Protein Degradation via CRL4CRBN Ubiquitin Ligase: Discovery and Structure-Activity Relationships of Novel Glutarimide Analogs That Promote Degradation of Aiolos and/or GSPT1

Article abstract of DOI:10.1021/acs.jmedchem.6b01911

We previously disclosed the identification of cereblon modulator 3 (CC-885), with potent antitumor activity mediated through the degradation of GSPT1. We describe herein the structure-activity relationships for analogs of 3 with exploration of the structurally related dioxoisoindoline class. The observed activity of protein degradation could in part be rationalized through docking into the previously disclosed 3-CRBN-GSPT1 cocrystal ternary complex. For SAR that could not be rationalized through the cocrystal complex, we sought to predict SAR through a QSAR model developed in house. Through these analyses, selective protein degradation could be achieved between the two proteins of interest, GSPT1 and Aiolos.

Full text of DOI:10.1021/acs.jmedchem.6b01911

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Article
Protein Degradation via CRL4CRBN Ubiquitin Ligase: Discovery
and Structure-Activity Relationships of Novel Glutarimide
Analogs that Promote Degradation of Aiolos and/or GSPT1
Joshua D. Hansen, Kevin R. Condroski, Matthew Correa, George W. Muller, Hon-Wah Man,
Alexander L Ruchelman, Weihong Zhang, Fan Vocanson, Tim Crea, Wei Liu, Gang Lu, Frans
Baculi, Laurie LeBrun, Afshin Mahmoudi, Gilles Carmel, Matthew Guy Hickman, and Chin-Chun Lu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01911 • Publication Date (Web): 30 Mar 2017
Downloaded from http://pubs.acs.org on March 31, 2017
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Protein Degradation via CRL4CRBN Ubiquitin
Ligase: Discovery and StructureꢀActivity
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Relationships of Novel Glutarimide Analogs that
Promote Degradation of Aiolos and/or GSPT1
Joshua D. Hansen,* Kevin Condroski, Matthew Correa, George Muller, Hon-Wah Man,
Alexander Ruchelman, Weihong Zhang, Fan Vocanson, Tim Crea, Wei Liu, Gang Lu, Frans
Baculi, Laurie LeBrun, Afshin Mahmoudi, Gilles Carmel, Matt Hickman, Chin-Chun Lu
Celgene Corporation, 10300 Campus Point Drive, Suite 100, San Diego, California 92121, USA.
KEYWORDS Aiolos, IKZF3, GSPT1, protein degradation, molecular glue, immunomodulatory.
ABSTRACT: We previously disclosed the identification of cereblon modulator 3 (CCꢀ885), with
potent antitumor activity mediated through the degradation of GSPT1. We describe herein the
structureꢀactivity relationships for analogs of 3 with exploration of the structurally related
dioxoisoindoline class. The observed activity of protein degradation could in part be rationalized
through docking into the previously disclosed 3ꢀCRBNꢀGSPT1 coꢀcrystal ternary complex.
SAR that could not be rationalized through the coꢀcrystal complex we sought to predict through
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a QSAR model developed in house. Through these analyses, selective protein degradation could
be achieved between the two proteins of interest, GSPT1, and Aiolos.
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INTRODUCTION
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Induction of protein degradation as a therapeutic strategy has been clinically validated by the
class of immunomodulatory drugs which include lenalidomide and pomalidomide (Figure 1).
The therapeutic benefits of immunomodulatory drug treatment are connected to their ability to
promote recruitment and ubiquitination of substrate proteins to the cullinꢀdamaged DNAꢀ
bindingꢀRING boxꢀdomain protein (CUL4ꢀDDB1ꢀRBX1ꢀCRBN) or simply (CRL4CRBN) E3
ubiquitin ligase, with the resulting ubiquitin tagged proteins directed to and subsequently
degraded by the 26S proteasome.1,2,3 Recently, we described the identification of 3 (CCꢀ885), a
novel cereblon (CRBN) modulator which was demonstrated to mediate antitumor effects through
the recruitment and degradation of G1 to S phase transition 1 protein (GSPT1).4 GSPT1 (also
named eRF3a) is a translation termination factor that binds eukaryotic translation termination
factor 1 (eFR1) to mediate stop codon recognition and nascent protein release from the
ribosome.5,6,7 Unlike 3, the currently marketed class of immunomodulatory drugs, which include
1 (lenalidomide) and 2 (pomalidomide), do not promote the degradation of GSPT1,
demonstrating selectivity favoring zinc finger transcription factors such as Aiolos (IKZF3) and
Ikaros (IKZF1).
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Figure 1. Aiolos and GSPT1 protein degradation by lenalidomide, pomalidomide, and 3.
Selectivity in protein degradation has also been reported between lenalidomide and
pomalidomide in the comparison of the protein kinase casein kinase 1 α (CK1α) where
recruitment and degradation is a key feature to the use of lenalidomide in 5qꢀdeletionꢀassociated
myelodysplastic syndrome.8 To investigate the structureꢀactivity requirements and constraints
governing the selectivity of protein recruitment and degradation for GSPT1 / Aiolos, we
examined protein degradation using a traditional measurement (EC50) as well as the measure of
maximal protein degradation that is induced by compound over a concentration range up to 10
ꢀM which is reported by Ymax (Figure 2) using an enhancedꢀProLable (ePL) degradation assay
(described in methods section). For analog comparison, the observed level of protein degradation
induced by 2 for Aiolos, and 3 for GSPT1 was used as our reference for comparison and was set
to Ymax = 0 respectively. Since the data was not normalized, a negative Ymax is possible and
indicates a greater extent of protein degradation than the control compound reference. The ePL
degradation assay was the general method used to generate and interpret SAR described herein.
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Additionally, protein degradation was examined via western blot analysis for select compounds
(Figure 3). All compounds tested demonstrated comparable efficiency and potency in the
degradation of ePLꢀtagged exogenous GSPT1 and /or Aiolos proteins versus degradation of
endogenous GSPT1 and Aiolos proteins measured by western blot analysis.
