Progress towards small molecule menin-mixed lineage leukemia (MLL) interaction inhibitors with in vivo utility

Article abstract of DOI:10.1016/j.bmcl.2015.04.026

A series of substituted hydroxymethyl piperidine small molecule inhibitors of the protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) are described. Initial members of the series showed good inhibitory disruption of the menin-MLL

Full text of DOI:10.1016/j.bmcl.2015.04.026

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2720–2725
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Progress towards small molecule menin-mixed lineage leukemia
(MLL) interaction inhibitors with in vivo utility
Timothy Senter a,b,c, Rocco D. Gogliotti a,c, Changho Han a,c, Charles W. Locuson a,c, Ryan Morrison a,c
,
J. Scott Daniels b,d, Tomasz Cierpicki d, Jolanta Grembecka d, Craig W. Lindsley b,c,d, Shaun R. Stauffer b,c,d,
⇑
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
c Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
d Department of Pathology, University of Michigan, Ann Arbor, 1150 West Medical Center Drive, MSRBI, Room 4510D, MI 48109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted hydroxymethyl piperidine small molecule inhibitors of the protein–protein inter-
action between menin and mixed lineage leukemia 1 (MLL1) are described. Initial members of the series
showed good inhibitory disruption of the menin-MLL1 interaction but demonstrated poor physicochem-
ical and DMPK properties. Utilizing a structure-guided and iterative optimization approach key sub-
stituents were optimized leading to inhibitors with cell-based activity, improved in vitro DMPK
parameters, and improved half-lives in rodent PK studies leading to MLPCN probe ML399. Ancillary
off-target activity remains a parameter for further optimization.
Received 14 March 2015
Revised 6 April 2015
Accepted 8 April 2015
Available online 25 April 2015
Keywords:
Menin
Mixed lineage leukemia (MLL)
Protein–protein interaction
ML399
Ó 2015 Published by Elsevier Ltd.
Misregulation or mutation of histone-modifying enzymes plays
a major role in the development of a wide-range of human
cancers.1,2 Mixed lineage leukemia 1 (MLL1) is one of six known
members of the SET1-family of human histone methyltransferases
(HMT) and participates in catalyzing the mono-, di-, and trimethy-
lation of histone H3 lysine 4 (H3K4).3 Genetic rearrangements of
MLL1 are associated with acute lymphoblastic leukemia (ALL)
and acute myeloid leukemia (AML) and can be found in 5–10% of
adult AML patients4 and up to 80% of infants with ALL.5 Patients
with acute leukemias harboring MLL translocations suffer a poor
prognosis with existing therapies providing only a ꢀ35% overall
5-yr survival rate.6,7 Thus, novel targeted therapies are urgently
needed to treat these leukemias and enhance patient long-term
outcome. The most common MLL1 rearrangements are balanced
MLL1 translocations, in which one MLL1 allele is truncated and
fused in frame with one of over 70 partners to produce oncogenic
fusion proteins. MLL fusion partners preserve the N-terminal frag-
ment of approximately 1400 amino acids and participate in a key
protein-protein interaction (PPI) with the oncogenic co-factor
menin. Menin is a ubiquitously expressed nuclear protein capable
of binding directly to both wild type MLL1, MLL2, and MLL1 fusion
protein complexes and is thought to function by targeting their
recruitment to the target genes which includes the homeotic genes
Hoxa9, Hoxc6, and Hoxc8.8,9 Menin contains a large and well
defined central cavity (human menin ꢀ5000 Å3) that represents
the MLL binding site. In addition, its architecture remains relatively
rigid upon binding of MLL, thus making menin a druggable and yet
challenging PPI interface for the development of small molecule
inhibitors. Current inhibitors of the menin-MLL interaction9
include peptidomimetics such as MCP-1 (1)10 and two classes of
small molecule inhibitors including the thienopyrimidines MI-2
(2) and MI-2-2 (3)11,12 and hydroxymethylpiperidines ML227
(4)13,14 (Fig. 1); the later class of which was discovered from an
NIH Molecular Libraries Production Center Network (MLPCN)
sponsored screen of the Molecular Libraries Small Molecule
Repository (MLSMR) of ꢀ280 K compounds. To date, systemically
available menin-MLL inhibitor tool compounds for studying menin
biology and proof-of-mechanism using MLL driven xenograft
leukemogenic animal models have not been described.15 The
development of potent small molecule inhibitors of the menin-
MLL interaction with good selectivity, metabolic stability, and
acceptable oral bioavailability will permit chronic systemic admin-
istration in animal models of MLL leukemia. These studies are crit-
ical in order to understand the anticipated efficacy, PK-PD
relationship, and therapeutic index for the mechanism.
