A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros and Aiolos

Article abstract of DOI:10.1021/acs.jmedchem.6b01921

The drugs lenalidomide and pomalidomide bind to the protein cereblon, directing the CRL4-CRBN E3 ligase toward the transcription factors Ikaros and Aiolos to cause their ubiquitination and degradation. Here we describe CC-220 (compound 6), a cereblon modu

Full text of DOI:10.1021/acs.jmedchem.6b01921

Article
pubs.acs.org/jmc
A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros
and Aiolos
Mary E. Matyskiela, Weihong Zhang, Hon-Wah Man, George Muller, Godrej Khambatta, Frans Baculi,
Matthew Hickman, Laurie LeBrun, Barbra Pagarigan, Gilles Carmel, Chin-Chun Lu, Gang Lu,
Mariko Riley, Yoshitaka Satoh, Peter Schafer, Thomas O. Daniel, James Carmichael, Brian E. Cathers,
and Philip P. Chamberlain*
1Celgene Corporation, 10300 Campus Point Drive, Suite 100, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: The drugs lenalidomide and pomalidomide bind
to the protein cereblon, directing the CRL4−CRBN E3 ligase
toward the transcription factors Ikaros and Aiolos to cause their
ubiquitination and degradation. Here we describe CC-220
(compound 6), a cereblon modulator in clinical development
for systemic lupus erythematosis and relapsed/refractory multi-
ple myeloma. Compound 6 binds cereblon with a higher affinity
than lenalidomide or pomalidomide. Consistent with this, the
cellular degradation of Ikaros and Aiolos is more potent and
the extent of substrate depletion is greater. The crystal struc-
ture of cereblon in complex with DDB1 and compound 6 reveals
that the increase in potency correlates with increased contacts
between compound 6 and cereblon away from the modeled
binding site for Ikaros/Aiolos. These results describe a new
cereblon modulator which achieves greater substrate degradation via tighter binding to the cereblon E3 ligase and provides
an example of the effect of E3 ligase binding affinity with relevance to other drug discovery efforts in targeted protein
degradation.
been associated with degradation of an additional substrate, the
protein kinase Ck1a.16 In addition, the translation termination
factor GSPT1 has now been described as a ligand-directed
substrate of the cereblon,8 highlighting the potential breadth of
activity of cereblon modulators, as now three unrelated classes
of substrate proteins, zinc finger transcription factors, protein
kinases, and protein translation factors, have been targeted. The
work on CK1a and GSPT1 further demonstrates the potential
for ligand-substrate specificity, as CK1a is targeted to cereblon
by lenalidomide but not by pomalidomide, and GSPT1 is
targeted by a recently reported cereblon modulator8b but not
by either lenalidomide or pomalidomide. Cereblon modulators
therefore provide an important proof-of-principle for the
further development of compounds targeting specific proteins
for destruction that may have previously been considered
undruggable.
INTRODUCTION
■
Lenalidomide and pomalidomide are immunomodulatory drugs
that are clinically effective in the treatment of cancer, including
newly diagnosed and relapsed multiple myeloma.1 These drugs
target cereblon (CRBN), a substrate receptor for the CRL4
(CUL4−RBX1−DDB1) ubiquitin ligase complex.2 Rather than
inhibiting cereblon, ligand binding confers neomorphic activity,
altering the substrate specificity of the ubiquitin ligase by
promoting the recruitment of substrate proteins. Bound sub-
strates are ubiquitinated by the cereblon−CRL4 complex,
leading to their degradation by the 26S proteasome, thereby
driving the clinical activity in myeloma.3 Ikaros and Aiolos
(encoded by the genes IKZF1 and IKZF3, respectively) were
the first cereblon substrates to be identified, and it was demon-
strated that the addition of cereblon modulating ligands trig-
gers their recruitment and degradation.4 Ikaros and Aiolos are
zinc finger transcription factors with critical roles in hematological
development and differentiation,5 and the downstream effects of
degrading these proteins mediate the antiproliferative and
immunomodulatory activities of lenalidomide and pomalido-
mide.6 Polymorphisms in both IKZF1 and IKZF3 are associated
with a risk of developing systemic lupus erythematosus (SLE).7
Lenalidomide is further approved for use in 5q-deletion-
associated myelodysplastic syndrome, where clinical activity has
Structural studies have shown that cereblon modulators bind
to cereblon through the glutarimide moiety, which docks into a
hydrophobic tritryptophan pocket formed by Trp380, Trp386,
Special Issue: Inducing Protein Degradation as a Therapeutic
Strategy
Received: January 13, 2017
© XXXX American Chemical Society
A
