation LiAlH4-AlCl3 complex, which was
described by K. May et al.76, that with AlCl3 adding into a
solution of LiAlH4 in THF and the reductive reaction should
perform at reflux temperature, successfully producing
analogue DC_YM9 in mild yield (Scheme 4).
l
C
Tf
O
O
iii
ii
i
N
N
oc
B
oc
B
N
6
7
oc
B
l
C
O
N
N
r
B
N
O
.
H
l
C
N
H
l
C
D
C
_
YM6
8
9
CN
N
N
C
N
i
ii
l
C
NH2
r
B
v
i
5
DC YM9
_
13
Cl
Cl
O
N
N
N
Scheme 4 Synthesis of Compound DC_YM9. Reagents and conditions: (i) 8, Na2CO3,
N
MeCN, 80°C, 4 h, 80%; (ii) LiAlH4-AlCl3, THF, r.t. to reflux, 7 h, 60%.
l
C
N
H
10
D YM7
C
_
10 | J. Name., 2012, 00, 1-3
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r
B
N
View Article Online
DOI: 10.1039/C6OB01248E
Conclusions
r
A
O
O
i
N
+
MLL1 protein is the core component of MLL1 complex,
possessing H3K4 methyltransferase activity. Translocation of
MLL1 genes in leukemia translates with MLL fusion proteins,
which are responsible for the upregulating expression of HOX
genes and MEIS1, resulting in enhanced proliferation and
blockage of hematopoietic differentiation30, 31. Menin interacts
with both wildtype MLL1 and MLL1 in MLL1-fusions and is
N
.
HCl
r
A
N
H
:
:
r =
r =
:
:
:
r =
r =
r =
e
M
DC YM11
_
A
CN
O
DC YM10
_
A
A
A
O
F
14
9
S
O
NH
DC YM13
_
A
DC YM12
_
O
:
:
:
r =
A
r =
A
r =
A
DC YM14
_
DC YM15
_
S
NH2
c
NHA
O
:
:
:
r =
r =
r =
DC YM16
_
DC YM17
_
A
A
A
CONH2
COOH
reported to act as a co-factor in leukemic transformation42, 47
.
DC YM18
_
DC YM19
_
Cl
N
Disrupting the interaction between menin and MLL1 by
genetic or biochemical methods could abolish the oncogenic
activity of MLL-fusion proteins42, 47. Utilizing a shape-based
scaffold hopping approach to analyze the recent small-
molecule inhibitors of menin-MLL, we identified the
antidiarrheals loperamide to possess, previously unreported,
mild inhibition of the menin-MLL interaction (IC50 = 69 ± 3 µM).
According to the binding mode analysis of loperamide at the
menin-MLL interface, Structure-activity relationship studies
were applied to obtain better inhibitors from this scaffold. In
this work, we explored several changes in the piperidin group
in the linker region, substitutions in the dimethylacetamide
group at the head region, modifications in the tail group region
as well as replacements of phenyl rings at the head region of
loperamide scaffold. Several loperamide-derived analogues
with nanomolar inhibitory activity for the menin-MLL
interaction were obtained, showing the same level activities
with the reported inhibitor MI-2-2 in our fluorescence
polarization assays and cell viability assays. Biochemical
experiments demonstrated that DC_YM21, one of the potent
novel scaffold molecules we developed from the antidiarrheals
loperamide, can specifically disrupt the menin-MLL interaction
in vitro and in vivo by fitting into the menin cavity and
competing with MLL. Treatment with DC_YM21 results in
cellular responses such as inhibition of proliferation, cell cycle
arrest, induction of differentiation and down-regulation of MLL
fusion target genes expression in human MLL leukemia cells,
emphasizing on-target effects of menin-MLL inhibitor. Thus,
we have provided molecules of a novel scaffold targeting
menin-MLL interaction, and they may serve as new potential
therapeutic agents for MLL leukemia. We also provided some
clues for further menin-MLL inhibitors design and
development in this work through repurposing the loperamide
DC YM20
_
O
Scheme 5 Synthesis of Compound DC_YM10~ DC_YM20. Reagents and conditions: (i) 9,
Na2CO3, MeCN, 80°C, 4~6 h, 62-81%.
The synthesis of compounds DC_YM10~DC_YM20
, with
multiple aryl-substituents on tetrahydropyridine ring is
depicted in Scheme 5. The 4-aryl substituted-1,2,3,6-
tetrahydropyridine intermediates (14) involving polar or less-
polar groups, polycyclic or heterocycle aromatic moieties were
synthesized via the previously described Suzuki coupling,
followed by substitution with the commercially available
furaniminium bromide
(9) to obtain the corresponding
loperamide-based analogues with tail group region modified.
R
R
Alk l
y
CN
R
CN
CN
iii
ii
i
R
Alk
l
CN
Alk l
y
y
OH
OTBS
R
19
15
17
18
R
R
Alk l
y
Alk l
y
N
CN
v
i
v
N
v
i
CN
NH2
r
A
r
A
Alk l
y
s
OT
~
DC YM21 DC YM27
21
_
_
20
Scheme 6 Synthesis of Compounds DC_YM21~DC_YM27. Reagents and conditions: (i)
Alkyl-Br 16, NaNH2, Et2O, reflux, 3 h, 83-90%; (ii) 2-(tert-butyldimethylsilyloxy)ethyl
bromide, NaNH2, PhMe, 120℃, 3 h, 74-85%; (iii) TBAF, THF, r.t., 2 h, 85-90%; (iv) TsCl,
pyridine, 0℃, 4 h, 85-88%; (v) aryl-substituted tetrahydropyridine 8 or 14, pyridine, 0℃,
4 h, 64-72%; (vi) LiAlH4-AlCl3, THF, r.t. to reflux, 7~15 h, 41-57%
In an attempt to further modify on head region, synthesis of
cyclopentyl aminomethyl tetrahydropyridine DC_YM21~
DC_YM27 is outlined in Scheme 6. Aryl acetonitriles 15 were
initial deprotonated and reacted with alkyl bromide to give
intermediate 17, which followed by a second deprotonation
and subjected to silyl ether-protected alkyl bromide, resulting
compound 18. Subsequent deprotection and tosylation to
afford tosylate intermediates 20 in good yield, which were
scaffold. However, the specific feature of menin in the
77
complex interaction network41,
and the important
physiological functions of MLL2 remain to be further studied.
Inhibitors for menin-MLL interactions are supposed to serve as
potential leads and powerful chemical biology probes for many
other diseases beyond leukemia.
further reacted with aryl-substituted tetrahydropyridine (14
)
via the same substitution condition. The nitriles 21 were also
reduced using LiAlH4-AlCl3 agent described for previous series
to offer the analogues DC_YM21~DC_YM27 with aminomethyl
moiety.