Recent menin-inhibitor co-crystal structures developed in these
⇑
Corresponding author.
E-mail address: shaun.stauffer@vanderbilt.edu (S.R. Stauffer).
laboratories for the individual enantiomers of
4 reveal key
http://dx.doi.org/10.1016/j.bmcl.2015.04.026
0960-894X/Ó 2015 Published by Elsevier Ltd.

T. Senter et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2720–2725
2721
S
N
S
N
N
N
N
N
HN
NH
NH2
F3C
N
N
H
N
S
S
N
N
N
3, MI-2-2
d = 22 nM
MV4;11 (MLL-AF4) GI50 = 3 μM
2, MI-2
Kd = 158 nM
O
O
O
HN
K
HN
O
O
H
N
H2N
N
HO
O
O
N
F
CN
F
4
4 R
Kd = 285 nM
MV4;11 (MLL-AF4) GI50 = 6.5 μM
, ML227
IC50= 390 nM
-
1, MCP-1
Ki = 4.7 nM
Figure 1. Small-molecule menin-MLL PPI inhibitors 1–5.
interactions that closely mimic the menin-MLL protein-protein
interaction.14 In cell based assays the slightly more potent R enan-
tiomer of 4 (4R, Kd = 285 nM, Fig. 1) was found to disrupt menin-
MLL fusion protein interactions in co-immunoprecipitation stud-
ies, dose-dependently reduce Hoxa9 expression levels by qRT-
PCR, and selectivity inhibit cell proliferation in MLL human leuke-
group containing a stereogenic tertiary carbinol, (2) a piperi-
dine core, (3) an propoxy linker, and (4) a benzonitrile tail
group. Within the binding pocket of menin, the lipophilic head
group of ML227 mimics key interactions normally occupied by
the F9 and P10 residues of MLL. The removal of either the
phenyl or cyclopentyl ring within ML227 results in >500-fold
loss of binding to menin. Similarly, removal of the tail group
cyano moiety, which participates in a unique hydrogen bond
accepting interaction with W341 results in loss of inhibitory
activity. Thus, these particular pharmacophore elements are
considered most critical to maintain high binding affinity to
menin.
Based on the high calculated LogP for ML227 (cLogP >5, Fig. 2)
structure-based design strategies predicted to attenuate lipophilic-
ity and maintain a favorable binding interaction with menin were
prioritized; specifically within the lipophilic carbinol and eastern
tail group region. In addition to IC50 measurements as determined
from our fluorescence polarization assay using a displaceable MLL
tagged substrate peptide,11,16 efficiency metrics, including ligand
efficiency (LE) and lipophilicity-dependent ligand efficiency
(LELP)17 were utilized to assess physicochemical impact from
structural modifications and to identify preferred enthalpy-driven
SAR.
mia cell lines with GI50s comparable to 3, between 6.5 and 7.4 lM
(MLL-AF4, MLL-AF9 containing).13,14 Although ML227 is a useful
in vitro tool compound, it possesses several key limitations pre-
cluding its use in animals (Fig. 2). These include poor metabolic
stability and off-target activity against multiple monoaminergic
and ion channel targets. Other challenges noted within the profile
of ML227 include moderate inhibition of human cytochrome P450
(CYP) enzymes 2D6 and 3A4 and high plasma protein binding
(Fig. 2). In an effort to develop a useful in vivo probe compound
within the series we initiated a Molecular Libraries extended probe
optimization campaign using an integrated approach focused on
menin binding affinity, tier one DMPK properties (e.g., predicted
hepatic clearance, plasma protein binding, and major CYP450 inhi-
bition), and ancillary screening for selected candidates. Herein our
progress towards reaching this goal is described.