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and Trp400.9 This binding mode leaves the insoindolinone/
phthalimide ring of the compounds exposed on the cereblon
surface, and it was predicted that this would form a hotspot of
unsatisfied hydrogen bonds that would mediate substrate
binding via direct protein−protein interactions.8a,9a Subsequent
structural and mutagenesis studies on cereblon−substrate com-
plexes have confirmed this hypothesis and surprisingly revealed
that unrelated proteins CK1a and GSPT1 bind to cereblon
through a common structural feature, thereby defining a
degron.8b,10 The enhancement of protein−protein interactions
by a small molecule is highly reminiscent of the “molecular
glue” mechanism described for the plant hormones auxin and
jasmonate.11 This mechanism contrasts with the linker-based
approaches, where heterobifunctional ligands recruit substrates
to a ligase via distinct small molecule binding events on each
side of a linker.12
Here we describe the cereblon modulator CC-220 (com-
pound 6),13 which is currently in phase 2 clinical trials for the
treatment of systematic lupus erythematosus (SLE) and phase
1b/2a clinical trials for relapsed and refractory multiple
myeloma (MM). We demonstrate that compound 6 has a
higher affinity for cereblon than lenalidomide or pomalidomide.
Consistent with these findings, compound 6 has a higher
potency for the cellular degradation of Ikaros and Aiolos.
The crystal structure of compound 6 bound to CRBN−DDB1
reveals that there are additional contacts with the surface of
cereblon compared to lenalidomide or pomalidomide. These
results provide a demonstration that increased target degrada-
tion can be achieved by alterations of the ligand binding affinity
for the ligase and supports the further evaluation of compound
6 in the clinic.
RESULTS
■
Compound 6 Is a Cereblon Modulator with Improved
Biochemical and Cellular Potencies. Like lenalidomide and
pomalidomide, compound 6 contains a glutarimide ring that
binds in the try-tryptophan pocket of cereblon and an
isoindolinone ring that can interact with both cereblon and
substrates (Figure 1a). In addition, the chemical structure of
compound 6 is extended compared to lenalidomide and
pomalidomide, containing additional phenyl and morpholino
moieties enabling further interactions with cereblon or
substrates. To determine the relative binding affinities between
lenalidomide, pomalidomide, and compound 6, we used a
TR-FRET cereblon binding assay to determine the IC50 values
for these compounds. This assay monitors the displacement of
a Cy5-conjugated cereblon modulating compound (compound 7)
from the tri-trp pocket of CRBN. Under these assay conditions,
the IC50 values for lenalidomide and pomalidomide were similar
at 1.5 and 1.2 μM, respectively, while compound 6 exhibited
significantly higher affinity with an IC50 of approximately 60 nM
(Figure 1b). IC50s for lenalidomide and pomalidomide have been
previously reported in multiple assay formats. The potencies
reported here are comparable to the values determined from
thermal shift studies, where lenalidomide and pomalidomide
showed similar potencies toward cereblon (both ∼3 μM).3a
A fluorescence polarization-based assay reported ∼10-fold more
potent binding (∼150 nM),9b however this is likely due to
the different assay formats, and lenalidomide and pomalidomide
were approximately equipotent which is consistent with this
study. A cereblon FRET assay has also been previously
reported,14 however the concentrations of protein used would
preclude assessment of potent compounds such as compound 6.
Figure 1. (a) Chemical structures of selected clinical stage cereblon
modulators including compound 6. (b) Determination of the relative
cereblon binding affinities for compound 6, lenalidomide, and
pomalidomide by TR-FRET. IC50s under these conditions were
1.5 μM for lenalidomide (circles), 1.2 μM for pomalidomide
(diamonds), and 60 nM for compound 6 (squares).
Consistent with increased biochemical binding affinity,
we also observe greater potency of compound 6 in cellular
degradation assays measuring the ligand-dependent depletion
of Ikaros or Aiolos. However, compound 6 does not signif-
icantly degrade GSPT1 or CK1α (Supporting Information,
Figure S1, and data not shown). To measure the effects of
compound treatment on Ikaros and Aiolos degradation in cells,
we used a cell-based assay that follows the chemiluminescent
signal of proteins incorporating an ePL tag. We find that
treatment with compound 6 results in the loss of Ikaros pro-
tein levels with an EC50 of 1 nM compared to 67 nM for
lenalidomide and 24 nM for pomalidomide. Compound 6 is
similarly potent toward Aiolos, with an EC50 of 0.5 nM com-
pared to 87 nM for lenalidomide and 22 nM for pomalidomide.