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Figure 2. (A) GSPT1 protein degradation curves of 2 (pomalidomide) and 3. (B) Aiolos protein
degradation curves of 2 (pomalidomide) and 3. (C) GSPT1 protein degradation curves of three
example compounds to illustrate; low levels of protein degradation (high Ymax, 14), or high level
of protein degradation (low or negative Ymax, comp 3 and 10).
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Figure 3. Comparison protein degradation for selected compounds between the ePL degradation
assay and Western blot. (A) OPM2 cells were treated with DMSO, 3, 8, 26, 13, 19 and 22 at the
indicated concentration for 8 hours, and whole cell extracts were subjected to immunoblot
analysis. (B) Table of Ymax values at 10 ꢀM of the selected compounds for comparison to the
western blot analysis.
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CHEMISTRY
The synthesis of urea analogs 7-17 in Scheme 1 began with the Fischer esterification of 4ꢀ
bromoꢀ2ꢀmethylbenzoic acid (4) followed by radical bromination of the tolyl methyl.9 Formation
of the bicyclic lactam was accomplished using 3ꢀaminopiperidineꢀ2,6ꢀdione as the nucleophile
for bromide displacement followed by further condensation onto the ester to form lactam 5. The
requisite aminomethyl functionality was installed via palladiumꢀmediated cyanide insertion to
the aryl bromide with subsequent reduction to yield 6. Oxoisoindoline 6 provided a convenient
scaffold from which to explore SAR through further elaborations such as aryl urea formation
shown in Scheme 1.
Scheme 1. Synthesis of 5-yl substituted oxoisoindolines.
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Reagents and conditions: (a) MeOHꢀH2SO4 (50ꢀ1), 65 °C, 18 h, 95%; (b) NBS, azoꢀ
isobutyronitrile, 85 °C, 18 h, ACN, 66%; (c) 3ꢀaminopiperidineꢀ2,6ꢀdione.HCl, triethylamine, rt
for 25 h, 44%; (d) 1,1'ꢀBis(diphenylphosphino)ferrocene, ZnCN, ZnOAc, Pd2(dba)3, 120 °C for
20 h, 57%; (e) MsOH, Pd/C, DMA, 50 psi, 40 °C for 20 h, 40%; (f) substituted anilines, CDI,
DMF, 80 °C for 16 h, 8ꢀ80%.
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RESULTS AND DISCUSSION
Concurrent with our oxoisoindoline SAR efforts, we explored alternative scaffolds such as the
dioxoisoindoline. In comparison of benchmark urea analogs, shown in Table 1, a clear trend
could be observed between the two scaffolds. For example, the unsubstituted phenyl urea 7 was
able to degrade GSPT1 with Ymax of 9 (Ymax = 9 corresponds to 91% of the protein was degraded
compared to control compound 3 [Figure 2]) while the unsubstituted phenyl urea in the
dioxoisoindoline series (8) was less efficient at GSPT1 protein degradation with Ymax = 34.
This same trend held for Aiolos, where 7 could promote ~7% more degradation than control
compound pomalidomide (2) giving it a negative numerical value (ꢀ7), while 8 could only
degrade 56% Aiolos protein (Ymax = 44) as compared to control. Further analysis of the 2, 3, or
4ꢀsubstituted chloroꢀphenyls of the oxoisoindolines (9, 11, and 13) compared to the
corresponding dioxoisoindolines (10, 12, and 14) suggests that dioxoisoindolines were in general
less efficient protein degraders of both GSPT1 and Aiolos. Comparison between the degree of
GSPT1 and Aiolos degradation in analogs 9, 11, and 13 revealed an interesting selectivity pattern
of maximal degradation between the two proteins. For example, while the 2ꢀchloro substitution
in 9 showed comparable GSPT1 and Aiolos degradation, selectivity was observed in the 3ꢀchloro
analog 11 which displayed increased GSPT1 degradation with 18 % more GSPT1 degraded than
Aiolos (standard error of measure (SEM) for 11 Ymax; GSPT1 = ꢀ6 + 1.0, Aiolos = 12 + 1.8; see
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supplemental for all SEM data). A reversal in selectively favoring Aiolos degradation was
observed with 4ꢀClꢀderivitive 13. To further explore the impact of substitution at the para
position, nitrile analog 15 was synthesized and demonstrated an unexpected reversal in
selectivity compared to 13, with significant loss in GSPT1 degradation. Although the apparent
alterations in selectivity caused by these subtle structural changes could not be readily
rationalized through analysis of the xꢀray structure, we were encouraged by the apparent
potential to discover selective protein degraders suggested by the differentiated selectivity
patterns revealed through this series of compounds.
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Table 1. SAR of GSPT1 and Aiolos protein degradation mediated by compounds in the
oxoisoindoline and dioxoisoindoline scaffolds.a,b,c
GSPT1
Aiolos
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Ymax
GSPT1
Ymax
Analog
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R2
EC50(nM)
EC50(nM)
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O
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3
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O
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H,H
aAll values are the mean of at least three separate assay determinations, SEM for data
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available in supplemental material, Ymax is the % protein remaining at 10 ꢀM, 4 h (see
methods).
With these encouraging results, we sought to further explore substitution on the aryl ring in the
oxoisoindoline series, probing potency and selectivity. Highlights of these efforts are outlined in
Table 2. When the 4ꢀsubstituent was changed from chloro (13) to fluoro (16) we observed that
the GSPT1 potency (EC50) was equivalent between 13 & 16, and 16 showed a slight loss in
ability to degrade protein (Ymax; ꢀ2.0 versus 4.8).