As shown in Figure 2, the lead structure ML227 possessed
four structural pharmacophore elements: (1) a lipophilic head
F9
cLogP
= 5.46
PPB fu human = 0.004
PPB rat
piperidine
fu
= 0.013
OH
lipophilic
core
head group
P13
high intrinsic clearance
(human, rat)
N
tail group
CYP450 (1A2, 2C9, 2D6, 3A4):
>30, >30, 1.29, 5.1 μM
P10
O
linker region
CN
Eurofins (>50% inhibition 10 M):
μ
4 (ML227)
α1D, β2, NET, L-channel, D3, D4.2, DAT,
H1, M1-3, Opiate μ, κ, hERG, 5-HT2B, SERT
IC50
=
390 nM
Figure 2. In vitro profile summary of MLPCN probe ML227 (4).

2722
T. Senter et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2720–2725
Table 2
O
O
HO
R1
In vitro tier one DMPK and rat IV pharmacokinetics of cyclopentyl hydrox-
ymethylpiperidines 4 (ML227), 9a, 9c18
a-b
c
Entrya
CLint (h, r)b
CLhep (h, r)b
PPB fu (h, m)
CLp
T1/2 (h)
Vss
c
d
N
N
4
9a
9c
124, 919
77.4, 558
16.2, 271
18, 65
16, 62
9.1, 57
0.004, 0.013
0.117, 0.192
0.291, 0.342
40
NT
293
1.6
NT
0.2
2.9
NT
4.3
Boc
Boc
5
6
HO R1
R2
NT = not tested.
a
Human (h), rat (r), mouse (m).
mL/min/kg, see Ref. 18.
mL/min/kg, rat IV dose 0.2 mg/kg.
Vss = volume distribution, L/kg.
b
c
HO R1
R2
d-e
N
d
HO
O
N
high yield. A second Grignard addition to the resulting ketone 6
provided the tertiary carbinols 7. Deprotection, followed by
sequential alkylation of the piperidine and desired phenol provided
the final products in good overall yields (42–78%), while allowing
for a high degree of control over three regions of interest for analog
synthesis.
Inhibitory potency and calculated properties for cyclopentyl
analogs 9a–h and ML227 (4) for comparison are shown in
Table 1. As previously reported,14 introduction of the correct hete-
rocycle moiety (e.g., 9a–9c) within the carbinol head group leads to
a slight increase in potency while reducing cLogP an order of mag-
nitude. Thus, with the retained potency, improvements in calcu-
lated LELP were observed. For example, the 2-pyridyl and
Boc
R3
7
8
9-10
R3
Scheme 1. Reagents and conditions: (a) N,O-dimethyl hydroxylamine-HCl, CDI,
Et3N, DCM, 75–90%; (b) R1-MgBr, THF, 0 °C to rt, 60–85%; (c) R2-MgBr, THF, 0 °C to
rt, 30–75%; (d) 4.0 M HCl/dioxanes, MeOH, rt, 2 h, 93–100%; (e) (i) K2CO3, DMF, 1-
bromo-3-chloropropane, rt, 3 h, (ii) 8, K2CO3, 40 °C, 2 h, 42–78%.