Experimental
Chemistry: General Information. Commercially available chemicals
were used without further purification. All products were
characterized by their NMR and MS spectra. Mass spectra and high-
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resolution mass spectra were measured on a Finnigan MAT-95 mass Step 3: A round-bottom flask was placed a solution of
spectrometer. Melting points (uncorrected) were determined using compound
7 (293 mg, 1 mmol) in ethyl acetate (5 mL),
1
a SGWX-4B micro melting point apparatus. H and 13C NMR spectra hydrogen chloride (gas) was bubbled through the solution and
were determined on Bruker AM-300, Bruker AM-400, Bruker AM- the resulting mixture was stirred for 1 h at room temperature.
500 instruments using tetramethylsilane as internal reference. Data The formed precipitate was collected by filtration and dried to
are presented as follows: chemical shift, multiplicity (s = singlet, br s yield 206 mg (90%) of a white solid
= broad singlet, d = doublet, br d = broad doublet, t = triplet, m = Step 4: A solution of compound (55 mg, 0.24 mmol) in CH3CN
multiplet), J = coupling constant in hertz (Hz). Silica gel 60H (200- (5 mL) was added dihydro-N,N-dimethyl-3,3-diphenyl-2(3H)-
300 mesh) manufactured by Qingdao Haiyang Chemical Group Co. furaniminium bromide ( , 65 mg, 0.19 mmol) and Na2CO3 (60
(China) was used for general chromatography. mg, 0.57 mmol), and the mixture was stirred at 80 for 4h.
8
; LRMS (EI) m/z 229 [M]+.
8
9
℃
Procedures for Synthesis of Compounds. Synthesis of presented Then the solvent was removed in vacuo. The residue was
compounds was performed according to Schemes 1-6. Full diluted with EtOAc and washed with water. The organic layers
experimental procedures, analytical data, and NMR spectra of were combined, washed with brine, dried over Na2SO4 and
compounds reported here can be found within Supporting concentrated under reduced pressure. The residue was
Information. Synthesis and analytical data for representative purified
by
flash
column
chromatography
compounds from N, N-dimethyl-2,2-diphenylbutanamide (DC_YM6 (dichloromethane/methanol
and DC_YM10 to DC_YM20) and 2-alkyl-2-arylbutan-1-amine DC_YM6 (80%);
= 20/1) to give compound
(DC_YM21 to DC_YM27) classes are provided below.
4-(4-(4-chlorophenyl)-3,6-dihydropyridin-1(2H)-yl)-N,N-
Typical Experimental Procedure A (step 1 to step 4) for Synthesis dimethyl-2,2-diphenylbutanamide (DC_YM6)
of DC_YM6 and DC_YM10 to DC_YM20 using various of 4-aryl Yield, 80%; mp 130 – 132 °C; 1H NMR (300 MHz, CDCl3) δ 7.45-
substituted-1,2,3,6-tetrahydropyridine (taking DC_YM6 as an 7.33 (m, 8H), 7.31-7.22 (m, 6H), 5.96-5.91 (m, 1H), 3.18-3.11
example):
(m, 2H), 2.98 (brs, 3H), 2.69 (t, J = 5.6 Hz, 2H), 2.60-2.46 (m,
Step 1: To a solution of diisopropylamine (0.7 mL) in THF (10 4H), 2.33 (brs, 3H), 2.32-2.21 (m, 2H); 13C NMR (100 MHz,
mL) was added n-butyllithium solution in hexane (3.1 mL, 1.6 CDCl3) δ 173.4, 140.7, 139.3, 133.7, 132.4, 128.3, 128.2, 128.0,
M in hexane) at -78°C. The mixture was stirred at -78°C for 15 126.7, 126.1, 122.7, 59.7, 55.4, 53.1, 50.2, 42.6, 39.1, 37.2,
min to get a LDA solution, and then 1-Boc-4-piperidone (1.0 g, 28.0; HRMS (EI) calcd. for C29H31ClN2O [M]+: 458.2125. Found:
5.02 mmol) in THF (10 mL) was slowly added to the reaction 458.2122.
mixture at -78°C. After 20 min, a solution of 1,1,1-trifluoro-N- DC_YM10 to DC_YM20 were prepared according to the
phenyl-N-(trifluoromethylsulfonyl) methane sulfonamide (1.8 g, procedure A, step 4. To a stirred equimolecular suspension of
5.02 mmol) in THF (10 mL) was slowly added to the mixture. dihydro-N,N-dimethyl-3,3-diphenyl-2(3H)-furaniminium
The reaction mixture was stirred at 0°C for 3 h. The reaction bromide
(9
,
0.2 mmol), 4-substituted aryl-1,2,3,6-
was quenched with saturated NH4Cl solution and extracted tetrahydropyridine intermediates (14, 0.25 mmol) and Na2CO3,
with EtOAc. The combined organic layers were washed with then the mixture was stirred at 80°C for 4-6 h. After general
brine, and dried over Na2SO4, and concentrated. The residue work up, the residue was purified by flash column
was purified by column chromatography with petroleum chromatography to give compound. 4-(4-methoxyphenyl)-
ether/ethyl acetate (10/1, v/v) to obtain the compound 6 (92% 1,2,3,6-tetrahydropyridine 14a for DC_YM10, 4-(1,2,3,6-
1
yield); LRMS (EI) m/z 331 [M]+; H NMR (300 MHz, CDCl3) δ tetrahydropyridin-4-yl)benzonitrile 14b for DC_YM11, 4-(4-
5.80-5.72 (m, 1H), 4.06-4.02 (m, 2H), 3.63 (t, J = 5.6 Hz, 2H), fluorophenyl)-1,2,3,6-tetrahydropyridine 14c for DC_YM12, N-
2.48-2.40 (m, 2H), 1.47 (s, 9H).
