Chemoselective Synthesis of Lenalidomide-Based PROTAC Library Using Alkylation Reaction

Article abstract of DOI:10.1021/acs.orglett.9b01326

An organic base-promoted chemoselective alkylation of lenalidomide with different halides was developed, which offers a novel approach to a highly functionalized lenalidomide-based PROTAC library under mild reaction conditions. DIPEA was found to act as an efficient base to trigger facile generation of arylamine alkylation products compared with inorganic bases. This library was successfully applied to BET PROTAC, which not only degraded BET protein but also effectively inhibited cancer cell proliferation.

Full text of DOI:10.1021/acs.orglett.9b01326

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Chemoselective Synthesis of Lenalidomide-Based PROTAC Library
Using Alkylation Reaction
Xing Qiu,†,§ Ning Sun,‡,§ Ying Kong,‡ Yan Li,‡ Xiaobao Yang,*,‡ and Biao Jiang*,†,‡
†CAS Key Laboratory of Synthetic Chemistry of Natural Substances, Shanghai Institute of Organic Chemistry, Chinese Academy of
Sciences, 345 Lingling Road, Shanghai 200032, China
‡Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China
S
* Supporting Information
ABSTRACT: An organic base-promoted chemoselective alkylation of lenalidomide with different halides was developed, which
offers a novel approach to a highly functionalized lenalidomide-based PROTAC library under mild reaction conditions. DIPEA
was found to act as an efficient base to trigger facile generation of arylamine alkylation products compared with inorganic bases.
This library was successfully applied to BET PROTAC, which not only degraded BET protein but also effectively inhibited
cancer cell proliferation.
mmunomodulatory drugs (Figure 1), including lenalidomide
I(1a), pomalidomide (1b), and thalidomide (1c), play an
important role in the treatment, response, and prognosis of
patients with multiple myeloma.1 Cereblon (CRBN), a
component of a cullin-RING ubiquitin ligase complex,2,3 was
identified as the target of 1c and its derivatives, 1b and 1a.
Mechanisms of action were confirmed by crystal structures of the
DDB1−CRBN complex E3 ubiquitin ligase in complex with 1c.2
Recently, PROTAC-directed protein-specific degradation using
bifunctional molecules (Figure 1) for the recruitment of E3
ubiquitin ligase complex was proven to be an efficacious
therapeutic strategy, particularly, for cancer,4 as illustrated by
degradation of AR,5 ERRα,6 BET,7 BCR-ABL,8 BTK,9 BRD9,10
and EGFR.11 CRBN ligands such as 1b have been widely used as
Figure 1. Reported E3 CRBN ligands and sketch of PROTACs
an E3 ubiquitin ligase ligand in PROTACs. It is reported that
1a lenalidomide, as an E3 ligand in BRD9 degrader, displayed a
betteroverallperformancethan1b.10 Furthermore,thelackofthe
one phthalimide keto group makes 1a more metabolically and
chemically stable.12 A powerful lenalidomide-based PROTAC
library is therefore critical in identifying potent PROTAC
degraders. However, no robust method has been reported to
build such a quality library.