Compound 17 was synthesized to confirm the expected loss in potency as predicted by the
observed binding mode of the glutarimide moiety in the triꢀtryptophan pocket (Figure 3),10
Methylation of the glutarimide would be expected to disrupt the hydrogen bond network in the
glutarimide moiety from (H378) to (W380), and indeed compound 17 showed a loss of binding
to CRBN (data not shown). This disruption of CRBN binding was manifest in reduced levels of
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protein degradation, demonstrating >1000ꢀfold loss of activity for both GSPT1 and Aiolos EC50
(16 versus 17). The SAR of diꢀsubstituted aryl chlorides (18, 19, 22, and 23) were also
examined. In comparison to the 2ꢀchloro 9, the 2,4ꢀdiꢀchloro 18 showed improved potency and
efficiency of degradation for both Aiolos and GSPT1, similar to that observed in the 4ꢀchloro
analog 13. The diꢀ2,6ꢀchloro 19 showed comparable GSPT1 potency to 18 but with a marked
reduction in maximal degradation. While the potency between 18 and 19 on GSPT1 were
comparable, it was interesting to note that 19 demonstrated a 100ꢀfold loss of Aiolos potency and
a large shift for Aiolos maximal degradation (Ymax = 70). A similar trend for GSPT1 degradation
selectivity was observed with 2,4,6ꢀtrichloro 20; the Aiolos Ymax is high for both 20 & 19 (61 /
70), although the 4ꢀchloro functionality in 20 might be responsible for the increased GSPT1
activity since both 13 and 18 are highly efficient GSPT1 degraders. This trend of potent GSPT1
activity with a 4ꢀchloro substituent was also observed in 23. While analog 22 with both meta
positions substituted also showed strong GSPT1 degradation, selectivity favoring GSPT1 over
Aiolos degradation was observed and to a greater extent than the 3ꢀCl analog 11. Comparison of
the Aiolos results for 19 and 20, which achieve similar levels of maximal degradation (Ymax= 70
and 61, respectively) but show ~13 fold potency differential (EC50 = 0.32 and 0.02 µM,
respectively), further highlights the SAR subtleties of protein degradation. When a large
substituent such as phenyl was placed in the ortho position (21), we observed selectivity toward
more complete GSPT1 degradation (Ymax = 2.2) compared to Aiolos (Ymax = 61). This trend was
also noted in compounds 19 & 20, which both contain diꢀortho-substitution at terminal phenyl
indicating a potential rationale for the selectivity based on an expected conformational restriction
of rotation about the phenylꢀNHꢀurea bond.
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Table 2. SAR of GSPT1 and Aiolos protein degradation.a,b,c
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GSPT1
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Analog
R1
R2
EC50(nM)
EC50(nM)
Ymax
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ꢀ3
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H
NCd
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5400
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H
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H
H
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ꢀ3
4
ꢀ1
aAll values are the mean of at least three separate assay determinations, SEM for data
b
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available in supplemental material, Ymax is the % protein remaining at 10 ꢀM, 4 h (see
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methods), NC (not calculated) due to minimal substrate degradation and lack of a
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sigmoidal curve.
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Our previously reported 3 bound coꢀcrystal structure with CRBN4 suggested the hydrogen
bonding network of the urea carbonyl to CRBN histidine (H353), as well as the urea NH to
glutamate (E377), were important binding features (Figure 4).
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Figure 4. Hydrogen bonding network between cereblon and 3 (W386 omitted for clarity).
Protein Data Bank accession code 5HXB.
To further explore this observation we made a series of capped urea derivatives (24-26) and
noted that methylation of the distal urea nitrogen (24) led to a 44ꢀfold loss in GSPT1 degradation
potency and significant loss in maximal degradation levels (Ymax = 50, compared to 3 Ymax = 0).
Substitution at the proximal urea nitrogen (25) was less disruptive showing only a 10ꢀfold shift
in GSPT1 potency with the ability to degrade 77% of protein levels compared to control. While
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both of these derivatives can maintain a portion of the observed Hꢀbond interactions with E377,
compound 26, with methylation at both urea nitrogens, cannot make this interaction with the
protein and we observed a complete lack of ability to degrade either substrate, even at
concentrations up to 10 ꢀM. The lack of activity for bisꢀmethylated urea 26 was consistent with
the cyclic urea analog 27 which also showed loss of activity on GSPT1. Interestingly, the Aiolos
potency was not significantly impacted in 27, providing further evidence for the potential for
Aiolos/GSPT1 selectivity. To further explore this result we synthesized the 5ꢀmembered cyclic
urea 28 which, surprisingly, displayed a selectivity toward GSPT1 degradation. SAR studies in
the cyclic ureas led to the identification of 29, which displayed enhanced GSPT1 potency and
substantially increased protein degradation (~88% compared to 28, 48%). Since the activity of
29 was difficult to explain via the crystal structure binding mode, we next explored
computational methods as a means to rationalize anomalous GSPT1 activity.
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Table 3. SAR of GSPT1 and Aiolos protein degradation.a,b,c
GSPT1
Aiolos
Aiolos
Ymax
GSPT1
Ymax
Analog
R1
R2
EC50(nM)
EC50(nM)
H
H
H
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0
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Me
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H
Me
10
23
100
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Me
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NCd
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aAll values are the mean of at least three separate assay determinations, SEM for data
b
c
available in supplemental material, Ymax is the % protein remaining at 10 ꢀM, 4 h (see
d
methods), NC (not calculated) due to minimal substrate degradation and lack of a
sigmoidal curve.
DOCKING STUDIES
To further understand the underlying structureꢀactivity relationships for GSPT1 recruitment by
small molecule CRBN modulators, we considered potential binding modes for analogs of 3. The
xꢀray crystallographic structure4 of the complex of human CRBN, DNA damage binding protein
1 (DDB1), GSPT1, and 3 (Protein Data Bank accession code 5HXB) was used for docking
studies using the Glide software package from Schrödinger, LLC. The docked pose of 3 in the
interfacial pocket between CRBN and GSPT1 was observed to reproduce the crystal structure
binding mode accurately (Figure 5).
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Figure 5. Poses of docked 3 (grey) and 3 from xꢀray crystallographic structure (green)
correspond well (Protein Data Bank accession code 5HXB).
The remaining compounds in Tables 1 and 2 were found to dock in a similar fashion to 3, each
forming a trio of hydrogen bonds that anchor the glutarimide moieties in the triꢀTrp binding site.
The one notable exception as discussed previously was the Nꢀmethylation of the glutarimide in
17, which disrupts the hydrogen bonding network, significantly reducing its ability to recruit and
degrade GSPT1.