(4-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)methanesulfonamide
Step 2: To a solution of enol triflate
(4 mL) were added 4-chlorobenzeneboronic acid (187 mg, 1.2 yl)phenyl)acetamide
6
(331 mg, 1 mmol) in THF 14d
for
DC_YM13
,
N-(4-(1,2,3,6-tetrahydropyridin-4-
for DC_YM14 4-(1,2,3,6-
14e
,
mmol), saturated NaHCO3 solution (1 mL) and PdCl2(PPh3)2 (14 tetrahydropyridin-4-yl)benzenesulfonamide 14f for DC_YM15
mg, 0.02 mmol), and the mixture was stirred at reflux for 2 h. 4-(1,2,3,6-tetrahydropyridin-4-yl)benzoic acid 14g for
The mixture was diluted with EtOAc and the aqueous layer was DC_YM16 4-(1,2,3,6-tetrahydropyridin-4-yl)benzamide 14h
separated and extracted with EtOAc. The organic layers were for DC_YM17 4-(4'-chloro-[1,1'-biphenyl]-4-yl)-1,2,3,6-
combined, washed with brine, dried over Na2SO4 and tetrahydropyridine 14i for DC_YM18 3-(4-(1,2,3,6-
,
,
,
,
concentrated under reduced pressure. The residue was tetrahydropyridin-4-yl)phenyl)quinolone 14j for DC_YM19, 4-
purified by flash column chromatography (petroleum (4-(benzofuran-2-yl)phenyl)-1,2,3,6-tetrahydropyridine 14k for
ether/ethyl acetate = 2/1) to give compound
7 (95%); LRMS (EI) DC_YM20. The purity of the synthesized compounds was
1
m/z 293 [M]+; H NMR (400 MHz, CDCl3) δ 7.34-7.27 (m, 4H), determined to be greater than 95% by HPLC analysis [HPLC:
6.01 (brs, 1H), 4.06 (brs, 2H), 3.63 (brs, 2H), 2.47 (brs, 2H), 1.48 column, YMC-Triart C18, 250 mm × 4.6 mm, 5 μ m; solvent
(s, 9H).
system, MeOH – 0.5 ‰ NH3.H2O and water – 0.5 ‰ NH3.H2O
with gradient elution; flow rate, 1.0 mL/min; UV detection, 210
12 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
1
or 254 nm; oven, 20 °C. Agilent 1100, Agilent Technologies, Yield, 63%; mp 245 – 248 °C; H NMR (300 MHz, DMSO-d ) δ
View Article Online
6
California, USA]. Detailed HPLC analysis data of the synthesized 7.74 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 HDz,O2I:H10),.170.3495/C–6O7B.20412(4m8E,
compounds are displayed in Table S3 Supporting Information.
4-(4-(4-methoxyphenyl)-5,6-dihydropyridin-1(2H)-yl)-N,N-
dimethyl-2,2-diphenylbutanamide (DC_YM10)
12H), 6.22 (s, 1H), 3.01-2.75 (m, 5H), 2.48-2.18 (m, 9H), 2.10-
1.99 (m, 2H); 13C NMR (125 MHz, DMSO-d6) δ 172.7, 143.8,
142.7, 141.6, 133.4, 128.9, 128.2, 127.0, 126.2, 125.3, 59.4,
Yield, 76%; mp 170 – 172 °C; 1H NMR (300 MHz, CDCl3) δ 7.43- 55.4, 53.3, 50.3, 41.7, 27.8; HRMS (EI) calcd. for C29H33N3O3S
7.33 (m, 8H), 7.31-7.23 (m, 4H), 6.85-6.77 (m, 2H), 5.81 (s, 1H), [M]+: 503.2243. Found: 503.2237.
3.76 (s, 3H), 3.42 (m, 2H), 2.94 (m, 5H), 2.73-2.60 (m, 4H), 2.52 4-(1-(4-(dimethylamino)-4-oxo-3,3-diphenylbutyl)-1,2,3,6-
(m, 2H), 2.29 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 173.4, 159.7, tetrahydropyridin-4-yl)benzamide (DC_YM16)
1
144.6, 135.1, 130.8, 129.7, 129.3, 129.1, 128.0, 127.9, 127.7, Yield, 73%; mp 143 – 145 °C; H NMR (300 MHz, DMSO-d6) δ
126.3, 113.9, 112.6, 59.8, 55.3, 53.83, 50.2, 48.8, 39.2, 39.1, 7.81 (d, J = 8.3 Hz, 2H), 7.47 – 7.25 (m, 14H), 6.18 (s, 1H), 3.00-
37.2, 23.5; HRMS (EI) calcd. for C30H34N2O2 [M]+ : 454.2620. 2.80 (m, 5H), 2.48-2.18 (m, 9H), 2.10-2.00 (m, 2H); 13C NMR
Found: 454.2615.
(125 MHz, DMSO-d6) δ 172.7, 168.0, 143.2, 141.6, 133.7, 132.9,
4-(4-(4-cyanophenyl)-5,6-dihydropyridin-1(2H)-yl)-N,N-
128.9, 128.2, 128.1, 127.0, 124.6, 124.3, 59.4, 55.4, 55.3, 53.4,
dimethyl-2,2-diphenylbutanamide (DC_YM11)
50.4, 41.7, 27.8; HRMS (EI) calcd. for C30H33N3O2 [M]+
:
1
Yield, 70%; mp 175 – 177 °C; H NMR (300 MHz, CDCl3) δ 7.55 467.2573. Found: 467.2576.
(d, J = 8.5 Hz, 2H), 7.46-7.33 (m, 10H), 7.31-7.25 (m, 2H), 6.12 4-(1-(4-(dimethylamino)-4-oxo-3,3-diphenylbutyl)-1,2,3,6-
(s, 1H), 3.13-3.07 (m, 2H), 2.99 (s, 3H), 2.62 (t, J = 5.7 Hz, 2H), tetrahydropyridin-4-yl)benzoic acid (DC_YM17)
1
2.55-2.41 (m, 4H), 2.33 (s, 3H), 2.23-2.13 (m, 2H); 13C NMR Yield, 71%; mp 238 – 240 °C; H NMR (300 MHz, DMSO-d6) δ
(125 MHz, CDCl3) δ 173.5, 145.3, 140.7, 133.6, 132.1, 128.4, 11.22 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H),
128.1, 126.8, 125.4, 119.1, 110.2, 59.8, 55.4, 53.2, 50.0, 42.8, 7.50 – 7.32 (m, 10H), 6.24 (s, 1H), 3.75-3.42 (m, 6H), 2.91 br(s,
+
39.2, 37.2, 27.8; HRMS (EI) calcd. for C30H31N3O [M]