alkylation of 1a and 1-bromopropane under inorganic base
(K2CO3) conditions (Scheme 1).13 To apply Li’s alkylation
methodtoefficientlyconstructthelenalidomide-basedlibrary,we
repeated the reaction and identified that 3a′ was the major
productinsteadof3aunderthesameconditions.14a Therefore,we
attempted a new synthetic route using 4b and different amine
linkers as the starting materials for nucleophilic aromatic
substitution reaction (SNAr), which was successfully used to
Recently, Bradner10 reported a reductive amination of 1a and
alkyl aldehyde substituted amines to synthesize a lenalidomide-
based CRBN ligand (2a), but the reactions require complex
operations, lack generality, and suffer from low overall yield
(46%). Li reported the synthetic method of 1a derivative 3a via
Received: April 16, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b01326
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Letter
Scheme 1. Reported Methods for the Synthesis Lenalidomide
Linkers
Table 1. Optimization of Reaction Conditions
a
a
entry
base
solvent time(h) yieldof6a(%) yieldof7a(%)
1
2
3
4
5
6
7
8
DIPEA
TEA
NMM
NMP
NMP
NMP
NMP
NMP
NMP
NMP
NMP
NMP
NMP
NMP
NMP
DMF
MeCN
NMP
12
12
12
12
12
12
2
74
40
32
0
9
1
7
7
2
0
1
DABCO
pyridine
DMAP
DBU
tBuOK
tBuONa
MeONa
LiHMDS
K2CO3
DIPEA
DIPEA
0
0
0
62
89
85
86
74
85
4
efficient construction of a pomalidomide-based library via a
2
microwave-mediated approach (Scheme 2).14b Initially, we
9
2
0
10
11
12
13
14
2
2
2
12
12
12
0
0
0
65
65
85
Scheme 2. Our Design and New Design
5
3
b
15
DIPEA
a
b
HPLC yield. reaction was carried out at 110 °C.
in the presence of 3 equiv of DIPEA in NMP at 100 °C for 12 h
(Table 1, entry 1). Desired 4-position alkylation product 6a was
obtained in 74% isolated yield with 7% glutarimide N-alkylated
7a. Other organic bases, such as Et3N, NMM, DABCO, pyridine,
and DMAP, were not effective in this reaction (entries 2−6);
however, when DBU was used as the base, the major alkylation
product of 7a was gained in 62% yield, possibly due to the strong
basicity of DBU (entry 7). Strong organic base or inorganic bases
such as tBuOK, tBuONa, MeONa, LiHMDS, and K2CO3, which
were proven to be efficient reagents in alkylation, afforded only
traces of the desired product 6a but with 74−89% yield of 7a
(entries 8−12). However, when MeCN or DMF was used as the
solvent,only65%of6awasformed(entries13and14).Increasing
the reaction temperature to 110 °C resulted in a higher product
yield of 85% with a same reaction time (entry 15). It was then
realized that the chemoselective favored 4-position instead of
glutarimide alkylation used different amine linkers and 1a as the
materials under metal-free organic base conditions.
Having optimized the reaction conditions of the alkylation, we
turned our attention to the scope of this method to build the
lenalidomide-based PROTAC library. A broad range of alkyl
smoothly synthesized starting material 4b by condensation
reaction under K2CO3 conditions. However, in the next step,
desiredalkylationproductwasnotobservedundercommonSNAr
conditions, including microwave assistance and regular thermal
conditions, and 4b remained (Scheme 2). Furthermore, as 1a is
commercially available, we redesigned the conditions for
optimizing alkylations to realize the construction of a variety of
4-position chemoselective alkylation instead of glutarimide N-
alkylation products. Based on the above research, we designed a
chemoselective synthesis of a lenalidomide-based PROTAC
library using alkylation under organic base conditions, which led
to a facile synthesis of PROTAC degraders. This is a generally
efficient and useful method.
t
bromides with different lengths bearing −CO2 Bu and −NHBoc
functional groups were well-tolerated under the optimized
reaction conditions (Table 1, entry 15), affording alkylated
productsingoodyields. AfterhydrolysisunderTFAconditions, a
series of PROTAC precursors were efficiently synthesized
(Schemes 3 and 4). 8b was obtained in moderate yield due to
the possibility of a competitive elimination reaction. Further-
more, when the desired product was given directly by freeze-
drying,itformsaTFAsalt;however,whenthedesiredproductwas
purified by preparative HPLC, it exists as a free base. A designed
terminalacidandamineCLB(CRBNligand-based)librarycanbe
used toreact with various smallmolecule drugsfor preparation of
PROTAC degraders to degrade different oncogenic proteins.
As the CLB library containing PEG parts is very useful in
PROTACdegraders,15 lenalidomide-basedPROTACprecursors
linked by (PEG)n were also designed. We first investigated the
To test the feasibility of our hypothesis, tert-butyl 5-
bromopentanoate 5a was chosen as a model substrate for
optimization of the reaction conditions. The results are shown in
Table 1. Due to the efficient performance of DIPEA in various
alkylationreactions,wefirstinvestigatedthereactionof1awith5a
B
DOI: 10.1021/acs.orglett.9b01326
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
following alkylation, which turned out to be a robust protocol for
the synthesis of PEG-containing PROTAC precursors 10a−10e.