Similar to our prior synthetic strategies, analogs within the
hydroxymethylpiperidine series 9 were synthesized according to
Scheme 1.14 Initial Grignard addition to the Weinreb amide of 1-
(t-butoxycarbonyl)piperidine-4-carboxylic acid (5) proceeded in
Table 1
Potency and physical properties of cyclopentyl hydroxymethyl piperidine menin-MLL inhibitors 4, 9a–h13,14
R2
HO
N
O
R3
Entry
R2
R3
IC50 (nM)
390
cLogP
LE/LELP
N
N
4
5.46
0.29/17.9
(ML227)
S
9a
9b
242
302
3.80
3.90
0.30/11.2
0.29/13.8
N
N
N
O
S
O
NH2
N
9c
114
2.78
5.37
0.28/9.9
N
9d
1100
0.25/21.74
O
F
N
9e
9f
437
226
5.74
6.16
0.26/21.77
0.28/21.69
F
N
N
Cl
F
9g
9h
197
5.59
4.60
0.29/19.53
0.2./22.90
N
28,000
NH

T. Senter et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2720–2725
2723
thiazolyl congeners 9a and 9b, demonstrated an LELP reduction
F
from ꢀ18 for ML227 to less than 14. In general LELP proved to
be a responsive indicator in lead optimization for this program
and informed prioritization of compounds for in vitro DMPK stud-
ies. As previously noted, analysis of 4 in complex with menin
revealed a hydrogen bond interaction between the eastern nitrile
of 4 with tryptophan residue 341, and this information was used
to investigate additional polar substituents that contained hydro-
gen bond acceptor moieties. Sulfonamide 9c (Table 1) proved par-
HO
N
NH
M
1 (major)
F
HO
ticularly successful, with an IC50 of 114 nM as
a racemic
O
CN
mixture.13,14 Relative to 9b this modification led to a further reduc-
tion in lipophilicity (cLogP = 2.78) and provided an inhibitor with
an attractive calculated LELP of ꢀ10.
10b
N
HO
(ML399)
N
Table 2 describes results from in vitro DMPK studies for the
cyclopentyl congeners 9a and 9c relative to lead 4. In addition,
parameters from IV pharmacokinetic studies in rat are summarized
for 4 versus 9c (IV 0.2 mg/kg: CLp, T1/2, Vss). Inhibitor 4 was found in
rat and human microsomal intrinsic clearance assays to demon-
strate high in vitro metabolism with predicted hepatic clearance
near the rate of blood flow in both human and rat,18 while the frac-
tion unbound from plasma proteins was ꢀ1% or lower (PPB
fu = 0.004, 0.013, human and rat, respectively). Due in part to the
high protein binding and weak base properties of 4, rat IV pharma-
cokinetics afforded a compound with moderate plasma clearance
and volume of distribution (Table 2). Replacement of the phenyl
to afford thiazole 9a (Table 2) led to a notable decrease in intrinsic
clearance whereas substitution of phenyl with a 2-pyridyl, and
replacement of the nitrile with a sulfonamide to afford 9c, further
M2
F
O
CN
O
N
HO
N
M3
Figure 3. Identification of major metabolites of 10b in rat S9.
10 residue by the cyclopentyl moiety was evident, X-ray structures
of the individual ML227 enantiomers bound to menin demon-
strated that aromatic replacements within the P10 pocket were
feasible. With respect to prior diaryl carbinol SAR this type of head
group appeared challenging for potency, as simple phenyl within
the P10 pocket13 was shown to lead to a >30-fold loss in IC50 rela-
tive to 4.14 By targeting a conserved water molecule found within
the P10 pocket the introduction of either a 2-pyridine or 2-thia-
zolyl to engage in a solvent mediated hydrogen bond with Y276
proved successful as a near equipotent replacement for the cyclo-
pentyl P10 mimic. Heteroaryl P10 cyclopentyl replacements, in
conjunction with a 3-fluorophenyl within the F9 pocket14 were
predicted to be favorable for potency while also mitigating sites
attenuated
intrinsic
and
predicted
hepatic
clearance.