449.2467. Found: 449.2462.
4-(4-(4-fluorophenyl)-5,6-dihydropyridin-1(2H)-yl)-N,N-
:
3H), 2.79-2.67 (m, 4H), 2.22 (brs, 3H); 13C NMR (125 MHz,
DMSO-d6) δ 172.6, 167.4, 142.7, 140.1, 139.9, 133.6, 130.4,
130.0, 129.3, 128.3, 128.2, 127.7, 12 5.3, 119.1, 59.6, 53.6,
50.1, 48.0, 39.0, 38.4, 37.1, 23.8; HRMS (EI) calcd. for
dimethyl-2,2-diphenylbutanamide (DC_YM12)
Yield, 81%; mp 134 – 136 °C; H NMR (300 MHz, CDCl3) δ 7.38 C30H32N2O3 [M]+ : 468.2413. Found: 468.2414
1
(m, 8H), 7.35-7.25 (m, 3H), 7.12-6.80 (m, 3H), 5.85 (s, 1H), 3.29 4-(4-(4'-chloro-[1,1'-biphenyl]-4-yl)-5,6-dihydropyridin-1(2H)-
(s, 2H), 2.98 (brs, 3H), 2.85 (t, J = 5.6 Hz, 2H), 2.58 (m, 4H), yl)-N,N-dimethyl-2,2-diphenylbutanamide (DC_YM18)
2.45-2.35 (m, 2H), 2.31 (brs, 3H); 13C NMR (125 MHz, CDCl3) δ Yield, 76%; mp 161 – 163 °C; 1H NMR (500 MHz, CDCl3) δ 7.54-
173.4, 162.0 (d, J = 244.1 Hz), 140.49, 136.7 (d, J = 3.0 Hz), 7.36 (m, 16H), 7.30 (t, J = 7.2 Hz, 2H), 6.06 (s, 1H), 3.11 (d, J =
134.1, 128.6, 128.5, 128.1, 126.8, 126.5, 126.4, 125.3, 120.9, 2.3 Hz, 2H), 3.01 (brs, 3H), 2.65 (t, J = 5.5 Hz, 2H), 2.59-2.47 (m,
115.1, 114.9, 59.7, 55.2, 52.8, 50.0, 42.1, 39.2, 37.1, 27.6; 4H), 2.36 (brs, 3H), 2.22 (m, 2H); 13C NMR (125 MHz, CDCl3) δ
HRMS (EI) calcd. for C29H31FN2O [M]+ : 442.2420. Found: 173.5, 140.9, 140.3, 139.3, 138.2, 134.3, 133.2, 128.9, 128.4,
442.2414.
128.2, 126.7, 126.7, 125.4, 122.6, 59.8, 55.5, 53.3, 50.4, 42.7,
N,N-dimethyl-4-(4-(4-(methylsulfonamido)phenyl)-5,6-
38.9, 37.3, 28.2; HRMS (EI) calcd. for C35H35ClN2O [M]+:
dihydropyridin-1(2H)-yl)-2,2-diphenylbutanamide (DC_YM13) 534.2438. Found: 534.2433.
1
Yield, 62%; mp 154 – 156 °C; H NMR (300 MHz, DMSO-d6) δ N,N-dimethyl-2,2-diphenyl-4-(4-(quinolin-3-yl)-5,6-
9.74 (brs, 1H), 7.45-7.21 (m, 12H), 7.12 (d, J = 8.7 Hz, 2H), 5.98 dihydropyridin-1(2H)-yl)butanamide (DC_YM19)
1
(s, 1H), 2.93 (s, 3H), 2.87 (m, 4H), 2.46-2.16 (m, 10H), 2.06-1.96 Yield, 75%; mp 132 – 134 °C; H NMR (300 MHz, DMSO-d6) δ
(m, 2H); 13C NMR (125 MHz, DMSO-d6) δ 172.7, 141.6, 137.6, 9.03 (s, 1H), 8.18 (s, 1H), 7.93 (t, J = 8.2 Hz, 2H), 7.66 (t, J = 7.5
136.2, 133.6, 128.8, 128.2, 127.0, 125.8, 121.9, 120.1, 59.4, Hz, 1H), 7.54 (t, J = 7.4 Hz, 1H), 7.46-7.20 (m, 10H), 6.38 (s, 1H),
55.4, 55.3, 53.3, 50.4, 41.7, 27.9; HRMS (EI) calcd. for 2.96 (m, 2H), 2.87 (brs, 3H), 2.49 (m, 4H), 2.40 (m, 2H), 2.27
C30H35N3O3S [M]+ : 517.2399. Found: 517.2396.
4-(4-(4-acetamidophenyl)-5,6-dihydropyridin-1(2H)-yl)-N,N-
(brs, 3H), 2.05 (m, 2H); 13C NMR (125 MHz, DMSO-d6) δ 172.7,
148.6, 146.9, 141.6, 132.9, 131.8, 130.4, 129.4, 129.0, 128.9,
128.7, 128.3, 127.9, 127.2, 127.0, 125.0, 59.4, 55.4, 55.4, 53.4,
dimethyl-2,2-diphenylbutanamide (DC_YM14)
1
Yield, 68%; mp 160 – 162 °C; H NMR (300 MHz, DMSO-d6) δ 50.3, 41.7, 27.6; HRMS (EI) calcd. for C32H33N3O [M]+: 475.2624.
9.92 (s, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.40-7.25 (m, 12H), 5.96 (s, Found: 475.2622.
1H), 2.93-2.73 (m, 5H), 2.45-2.21 (m, 9H), 2.05-1.95 (m, 5H); 4-(4-(benzofuran-2-yl)-5,6-dihydropyridin-1(2H)-yl)-N,N-
13C NMR (125 MHz, DMSO-d6) δ 172.7, 168.6, 141.7, 138.7, dimethyl-2,2-diphenylbutanamide (DC_YM20)
135.2, 133.7, 128.8, 128.2, 127.0, 125.1, 121.3, 119.2, 59.4, Yield, 65%; mp 140 – 142 °C; 1H NMR (300 MHz, CDCl3) δ 7.49-
55.5, 55.4, 53.4, 50.5, 41.7, 27.9, 24.5; HRMS (EI) calcd.for 7.33 (m, 10H), 7.32 – 7.24 (m, 2H), 7.23 – 7.10 (m, 2H), 6.47 (s,
C31H35N3O2 [M]+ : 481.2729. Found: 481.2731.
N,N-dimethyl-2,2-diphenyl-4-(4-(4-sulfamoylphenyl)-5,6-
dihydropyridin-1(2H)-yl)butanamide (DC_YM15)
1H), 6.41 (s, 1H), 3.19-3.11 (m, 2H), 2.99 (brs, 3H), 2.65 (t, J =
5.6 Hz, 2H), 2.58 – 2.42 (m, 4H), 2.34 (brs, 3H), 2.26-2.18 (m,
2H); 13C NMR (125 MHz, CDCl3) δ 173.5, 156.4, 154.5, 140.7,
129.0, 128.5, 128.1, 126.8, 125.5, 124.0, 122.6, 120.7, 110.8,
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Organic & Biomolecular Chemistry
Page 14 of 20
Journal Name
View Article Online
DOI: 10.1039/C6OB01248E
100.8, 59.8, 55.4, 52.7, 49.6, 42.5, 39.2, 37.2, 25.8; HRMS (EI) (318 mg, 3mmol), and the mixture was stirred at 80°C for 4h.
calcd. for C31H32N2O2 [M]+: 464.2464. Found: 464.2467.
