Discovery of CRBN E3 Ligase Modulator CC-92480 for the Treatment of Relapsed and Refractory Multiple Myeloma

Article abstract of DOI:10.1021/acs.jmedchem.9b01928

Many patients with multiple myeloma (MM) initially respond to treatment with modern combination regimens including immunomodulatory agents (lenalidomide and pomalidomide) and proteasome inhibitors. However, some patients lack an initial response to therapy (i.e., are refractory), and although the mean survival of MM patients has more than doubled in recent years, most patients will eventually relapse. To address this need, we explored the potential of novel cereblon E3 ligase modulators (CELMoDs) for the treatment of patients with relapsed or refractory multiple myeloma (RRMM). We found that optimization beyond potency of degradation, including degradation efficiency and kinetics, could provide efficacy in a lenalidomide-resistant setting. Guided by both phenotypic and protein degradation data, we describe a series of CELMoDs for the treatment of RRMM, culminating in the discovery of CC-92480, a novel protein degrader and the first CELMoD to enter clinical development that was specifically designed for efficient and rapid protein degradation kinetics.

Full text of DOI:10.1021/acs.jmedchem.9b01928

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Drug Annotation
Discovery of CRBN E3 Ligase Modulator CC-92480 for the
Treatment of Relapsed and Refractory Multiple Myeloma
Joshua D. Hansen, Matthew Correa, Mark Nagy, Matt Alexander, Veronique Plantevin, Virginia Grant,
Brandon Whitefield, Dehua Huang, Timothy Kercher, Roy Harris, Rama Krishna Narla, Jim Leisten,
Yang Tang, Mehran Moghaddam, Katalin Ebinger, Joseph Piccotti, Courtney G. Havens, Brian E.
Cathers, James Carmichael, Thomas O Daniel, Rupert Vessey, Lawrence G. Hamann, Katerina
Leftheris, Derek Mendy, Frans Baculi, Laurie LeBrun, Gody Khambatta, and Antonia Lopez-Girona
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01928 • Publication Date (Web): 04 Mar 2020
Downloaded from pubs.acs.org on March 5, 2020
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Discovery of CRBN E3 Ligase Modulator CC-
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2480 for the Treatment of Relapsed and Refractory
Multiple Myeloma
Joshua D. Hansen,* Matthew Correa, Mark A. Nagy, Matt Alexander, Veronique
Plantevin, Virginia Grant, Brandon Whitefield, Dehua Huang, Timothy Kercher, Roy
Harris, Rama Krishna Narla, Jim Leisten, Yang Tang, Mehran Moghaddam, Katalin
Ebinger, Joseph Piccotti, Courtney G. Havens, Brian Cathers, James Carmichael,
Thomas Daniel, Rupert Vessey, Lawrence G. Hamann, Katerina Leftheris, Derek
Mendy , Frans Baculi, Laurie A. LeBrun, Gody Khambatta, Antonia Lopez-Girona
Celgene Corporation, 10300 Campus Point Drive, Suite 100, San Diego, California
92121, USA.
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KEYWORDS Ikaros, IKZF1, Aiolos, IKZF3, protein degradation, molecular glue,
immunomodulatory, CELMoD, lenalidomide-resistant, relapsed refractory multiple
myeloma.
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ABSTRACT: Many patients with multiple myeloma (MM) initially respond to treatment with
modern combination regimens including immunomodulatory agents (lenalidomide and
pomalidomide) and proteasome inhibitors. However, some patients lack an initial
response to therapy (i.e., are refractory) and, although the mean survival of MM patients
has more than doubled in recent years, most patients will eventually relapse. To address
this need, we explored the potential of novel Cereblon E3 Ligase Modulators (CELMoDs)
for the treatment of patients with relapsed or refractory multiple myeloma (RRMM). We
found that optimization beyond potency of degradation, including degradation efficiency
and kinetics, could provide efficacy in a lenalidomide-resistant setting. Guided by both
phenotypic and protein degradation data, we describe a series of CELMoDs for the
treatment of RRMM, culminating in the discovery of CC-92480, a novel protein degrader
and the first CELMoD to enter clinical development that was specifically designed for
efficient and rapid protein degradation kinetics.