In addition, the extent of substrate depletion is more dramatic
with compound 6, indicating more efficient substrate degrada-
tion relative to the rate of protein resynthesis. The increase in
the cellular degradation potency of compound 6 corresponds
with the increase in binding affinity observed in the biochemical
assay, suggesting that the increase in cereblon affinity likely
contributes to the increase in cellular potency. In contrast, the
3−4-fold increase in cellular potency observed for pomalido-
mide compared to lenalidomide is more likely driven by other
B
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Figure 2. (a) Quantitation of Ikaros degradation with chemiluminescent cell-based assay. EC50s determined were 67 nM for lenalidomide (circles),
24 nM for pomalidomide (diamonds), and 1 nM for compound 6 (squares). (b) Quantitation of Aiolos degradation with chemiluminescent cell-
based assay. EC50s determined were 87 nM for lenalidomide (circles), 22 nM for pomalidomide (diamonds), and 0.5 nM for compound 6 (squares).
(c) Immunoblot analysis of whole-cell extracts of DF15 and OPM2 cells incubated for 5 h with DMSO, lenalidomide, pomalidomide, or compound
6 at the indicated concentrations.
factors, such as the alteration of substrate recruitment affinity or
physicochemical properties of the compound.
The glutarimide ring of compound 6 binds in the tri-trp pocket
in a similar binding mode to the previously reported structures,
and the isoindolinone ring is presented on the surface in a similar
binding mode to lenalidomide and other reported analogues8b,9,10
(Figure 3, for a detailed structural comparison of compound 6
and lenalidomide binding modes, see Supporting Information,
Figure S2). Compound 6 has an extended structure compared to
lenalidomide and pomalidomide (Figure 1a), and the crystal
structure shows that the phenyl ring of compound 6 is positioned
inside a groove on the cereblon surface formed by E377, H378,
P352, and H353 (Figure 3a). The morpholine ring is oriented
toward a hydrophobic pocket formed of residues I154 and F102,
however this moiety is poorly defined in the electron density,
possibly due to motion (Supporting Information, Figure S3).
F150 is positioned adjacent to the morpholino group of
compound 6, in contrast to some other structures, where this is
found in an extended conformation (Figure 3b8b,9a). However,
this conformation should be interpreted with caution as F150
occurs at the apex of a loop that has exhibited mobility in other
structures, and often participates in crystal lattice contacts.
To confirm these effects are relevant with respect to the
endogenous proteins, we treated DF15 and OPM2 myeloma
cell lines3a,15 with lenalidomide, pomalidomide, and compound
6 for 5 h (Figure 2c). Compound 6 treatment led to greater
degradation of Ikaros and Aiolos compared to lenalidomide and
pomalidomide, consistent with the results from the chem-
iluminescence-based cellular degradation assay.
Crystal Structure of Cereblon in Complex with DDB1
and Compound 6. To examine the mechanism of higher
affinity binding by compound 6, we determined the crystal
structure of the ternary complex of compound 6 bound to
human cereblon (aa 40−442) and human DDB1 (full length)
at 3.2 Å resolution. Data collection statistics are in Supporting
Information, along with sample electron density (Supporting
Information, Table S1 and Figure S3). The overall structure was
highly similar to the previously reported cereblon−DDB1
structures, with the binding site for cereblon modulators on the
opposite face of cereblon compared to the DDB1 interaction site.
C
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The extended chemical moieties relative to lenalidomide and
pomalidomide appear to enhance the surface area of compound
6 that contacts cereblon, which correlates with the observed
binding affinity increase in biochemical assays. Furthermore,
the 20-fold increase in binding affinity in vitro observed
compared to lenalidomide correlates well with the increased
potency of compound 6 in cellular degradation of Ikaros and
Aiolos. However, potency increases are not always driven by
enhanced cereblon binding affinity. For example, lenalidomide
and pomalidomide have similar IC50 values in the TR-FRET
assay (Figure 1) and yet pomalidomide is 3−4-fold more
potent than lenalidomide in the cell-based chemiluminescent
degradation assays. In this case, additional factors including
increased affinity for substrates, physcicochemical properties, or
other pharmacological differences may be contributing to the
differences in compound potency.