Then the solvent was removed in vacuo. The residue was
General procedure B (step 1 to step 6) for the synthesis of diluted with EtOAc and washed with water. The organic layers
DC_YM21 to DC_YM27 (taking DC_YM21 as an example): were combined, washed with brine, dried over Na2SO4 and
Step 1: A solution of 2-phenylacetonitrile (15a, 3 g, 25 mmol) concentrated under reduced pressure. The residue was
in Et2O (50 mL) was added bromocyclopentane (16a 2.7 mL) purified by flash column chromatography (petroleum ether
and NaNH2 (1 g, 25 mmol), the mixture was refluxed for 3 h /acetone = 3/1) to give compound 21a (70%); LRMS (EI) m/z
then quenched with water and extracted with EtOAc. The 404 [M]+; 1H NMR (300 MHz, CDCl3) δ 7.49-7.14 (m, 13H), 5.98
combined organic layers were washed with brine, and dried (s, 1H), 3.09-3.01 (m, 2H), 2.67-2.55 (m, 2H), 2.50-2.43 (m, 2H),
over Na2SO4, and concentrated. The residue was purified by 2.40-2.31 (m, 2H), 2.24-2.10 (m, 2H), 2.07-1.93 (m, 2H), 1.64-
column chromatography with petroleum ether/ethyl acetate 1.55 (m, 3H), 1.46-1.38 (m, 1H), 1.31-1.20 (m, 3H).
(30/1, v/v) to obtain the compound 17a (90% yield); LRMS (EI) Step 6: LAH (0.15 mL, 2.4 M in THF) was slurried in anhydrous
1
m/z 185 [M]+; H NMR (300 MHz, CDCl3) δ 7.41-7.27 (m, 5H), THF (5 mL), and the solution was cooled in an ice bath to 0°C
3.71 (d, J = 7.7 Hz, 1H), 2.38-2.24 (m, 1H), 1.94-1.80 (m, 1H), under nitrogen. AlCl3 (49 mg, 0.37 mmol) was dissolved in
1.76-1.63 (m, 3H), 1.62-1.48 (m, 3H), 1.40-1.28 (m, 1H).
anhydrous THF (5mL). The AlCl3 solution was added to the LAH
Step 2: To a solution of compound 17a (253 mg, 1.4 mmol) in slurry via an addition funnel over 15 min. Compound 21a (50
PhMe (10 mL) was added 2-(tert-butyldimethylsilyloxy)ethyl mg, 0.12 mmol) was dissolved in anhydrous THF (5mL). This
bromide (327 g, 1.4 mmol) and NaNH2 (164 mg, 4.2 mmol), the solution was added to the LAH/AlCl3 mixture slowly. The
mixture was stirred at 120°C for 3 h then quenched with water resulting mixture was stirred at ambient temperature for 1 h
and extracted with EtOAc. The combined organic layers were and then heated to reflux for 7 h. When cooled to 0°C the
washed with brine, and dried over Na2SO4, and concentrated. reaction was quenched with water and extracted with Et2O.
The residue was purified by column chromatography with The combined organic layers were washed with brine, and
petroleum ether/ethyl acetate (30/1, v/v) to obtain the dried over Na2SO4, and concentrated. The residue was purified
1
compound 18a (85% yield); LRMS (EI) m/z 343 [M]+; H NMR by column chromatography with dichloromethane/methanol
(300 MHz, CDCl3) δ 7.44-7.24 (m, 5H), 3.66-3.56 (m, 1H), 3.35- (15/1, v/v) to obtain the compound DC_YM21 (53%).
3.26 (m, 1H), 2.41-2.28 (m, 2H), 2.21-2.10 (m, 1H), 2.07-1.93 4-(4-(4-chlorophenyl)-3,6-dihydropyridin-1(2H)-yl)-2-
(m, 1H), 1.82-1.70 (m, 1H), 1.69-1.56 (m, 3H), 1.51-1.37 (m, cyclopentyl-2-phenylbutan-1-amine (DC_YM21)
1H), 1.31-1.21 (m, 2H), 0.82 (s, 9H), -0.07 (d, J = 4.2 Hz, 6H).
Mp 195 – 197 °C; 1H NMR (300 MHz, CDCl3) δ 7.49-7.20 (m, 9H),
Step 3: To a stirred solution of compound 18a (343 mg, 1 5.99 (s, 1H), 3.06 (s, 2H), 2.72-2.50 (m, 3H), 2.49 (m, 2H), 2.44-
mmol) in THF (5 mL) was added tetrabutylammonium fluoride 2.29 (m, 2H), 2.13 (d, J = 9.5 Hz, 3H), 1.65 (d, J = 12.3 Hz, 5H),
(1 mL, 1M in THF), the reaction was stirred at room 1.44 (s, 1H), 1.34-1.20 (m, 3H); 13C NMR (125 MHz, CDCl3) δ
temperature for 2 h. The mixture was diluted with EtOAc and 142.2, 139.2, 134.1, 132.7, 128.4, 128.1, 127.9, 126.2, 125.9,
washed with water. The organic layers were combined, 122.2, 62.9, 53.7, 53.5, 50.6, 47.7, 47.0, 46.6, 31.3, 28.0, 27.1,
washed with brine, dried over Na2SO4 and concentrated under 27.0, 24.5, 24.4; HRMS (EI) calcd. for C26H33ClN2 [M]+, 408.2332.
reduced pressure. The residue was purified by flash column Found, 408.2325.
chromatography (petroleum ether /acetone = 2/1) to give DC_YM22 to DC_YM27 were prepared according to the
compound 19a (90%); LRMS (EI) m/z 229 [M]+.