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INTRODUCTION
Multiple myeloma (MM) is the second most common hematological malignancy in the
US but constitutes less than 1% of all cancer types (~32,000 expected new diagnosed
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cases in 2020). It is considered an orphan disease by the US Food and Drug
Administration. Lenalidomide (1, Figure 1) has demonstrated significant improvement of
overall survival rates (more than double compared to placebo + dexamethasone) and is
approved in combination with dexamethasone for the treatment of MM. The widespread
use of immunomodulatory drugs and proteasome inhibitors has come to represent the
backbone of current standard-of-care therapy for MM patients. Despite these advances
in the overall survival rate, there remains significant unmet medical need in both refractory
and relapsed patient populations. 2
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 by the cullin-
damaged DNA-binding-RING box-domain protein (CUL4-DDB1-RBX1-CRBN), or simply
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(
CRL4CRBN) E3 ubiquitin ligase, with the resulting ubiquitin-tagged proteins directed to
and subsequently degraded by the 26S proteasome (Figure 2).3,4,5 In this manner,
lenalidomide directs cereblon (CRBN) to degrade Ikaros (IKZF1) and Aiolos (IKZF3),
initiating the downstream effects6,7,8 which were shown to be associated with the
antitumor and immunomodulatory properties of lenalidomide.9,10
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NH2
(lenalidomide)
NH2
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O
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3 (CC-885)
Figure 1. Structures of lenalidomide, pomalidomide, and CC-885.
Figure 2 a)
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Figure 2 b)
Figure 2. a) Overview of the ubiquitin proteasome system. 1) The E1 enzyme catalyzes the activation of
ubiquitin, leading to ubiquitin transfer to E2 conjugating enzyme. 2) Target substrate ubiquitination occurs
after E2 transfers ubiquitin via the E3 ligase complex, to which the target is bound. 3) After ubiquitination,
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the substrate is recognized by the proteasome for degradation. b) Magnified view of the E3 Ligase complex
model. Lenalidomide acts as a “molecular glue” to induce the binding of S (substrate; Ikaros/Aiolos) to
CRBN and drive the ubiquitination and ultimate degradation by the proteasome. Figure modified from ACS
Med. Chem. Lett. 2019, 10, 1592-1602.
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Focused on a patient-oriented outcome, our goal was to develop a deeper knowledge
surrounding the degradation of Ikaros and Aiolos, the aspects of which we believed could
provide insight into the development of new protein degraders with enhanced efficacy in
MM and particularly in lenalidomide-refractory settings. The establishment and
characterization of a lenalidomide-resistant MM cell line, H929 R10-1 has been previously
reported, which in brief, results from the continuous treatment of H929 cells with
increasing doses of lenalidomide. In order to widen the possibility of finding novel
chemical matter as a starting point for investigation, we employed a phenotypic screen of
our CRBN modulator library using the lenalidomide-resistant H929 cell line. In conjunction
to the phenotypic approach, we screened for protein degradation, and since degradation
of Aiolos and Ikaros showed high correlation in previous test sets, and parallel results
would be expected, Aiolos was used as the representative target.
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Recently, we described the structure-activity relationships of a series of urea-containing
compounds represented by 3 (CC-885, Figure 1) which were observed to degrade the
key proteins Ikaros and Aiolos, but also effected degradation of GSPT1 (eRF3a) with
variable levels of selectivity.11 Urea 3 was included in the phenotypic screen and
displayed potent activity against H929 R10-1. Compound 3 however, was not suitable
for further development due to concerns about its toxicity profile. Compound 3 was not
tolerated after a single dose in cynomolgus monkeys (data not shown), which we
correlated to the lack of an in vitro selectivity ratio between the viability of normal
peripheral blood mononuclear cells (PBMCs) from healthy donors versus the target
lenalidomide-resistant cells (Figure 3). We removed from consideration compounds like
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that had little to no in vitro selectivity and focused on the leads with high cell viability in
PBMCs (IC > 5 M). As expected, lenalidomide demonstrated no activity against H929
50
R10-1, and pomalidomide showed poor activity clustering, with a group of analogs in a
half-micromolar potency range. On the other extreme, we identified compound 13 which
showed little effect on PBMC viability yet displayed potent single digit-nanomolar
antiproliferative activity against the lenalidomide-resistant cell line (Figure 3).