The crystal structures of cereblon−DDB1 in complex with
the bound substrates CK1a or GSPT1 revealed a common site
of interaction on the cereblon surface, which docking and
mutagenesis studies indicated are shared by Ikaros. It is
interesting, therefore, to consider whether the modified struc-
ture of compound 6 would in some way contribute to the
cereblon−Ikaros interaction. As shown in Figure 3b, the phenyl
and morpholino groups of compound 6 are positioned in an
orientation away from the site where the zinc finger domain of
Ikaros is predicted to interact on the surface of cereblon,
indicating that the observed differences in potency are more
likely to be driven by the improved compound 6−cereblon
interactions than by additional substrate interactions. This
system will benefit from a kinetic analysis of substrate and
compound binding in order to fully characterize the contribu-
tions of cereblon versus substrate binding and ubiquitination to
the increased potency of the compound.
Robust ligand-directed substrate degradation requires many
sequential steps (ligand binding, substrate recruitment,
ubiquitination, release, proteosomal degradation), representing
a potentially complex system for medicinal chemistry optimiza-
tion compared to classical small molecule inhibitors. As such,
there are many factors that could lead to alteration of cellular
efficacy of a ligase modulator. This study describes one strategy
for improving substrate degradation rates by enhancing ligand
interactions and affinity with the ligase while describing a next-
generation clinical stage cereblon modulator with improved
activity against Ikaros and Aiolos. The resulting increase in
potency is expected to contribute to clinical outcomes in the
treatment of both SLE and relapsed/refractory MM.
Figure 3. (a) The crystal structure of cereblon (blue) in complex with
DDB1 (green) and compound 6 (yellow sticks). Inset: detail of bound
compound 6, with the glutarimide ring docked in the tri-trp pocket
and the phenyl ring extending from the isoindolinone positioned
in a groove formed by E377, H378, P352, and H353 of cereblon.
(b) Comparison of the binding surface interactions of lenalidomide
(yellow, PDB 4TZ4, left panel) and compound 6 (yellow, this study,
right panel) with cereblon (blue, surface representation). Cereblon
surface residues that were previously shown to mediate binding to
Ikaros by mutational studies are shown in orange.8b The amino acid
side chain F150 is labeled.
The structures of cereblon in complex with CK1a and
GSPT1 revealed that the primary site of cereblon substrate
interactions is shared between the characterized substrates, and
docking and mutagenesis studies predicted the same site of
interaction for Ikaros and Aiolos.8b,10 This site of interaction is
formed by three hydrogen bond donors on cereblon, N351,
H357, and W400, which are positioned proximal to the iso-
indolinone or phthalimide rings of bound cereblon modulators
(Figure 3a). These residues (shown in orange surface repre-
sentation in Figure 3b) are positioned away from the phenyl
and morpholine rings of compound 6, such that differences in
compound activity are unlikely to be the result of direct
substrate interactions of these moieties with Ikaros or Aiolos.
Instead, a comparison of the crystal structures of lenalidomide
and compound 6 bound to cereblon−DDB1 reveals that
the observed increase in affinity correlates well with the
increased surface interactions between compound 6 and cer-
eblon (Figure 3b).
EXPERIMENTAL SECTION
■
Chemistry. Proton and carbon magnetic resonance (1H NMR and
13C NMR) spectra were recorded on a Bruker UltraShield 300
spectrometer and are reported in ppm relative to the reference solvent
of the sample in which they were run. HPLC and LC-MS analyses
were conducted using a Waters Aquity HPLC system and Water
Aquity PDA detector at 240 nm with the MS detection using Waters
Aquity SQ spectrometer. All flash column chromatography was
performed on EM Science silica gel 60 (particle size of 40−60 μm). All
reagents were purchased from commercial sources and used without
further purification unless otherwise noted. All reactions were
performed under an inert atmosphere. HPLC analyses were performed
using the following conditions.
DISCUSSION AND CONCLUSIONS
■
These results describe a new cereblon modulator which
achieves potent cellular degradation of the transcription factors
Ikaros and Aiolos. We find that the cellular potency increase
correlates with a higher affinity between compound 6 and
cereblon, which is likely driving higher occupancy of cer-
eblon in the cell and effectively increasing the active fraction
of cereblon−CRL4 enzyme capable of binding to substrate.
All final compounds had an HPLC purity of ≥95% unless spe-
cifically mentioned.