procedure B (step 1 to step 6). 2-(3-fluorophenyl) acetonitrile
Step 4: The p-toluenesulfonyl chloride (191 mg, 1mmol) was 15b for DC_YM22, 2-(4-fluorophenyl) acetonitrile 15c for
added to the solution of the compound 19a (206 mg, 0.9mmol) DC_YM23 1-bromopentane 16b for DC_YM24 4-(4-
,
,
in dry pyridine (5mL) at 0°C under atomosphere of argon, and fluorophenyl)-1,2,3,6-tetrahydropyridine 14c for DC_YM25, N-
the mixture was stirred at 0°C for 4 h. The mixture was diluted (4-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)methanesulfonamide
with EtOAc and washed with hydrochloric acid (2M), the 14d
for
DC_YM26
,
N-(3-(1,2,3,6-tetrahydropyridin-4-
combined organic layers were washed with brine, and dried yl)phenyl)methanesulfonamide 14l for DC_YM27. The purity of
over Na2SO4, and concentrated. The residue was purified by the synthesized compounds was determined to be greater
column chromatography with petroleum ether/ethyl acetate than 95% by HPLC analysis [HPLC: column, YMC-Triart C18, 250
(6/1, v/v) to obtain the compound 20a (85% yield); LRMS (EI) mm × 4.6 mm, 5 μ m; MeOH – 0.5 ‰ NH3.H2O and water –
1
m/z 383 [M]+; H NMR (300 MHz, CDCl3) δ 7.65 (d, J = 8.3 Hz, 0.5 ‰NH3.H2O with gradient elution; flow rate, 1.0 mL/min;
2H), 7.37-7.25 (m, 7H), 3.96 (td, J = 9.8, 6.0 Hz, 1H), 3.75 (td, J UV detection, 210 or 254 nm; oven, 20 °C. Agilent 1100,
= 9.8, 5.5 Hz, 1H), 2.53-2.41 (m, 4H), 2.38-2.25 (m, 2H), 1.99- Agilent Technologies, California, USA]. Detailed HPLC analysis
1.87 (m, 1H), 1.81-1.69 (m, 1H), 1.62-1.53 (m, 3H), 1.49-1.35 data of the synthesized compounds are displayed in Table S3
(m, 1H), 1.28-1.20 (m, 2H).
Supporting Information.
Step 5: A solution of compound 20a (383 mg, 1mmol) in CH3CN 4-(4-(4-chlorophenyl)-5,6-dihydropyridin-1(2H)-yl)-2-
(5mL) was added compound
8
(229 mg, 1mmol) and Na2CO3 cyclopentyl-2-(3-fluorophenyl) butan-1-amine (DC_YM22)
14 | J. Name., 2012, 00, 1-3
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Page 15 of 20
Organic & Biomolecular Chemistry
Mp 190 – 192 °C; 1H NMR (300 MHz, CDCl3) δ 7.31-7.23 (m, 5H), N-(3-(1-(4-amino-3-cyclopentyl-3-phenylbutyl)-1,V2i,e3w,A6r-ticle Online
DOI: 10.1039/C6OB01248E
7.06 (m, 2H), 6.90 (m, 1H), 6.03 (s, 1H), 3.20-3.00 (m, 4H), 2.70 tetrahydropyridin-4-yl)phenyl)methanesulfonamide
(m, 2H), 2.54 (m, 2H), 2.41 (m, 2H), 2.10 (m, 2H), 1.73-1.49 (m, (DC_YM27)
3H), 1.39 (m, 5H), 1.01 (m, 3H); 13C NMR (125 MHz, CDCl3) δ Mp 210 – 213 °C; 1H NMR (400 MHz, CDCl3) δ 7.40 (t, J = 7.7 Hz,
162.9 (d, J = 242.9 Hz), 145.7 (d, J = 6.25 Hz), 139.2, 134.2, 2H), 7.28 (d, J = 7.3 Hz, 1H), 7.26 – 7.19 (m, 3H), 7.17 – 7.09 (m,
132.8, 129.2 (d, J = 8.12 Hz), 128.5, 126.3, 123.7 (d, J = 2.12 Hz), 1H), 7.11 – 6.96 (m, 2H), 5.90 (s, 1H), 3.58 (d, J = 13.8 Hz, 1H),
122.3, 115.3 (d, J = 21.6 Hz), 112.8 (d, J = 20.7Hz),53.7, 53.6, 3.44 (s, 3H), 3.22 (m, 1H), 3.17 (d, J = 13.8 Hz, 1H), 3.06-2.89
50.8, 47.5, 47.2, 46.6, 31.4, 28.1, 27.1, 27.1, 24.5, 24.5; HRMS (m, 2H), 2.86 (s, 3H), 2.54 (m, 4H), 2.39 (m, 1H), 2.05 (m, 2H),
(EI) calcd. for C26H32ClFN2 [M]+, 426.2238. Found, 426.2240.
4-(4-(4-chlorophenyl)-5,6-dihydropyridin-1(2H)-yl)-2-
1.54 (m, 2H), 1.40 (m, 3H), 1.10 (m, 2H); 13C NMR (125 MHz,
CDCl3) δ 129.2, 128.9, 127.8, 126.9, 121.5, 120.5, 119.7,
118.0,52.8, 52.4, 51.3, 49.6, 47.3, 44.8, 38.9, 32.2, 27.2, 27.1,
cyclopentyl-2-(4-fluorophenyl)butan-1-amine (DC_YM23)
1
Mp 195 – 197 °C; H NMR (300 MHz, CDCl3) δ 7.36 – 7.23 (m, 27.1, 24.7, 24.5; HRMS (EI) calcd. for C27H37N3O2S[M]+,
6H), 7.03 (t, J = 8.3 Hz, 2H), 6.01 (d, J = 6.9 Hz, 1H), 3.13 (m, 3H), 467.2606. Found, 467.2601.
2.69 (m, 2H), 2.56 (m, 3H), 2.41 (m, 2H), 2.02 (m, 2H), 1.85 (m, Scaffold Hopping
1H), 1.63 (m, 3H), 1.40 (m, 4H), 1.15 (m, 3H); 13C NMR (125
MHz, CDCl3) δ 161.1 (d, J = 243.7 Hz), 139.2, 138.1 (d, J = 2.5
Hz), 134.1, 132.7, 129.5 (d, J = 8.12 Hz), 128.4, 126.2, 122.2,
114.5 (d, J = 20.6 Hz), 53.7, 53.6, 50.8, 47.2, 46.7, 46.6, 31.4,
Taking the reported bioactive conformation of MI-2-2 and
MIV-6R in the crystal structure (PDB code 4GQ4 and 4GO8) as
the query, 3D-similarity search was performed using SHFATS67,
68.SHAFTS method combines the strength of molecular shape
28.1, 27.0, 26.9, 24.4, 24.3; HRMS (EI) calcd. for C26H32ClFN2
superposition and chemical feature matching for the 3D
similarity calculation and ranking. Considering the drug-like
properties of candidates, we built an in-house library
comprising ~1600 compounds from existing drugs for 3D
[M]+, 426.2238. Found, 426.2242.