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Using 13 as a lead structure, we resolved to explore the SAR of this series (Figure 4)
as it related to both lenalidomide-resistance activity as well as preservation of selectivity
against PBMCs. Herein, we describe the design and synthesis of a series of potent aryl
piperazine-containing compounds that exhibit deep levels of protein degradation with
rapid degradation kinetics, leading to preclinical activity for the treatment of RRMM. The
optimization of degradation efficiency in this series which translated to strong induction of
apoptosis, culminated in the identification of 4 (CC-92480), the first CELMoD entering
clinical development that was specifically designed for high efficiency and rapid kinetics
of protein degradation.
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Figure 3. Phenotypic screen of CELMoDs in which in vitro sensitivity toward PBMC viability is plotted against
lenalidomide-resistant MM cell viability (H929 R10-1). Compounds above the diagonal line have stronger
activity in H929-1051 cells than in PBMC. Lenalidomide (Len), pomalidomide (Pom), and CC-885 are
colored in yellow.
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SAR explored
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O
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F
4 (CC-92480)
N
Figure 4. Structures showing the scope of SAR described, leading to 4 (CC-92480).
CHEMISTRY
The chemistry efforts toward CC-92480 (4) involved the preparation of several analogs
accessed through varying synthetic routes, which are outlined in the following schemes
and Supporting Information. Synthesis of key amine 8 (Scheme 1) began with
regioselective methyl esterification of the gamma carboxylic acid group of glutamic acid
(5), followed by boc protection of the amine to give 6. Conversion of the alpha carboxylic
acid to a primary amide followed by acidic boc deprotection gave amine 8.
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The synthesis of the key intermediates 12a-b (Scheme 2) began with Fischer
esterification of 3-hydroxy-2-methylbenzoic acid (9), followed by radical bromination of
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the tolyl-methyl group. Formation of the isoindolinone 11 was accomplished via bromide
displacement using methyl 4,5-diamino-5-oxopentanoate (8) as the nucleophile, followed
by base-mediated cyclization. After silyl deprotection, the requisite benzyl spacer could
be installed either via phenol alkylation with 1,4-bis(bromomethyl)benzene or by
Mitsunobu reaction with (4-(chloromethyl)phenyl)methanol. In the case of benzyl bromide
intermediate 12a, the glutarimide functionality was obtained through treatment of the
primary amide with potassium tert-butoxide at low temperature. Alkylation of the benzyl
bromide with amines 13-33 afforded the final compounds. For instances involving benzyl
chloride intermediate 12b, alkylation of the chloride with the desired amine nucleophile
occurred first and was followed by closure of the glutarimide ring using the low
temperature, potassium tert-butoxide conditions mentioned above.
To evaluate the dependence of CRBN-binding, the N-methyl analog of 13 (39) was
accessed through alkylation of the glutarimide functionality as outlined in Scheme 3.
Benzyl intermediate 36 was synthesized by silyl protection of benzyl alcohol 35, followed
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by reduction of the ester and subsequent conversion of the resulting benzyl alcohol to
benzyl bromide 36. Closure of the glutarimide ring with potassium tert-butoxide was
followed by silyl deprotection and alkylation of the glutarimide nitrogen with methyl iodide
to provide 38. Conversion of the benzyl alcohol of 38 to the corresponding mesylate
allowed for alkylation with 1-(2,4-difluorophenyl)piperazine to give compound 39.
SAR exploration of analogs containing the phthalimide core was supported by the
synthesis described in Scheme 4 which began with the reaction of phthalic anhydride 41
and 3-aminoglutarimide to give compound 42. Alkylation of phenol 42 with previously
described benzyl bromide 36 was followed by acidic silyl deprotection and conversion of
the resulting benzyl alcohol to mesylate 44. This penultimate intermediate provided a
common access point to compounds 45-47.
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For purposes of linker SAR, the nitrogen-linked analog of lead compound 13 was
obtained in convergent fashion by reductive amination of lenalidomide (1) with
functionalized aldehyde 49 which was obtained in one step from commercial 4-
(bromomethyl)benzaldehyde (Scheme 5).
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The synthesis of chiral alpha-methyl glutarimide analog 58 shown in Scheme 6 began
with formation of imine 52 from methyl L-alaninate (51) and benzaldehyde. Ensuing
addition of prop-2-enamide followed by acidic imine hydrolysis provided racemic 3-amino-
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3
4
5
6
7
8
9
0
3-methylpiperidine-2,6-dione (53). Addition of menthol derivative 54 to the amine provided
the necessary chiral auxiliary to enrich for the desired S-enantiomer. Compound 56 was
obtained in high enantiomeric excess after crystallization of 55 followed by carbamate
hydrolysis. Formation of the isoindolinone core was achieved by bromide displacement
of 10 and subsequent trimethylaluminum-mediated condensation to give lactam 57. A
one-pot base-mediated silyl deprotection and alkylation of the liberated phenol with
benzyl bromide 63 provided the target alpha-methyl glutarimide analog 58.