Compounds in Table 3 investigate the effects of N-alkylation of the urea, which are all
deleterious to GSPT1 degradation activity to varying extents. Docking studies demonstrated
either significantly altered binding modes due to disruption of one or more hydrogen bonds
between the urea moiety and residues E377 and H353. As previously noted, compound 29
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recovered significant GSPT1 degradation ability with the incorporation of the mꢀphenoxyphenyl
ether, and docking studies did not adequately explain this observation.
7
8
9
QUANTITATIVE STRUCTUREꢀACTIVITY RELATIONSHIP (QSAR) MODEL
CONSTRUCTION
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While docking studies utilizing the existing GSPT1 complex coꢀcrystal structure provides
insights into the SAR revealed by this congeneric series of molecules, there is currently no
analogous structure of the complex of Aiolos and CRBN with a small molecule bound. Since we
desired to construct models that could serve our efforts in a predictive capacity for both GSPT1
and Aiolos degradation we turned to the wealth of GSPT1 and Aiolos assay data collected for
our inꢀhouse compound library and used it to develop statistical models.
An initial collection of more than 1,500 GSPT1 and Aiolos EC50 and Ymax pairs of assay
measurements were used to construct a categorical QSAR model using the AutoꢀModeler
module in Optibrium’s StarDrop software.11 We began by dividing the data sets into training
(70%), validation (15%), and test sets (15%) of compounds, each chosen randomly to eliminate
any compound selection bias. A library of 321 SMARTS based descriptors (counts of atom
types and functionalities),12 and 9 wholeꢀmolecule properties were generated within StarDrop for
each compound in these data sets.
Category models were then constructed using random forest (RF) ensemble methods with
predictions based upon the output of a collection of 100 random trees;13 these were found to
provide superior accuracy for predictions of GSPT1 and Aiolos EC50 and Ymax when compared
to models built with recursive partitioning approaches using decision trees14 and Gaussian
processes.15
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QSAR MODEL RESULTS
5
6
7
8
Binning cutoff values for the categorical models were chosen in order to maximize the
accuracy, sensitivity, and specificity of each model over the activity ranges of our data sets
(Table 4). The category matrix for the RF model for Aiolos EC50 with a binning cutoff of 21 nM
is shown in Table 5, with predicted categories on the yꢀaxis compared with experimentally
observed categories on the xꢀaxis. The overall accuracy is 82% when the model is applied to the
test set. A similar plot for Aiolos Ymax data demonstrates an overall accuracy of 80% with a
binning cutoff of 10% protein remaining (Table 6).
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Table 4. QSAR models constructed for Aiolos and GSPT1 EC50 and Ymax
.
Model
Binning Cutoff
Units
nM
Overall Accuracy (test set)
Aiolos EC50
Aiolos Ymax
GSPT1 EC50
GSPT1 Ymax
21
10
71
29
82%
80%
79%
79%
%Ctrl at 4hrs
nM
%Ctrl at 4hrs
Table 5. RF model for Aiolos EC50 with a binning cutoff of 21 nM (test set).
Observed > 21 nM Observed < 21 nM
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Predicted > 21 nM
Predicted < 21 nM
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Table 6. RF model for Aiolos Ymax with binning cutoff of 10% (test set).
Observed < 10%
Observed > 10%
103
31
48
Predicted < 10%
Predicted > 10%
219
In the case of GSPT1 (Table 7 and 8), the overall accuracies for EC50 and Ymax were 79%,
slightly lower in accuracy than the corresponding Aiolos models.
Table 7. RF model for GSPT1 EC50 with a binning cutoff of 71 nM (test set).
Observed > 71 nM Observed < 71 nM
60
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Predicted > 71 nM
Predicted < 71 nM
122
Table 8. RF model for GSPT1 Ymax with binning cutoff of 29% (test set).
Observed < 29%
Observed > 29%
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Predicted < 29%
Predicted > 29%
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The successful application of these models was demonstrated as they were applied to more
than 1,000 additional analogs prepared subsequent to model construction. Aiolos EC50 and Ymax
predictions demonstrated similar performance to our earlier test set assessments, as did those for
GSPT1 Ymax, while GSPT1 EC50 predictions demonstrated modest accuracy (see supplemental
information). The predictive capacity of these models has allowed us to thoughtfully and
efficiently prioritize the synthesis of selected analogs from large enumerated libraries of
proposed compounds.
CONCLUSIONS
The ability to affect protein homeostasis and its association with disease state through targeted
protein degradation has exciting implications for drug discovery. In contrast to chimericallyꢀ
linked protein degraders16,17,18 as a method to utilize the CRBN ligase system, ureaꢀbased
analogs of 3 can be used to create an interaction hotspot on the surface of CRBN for direct
proteinꢀprotein interactions.4 We employed a dual strategy of structureꢀbased design and QSAR
modeling to more completely rationalize SAR investigations into two series of urea analogs that
both demonstrated the ability to recruit and induce the degradation of Aiolos and/or GSPT1
protein. During the course of these SAR explorations we observed several categories of activity.
This included compounds where Aiolos and GSPT1 were equally degraded but with either low
or high potency, as well as profiles that displayed comparable potency but differentiated levels of
protein degradation (Aiolos degradation of 7 versus 9). The apparent disconnect between
potency and efficiency of protein degradation was not an uncommon observation across the
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series, and may reveal insights into the inherent complexity in the SAR of protein degradation.
For example, we measure protein degradation as the sum endpoint of a process that includes
multiple variable steps including: 1) compound binding to CRBN, forming the “protein hotspot”
or basis for “molecular glue”19,20,21, 2) formation of the ternary complex between the CRBN
bound compound and substrate such as GSPT14, 3) transfer of ubiquitin from the E2, 4)
disassociation of ubiquitinated substrate from the ternary complex, 5) potential reverse
competition with deubiquitinating enzymes (DUBs), and 6) trafficking to the 26S proteasome for
degradation. These steps underpin the currently understood mechanism of action for UPSꢀ
dependent protein degradation3,22, 23, and 3 was previously shown to induce protein degradation
in a CRBN as well as UPSꢀdependent manner.4 Compounds with selectivity toward either Aiolos
or GSPT1 degradation were noted, also across an activity continuum. The subtleties of
substitution pattern SAR offer opportunity to discover compounds across a varied spectrum of
maximal degradation, potency, as well as selectivity; which we hope can lead to profiles of tuned
degradation profiles to derive maximal clinical benefit.