HPLC Method. A linear gradient (t = 0 min, 5% B; t = 15 min,
95% B) using 5% acetonitrile, 95% water with 0.1% phorsphoric acid
D
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Scheme 1
(solvent A) and 95% acetonitrile, 5% water with 0.1% phorsphoric
acid (solvent B) was employed on a Waters Symmetry C18, 5 μm,
3.9 mm × 150 mm column. Flow rate was 1 mL/min, and UV
detection was set to 240 nm. The LC column was maintained at
ambient temperature.
Chiral HPLC Method. An isocratic gradient using 50% 10 mM
NH4OAc in H2O (sovlent A) and 50% acetonitrile (solvent B) was
employed on a CHIRALPAK AGP, 5 μm, 4.0 mm × 150 mm column.
Flow rate was 1 mL/min, and UV detection was set to 240 nm. The
LC column was maintained at ambient temperature.
Synthesis of Compound 6 (Scheme 1). As shown in Scheme 1,
the synthesis started with optically pure (S)-3-(4-hydroxy-1-oxo-1,3-
dihydroisoindol-2-yl)piperidine-2,6-dione (1), which was alkylated
with 1,4-bis(bromomethyl)benzene (2) in the presence of potassium
carbonate to furnish bromide (3). Reaction of the bromide (3) with
morpholine (4) afforded 5. Subsequent cyclization of 5 with potassium
tert-butoxide at −78 °C proceeded to give imide (compound 6) in a
yield of 82% with a HPLC purity of 98.5%. The configuration of
compound 1 was assigned based on the commercially available
startling material, (S)-methyl 4,5-diamino-5-oxopentanoate hydro-
chloride. The configuration and chiral purity of 3 and 5 were not
measured. However, the chiral purity of compound 6 was determined
to be greater than 99% by chiral HPLC. Thus, the configuration of
compound 6 was assigned as the S-isomer. Compound 6 is chirally
stable in vivo. During DMPK study dosing with compound 6
(ee >99%), R-enantiomer was observed at low levels (<3% in monkey
and <6% in rat).
1 H), 4.49 (d, J = 17.42 Hz, 1 H), 4.72 (dd, J = 10.22, 4.26 Hz, 1 H),
6.92−7.08 (m, 1 H), 7.09−7.26 (m, 2 H), 7.26−7.47 (m, 1 H), 7.57
(s, 1 H), 10.04 (s, 1 H). 13C NMR (DMSO-d6) δ 24.86, 30.32, 44.76,
51.24, 53.30, 113.63, 117.78, 128.15, 128.20, 133.51, 152.44, 168.13,
171.76, 172.48. LCMS: M + 1 279. Anal. Calcd for C14H16N2O5:
C, 57.53; H, 5.52; N, 9.58. Found: C, 57.35; H, 5.27; N, 9.45.
4-[4-(4-Bromomethylbenzyloxy)-l-oxo-1,3-dihydro-isoindol-2-yl]-
4-carbamoylbutyric Acid Methyl Ester (3). In a 2 L round-bottom
flask were charged (S)-methyl 5-amino-4-(4-hydroxy-l-oxoisoindolin-
2-yl)-5-oxopentanoate (1) (30 g, 103 mmol), 1,4-bis(bromomethyl)-
benzene (2) (81 g, 308 mmol), potassium carbonate (14.19 g,
103 mmol), and acetonitrile (1.2 L). The mixture was stirred at 50 °C
for 12 h then cooled to room temperature. The mixture was filtered,
and the filtrate was concentrated on rota-vap. The resulting solid was
dissolved in CH2Cl2 and purified on silica gel columns eluted with
CH2Cl2/MeOH to give 4-[4-(4-bromomethylbenzyloxy)-l-oxo-1,3-
dihydro-isoindol-2-yl]-4-carbamoylbutyric acid methyl ester (3) as
1
white solid (40 g, 82% yield). H NMR (DMSO-d6) δ 1.98−2.13 (m,
1H, CHH), 2.14−2.23 (m, 1H, CHH), 2.23−2.32 (m, 2H, CHH,
CHH), 3.50 (s, 3H, CH3), 4.34−4.63 (m, 2H, CH2), 4.67−4.80 (m,
3H, CH2, NCH), 5.25 (s, 4H, CH2), 7.19 (s, 1H, NHH), 7.24−7.34
(m, 2H, Ar), 7.41−7.54 (m, 5H, Ar), 7.58 (br s, 1H, NHH). LCMS:
M + 1 475.