Synthesis of amide-linked piperazine analogs shown in Scheme 7 was achieved
through a slight modification of the route described in Scheme 2. Alkylation of lactam 11
with benzyl bromide ester 64, followed by formation of the glutarimide ring with potassium
tert-butoxide and acidic hydrolysis of the tert-butyl ester provided the perfluorophenyl
acetate 67 which afforded a common intermediate from which compounds 68-71 were
accessed through amide formation with their respective N-aryl piperazines.
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Scheme 8 depicts the asymmetric synthesis of 4 that began with intermediate 10 where
formation of the lactam proceeded with tert-butyl (4S)-4,5-diamino-5-oxo-pentanoate and
ensuing silyl deprotection gave hydroxyl 74. Aryl piperazine intermediate 73 was made
through mono-alkylation of 1,4-bis(chloromethyl)benzene with 3-fluoro-4-(piperazin-1-
yl)benzonitrile (72). The route converged through alkylation of intermediate 74 with benzyl
chloride 73. Formation of the glutarimide ring proceeded via dehydration under acidic
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
conditions at 85 ºC to provide 4 with >98 % enantiomeric excess.
Scheme 1. Synthesis of amine intermediate 8.
COOH
COOH
a, b
c, d
H N
2
COOH
NH2
7
BocHN
e
COOMe
5
6
O
O
NH2
BocHN
COOMe
H N
2
COOMe
8
Reagents and conditions: (a) TMSCl, MeOH, rt, 30 min; (b) Boc
Boc O, pyridine, rt, 30 min, 1,4-dioxane; (d) NH HCO , rt, 16 h, 90% over 2 steps; (e) HCl, rt, 12 h, EtOAc,
5%.
2
O, Et
3
N, rt, 2 h, 56% over 2 steps; (c)
2
4
3
9
Scheme 2. Synthesis of benzyl piperazine and piperidine analogs.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
NH2
d, e
8
HCl
H N
COOMe
2
O
O
COOH
COOMe
Br
a,b,c
NH2
N
1
0
9
11
OH
OTBS
COOMe
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
NH2
NH
N
N
O
f, or g
COOMe
h, i
O
O
1
1
2a X = Br
2b X = Cl
or j, k
X
R
R =
13
14
15
16
17
18
19
N
N
N
N
N
N
N
N
N
N
N
N
N
N
F
F
F
F
Cl
F
F
F
21
N
Cl
25
N
Cl
26
N
20
22
N
23
24
N
N
N
N
O
N
N
F
F
F
F
F
2
7
28
N
29
30
31
32
N
33
N
34
N
N
O
N
N
N
N
N
N
N
N
N
N
N
Cl
F
N
N
N
N
H N
Cl
F
O
2
N
Reagents and conditions: (a) H
5%; (c) AIBN, NBS, 65 ºC, 2 h, EtOAc, 98%; (d) 8, DIEA, 60 ºC, 16 h, ACN, 76%; (e) K
h, DMF, then HCl, 77%; (f) 1,4-bis(bromomethyl)benzene, K CO , 60 ºC, 16 h, ACN, 63%; (g) (4-
chloromethyl)phenyl)methanol, PPh , DIAD, 0 ºC, 1.5 h, THF, 92%; (h) t-BuOK, -78 ºC, 2 h, THF, 93%; (i)
2
SO
4
, MeOH, 65 ºC, 17 h, 88%; (b) TBSCl, imidazole, 25 ºC, 1 h, DCM,
7
2
CO , H O, RT, 15
3
2
2
3
(
3
Substituted phenyl/alkyl piperazines/piperidines, DIEA, 40 ºC, 18 h, ACN, 18%-90%; (j) Substituted
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phenyl/alkyl piperazines/piperidines, DIEA, 40 ºC, 18 h, ACN, 45%-60%; (k) t-BuOK, 0 ºC, 5 min, THF, 36-
50%.
Scheme 3. Synthesis of the N-methyl glutarimide compound 39.