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EXPERIMENTAL SECTION
General. Compounds were named using ChemDraw Ultra. All materials were obtained
from commercial sources and used without further purification, unless otherwise noted.
Chromatography solvents were HPLC grade and used as purchased. All airꢀsensitive reactions
were carried out under a positive pressure of an inert nitrogen atmosphere. Chemical shifts (δ)
are reported in ppm downfield of TMS and coupling constants (J) are given in Hz. Thin Layer
Chromatography (TLC) analysis was performed on Whatman thin layer plates. The purity of
final tested compounds was typically determined to be > 95% by HPLC. Compounds were
analyzed for purity using the following method: gradient (5ꢀ95% acetonitrile + 0.075% formic
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acid in water + 0.1% formic acid over 8 min, followed by 95% acetonitrile + 0.075% formic acid
for 2 min); flow rate 1 mL/min, column Phenomenex Luna 5µ PFP(2) 100A (150 mm x 4.60
mm). Elemental analysis was performed at Robertson Microlit Laboratories, Ledgewood, New
Jersey.
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Synthesis. Compounds 4-16, 18-20, and 22 have been previously described in the patent
literature.24,25
1-(4-Fluorophenyl)-3-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)
urea (17). To a 0 °C stirred solution of 15 (295 mg, 0.71 mmol) in DMF (15 mL) was added
cesium carbonate (468 mg, 1.43 mmol) followed by dropꢀwise addition of methyl iodide (306
mg, 2.15 mmol). The reaction mixture was stirred for 16 h at room temperature. The reaction
mixture was poured into ice cold water (100 mL) and stirred for 30 min. The precipitate was
filtered and dried under vacuum. The crude product was purified by reverse phase HPLC to give
1
the title compound (110 mg, 0.26 mmol, 36% yield, HPLC purity >99%). H NMR (400 MHz,
DMSOꢀd6) δ 8.66 (s, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.47 – 7.38 (m, 3H), 7.06 (t, J =
8.9 Hz, 2H), 6.73 (t, J = 6.1 Hz, 1H), 5.17 (dd, J = 13.4, 5.0 Hz, 1H), 4.55 – 4.16 (m, 4H), 3.02 –
2.92 (m, 4H), 2.62 – 2.56 (m, 1H), 2.48 – 2.26 (m, 1H), 2.03 – 1.91 (m, 1H); MS (ESI) m/z
425.19 [M+1]+.
1-([1,1'-Biphenyl]-2-yl)-3-((2-(2,6-Dioxopiperidin-3-yl)-1-Oxoisoindolin-5-yl)methyl)urea
(21). Step A. To a stirred solution of [1,1'ꢀbiphenyl]ꢀ2ꢀamine (0.5 g, 2.95 mmol) in
dichloromethane (30 mL) was added pyridine (0.6 mL, 8.85 mmol)
and phenyl
carbonochloridate (0.4 mL, 3.54 mmol) at 0 °C. The reaction was stirred at room temperature for
30 min. The reaction was diluted with water and extracted with dichloromethane. The combined
organic layers were dried over anhydrous sodium sulphate, concentrated under reduced pressure
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and the residue obtained was purified by silica gel chromatography to give phenyl [1,1'ꢀ
biphenyl]ꢀ2ꢀylcarbamate (450 mg, 1.33 mmol, 60% yield). MS (ESI) m/z 290.30[M+1]+.
Step B. To a stirred solution of phenyl [1,1'ꢀbiphenyl]ꢀ2ꢀylcarbamate (0.3 g, 1.00 mmol) in
DMF (10 mL) was added 5 (309 mg, 0.84 mmol) at 0 °C. The reaction was stirred at 100 °C for
1 h. The reaction mixture was diluted with water and the resulting precipitate was collected by
vacuum filtration. The obtained crude compound was purified by reverse phase HPLC to give 1ꢀ
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([1,1'ꢀbiphenyl]ꢀ2ꢀyl)ꢀ3ꢀ((2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1ꢀoxoisoindolinꢀ5ꢀyl)methyl)urea
(21).
(80 mg, 0.17 mmol, 20% yield, HPLC purity >99%). 1H NMR (300 MHz, DMSOꢀd6) δ 10.98 (s,
1H), 7.87 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.55 – 7.33 (m, 7H), 7.32 – 7.23 (m, 1H),
7.21 – 7.03 (m, 3H), 5.11 (dd, J = 13.2, 5.1 Hz, 1H), 4.64 – 4.11 (m, 4H), 2.89 – 2.87 (m, 1H),
2.60 – 2.58 (m, 1H), 2.41 – 2.39 (m, 1H), 2.01 – 1.99 (m, 1H); MS (ESI) m/z 469.041[M+1]+.
1-(3-Chloro-4-methylphenyl)-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-
yl)methyl)-1-methylurea (24). To stirred solution of 6 (0.4 g, 1.08 mmol) in DMF (10 mL) at
room temperature was added 1,1’ꢀcarbonyldiimidazole (210 mg, 1.30 mmol) and stirred for 1 h.
To the reaction mixture was added 3ꢀchloroꢀN,4ꢀdimethylaniline (201 mg, 1.30 mmol) and was
heated at 80 °C for 12 h. The reaction mixture was concentrated under reduced pressure and
resultant residue was purified by reverse phase HPLC to give the title compound (90 mg, 0.19
mmol, 18% yield, HPLC purity >99%). 1H NMR (400 MHz, DMSOꢀd6) δ 10.98 (s, 1H), 7.66 (d,
J = 7.8 Hz, 1H), 7.46 (s, 1H), 7.42 – 7.30 (m, 3H), 7.17 (dd, J = 8.1, 2.2 Hz, 1H), 6.86 (t, J = 5.9
Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.57 – 4.08 (m, 4H), 3.16 (s, 3H), 2.91 (ddd, J = 17.7,
13.5, 5.3 Hz, 1H), 2.65 – 2.54 (m, 1H), 2.45 – 2.34 (m, 1H), 2.31 (s, 3H), 2.05 – 1.95 (m, 1H);
MS (ESI) m/z 454.03 [M+1]+.
