may promote reader selectivity. Noteworthy, replacement of the piperidine ring with
a (pyrrolidinyl)azepane (analog #19, Fig. 12) or an azabicyclooctane (analog #20,
Fig. 12) introduced an increase in hydrophobicity, rigidity, and size to the ligand,
resulting in enhanced potency and selectivity for the L3MBTL3 reader (L3MBTL3:
IC 50 values ¼ 6.7 μM and 3.6 μM, respectively; L3MBTL1: IC 50 values ¼ 28.9 μM
and 23.6 μM, respectively) [177]. Modifications to the linker region connecting
the phenyl and piperidine rings did not increase potency toward L3MBTL1 when
compared to UNC926. Nevertheless, several of these analogs were modest inhibitors
of L3MBTL3 and MBTD1 readers. Sulfonamide analog #21 maintained potency
against L3MBTL3 (IC 50 ¼ 3.7 μM) and MBTD1 (IC 50 ¼ 21.9 μM) but weakly
bound L3MBTL1 at 48% inhibition. Similarly, both removal of the carbonyl (analog
#22, Fig. 12) or its replacement with a methyl group (analog #23) resulted
in compounds that demonstrated modest potency against L3MBTL3 (IC 50
values ¼ 13.3 μM and 16.0 μM, respectively) and MBTD1 (IC 50 values ¼ 18.6 μM
and 9.0 μM, respectively), but not against L3MBTL1 (IC 50 > 30 μM). Finally, the
addition of a phenyl ring (analog #24) yielded an analog with approximately
eightfold selectivity for L3MBTL3 (IC 50 ¼ 2.4 μM) compared to L3MBTL1
(IC 50 ¼ 20.3 μM) [177].
4.4.2.2 Targeting L3MBTL3
L3MBTL3 reader antagonists based on the 4-(pyrrolidin-yl)-piperidine scaffold
have been recently identified during a campaign aimed to develop small-molecule
probes of Tudor domain-containing p53-binding protein 1 (53BP1, see above),
which binds Kme2 residues adjacent to lysine or arginine [178, 179]. The primary
design strategy for these compounds consisted of appending an additional basic
amine to the phenyl ring of the UNC926 scaffold to provide UNC928 (Fig. 13)
[179]. Interestingly, while UNC928 was inactive against 53BP1, it demonstrated
submicromolar activity against L3MBTL3 (AlphaScreen™ IC 50 ¼ 0.36 μM).
A LANCE (Lanthanide chelate excite) time-resolved fluorescence resonance
energy transfer (TR-FRET) assay was used to confirm the AlphaScreen™ results
and found UNC928 had an analogous binding affinity of IC 50 ¼ 0.29 μM. In
addition, UNC928 was moderately active only against L3MBTL1 (IC 50 ¼ 2.8 μM),
suggesting the potential for developing selective probes of the L3MBTL3 reader.
To further probe SAR for this “dimeric” scaffold with regard to both potency and
selectivity for the L3MBTL3 reader, an extensive series of small molecules that
contained modifications to either the “left-side” amine, the linker, or aromatic core of
UNC928 (Fig. 13) were synthesized and evaluated [179]. Repositioning the amine in
the para position with respect to the original “right-side” 4-(pyrrolidin-yl)-piperidine
resulted in a significant improvement in activity against L3MBTL3 (UNC1021,
LANCE IC 50 ¼ 0.048 μM and AlphaScreen™ IC 50 ¼ 0.071 μM), without increased
affinity for L3MBTL1 (AlphaScreen™ IC 50 ¼ 2.9 μM). To explore the central
aromatic core, a variety of functional groups were appended to the phenyl
ring of UNC1021 [179]. While the majority of compounds evaluated did not
368
G. Sbardella
a (pyrrolidinyl)azepane (analog #19, Fig. 12) or an azabicyclooctane (analog #20,
Fig. 12) introduced an increase in hydrophobicity, rigidity, and size to the ligand,
resulting in enhanced potency and selectivity for the L3MBTL3 reader (L3MBTL3:
IC 50 values ¼ 6.7 μM and 3.6 μM, respectively; L3MBTL1: IC 50 values ¼ 28.9 μM
and 23.6 μM, respectively) [177]. Modifications to the linker region connecting
the phenyl and piperidine rings did not increase potency toward L3MBTL1 when
compared to UNC926. Nevertheless, several of these analogs were modest inhibitors
of L3MBTL3 and MBTD1 readers. Sulfonamide analog #21 maintained potency
against L3MBTL3 (IC 50 ¼ 3.7 μM) and MBTD1 (IC 50 ¼ 21.9 μM) but weakly
bound L3MBTL1 at 48% inhibition. Similarly, both removal of the carbonyl (analog
#22, Fig. 12) or its replacement with a methyl group (analog #23) resulted
in compounds that demonstrated modest potency against L3MBTL3 (IC 50
values ¼ 13.3 μM and 16.0 μM, respectively) and MBTD1 (IC 50 values ¼ 18.6 μM
and 9.0 μM, respectively), but not against L3MBTL1 (IC 50 > 30 μM). Finally, the
addition of a phenyl ring (analog #24) yielded an analog with approximately
eightfold selectivity for L3MBTL3 (IC 50 ¼ 2.4 μM) compared to L3MBTL1
(IC 50 ¼ 20.3 μM) [177].
4.4.2.2 Targeting L3MBTL3
L3MBTL3 reader antagonists based on the 4-(pyrrolidin-yl)-piperidine scaffold
have been recently identified during a campaign aimed to develop small-molecule
probes of Tudor domain-containing p53-binding protein 1 (53BP1, see above),
which binds Kme2 residues adjacent to lysine or arginine [178, 179]. The primary
design strategy for these compounds consisted of appending an additional basic
amine to the phenyl ring of the UNC926 scaffold to provide UNC928 (Fig. 13)
[179]. Interestingly, while UNC928 was inactive against 53BP1, it demonstrated
submicromolar activity against L3MBTL3 (AlphaScreen™ IC 50 ¼ 0.36 μM).
A LANCE (Lanthanide chelate excite) time-resolved fluorescence resonance
energy transfer (TR-FRET) assay was used to confirm the AlphaScreen™ results
and found UNC928 had an analogous binding affinity of IC 50 ¼ 0.29 μM. In
addition, UNC928 was moderately active only against L3MBTL1 (IC 50 ¼ 2.8 μM),
suggesting the potential for developing selective probes of the L3MBTL3 reader.
To further probe SAR for this “dimeric” scaffold with regard to both potency and
selectivity for the L3MBTL3 reader, an extensive series of small molecules that
contained modifications to either the “left-side” amine, the linker, or aromatic core of
UNC928 (Fig. 13) were synthesized and evaluated [179]. Repositioning the amine in
the para position with respect to the original “right-side” 4-(pyrrolidin-yl)-piperidine
resulted in a significant improvement in activity against L3MBTL3 (UNC1021,
LANCE IC 50 ¼ 0.048 μM and AlphaScreen™ IC 50 ¼ 0.071 μM), without increased
affinity for L3MBTL1 (AlphaScreen™ IC 50 ¼ 2.9 μM). To explore the central
aromatic core, a variety of functional groups were appended to the phenyl
ring of UNC1021 [179]. While the majority of compounds evaluated did not
368
G. Sbardella