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
35
OH
a, b, c
O
O
NH2
Br
N
OTBS
O
O
36
NH2
O
O
O
e, f, g
N
d
3
7
O
11
O
O
OH
TBSO
O
O
N
O
N
O
N
N
O
O
O
h, i
38
39
F
N
N
HO
F
Reagents and conditions: (a) TBSCl, imidazole, 0-25 ºC, 16 h, DMF; (b) LiAlH
steps; (c) NBS, Me S, -20-25 ºC, 2 h, DCM; (d) 37, K CO , 30 ºC, 4 h, DMF, 44%; (e) t-BuOK, 0 ºC, 1 h,
THF; (f) H SO , 20 ºC, 2 h, THF, 84% over 2 steps; (g) MeI, K CO , rt, 5 h, DMF, then HCl, 72%; (h) MsCl,
4
, 0 ºC, 1 h, THF, 80% over
2
2
2
3
2
4
2
3
DIEA, -5 ºC, 1 h, DMF; (i) 1-(2,4-difluorophenyl)piperazine, DIEA, 30 ºC, 16 h, DMF, 25% over 2 steps.
Scheme 4. Synthesis of analogs containing the phthalimide core.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Br
36
OTBS
O
O
O
NH
a
b
O
N
O
4
1
42
O
O
OH
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
O
O
NH
NH
NH
N
O
N
O
N
O
e
c, d
O
O
O
O
43
O
44
O
4
4
4
5; R1 = 2,4-diF phenyl
6; R1 = 4-F phenyl
7; R1 = 2,4-diCl phenyl
TBSO
MsO
N
N
R
Reagents and conditions: (a) 3-aminopiperidine-2,6-dione, AcONa, 100 ºC, 2 h AcOH, 71%; (b) 36, K
20 ºC, 3 h, DMF, 38% over 2 steps; (c) H SO , 24 ºC, 30 min, THF, 85%; (d) MsCl, DIEA, 20 ºC, 2 h, DMF
e) Substituted phenyl piperazines, DIEA, 24 ºC, 16 h, DMF, 33-36%.
2
CO
3
,
2
4
(
Scheme 5. Synthesis of the N-linked compound 50.
O
N
O
O
NH
O
O
NH
a
b
5
0
F
N
48
N
49
Br
N
F
N
F
F
Reagents and conditions: (a) 1-(2,4-difluorophenyl)piperazine, TEA, RT, 16 h, ACN, 95%; (b) Lenalidomide
1), NaBH , 100 ºC – 25 ºC, 3 h, AcOH, 48% over 2 steps.
(
4
Scheme 6. Synthesis of the chiral-methyl glutarimide compound 58.
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O
Cl
O
d
O
54
O
H N
2
a
O
b, c
O
(
S)
O
O
O
N
N
NH2
O
H
HN
O
O
Ph
55
NH
NH
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
51
52
53
O
N
O
O
Br
O
O
10
H N
O
2
j
e
TBS
f, g
N
O
HBr
58
O
NH
N
O
H
O
57
O
TBS
56
N
N
F
F
F
F
F
F
F
F
h
i
N
O
N
OH
N
Br
N
N
62
N
4
9
63
Reagents and conditions: (a) MgSO
ºC, 30 min, THF, 75% over 2 steps; (c) HCl, 10 ºC, 3 h, THF, 96%; (d) 54, NaHCO
%; (e) HBr, AcOH, 90 ºC, 6 h, 87%; (f) 10, DIEA, rt, 16 h, ACN; (g) AlMe , 110 ºC, 2 h, toluene, 54% over
2 steps; (h) NaBH , 0 ºC, 2 h, MeOH, 87%; (i) SOBr , rt, 2 h, DCM, 91%; (j) 63, K CO , rt, 16 h, DMF, 73%.
4
, TEA, PhCHO, rt, 16 h, DCM; (b) prop-2-enamide, t-BuOK, NH
4
Cl, 0
3
, 0 ºC, 2 h, THF, water,
9
3
4
2
2
3
Scheme 7. Synthesis of amide-linked piperazine analogs.