4-Carbamoyl-4-[4-(4-morpholin-4-ylmethyl-benzyloxy)-l -oxo-
1,3-dihydro-isoindol-2-yl]-butyric Acid Methyl Ester (5). Morpholine
(4) (14.72 mL, 169 mmol) was added to the CH2Cl2 solution of
methyl 5-amino- 4-(4-(4-(bromomethyl)benzyloxy)-l-oxoisoindolin-2-
yl)-5-oxopentanoate (3) (36.5 g, 77 mmol) at room temperature. The
mixture was stirred at room temperature for 1 h. The resulting
suspension was filtered, and the filtrate was concentrated on rota-vap.
The resulting oil was dissolved in EtOAc and washed with water
(50 mL × 3). The organic layer was concentrated on rota-vap to give
4-carbamoyl-4-[4-(4-morpholin-4-ylmethyl-benzyloxy)-l-oxo-1,3-dihy-
dro-isoindol-2-yl]-butyric acid methyl ester (5) as a foamy solid (39 g,
(S)-3-(4-Hydroxy-1-oxo-1,3-dihydroisoindol-2-yl)piperidine-2,6-
dione (1). (S)-methyl 4,5-diamino-5-oxopentanoate hydrochloride
(58.7 g, 299 mmol) was added to a solution of methyl 2-(bromo-
methyl)-3-(tert-butyldimethylsilyloxy)benzoate (113 g, 299 mmol) in
acetonitrile (1 L) to form a suspension. N-Ethyl-N-isopropylpropan-2-
amine (104 mL, 597 mmol) was then added, and the resulting mixture
was stirred at 40 °C overnight. The reaction mixture was concentrated
under vacuum to give yellow solid. The solid was dissolved in ether
(900 mL) and washed by aq HCl (1N, 750 mL), satd aq NaHCO3
(2 × 350 mL), and brine (600 mL). The organic layer was con-
centrated to give (S)-methyl 5-amino-4-(4-(tert-butyldimethylsily-
loxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate as yellow oil. Without
further purification, to the oil was added DMF (500 mL) and water
(55 mL). The solution was added to K2CO3 (16.18 g, 117 mmol). The
resulting suspension was stirred at room temperature for 1 h. HCl
(12N, 19.5 mL) and acetonitrile (290 mL) were then added to the
suspension. The resulting suspension was filtered, and the filtrate was
concentrated in vacuo. Then 70 mL of acetonitrile was added to the
resulting oil and heated to 50 °C until a solution formed. The solution
was cooled gradually to room temperature, and the resulting sus-
pension was filtered. Solid was dried to give compound 1 (25 g, 30%);
1
95% yield); mp 66−68 °C. H NMR (DMSO-d6) δ 2.00−2.12 (m,
1H, CHH), 2.14−2.22 (m, 1H, CHH), 2.22−2.29 (m, 2H, CHH,
CHH), 2.30−2.39 (m, 4H, CH2, CH2), 3.46 (s, 2H, CH2), 3.50 (s, 3H,
CH3), 3.53−3.63 (m, 4H, CH2, CH2), 4.28−4.59 (m, 2H, CH2), 4.73
(dd, J = 4.7, 10.2 Hz, 1H, NCH), 5.22 (s, 2H, CH2), 7.14−7.23 (m,
1H, NHH), 7.26−7.39 (m, 4H, Ar), 7.41−7.51 (m, 3H, Ar), 7.58 (s,
1H, NHH). 13C NMR (DMSO-d6) δ 24.82, 30.34, 44.78, 51.24, 53.12,
53.39, 62.09, 66.15, 69.35, 114.67, 115.13, 127.60, 129.00, 129.56,
130.18, 133.43, 135.32, 137.66, 153.43, 167.85, 171.74, 172.47. LCMS:
M + 1 482.2. Anal. Calcd for C23H31N3O6 + 0.3H2O: C, 64.13; H,
6.54; N, 8.63. Found: C, 63.89; H, 6.39; N, 8.56.