Unfortunately 9c suffered from a short half-life in vivo (IV rat,
T1/2 <0.2 h, Table 2) probably owing to the greater than ten-fold
improvement in fraction unbound that accompanied these struc-
tural changes (PPB fu 29–34%). Hence, further effort was spent on
reducing metabolic clearance. Alcohol or primary sulfonamide con-
jugation were suspected to contribute to the high clearance of 9c;
however, soft-spot metabolite analysis in microsomal incubations
implicated the cyclopentyl ring was a retained liability for exten-
sive oxidative metabolism. Although mimicry of the MLL proline
Table 3
In vitro, calculated, and in vivo characterization of advanced aryl/heteroaryl hydroxymethyl piperidine menin-MLL inhibitors
F
HO
R1
N
O
R2
CLhep (h, r)b
c
d
Entrya
R2
R3
IC50
460
cLogP
LE/LELP
CLint (h, r)b
PPB fu (h, r, m)
CLp
T1/2 (h)
Vss
S
N
N
10a
3.62
0.27/13.4
69, 273
20, 87
16, 56
10, 39
0.04, 0.108, NT
32
2.8
7.2
7.4
N
10b (ML399) 1st peak SFC (R)
90
3.57
2.36
0.30/12.2
0.29/11.6
0.062, 0.074, 0.079
0.170, 0.322, 0.166
12
26
10.1
1.4
N
S
O
S
O
O
S
NH2
NH2
10c
354
50, 206
17, 21
15, 52
14, 11
4.4
4.6
N
10d
246
3.00
0.28/9.1
0.267, NT, 0.178
32
2.5
N
O
NT = not tested.
a
Human (h), rat (r), mouse (m).
mL/min/kg, see Ref. 18.
mL/min/kg, rat IV dose 0.2 mg/kg.
Vss = volume distribution, L/kg.
b
c
d

2724
T. Senter et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2720–2725
on proliferation using murine bone marrow cells (MBC) trans-
formed with MLL-AF9, a prevalent fusion protein observed in
both AML and ALL patients.19 Using this leukemic cell line with
a 72 h incubation protocol14 significant growth inhibition was
observed for 10b with a GI50 of 4 lM. These results demon-
F9
strate an association between in vitro inhibition of the menin-
MLL interaction and inhibition of cell growth in MLL leukemia
cells; however, a broader assessment of anti-proliferative activ-
ity in additional leukemia and non-MLL containing cell lines is
needed.
P10
Based on the overall in vitro and in vivo profile obtained,
menin-MLL inhibitor 10b was declared second generation probe
ML399. In a Eurofins radioligand binding panel of over 60 G pro-
tein-coupled receptors, ion channels, and transporters at a concen-
tration of 10 lM several significant activities at ancillary targets
were found for ML399, similar to ML227 (Fig. 2). These included
binding to the hERG potassium ion channel, a known off-target
activity for Terfenadine-like scaffolds.20,21 Although diminished,
notable hERG binding was also observed for the more polar sulfon-
amide 10d. Efforts to address hERG binding and related off-target
activity continue. Strategies include attenuation of basicity of the
piperidine moiety and introduction of alternative polar groups,
including carboxylate to engender zwitterionic character.21
Structural studies of 9b in complex with menin14 revealed that
the propyl linker region occupied a relatively unhindered region
of the binding pocket (Fig. 4), and might readily tolerate further
derivatization. Thus, hydroxyl-substituted analog 13 was synthe-
sized according to Scheme 2. Piperidine starting material 11 was
accessed as before according to Scheme 1. Subsequent opening of
epoxide 12 using 11 was achieved in good yield leading to 13.
When tested as a mixture of four diastereomers piperidine 13
was found to inhibit the menin-MLL interaction with an IC50 of
222 nM, approximately two-fold less potent than ML399 (10b),
demonstrating that polar functionality within this region of the lin-
ker is well tolerated. In addition, secondary alcohol 13 may provide
a useful synthetic handle for introduction of a b-difluoro or car-
boxylic acid modification as proposed (see Fig. 4). Efforts to capital-
ize on this strategy and its potential impact continue to be
investigated.
Ongoing efforts are required in order to develop potent, selec-
tive, and orally available menin inhibitors to test in animal models
of mixed lineage leukemia.22 In summary, starting from ML227 an
iterative strategy incorporating analysis of physical properties and
metrics, as well as rational design informed by metabolite identifi-
cation studies were used to develop inhibitors of the menin-MLL
interaction with improved potency and stability in vivo, leading
to ML399.23 Efforts to enhance selectivity within the series, as well
as oral dosing studies using ML399 in naïve animals to assess
bioavailability and tolerability are in progress and will be reported
in due course.
di-F or -CO2H
Figure 4. X-ray crystal structure of 9b in complex with menin (PDB: 4OGS) and
proposed modification to central propyl linker carbon.