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
O
O
O
NH2
NH2
Br
64
O
N
N
a
O
11
COOMe
O
65
COOMe
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
F
F
O
O
NH
F
F
F
NH
O
b
c, d
O
N
O
N
O
O
O
O
66
O
67
O
O
NH
O
68; R = 2,4-difluorophenyl
N
O
e
69
; R = 4-fluorophenyl
R = 4-chlorophenyl
N
70
;
N
O
71; R = 2,4-dichlorophenyl
R
Reagents and conditions: (a) 64, K
2
CO , 25 ºC, 4 h, NMP, 57%; (b) t-BuOK, -70 ºC, 90 min, THF, 73%; (c)
3
TFA, 25 ºC, 12 h, DCM, 100%; (d) Perfluorophenyl 2,2,2-trifluoroacetate, DIEA, THF/DMF, 57%; (e)
Substituted phenyl piperazines, DIEA, RT, 10 min, DMF, 50-74%.
Scheme 8. Asymmetric synthesis of 4.
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F
NH
N
N
72
Cl
c
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
N
7
3
N
O
O
N
Me
O
a, b
O
O
d
Br
N
10
O
NH2
TBS
O
OH
7
4
O
O
O
O
N
O
NH
N
O
NH2
O
O
O
e
4
7
5
F
N
N
F
N
N
N
N
Reagents and conditions: (a) tert-butyl (4S)-4,5-diamino-5-oxo-pentanoate, DIEA, 50 ºC, 16 h, ACN; (b)
TBAF, rt, 16 h, MeOH, 60% over 2 steps; (c) 1,4-bis(chloromethyl)benzene, DIEA, 60 ºC, 1 h, ACN/DMF,
6
7%; (d) 73, K
2
CO , 45 ºC, 16 h, DMF, 100%; (e) benzenesulfonic acid, 85 ºC, 18 h, ACN, 72%, 98% e.e..
3
RESULTS AND DISCUSSION
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Since the mode of action of targeted protein degraders (molecular glue and
heterobifunctional molecules/PROTACs) hinges upon removal of a disease-associated
protein through an event-driven process (formation of the ternary complex to facilitate
ubiquitin transfer) whereby the degrader can facilitate multiple rounds of degradation, the
ratio of the number of molecules of target proteins degraded per molecule of degrader
compound should be > 1:1. After the protein is tagged with ubiquitin and degraded, the
degrader molecule becomes available to engage in the formation of a ternary complex
with additional protein, thus degraders have the potential for catalytic-like efficiency. The
degradation efficiency is inherent to and reflected by the level of protein remaining over
a time course of measurement, generally 4 h in the case of work described in this
communication. The level of protein remaining is denoted by percent of control on the Y-
axis of the degradation curve, and the point at which the curve reaches a minimum (Ymin)
depicts the minimum percent protein remaining (Figure 5). Percent protein remaining
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Y ) is reached when degradation and protein synthesis reach an equilibrium, and thus
min
comparisons of Ymin values would reveal which compounds can more effectively induce
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protein degradation (lower Y ). It is worth pointing out that for targets with very fast
min
protein resynthesis, a low Ymin requires fast degradation kinetics.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Comparison of degradation efficiency can be drawn across groups of compounds
assayed under the same conditions and timeframe. As a relevant example, lenalidomide
and pomalidomide display a differential in Aiolos degradation efficiency (Figure 5). After
4
hours lenalidomide demonstrates the least efficient degradation at Ymin = 35 (35 %
protein remaining), and pomalidomide shows improved degradation efficiency with Ymin
=
18. This differentiated pharmacology is clinically correlated with a greater efficacy of
pomalidomide which can achieve responses in lenalidomide-refractory patients.13 The
novel degrader CC-92480 (4), in addition to a dramatic increase in potency shows
significant improvement in degradation efficiency as measured by the depth of protein
degradation (Ymin = 5), a parameter we expect to translate into higher clinical efficacy in
RRMM patients.
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Aiolos degradation curves (4h) of pomalidomide (Pom), lenalidomide (Len), and CC-92840 (4).
Y
min is the lowest point of the dose-response degradation curve and denotes the minimum % protein
remaining.
The identified lead 13, in addition to potent antiproliferative activity in the lenalidomide-
resistant line (H929 R10-1), also displays potent and efficient degradation of Aiolos.
(Figure 6). To assess the minimum pharmacophore, we explored the effect of a
systematic truncation/simplification of the terminal aryl in lead 13. To begin, the 2,4-
difluoro substitution was removed yielding the phenyl analog 21, which retained both
excellent antiproliferative activity as well as Aiolos degradation potency and efficiency.
Replacement of phenyl with i-propyl, as exemplified in analog 22, led to a loss in desired
antiproliferative activity. It is worth noting that the loss in antiproliferative activity (~20x
compared to 13) was not mirrored by the same loss (3x) of Aiolos degradation potency.