Finally, a new lenalidomide-based PROTAC library containing
PEG parts was efficiently constructed (Scheme 6).
Scheme 3. Synthesis of PROTAC Carboxylic Acid Precursors
Scheme 6. Synthesis of PROTAC (PEG)n Carboxylic Acid
Precursors
Scheme 4. Synthesis of PROTAC Amine Precursors
We applied our library to produce PROTAC first to target the
BET (bromodomain and extra-terminal) protein. BET is an
epigenetic “reader” by binding to acetyl lysine residues in the
histone tail and regulate gene expression and has been implicated
in a variety of human diseases to drive cancer formation.16 In this
study, wemainlyuseJQ1-acid(generatedfromJQ1byhydrolysis
oftert-butylester)asthesmallmoleculebinderand9casthelinker
to highly screen PROTAC SIAIS213110 to specifically target the
BET protein (Scheme 7).
Scheme 7. Synthesis of BET PROTAC Degrader
reactivity of 4-methylbenzenesulfonate 12a. Only a trace of
desired product was observed under the standard reaction
condition, and 43% of desired product was obtained when the
reaction was carried out at 80 °C (Scheme 5A). Alkyl iodide 11a
and alkyl bromide 11b underwent smooth alkylation to furnish
the desired product in excellent yields (Scheme 5B). Guided by
these observations, we chose to convert OTs to I to facilitate the
Scheme 5. Optimized Reaction Conditions for Lenalidomide-
Based PROTAC Precursors Linked by (PEG)n
Next, three cell lines (multiple myeloma cell line MM.1S,
leukemia cell line MV-4-11, triple-negative breast cancer cell line
MDA-MB-468) which overexpressed the BET protein were
chosen to test the biological efficacy of the synthesized
SIAIS213110 (Table 2). SIAIS213110 shows better inhibition
oftheproliferationcomparedtoreportedPROTACdBET17a and
JQ1 in three different cancer cell lines. Moreover, SIAIS213110
degradedthetargetBETproteininadose-dependentmanner,10-
fold(DC50 <0.5nMvs5.0nM)betterthanwithdBET1.Asc-Myc
Table 2. Cell Proliferation Assay in Three Cell Lines
a
IC50 (nM) (mean SD)
cell line
JQ1
dBET1
MM.1S
MV-4-11
MDA-MB-468
54.2 17.1
115.3 45.9
6.7 0.5
75.8 12.7
11.5 1.6
0.9 0.01
501.8 68.1
863.7 201.0
125.4 11.6
SIAIS213110
a
IC50 values were obtained from three independent experiments.
C
DOI: 10.1021/acs.orglett.9b01326
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
protein expression is regulated by BET proteins, the potent BET
degraderSIAIS213110downregulatesthelevelsofc-Mycprotein
(Figure2).ThisevidencecollectivelydemonstratesthattheBET-
Foundation (2018M632181 to N.S. and 2018M642110 to L.L.),
and Jing Medicine for financial support.
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ASSOCIATED CONTENT
* Supporting Information
■
S
TheSupportingInformationisavailablefreeofchargeontheACS
Publications website at DOI: 10.1021/acs.orglett.9b01326.
Experimental details, characterization data, and NMR
spectra for all new compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: yangxb@shanghaitech.edu.cn.
*E-mail: jiangbiao@shanghaitech.edu.cn.
ORCID
Xiaobao Yang: 0000-0001-5266-7673
Biao Jiang: 0000-0002-4292-7811
Author Contributions
§X.Q. and N.S. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the National Natural Science Foundation of China
(Grant No. 21502114 to B.J.), China Postdoctoral Science
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DOI: 10.1021/acs.orglett.9b01326
Org. Lett. XXXX, XXX, XXX−XXX