3-[4-(4-Morpholin-4-ylmethyl-benzyloxy)-oxo-1,3-dihydro-isoin-
dol-2-yl]-piperidine-2,6-dione (6). To the THF solution of methyl
5-amino-4-(4-(4-(morpholinomethyl)benzyloxy)-yl-oxoisoindolin-2-
yl)-5-oxopentanoate (5) (40 g, 83 mmol) was added potassium tert-
butoxide (9.80 g, 87 mmol) at −78 °C. The mixture was stirred at this
1
HPLC purity 97.6%; mp 163−165 °C. H NMR (DMSO-d6) δ 1.33
(s, 9 H), 1.88−2.08 (m, 1 H), 2.08−2.27 (m, 3 H), 4.32 (d, J = 17.42 Hz,
E
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The compound used in the FRET assay (compound 7) is:
2-((1E,3E,5Z)-5-(1-(6-((4-(2-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoi-
soindolin-4-yl)oxy)acetamido)butyl)amino)-6-oxohexyl)-3,3-dime-
thylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-
1-ium (Compound 7). Cell-Based Chemiluminescent Substrate
Degradation Assays. DF15 multiple myeloma cells expressing
cereblon substrates Ikaros, Aiolos, and GSPT1 fused to an ePL tag
(DiscoverX) were dispensed into a 384-well plate (Corning no. 3712)
prespotted with compound. Compounds were dispensed by an
acoustic despenser (ATS acoustic transfer system from EDC Bio-
systems) into a 384-well plate in a 10-point dose response curve using
3-fold dilutions starting at 10 μM and going down to 0.0005 μM. Then
25 μL of media (RPMI-1640 + 10% heat inactivated FBS + 25 mM
Hepes + 1 mM Na pyruvate + 1× NEAA + 0.1% Pluronic F-68 + 1×
Pen Strep Glutamine) containing 5000 cells was dispensed per well.
Assay plates were incubated at 37 °C with 5% CO2 for 4 h. After
incubation, 25 μL of the InCELL Hunter detection reagent working
solution (DiscoverX, catalogue no. 96-0002, Fremont, CA) was added
to each well and incubated at room temperature for 30 min protected
from light. After 30 min, luminescence was read on a PHERAstar
luminometer (Cary, NC). To determine the EC50 value of a com-
pound for the degradation of a given substrate (the half-maximum
effective concentration), a four-parameter logistic model (sigmoidal
dose−response model) (FIT = (A + {(B − A)/1 + [(C/x)D]})) where
C is the inflection point (EC50), D is the correlation coefficient, and
A and B are the low and high limits of the fit, respectively) was used.
All substrate degradation curves were processed and evaluated using
ActivityBase (IDBS), a data analysis software package.
temperature for 30 min, then 45 mL of 1 N HCl solution was added
to the reaction mixture, followed by 200 mL of saturated NaHCO3
solution. The mixture was diluted with 500 mL of EtOAc at 0 °C,
stirred for 5 min, and separated. The organic layer was washed with
water (50 mL × 3), brine (100 mL) and concentrated on rota-vap to
give a white solid, which was stirred in diethyl ether (300 mL) to give a
suspension. The suspension was filtered to give 3-[4-(4-morpholin-4-
ylmethyl-benzyloxy)-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-
dione (6) as white solid (28.5 g, 72% yield). HPLC: Waters Symmetry
C18, 5 μm, 3.9 mm × 150 mm, 1 mL/min, 240 nm, gradient to 95/5
acetonitrile/0.1% H3PO4 in 5 min, RT = 4.78 min (98.5%); Chiral
HPLC (Chiralpak AGP column, 4.0 mm × 150 mm, 5 μm particle
size, isocratic 50% 10 mM NH4OAc in H2O and 50% acetonitrile)
RT = 8.15 min (0.5%, R-isomer), RT = 10.33 min (99.5%, S-isomer);
1
mp 209−211 °C. H NMR (DMSO-d6) δ 1.86−2.09 (m, 1H, CHH),
Immunoblot Analysis of Endogenous Substrate Protein
Levels. First, 1 ×106 cells (either DF15 and OPM2) were treated with
lenalidomide, pomalidomie, or compound 6 for 5 h before harvest.