However, the degradation efficiency of 22 is lower (13% remaining as compared to 6%
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6
7
8
9
for 13) and may help rationalize the large loss in antiproliferative potency. A further
reduction of size, in which the terminal aryl found in 13 was replaced with methyl (34), led
to a sharp activity drop (300x) in antiproliferative activity which correlates with
concomitant loss in Aiolos degradation potency and efficiency. Finally, removal of the
piperazine group in 40, where the O-benzyl substituent becomes the terminus abolished
antiproliferative activity in the H929 R10-1 cell line.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
F
N
N
N
N
N
O
O
O
N
N
O
N
O
N
O
NH
NH
NH
O
O
O
O
O
O
13
21
22
H929 R10-1 IC50 = 0.0017 M
Aiolos EC50 = 0.00010 M
Aiolos Ymin = 6
H929 R10-1 IC50 = 0.0021 M
Aiolos EC50 = 0.00010 M
Aiolos Ymin = 5
H929 R10-1 IC50 = 0.035 M
Aiolos EC50 = 0.0003 M
Aiolos Ymin = 13
N
N
O
N
O
O
N
O
NH
NH
O
O
O
O
34
40
H929 R10-1 IC50 = 0.51 M
Aiolos EC50 = 0.003 M
Aiolos Ymin = 28
H929 R10-1 IC50 = > 10 M
Aiolos EC50 = 0.0075 M
Aiolos Ymin = 62
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Journal of Medicinal Chemistry
Page 24 of 134
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 6. An SAR exploration in which systematic reduction in size from lead 13, defines the minimum
pharmacophore to include an aryl substituted piperazine.
The requirement of the 2,4-difluoro substitution present in 13 was next examined,
represented by a selected set of data in Table 1. It is readily apparent that changing the
substitution pattern of aryl-fluoro groups (13-17) made little impact on either
antiproliferative activity, or degradation potency/efficiency. Compound 18, the chloro
analog of 13 maintained potency showing only a minimal loss (5x) in antiproliferative
activity. Electron donating groups were also assessed as exemplified by 20 and
maintained comparable activity to 13. Taken together, we found the substitution at the
terminal aryl to have little impact on antiproliferative activity or Aiolos degradation.
Analogs of 13 maintained a similar PBMC viability profile and additionally when measured
for degradation selectivity, piperazine 13 and its closely related analogs were devoid of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
GSPT1 degradation activity.
Table 1. Effect of substitution at the terminal aryl group.a,b,c
R
N
O
O
NH
N
N
O
O
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Page 25 of 134
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
H929 R10-1
IC (nM)
Aiolos
Aiolos
Ymin
GSPT1
Compd
R
EC (nM)
EC (nM)
50
50
50
F
13
1.7
0.1
6
>10,000
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
1
4
5
2.8
3.7
0.1
0.1
5
6
>10,000
>10,000
F
F
1
F
16
5.0
4.0
9.2
0.2
0.3
0.2
6
7
8
>10,000
>10,000
>10,000
F
17
F
Cl
1
8
9
Cl
Cl
1
3.6
7.1
2.1
0.1
0.5
0.1
6
9
5
>10,000
>10,000
>10,000
20
O
21
a
b
All values are the mean of at least three separate assay determinations, SEM for data available in
supplemental material, cYmin is the % protein remaining followed by treatment with compound
concentrations up to 10 M, and measured after 4 h (see methods).
It was anticipated based on previously established SAR, and confirmed by
14
crystallography, that the glutarimide moiety is an important binding partner to CRBN,
and the glutarimide N-H makes a hydrogen-bond interaction with His378 contributing to
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Journal of Medicinal Chemistry
Page 26 of 134
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CRBN binding affinity. To this end, we synthesized the N-methyl glutarimide analog of 13
(39). As expected, methylation of the glutarimide disrupts CRBN binding and translated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
into a >370-fold loss of activity in both degradation and proliferation (Table 2).
Table 2. Effect of N-methyl cap at glutarimide. a,b,c
O
O
R
N
N
O
F
N
N
O
F
Aiolo
s
CRBN
H929 R10-1
IC (nM)
Aiolos
GSPT1
Compd
R
Binding
EC (nM)
EC (nM)
50
50
50
Ymin
IC (nM)
50
13
H
1.7
0.1
6
>10,000
55.1
39
Methyl
632
>10,000
93
>10,000
28,000
a
b
All values are the mean of at least three separate assay determinations, SEM for data available in
supplemental material, cYmin is the % protein remaining followed by treatment with compound
concentrations up to 10 M, and measured after 4 h (see methods).