for oxidative metabolism. Thus, hybrid compounds 10a–10d con-
taining a heteroaryl and aryl moiety within the carbinol head
group, in addition to either a 4-cyano or 4-sulfonamide substituent
within the tail group were prepared according to methods in
Scheme 1. As shown in Table 3 these compounds consistently low-
ered cLogP from ꢀ5 to <3.6, while improving LELP to as low as 9.1
(e.g., 10d), affording inhibitors with comparable or improved
potency. In the case of single enantiomer 10b, an IC50 less
100 nM was measured (IC50 = 90). Efforts to resolve sulfonamide
10d using chiral SFC were unsuccessful. Overall improvements in
these metrics were reflected well in in vitro DMPK studies, provid-
ing menin-MLL inhibitors with predicted hepatic clearance less
than half of liver blood flow based on human and rat microsomal
incubation experiments. Analogs 10a–10d were subsequently
advanced to an in vivo IV cassette study in rat (Table 3).
Importantly, replacement of the cyclopentyl group, an identi-
fied liability in soft-spot metabolite studies, led to compounds
with improved half-lives in vivo (T1/2 from 1.4–10 h). Of partic-
ular interest was 10b, which demonstrated a 10 h half-life in
rat and 5.5 h half-life in mouse (mouse data not presented),
with
a
somewhat high volume of distribution in rat
(Vss = 7.4 L/kg). Metabolite identification experiments using 10b
in rat hepatic S9 fractions revealed N-dealkylation at the piper-
idine core as the major metabolite, followed by oxidation of the
3-fluorophenyl substituent (Fig. 3). Menin-MLL inhibitor 10b
was subsequently tested in a cell viability assay for its impact
F
F
HO
HO
a
IC
50 = 222 nM
N
N
cLogP
LE/LELP
= 2.56
= 0.27/9.56
NC
NH
N
OH
O
11
O
O
12
13
CN
Scheme 2. Reagents and conditions: (a) acetonitrile, 12, K2CO3, 30 °C, 4 h, 67%.

T. Senter et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2720–2725
2725
In Probe Reports from the NIH Molecular Libraries Program, Bethesda (MD), 2010,
http://www.ncbi.nlm.nih.gov/pubmed/23658959.
Acknowledgments
14. He, S.; Senter, T. J.; Pollock, J.; Han, C.; Upadhyay, S. K.; Purohit, T.; Gogliotti, R.
D.; Lindsley, C. W.; Cierpicki, T.; Stauffer, S. R.; Grembecka, J. J. Med. Chem.
2014, 57, 1543.
15. Daigle, S. R.; Olhava, E. J.; Therkelsen, C. A.; Basavapathruni, A.; Jin, L.; Boriack-
Sjodin, P. A.; Allain, C. J.; Klaus, C. R.; Raimondi, A.; Scott, M. P.; Waters, N. J.;
Chesworth, R.; Moyer, M. P.; Copeland, R. A.; Richon, V. M.; Pollock, R. M. Blood
2013, 122, 1017.
The authors thank the MLPCN (1U54 MH084659 and R03
MH084875) for funding support. Nathan Kett for preparative chiral
SFC (VSCC) and Julie Engers for project team management (VSCC).
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22. During the review of this manuscript
development of acute leukemia in a mouse model via menin-MLL inhibition
with small molecule was reported, see: Borkin, D.; He, S.; Miao, H.;
a study demonstrating suppressed
a
Kempinska, K.; Pollock, J.; Chase, J.; Purohit, T.; Malik, B.; Zhao, T.; Wang, J.;
Wen, B.; Zong, H.; Jones, M.; Danet-Desnoyers, G.; Guzman, M. L.; Talpaz, M.;
Bixby, D. L.; Sun, D.; Hess, J. L.; Muntean, A. G.; Maillard, I.; Cierpicki, T.;
Grembecka, J. Cancer Cell 2015. http://dx.doi.org/10.1016/j.ccell.2015.02.016.
in press.
23. For information on the MLPCN and information on how to request probe
compounds, such as ML399, see: http://mli.nih.gov/mli/mlpcn/.