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3-(3-Chloro-4-methylphenyl)-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-
yl)methyl)-1-methylurea (25). Step A. To a stirred solution of 1ꢀoxoꢀ1,3ꢀdihydroisobenzofuranꢀ
5ꢀcarbonitrile (10.0 g, 62.85 mmol) in N-methylꢀ2ꢀpyrrolidone (100 mL) was added diꢀtertꢀbutyl
dicarbonate (27.0 g, 125.70 mmol), sodium borohydride (7.1 g, 188.55 mmol) and nickel
chloride hexahydrate (60.0 g, 251.40 mmol) at 0 °C and stirred at room temperature for 12 h.
The reaction mixture was diluted with water and extracted with ethyl acetate. The combined
organic layers were washed with water, brine, dried over sodium sulphate and concentrated
under reduced pressure. The resultant residue was purified by silica gel chromatography to give
tertꢀbutyl ((1ꢀoxoꢀ1,3ꢀdihydroisobenzofuranꢀ5ꢀyl)methyl)carbamate (9.0 g, 34.22 mmol, 56%
yield). MS (ESI) m/z 264.22 [M+1]+.
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Step B. To
a
stirred solution of tertꢀbutyl (1ꢀoxoꢀ1,3ꢀdihydroisobenzofuranꢀ5ꢀ
yl)methylcarbamate (5.0 g, 19.04 mmol) in DMF (50 mL) was added sodium hydride (685 mg,
28.56 mmol) at 0 °C and stirred for 30 min. Methyl iodide (3.5 mL, 57.0 mmol) in DMF was
added and stirred at room temperature for 2 h. The reaction mixture was diluted with water and
extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried
over sodium sulphate and concentrated under reduced pressure. The resultant residue was
purified by silica gel chromatography to give tertꢀbutyl methyl((1ꢀoxoꢀ1,3ꢀ
dihydroisobenzofuranꢀ5ꢀyl)methyl)carbamate (3.0 g, 10.80 mmol, 57% yield). MS (ESI) m/z
278.26[M+1]+.
Step C. To a stirred solution of tertꢀbutyl methyl((1ꢀoxoꢀ1,3ꢀdihydroisobenzofuranꢀ5ꢀ
yl)methyl)carbamate (1.0 g, 3.61 mmol) in THF (10 mL) was added 2 M solution of sodium
hydroxide (10 mL) at 0 °C and stirred at room temperature for 5 h. The reaction mixture was
concentrated
under
reduce
pressure
to
give
sodium
4ꢀ((tertꢀ
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butoxycarbonyl(methyl)amino)methyl)ꢀ2ꢀ(hydroxymethyl)benzoate (1.0 g, 3.15 mmol, 80%
yield). MS (ESI) m/z 318.34[M+1]+.
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Step D. To a stirred solution of sodium 4ꢀ((tertꢀbutoxycarbonyl(methyl)amino)methyl)ꢀ2ꢀ
(hydroxymethyl) benzoate (500 mg, 1.57 mmol) in DMF (50 mL) was added ethyl iodide (0.1
mL, 1.73 mmol) at 0 °C and stirred at room temperature for 12 h. The reaction mixture was
diluted with water and extracted with ethyl acetate. The combined organic layers were washed
with water, brine, dried over sodium sulphate and concentrated to give ethyl 4ꢀ((tertꢀ
butoxycarbonyl(methyl)amino)methyl)ꢀ2ꢀ(hydroxymethyl)benzoate (0.3 g, 0.92 mmol, 60%
yield). 1H NMR (300 MHz, DMSOꢀd6) δ 7.83 (dd, J = 7.9, 3.5 Hz, 1H), 7.48 (s, 1H), 7.18 (d, J =
8.0 Hz, 1H), 5.23 (s, 1H), 4.81 (d, J = 4.2 Hz, 2H), 4.43 (s, 2H), 4.34 – 4.18 (m, 2H), 2.79 (d, J =
13.3 Hz, 3H), 1.41 (d, J = 11.1 Hz, 9H), 1.33 – 1.26 (m, 3H).
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Step E. To a stirred solution of ethyl 4ꢀ((tertꢀbutoxycarbonyl(methyl)amino)methyl)ꢀ2ꢀ
(hydroxymethyl)benzoate (0.3 g, 0.92 mmol) in dichloromethane (20 mL) was added carbon
tetrabromide (460 mg, 1.39 mmol) and triphenyl phosphine (364 mg, 1.39 mmol) at 0 °C and
stirred for 2 h. The reaction mixture was diluted with water and extracted with dichloromethane.
The combined organic layers were washed with water, brine, dried over sodium sulphate and
concentrated under reduced pressure. The resultant residue was purified by silica gel
chromatography
to
give
ethyl
2ꢀ(bromomethyl)ꢀ4ꢀ((tertꢀ
butoxycarbonyl(methyl)amino)methyl)benzoate (150 mg, 0.40 mmol, 50% yield). 1H NMR (300
MHz, DMSOꢀd6) δ 7.87 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H),
5.02 (s, 2H), 4.42 (s, 2H), 4.32 (t, J = 7.1 Hz, 2H), 2.81 (s, 3H), 1.46 – 1.28 (m, 12H).
Step
F.
To
a
stirred
solution
of
ethyl
2ꢀ(bromomethyl)ꢀ4ꢀ((tertꢀ
butoxycarbonyl(methyl)amino)methyl) benzoate (0.7 g, 1.88 mmol) in DMF (20 mL) was added
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3ꢀaminopiperidineꢀ2,6ꢀdione hydrochloride (0.3 g, 1.88 mmol) and triethylamine (0.7 mL, 4.70
mmol). The reaction was stirred at 80 °C for 12 h. The reaction mixture was diluted with water
and extracted with ethyl acetate. The combined organic layers were washed with water, brine,
dried over sodium sulphate and concentrated under reduced pressure. The resultant residue was
purified by silica gel chromatography to give tertꢀbutyl (2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1ꢀ
oxoisoindolinꢀ5ꢀyl)methyl(methyl)carbamate (0.4 g, 1.23 mmol, 60% yield). MS (ESI) m/z
388.35 [M+1]+.