The structural difference between lenalidomide and pomalidomide (Figure 1) is an
additional carbonyl group in the scaffold of pomalidomide (oxoisoindoline versus
dioxoisoindoline), the presence of which in pomalidomide favorably impacts the efficiency
and potency of Aiolos degradation (Figure 5). However, in the context of lead 13, we
found that the dioxoisoindoline core led to the opposite activity profile (Table 3).
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Page 27 of 134
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Comparison of 13 to 45 reveals that while GSPT1 selectivity was retained, there was a
24-fold loss in Aiolos degradation potency in combination with reduced degradation
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
efficiency by 17%. The loss in degradation efficiency apparently drove the nearly 100-fold
loss in H929 R10-1 antiproliferative potency. The trend was similar for related analogs 46
and 47 in which introduction of the second carbonyl into the scaffold had a detrimental
effect on activity.
Table 3. Effect of dioxoisoindoline scaffold in terms of antiproliferative activity in the lenalidomide-resistant
cell line H929 R10-1, CRBN binding, and the degradation of Aiolos and GSPT1. a,b,c
R
N
O
O
NH
N
N
O
O
O
H929 R10-
1
CRBN
Aiolos
Aiolos
GSPT1
Compd
R
Binding
EC (nM)
Ymin EC (nM)
50
50
IC (nM)
IC (nM)
50
50
F
45
46
159
146
2.4
2.1
23
18
>10,000
>10,000
502
322
F
F
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Journal of Medicinal Chemistry
Page 28 of 134
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cl
47
170
3.3
30
>10,000
877
Cl
a
b
All values are the mean of at least three separate assay determinations, SEM for data available in
c
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
supplemental material, Ymin is the % protein remaining following treatment with compound concentrations
up to 10 M, and measured after 4 h (see methods).
Again, in reference to the structural features of pomalidomide and lenalidomide (Figure
1
), both drugs contain an NH functional group at the 4-position of the isoindoline scaffold.
2
To assess the effect exerted by either nitrogen or oxygen as a tether point to the
isoindoline core, we synthesized 50 (Table 4) as a direct comparator to 13. The difference
between 13 and 50 asserts the view that the action of binding to CRBN does not directly
translate to degradation potency, but rather, is just one of the necessary steps toward
formation of the ternary complex requisite for ubiquitination of substrate. In this case, the
CRBN binding IC of the N-linked compound 50 was only 2-fold lower than that of 13, yet
50
the Aiolos degradation potency was reduced by 15-fold and level of protein remaining
increased from 6% to 15%, ultimately leading to a 15-fold loss in antiproliferative potency.
Table 4. Effect of oxygen versus nitrogen-linked oxoisoindoline scaffolds. a,b,c
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Page 29 of 134
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
O
NH
O
N
F
N
N
X
F
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H929 R10-1
Aiolos
Aiolos
GSPT1
GSPT1 CRBN Binding
Compd
R
IC (nM)
EC (nM)
Ymin
EC (nM)
Ymin
IC (nM)
50
50
50
50
13
O
N
1.7
0.1
1.5
6
>10,000
>10,000
93
502
194
50
26.3
15
90
a
b
All values are the mean of at least three separate assay determinations, SEM for data available in
c
supplemental material, Ymin is the % protein remaining following treatment with compound concentrations
up to 10 M, and measured after 4 h (see methods).
Pomalidomide, lenalidomide, and thalidomide (Figure 1), contain a chiral center at the
tertiary glutarimide carbon and this chiral center is known to be unstable and can undergo
racemization.15,16 In the case of thalidomide, crystallographic and binding data supports
17
that the S-enantiomer has stronger binding to CRBN. To identify if a single enantiomer
could be largely responsible for the activity, both enantiomers of 13 were independently
evaluated (Table 5). In the proliferation assay, we found there was no appreciable
difference in activity when comparison was drawn between the two enantiomers (60 and
6
1), or a single enantiomer and the racemate. Additionally, we found that while either
enantiomer could degrade equivalent total levels of Aiolos (Y ), the S-enantiomer 60 did
min
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