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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%.