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Step G. A solution of ethyl tertꢀbutyl (2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1ꢀoxoisoindolinꢀ5ꢀyl)
methyl(methyl) carbamate (0.2 g, 0.61 mmol) in 4 M hydrochloric acid in 1,4ꢀdioxane (5 mL)
was stirred at 0 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give
3ꢀ(5ꢀ((methylamino)methyl)ꢀ1ꢀoxoisoindolinꢀ2ꢀyl)piperidineꢀ2,6ꢀdione hydrochloride (130 mg,
0.58 mmol, 90% yield). 1H NMR (300 MHz, DMSOꢀd6) δ 11.00 (s, 1H), 9.11 (s, 2H), 7.81 (d, J
= 7.9 Hz, 1H), 7.74 (s, 1H), 7.65 (dd, J = 7.7, 1.5 Hz, 1H), 5.14 (dd, J = 13.2, 5.1 Hz, 1H), 4.56 –
4.30 (m, 2H), 4.25 (s, 2H), 2.93 (ddd, J = 17.9, 13.4, 5.3 Hz, 1H), 2.56 (s, 4H), 2.40 (dd, J =
13.4, 9.1 Hz, 1H), 2.08 – 1.91 (m, 1H); MS (ESI) m/z 288.4 [M+1]+.
Step H. To a 0 °C solution of 3ꢀchloroꢀ4ꢀmethylaniline (2.0 g, 14.12 mmol) and phenyl
carbonochloridate (2.1 mL, 42.37 mmol) in dichloromethane (30 mL) was added pyridine (3 mL,
16.24 mmol) and stirred at 0 °C for 2 h. The reaction mixture was diluted with water and
extracted with dichloromethane. The combined organic layers were washed with water, brine,
dried over sodium sulphate and concentrated. The resultant residue was purified by silica gel
chromatography to give phenyl 3ꢀchloroꢀ4ꢀmethylphenylcarbamate. (1.2 g, 4.59 mmol, 37%
yield). MS (ESI) m/z 262.18 [M+1]+.
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Step I. To a stirred solution of 3ꢀ(5ꢀ((methylamino)methyl)ꢀ1ꢀoxoisoindolinꢀ2ꢀyl)piperidineꢀ
2,6ꢀdione hydrochloride (0.1 g, 0.30 mmol) in DMF (5 mL) was added the phenyl 3ꢀchloroꢀ4ꢀ
methylphenylcarbamate (50 mg, 0.36 mmol) and triethylamine (0.1 mL, 0.90 mmol). The
reaction was stirred at 80 °C for 2 h. The reaction mixture was poured in ice cold water and solid
precipitate was collected by vacuum filtration. The collected precipitate was purified by reverse
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HPLC
to
give
3ꢀ(3ꢀchloroꢀ4ꢀmethylphenyl)ꢀ1ꢀ((2ꢀ(2,6ꢀdioxopiperidinꢀ3ꢀyl)ꢀ1ꢀ
oxoisoindolinꢀ5ꢀyl)methyl)ꢀ1ꢀmethylurea (25) (20 mg, 0.04 mmol, 14% yield, HPLC purity
>99%). 1H NMR (400 MHz, DMSOꢀd6) δ 10.98 (s, 1H), 8.53 (s, 1H), 7.89 – 7.63 (m, 2H), 7.48
(s, 1H), 7.39 (dd, J = 14.5, 8.1 Hz, 2H), 7.20 (d, J = 8.4 Hz, 1H), 5.11 (dd, J = 13.7, 5.1 Hz, 1H),
4.66 (s, 2H), 4.46 (d, J = 17.4 Hz, 1H), 4.32 (d, J = 17.2 Hz, 1H), 2.95 (s, 4H), 2.60 (d, J = 18.5
Hz, 1H), 2.44 – 2.30 (m, 1H), 2.25 (s, 3H), 2.00 (d, J = 12.4 Hz, 1H); MS (ESI) m/z 455.16
[M+1]+.
1-(3-Chloro-4-methylphenyl)-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-
yl)methyl)-1,3-dimethylurea (26). To a stirred solution of 3ꢀchloroꢀN,4ꢀdimethylaniline (120
mg, 0.37 mmol) in tetrahydrofuran (10 mL) was added triethylamine (0.2 mL, 1.11 mmol) and
triphosgene (43 mg, 0.14 mmol) at 0 °C and stirred at room temperature for 2 h. To the reaction
mixture was added 3ꢀ(5ꢀ((methylamino)methyl)ꢀ1ꢀoxoisoindolinꢀ2ꢀyl)piperidineꢀ2,6ꢀdione
hydrochloride (43 mg, 0.14 mmol) and stirred at room temperature for 5 h. The reaction mixture
was poured into ice cold water and the desired product precipitated as a solid. The resulting
precipitate was dried and purified by reverse phase HPLC to give the title compound (20 mg,
1
0.04 mmol, 12% yield, HPLC purity >96%) H NMR (300 MHz, DMSOꢀd6) δ 10.98 (s, 1H),
7.69 (d, J = 7.8 Hz, 1H), 7.44 (s, 1H), 7.39 – 7.33 (m, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.12 (d, J =
2.3 Hz, 1H), 6.99 (dd, J = 8.2, 2.3 Hz, 1H), 5.11 (dd, J = 13.2, 5.1 Hz, 1H), 4.69 – 4.18 (m, 4H),
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3.32 (s, 3H), 3.10 (s, 3H), 2.92 (m, 1H), 2.60 (d, J = 18.1 Hz, 1H), 2.40 (dd, J = 13.2, 4.4 Hz,
1H), 2.25 (s, 3H), 2.07 – 1.91 (m, 1H); MS (ESI) m/z 468.97 [M+1]+.