Then after quick 1× cold PBS rinse, cells were lysed by cell lysis buffer
(Cell Signaling’s Technologies). Then 10 μg of total lysate was loaded
in each lane. Proteins were transferred to nitrocellulose membrane
using Bio-Rad’s Turbo System. Antibodies used in the Western
included: Ikaros (14859S, Cell Signaling Technologies), Aiolos
(15103S, Cell Signaling Technologies), actin (A5316, Sigma), and
CRBN (in house antibody from Celgene).3a
2.29−2.38 (m, 4H, CH2, CH2), 2.44 (dd, J = 4.3, 13.0 Hz, 1H, CHH),
2.53−2.64 (m, 1H, CHH), 2.82−2.99 (m, 1H, CHH), 3.46 (s, 2H,
CH2), 3.52−3.61 (m, 4H, CH2, CH2), 4.18−4.51 (m, 2H, CH2), 5.11
(dd, J = 5.0,13.3 Hz, 1H, NCH), 5.22 (s, 2H, CH2), 7.27−7.38 (m,
5H, Ar), 7.40−7.53 (m, 3H, Ar), 10.98 (s, 1H, NH). 13C NMR
(DMSO-d6) δ 22.36, 31.21, 45.09, 51.58, 53.14, 62.10, 66.17,
69.41,114.97, 115.23, 127.64, 128.99, 129.81, 129.95, 133.31, 135.29,
137.68, 153.50, 168.01, 170.98, 172.83. LCMS: M + 1 465. Anal. Calcd
for C25H27N3O5 + 0.86H2O: C, 64.63; H, 6.22; N, 9.04. Found: C,
64.39; H, 6.11; N, 8.89; H2O, 3.24.
Crystallography. The purification of CRBN−DDB1 for crystal-
lization was performed as previously described.8b Crystallization of
CRBN−DDB1 with compound 6 was performed using sitting-drop
vapor diffusion. Thirty mg/mL CRBN−DDB1 in the presence of
1 mM compound 6 was mixed 1:1 with, and subsequently equilibrated
against, a mother liquor containing 200 mM sodium fluoride and
20% PEG 3350 at 20 C. Crystals were cryoprotected in the reservoir
solution supplemented with 20% ethylene glycol and cooled under
liquid nitrogen. Data was collected at the Advance Photon Source
beamline LS-CAT 21ID-F. The complex crystal structure was solved
by molecular replacement using Phaser with search model of 4TZ4.9a
Subsequent manual model building using Coot and refinement were
performed using Refmac5.16 Data collection statistics and sample
electron density can be found in the Supporting Information.
Coordinates have been deposited in the Protein Data Bank with the
accession code 5V3O.
TR-FRET Assay. The 6XHis-tagged full length human CRBN
bound to full length human DDB1 used in the assay was purified as
described elsewhere with the exception that the thrombin cleavage/
ortho nickle step was removed.8b In the assay, 60 nM 6Xhis-tagged
CRBN-DDB1 was combined with 30 nM cy5-conjugated cereblon
modulator (compound 7) and 3 nM LanthaScreen Eu-anti-His Tag
antibody (ThermoFisher catalogue no. PV5596) in 20 mM HEPES
pH 7, 150 mM NaCl, 0.005% Tween-20 assay buffer. FRET was
observed by exciting at 340 nm and monitoring emission at 615 nm
(non-FRET emission) and 665 nm (FRET emission), and FRET
efficiency was determined by the ratio of FRET to non-FRET emission
(665 nm/615 nm). Competing cereblon modulating compound or
DMSO carrier was titrated and incubated for 10 min before scanning
on the Envision plate reader with a 60 μs delay to quantitate loss of
FRET efficiency. All FRET competition curves were processed and
evaluated using ActivityBase (IDBS), a data analysis software package.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.6b01921.
Quantitation of GSPT1 degradation with chemilumines-
cent cell-based assay; comparison of lenalidomide and
compound 6 binding modes; sample electron density
from the CRBN-DDB1-compound 6 crystal structure;
X-ray data and refinement statistics (PDF)
Molecular formula strings (CSV)
Accession Codes
Coordinates for the structure of CRBN-DDB1- 6 have been
deposited in the Protein Data Bank with the accession code
5V3O. Authors will release the atomic coordinates and experi-
mental data upon article publication.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 858-795-4728. E-mail: pchamberlain@celgene.com.
ORCID
Philip P. Chamberlain: 0000-0002-6407-7344
Notes
The authors declare the following competing financial inter-
est(s): Authors are or have been employees, consultants, or
collaborators of Celgene.
F
DOI: 10.1021/acs.jmedchem.6b01921
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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ACKNOWLEDGMENTS
■
We thank Shamrock Biostructures for data collection services.
This research used resources of the Advanced Photon Source, a
U.S. Department of Energy (DOE) Office of Science User
Facility operated for the DOE Office of Science by Argonne
National Laboratory under contract no. DE-AC02-06CH11357.
ABBREVIATIONS USED
■
CRBN, cereblon; IKZF1, Ikaros; IKZF3, Aiolos; SLE, system-
atic lupus erythematosus; MM, multiple myeloma
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