2-(2-(4-(4-chlorophenyl)-5,6-dihydropyridin-1(2H)-yl)ethyl)-2-
phenylhexan-1-amine (DC_YM24)
Mp 170 – 173 °C; 1H NMR (400 MHz, CDCl3) δ 7.36-7.31 (m, 4H),
similarity searching. Before SHAFTS calculation, diverse
7.28 (m, 3H), 7.21 (m, 2H), 6.03 (m, 1H), 3.17-3.04 (m, 2H),
conformation generation for each molecule in the database
was performed by Discovery Studio 2.572. Subsequently, each
conformer was aligned onto the query and calculated the 3D
2.91 (s, 2H), 2.66 (t, J = 5.7 Hz, 2H), 2.52 (m, 2H), 2.36-2.20 (m,
2H), 1.98 (m, 2H), 1.72-1.64 (m, 2H), 1.29 (m, 4H), 1.17-1.07 (m,
2H), 0.88 (t, J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 145.5,
similarity score by SHAFTS, the top-ranked molecules with
139.4, 134.2, 132.8, 128.5, 126.8, 126.3, 126.0, 122.5,53.6,
SHAFTS similarities >1.2 (maximum 2.0) were selected for
53.5, 50.7, 49.2, 44.8, 34.9, 32.2, 28.2, 25.8, 23.6, 14.2; HRMS
further validation.
Molecular Docking
(EI) calcd. for C25H33ClN2 [M]+, 396.2332. Found, 396.2321.
2-cyclopentyl-4-(4-(4-fluorophenyl)-5,6-dihydropyridin-1(2H)-
The molecular docking was performed by Glide program with
yl)-2-phenylbutan-1-amine (DC_YM25)
Maestro v9.0 (Schrodinger, Inc.) 69, 70. The crystal structure of
1
Mp 210 – 212 °C; H NMR (400 MHz, CDCl3) δ 7.36 (m, 4H),
menin in complex with MIV-6R (PDB code4GO8) was used as a
target for the following molecular docking. Hydrogen atoms
and charges of receptor were added using the "Protein
Preparation Wizard" module in Maestro. To relieve steric clash,
a partial minimization of the target was terminated when the
root-mean-square deviation (rmsd) reached a maximum value
of 0.3 Å. Amino acid residues within a distance of 14 Å around
MIV-6R were defined as the binding site. For receptor grid
7.24 (m, 3H), 6.95 (t, J = 8.6 Hz, 2H), 6.51 (brs, 2H), 5.84 (s, 1H),
3.33 (d, J = 13.6 Hz, 1H), 3.17 (dd, J = 32.6, 15.1 Hz, 2H), 3.03 (d,
J = 16.0 Hz, 1H), 2.88 (m, 1H), 2.68 (m, 1H), 2.60-2.44 (m, 4H),
2.34 (d, J = 15.7 Hz, 1H), 2.12-2.00 (m, 2H), 1.36 (m, 6H), 1.04
(m, 2H); 13C NMR (125 MHz, CDCl3) δ 162.2 (d, J = 245 Hz),
139.9, 136.8 (d, J = 3.75 Hz), 134.7, 128.6, 127.8, 126.9 (d, J =
6.25 Hz), 126.7, 120.3, 115.3, 115.1,52.9, 52.6, 50.4, 49.8, 47.2,
44.7, 32.4, 27.4, 27.2, 27.0, 24.6, 24.5; HRMS (EI) calcd. for
C26H33FN2 [M]+, 392.2628. Found, 392.2623.
generation, a scaling factor of 1.0 was set to van der Waals
(VDW) radii of receptor atoms with the partial charge less than
N-(4-(1-(4-amino-3-cyclopentyl-3-phenylbutyl)-1,2,3,6-
tetrahydropyridin-4-yl)phenyl)methanesulfonamide
0.25. Ligands were prepared by "LigPrep" module in Maestro,
the generation of ionization states were carried outwith Epik78,
(DC_YM26)
1
a tools that are part of LigPrep. For ligand docking, extra-
precision (XP) was adopted, at most 10 poses per ligand were
Mp 215 – 217 °C; H NMR (500 MHz, CDCl3) δ 7.32 (m, 4H),
7.18 (m, 5H), 5.97 (s, 1H), 3.31 (d, J = 13.4 Hz, 1H), 3.17 (dt, J =
written out, and the Glide scoring function (G-Score) was used
27.1, 13.8 Hz, 3H), 2.93 (s, 3H), 2.76 (s, 1H), 2.65 (s, 1H), 2.49
(m, 4H), 2.33 – 1.94 (m, 4H), 1.39 (m, 5H), 1.08 (m, 3H); 13C
to select the final poses for each ligand.
Menin Expression and Purification
NMR (125 MHz, CDCl3) δ 141.5, 136.7, 136.6, 133.9, 128.0,
127.9, 125.9, 125.7, 120.7, 120.6, 53.5, 53.3, 50.4, 48.3, 47.1, The full-length of human menin were expressed and purified
46.0, 39.0, 31.0, 27.7, 26.9, 26.7, 24.3, 24.2; HRMS (EI) calcd. as described previously54. For the further purification, a Q-
for C27H37N3O2S[M]+, 467.2606. Found, 467.2603.
Sepharose protocol (GE Healthcare) was performed after
removing of the His6-SUMO tag.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 15

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Organic & Biomolecular Chemistry
Page 16 of 20
Journal Name
View Article Online
DOI: 10.1039/C6OB01248E
Fluorescence polarization (FP) assays of inhibition of menin-MLL
interaction
PH 7.4, 150mM NaCl, 3mM EDTA, 0.05% (v/v) surfactant P20,
and 0.1% (v/v) DMSO) overnight. The compound was serially
diluted with HBS-EP buffer and injected for 120s (contact
phase), followed by 120s dissociation. KD values of the
compound to menin were determined by Biacore T200
evaluation softerware (GE Healhcare).
The fluorescence polarization reaction contains N-terminal
FITC-labeled MLL4-15 peptide (FITC-MBM1, ChinaPeptides Co.,
Ltd.)43, 45 at 30nM and menin at 600 nM in the FP buffer (50
mM HEPES, pH 7.5; 50 mM NaCl; 1mM DTT; 0.1mg/ml BSA).
Compounds were added immediately after the mixture and
Cell Viability Assays
incubated for 2 h in the darker at 4°C. Change in fluorescence MV4;11, KOPN-8, HL60 and Jurkat cells were cultured in RPMI-
polarization was monitored at 535 nm after excitations at 480 1640 medium supplemented with 10% FBSand 1%
nm using the Envison protocol and applied to determine IC50 penicillin/streptomycin (Invitrogen). For the 7-d proliferation
values with the GraphPad Prism 5.0 program. Ki calculations assay, plating densities were determined for each cell line on
were performed with the website The Ki Calculator website the basis of linear log-phase growth. Cells were counted and
http://sw16.im.med.umich.edu/software/calc_ki/.
split back to the original plating density in fresh medium with
compound resupplied on days 4. An AlamarBlue cell viability
assay kit (Invitrogen) was employed. Plates were read for
fluorescence intensity at 590 nm after excitations at 544 nm
using the PHERAstar BMG microplate reader.