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3-(5-((3-(3-Chloro-4-methylphenyl)-2-oxotetrahydropyrimidin-1(2H)-yl)methyl)-1-
oxoisoindolin-2-yl)piperidine-2,6-dione (27). Step A. To a solution of 3ꢀchloroꢀ4ꢀmethylaniline
(2.0 g, 11.23 mmol) in acetonitrile (20 mL) was added bromopropanol (1.7 g, 12.23 mmol)
followed by sodium bicarbonate (1.8 g, 21.42 mmol). The reaction was heated in a microwave
reactor for 1 h at 90 °C. The reaction mixture was filtered and the filtrate was concentrated under
reduced pressure. The resulting residue was purified by silica gel chromatography to give 3ꢀ(3ꢀ
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chloroꢀ4ꢀmethylphenylamino)propanꢀ1ꢀol (0.9 g, 4.52 mmol, 40% yield). H NMR (300 MHz,
CDCl3) δ 6.99 (d, J = 8.2 Hz, 1H), 6.64 (d, J = 2.4 Hz, 1H), 6.45 (dd, J = 8.2, 2.5 Hz, 1H), 3.81
(t, J = 5.9 Hz, 2H), 3.24 (t, J = 6.5 Hz, 2H), 2.24 (s, 3H), 1.87 (p, J = 6.3 Hz, 2H).
Step B. To a stirred solution of 3ꢀ(3ꢀchloroꢀ4ꢀmethylphenylamino)propanꢀ1ꢀol (0.9 g, 4.52
mmol) in dichloromethane (25 mL) was added tetrabromomethane (2.4 g, 7.23 mmol) followed
by triphenylphosphine (1.9 g, 7.23 mmol) at 0 °C and stirred at room temperature for 12 h. The
reaction mixture was concentrated under reduced pressure and the obtained residue was purified
by silica gel chromatography to give Nꢀ(3ꢀbromopropyl)ꢀ3ꢀchloroꢀ4ꢀmethylaniline (0.9 g, 3.43
mmol, 77% yield). 1H NMR (300 MHz, CDCl3) δ 7.03 – 6.93 (m, 1H), 6.63 (d, J = 2.5 Hz, 1H),
6.44 (dd, J = 8.2, 2.5 Hz, 1H), 3.50 (t, J = 6.4 Hz, 2H), 3.28 (d, J = 6.5 Hz, 2H), 2.24 (s, 3H),
2.17 – 2.07 (m, 2H).
Step C. To a stirred solution of Nꢀ(3ꢀbromopropyl)ꢀ3ꢀchloroꢀ4ꢀmethylaniline (50 mg, 0.13
mmol) in acetonitrile (5 mL) was added 6 (40 mg, 0.16 mmol) followed by sodium carbonate (28
mg, 0.27 mmol) and potassium iodide (44 mg, 0.02 mmol). The reaction was stirred at 80 °C for
12 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic
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layer was dried over sodium sulphate, filtered, and the organic phase was concentrated under
reduced pressure. The resulting residue was purified by silica gel chromatography to give 3ꢀ(5ꢀ
((3ꢀ(3ꢀchloroꢀ4ꢀmethylphenylamino)propylamino)methyl)ꢀ1ꢀoxoisoindolinꢀ2ꢀyl)piperidineꢀ2,6ꢀ
dione (10 mg, 0.02 mmol, 17% yield). MS (ESI) m/z 455.08 [M+1]+.
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Step D. To a stirred solution of 3ꢀ(5ꢀ((3ꢀ(3ꢀchloroꢀ4ꢀmethylphenylamino)propylamino)methylꢀ
1ꢀoxoisoindolinꢀ2ꢀyl)piperidineꢀ2,6ꢀdione (20 mg, 0.04 mmol) in DMF (1 mL) was added
triethylamine (13 mg, 0.13 mmol) and 1,1’ꢀcarbonyldiimidazole (8 mg, 0.05 mmol). The reaction
was stirred at 80 °C for 12 h. The reaction mixture was diluted with ice water, extracted with
ethyl acetate, dried over sodium sulphate, filtered, and concentrated. The obtained residue was
purified by silica gel chromatography to give 3ꢀ(5ꢀ((3ꢀ(3ꢀchloroꢀ4ꢀmethylphenyl)ꢀ2ꢀ
oxotetrahydropyrimidinꢀ1(2H)ꢀyl)methyl)ꢀ1ꢀoxoisoindolinꢀ2ꢀyl)piperidineꢀ2,6ꢀdione (27) (5 mg,
0.01 mmol, 9% yield, HPLC purity >97%). 1H NMR (400 MHz, DMSOꢀd6) δ 10.98 (s, 1H), 7.71
(d, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.46 – 7.36 (m, 2H), 7.29 (d, J = 8.3 Hz, 1H), 7.19 (dd, J = 8.3,
2.3 Hz, 1H), 5.11 (dd, J = 13.2, 5.1 Hz, 1H), 4.62 (s, 2H), 4.50 – 4.26 (m, 2H), 3.67 (t, J = 5.7
Hz, 2H), 2.92 – 2.88 (m, 1H), 2.67 – 2.57 (m, 2H), 2.41 – 2.33 (m, 2H), 2.30 (s, 4H), 2.03 – 1.99
(m, 3H).
3-(5-((3-(3-Chloro-4-methyl phenyl)-2-oxoimidazolidin-1-yl) methyl)-1-oxoisoindolin-2-
yl) piperidine-2,6-dione (28). Step A. To 3ꢀchloroꢀ4ꢀmethylaniline (3.0 g, 21.27 mmol) in
acetonitrile (30 mL), was added bromoethanol (2.9 g, 23.40 mmol) followed by sodium
bicarbonate (3.6 g, 42.54 mmol). The reaction was heated in a microwave reactor for 1 h at
90 °C. The reaction mixture was filtered and the filtrate was concentrated resulting in a residue
which was purified by silica gel chromatography to give 2ꢀ(3ꢀchloroꢀ4ꢀ
1
methylphenylamino)ethanol (1.5 g, 8.10 mmol, 38% yield) H NMR (300 MHz, DMSOꢀd6) δ
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