NMR spectroscopy
Both Carr-Purcell-Meiboom-Gill (CPMG) and saturation
transfer difference (STD) experiments contained 5 µM menin
in 50 mM phosphate buffer, pH7.5, dissolved in D2O.
Compounds in DMSO-D6 were added to a final concentration
of 200 µM with 2-5% DMSO-D6. All NMR experiments were
Inhibitor effects on cell cycle of MV4;11 leukemia cells
5×
105 mL-1 MV4;11 cell were plated in 12-well plates and
treated with compounds or 0.2% DMSO and incubated for 48h
at 37°C in a 5% CO2 incubator. Cells were harvested, washed
with PBS buffer, and resuspended in cooled PBS buffer
containing 70% ethanol. After fixation at 4°C for 24 h, samples
were washed and resuspended in FACS buffer, and then
incubated with RNase and propidium iodide (BD Pharmingen)
at room temperature for 30min before flow cytometry.
Expression of CD11b
performed at 25
spectrometer with a cryogenically cooled probe Brukerbiosp-in,
Germany). Solvent-suppressed 1D H CPMG (cpmgPr1d) were
acquired using the pulse sequence [RD-90°-(τ-180°-τ)n-ACQ].
Water resonance was suppressed by presaturation using a
54.87 dB pulse during the recycle delay (RD) of 4s. The 90°
pulse length was adjusted to about 11.82 μs. A total of 4
dummy scans and 64 free induction decays (FIDs) were
℃
using 600 MHz BrukerAvance III
1
collected into 64K acquisition points, covering a spectral width MV4;11 and KOPN-8 cells were plated in 12-well plates at an
of 12 KHz (20ppm) and giving an acquisition time (ACQ) of initial concentration of 3
105 cells mL-1 and treated with
2.73s. compounds or DMSO. At day three, cells were replanted with
The STD experiment was acquired using the acquisition time of cell concentration adjusted to 3
105cells mL-1and compounds
×
×
1.71s, 4 dummy scans, and relaxation delay of 3s, followed by resupplied. After 7 days, cells were harvested, washed and
a 40 dB pulse with the irradiation frequency at 0.25 ppm or - resuspended with PBS Buffer with 1% BSA. PE mouse anti-
1000 ppm alternatively. The total acquisition time was 31 human CD11b Ab (BD Phamingen) were added and incubated
minutes with 192 scans.
at 4
℃ for 30min before being analyzed by flow cytometry.
Stability Studies by DSF
Cytospin/Wright-Giemsa staining
Differential scanning fluorimetry were performed on a Quant MV4;11 and KOPN-8 cells were plated in 12-well plates at an
Studio 6 Flex Real-Time PCR system (ABI). Each reaction were initial concentration of 3
105cells mL-1 and treated with
heated from 25 to 95 °C in 20 L thermal shift buffer (50 mM compounds or DMSO. Cells were counted and split back to the
Mops, pH6.48; 50 mM NaCl; 1 mM DTT) with 2.5 M menin, original plating density in fresh medium with compound
5×SYPRO orange (Invitrogen) and series of diluted resupplied every three-four days. Cells were harvested and
×
µ
µ
a
compounds. All samples were tested in triplicate. centrifuged at 500rpm for 5min in Shandon cytospin3 (Thermo
ABsoluteqPCR Plate Seals (Thermo Scientific) were used to Scientific Cytospin) after 10 days of treatment. The slides were
limit evaporation. Change of fluorescence signal of SYPRO air-dried before staining with Wright-Giemsastain (Baso).
orange was monitored and applied to determine the Tm of Co-immunoprecipitation
menin with Protein Thermal Shift Software Version 1.1 (ABI).
Surface Plasmon Resonance (SPR) Based Binding Assays
HEK293 cells were plated and transfected with Flag-MLLN
plasmid using PEI (Sigma-Aldrich). After transfection, cells
The SPR binding assays were performed on the Biacore T200
were treated with compounds or DMSO for 12h. Whole cell
instrument (GE Healthcare) at 25
℃. Menin protein was
lysates were prepared by cell lysis buffer (Cell Signaling
Technology) containing Protease Inhibitor Cocktail Roche) and
covalently immobilized on a CM5 chip using a standard amine-
coupling procedure in 10 mM sodium acetate (PH 5.0). The
chip was first equilibrated with HBS-EP buffer (10mM HEPES
compounds.
Samples
were
centrifuged
and
16 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 17 of 20
Organic & Biomolecular Chemistry
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immunoprecipitated with ANTI-FLAG M-2 Magnetic beads
DOI: 10.1039/C6OB01248E
(Sigma-Aldrich) at 4 °C for 2h. The immunoprecipitation
samples were applied to SDS-PAGE electrophoresis and
Western blotting after extensive PBS washing. Antibodies used
include rabbit anti-menin (Cell Signaling Technology, Cat.6891),
rabbit DYDDDK tag antibody (Cell Signaling Technology,
Cat.2368).
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AUTHOR INFORMATION
Corresponding Author
*Yaxi Yang, telephone 86-21-50806600-3508, email:
yangyaxi@simm.ac.cn.
*Yuanyuan Zhang, telephone 86-21-50806600-5307, email:
10110700070@fudan.edu.cn.
15.
16.
* Cheng Luo, telephone: 86-21-50806600, email:
cluo@simm.ac.cn.
Present Addresses
17.
R. Zhou, Y. Xie, H. Hu, G. Hu, V. S. Patel, J. Zhang, K. Yu, Y.
Huang, H. Jiang, Z. Liang, Y. G. Zheng and C. Luo, J Chem
Inf Model, 2015, 55, 2623-2632.
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
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Author Contributions
Liyan Yue,‡a,b Juanjuan Du,‡c and Fei Ye,‡d contributed equally
to this work.
Notes
The authors declare no competing financial interests.
Acknowledgements
23.
24.
This work was supported by the National Basic Research
Program (2015CB910304), Hi-Tech Research and Development
Program of China (2014AA01A302), the National Natural
Science Foundation of China (21210003, 81430084, 81202398,
81402849 and 21272246), Zhejiang Province Natural Science
Foundation (LQ14H300003), Public Projects of Zhejiang
Province (2015C33159), Zhejiang Provincial Top Key Discipline
of Biology and CAS Strategic Priority Research Program
(XDA12020333)
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J. Name., 2013, 00, 1-3 | 19

Organic & Biomolecular Chemistry
Page 20 of 20
View Article Online
DOI: 10.1039/C6OB01248E
Graphical abstract
Scaffold hopping combines with biochemical studies and medicinal chemistry optimizations, leading to
potent inhibitors for menin-MLL interaction.