Rational Design and Synthesis of Novel Dual PROTACs for Simultaneous Degradation of EGFR and PARP

Article abstract of DOI:10.1021/acs.jmedchem.1c00649

Inspired by the success of dual-targeting drugs, especially bispecific antibodies, we propose to combine the concept of proteolysis targeting chimera (PROTAC) and dual targeting to design and synthesize dual PROTAC molecules with the function of degrading two completely different types of targets simultaneously. A library of novel dual-targeting PROTAC molecules has been rationally designed and prepared. A convergent synthetic strategy has been utilized to achieve high synthetic efficiency. These dual PROTAC structures are characterized using trifunctional natural amino acids as star-type core linkers to connect two independent inhibitors and E3 ligands together. In this study, gefitinib, olaparib, and CRBN or VHL E3 ligands were used as substrates to synthesize novel dual PROTACs. They successfully degraded both the epidermal growth factor receptor (EGFR) and poly(ADP-ribose) polymerase (PARP) simultaneously in cancer cells. Being the first successful example of dual PROTACs, this technique will greatly widen the range of application of the PROTAC method and open up a new field for drug discovery.

Full text of DOI:10.1021/acs.jmedchem.1c00649

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Article
Rational Design and Synthesis of Novel Dual PROTACs for
Simultaneous Degradation of EGFR and PARP
Mengzhu Zheng,§ Junfeng Huo,§ Xiaoxia Gu,§ Yali Wang, Canrong Wu, Qingzhe Zhang, Wang Wang,
Yang Liu, Yu Liu, Xuechen Zhou, Lixia Chen,* Yirong Zhou,* and Hua Li*
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ABSTRACT: Inspired by the success of dual-targeting drugs,
especially bispecific antibodies, we propose to combine the concept
of proteolysis targeting chimera (PROTAC) and dual targeting to
design and synthesize dual PROTAC molecules with the function
of degrading two completely different types of targets simulta-
neously. A library of novel dual-targeting PROTAC molecules has
been rationally designed and prepared. A convergent synthetic
strategy has been utilized to achieve high synthetic efficiency.
These dual PROTAC structures are characterized using trifunc-
tional natural amino acids as star-type core linkers to connect two
independent inhibitors and E3 ligands together. In this study,
gefitinib, olaparib, and CRBN or VHL E3 ligands were used as substrates to synthesize novel dual PROTACs. They successfully
degraded both the epidermal growth factor receptor (EGFR) and poly(ADP-ribose) polymerase (PARP) simultaneously in cancer
cells. Being the first successful example of dual PROTACs, this technique will greatly widen the range of application of the PROTAC
method and open up a new field for drug discovery.
complex diseases. They have gradually become an alternative
to combination therapy or the use of mixtures.6,7
INTRODUCTION
■
In cancer, tumor cells often upregulate different growth-
promoting factors, which can act independently or interfere
with each other through a signal network.1 It is easy for tumor
cells to acquire drug resistance by resorting to the
compensatory factors or switching the signal pathway that
promotes proliferation.2 Therefore, the treatment with drugs
targeting only one single target inevitably exhibits limitations.
In addition to drug resistance, the side effects and the tissue
toxicity of single-target drugs often lead to reduced efficacy and
decreased life quality of patients.3 To overcome these
shortcomings of single-target drugs, drug combination
targeting two or more different signal pathways of tumor has
become a well-recognized effective method.4 Due to the
synergistic effects of the drug combination, a smaller dose of
each single drug is needed, thus reducing the side effects.
Another approach to improve therapeutic efficacy is to
design a single hybrid molecule merged by two or more
pharmacophores to target two or more antitumor epitopes or
targets simultaneously.3 Regulating multiple targets or path-
ways at the same time, these hybrid molecules usually exhibit
better efficacy while causing fewer side effects. In the past few
decades, these hybrid molecules, including bispecific antibodies
and other small molecule drugs with double or multiple
targets,3,5 have attracted great interest and achieved consid-
erable success due to their superiority in the treatment of
Proteolysis targeting chimera (PROTAC) is a kind of
bifunctional small molecule, in which the target protein ligand
and the E3 ubiquitin ligase ligand are linked together through a
linker to form a triplet compound.8 Compared with traditional
small molecule inhibitors, PROTAC has several advantages. It
no more needs to bind to the active site of the target protein to
exert effects, while it is capable of the degradation of
“nondruggable” targets. There exists an event-driven mecha-
nism rendering its catalytic properties, and it works at lower
doses while providing great opportunities for the development
of anticancer drugs.9 However, the majority of reported
PROTAC molecules only connect one inhibitor with one E3
ligand, which often degrades only a single-target protein and
not exceeding the limit of two or more similar proteins.
RESULTS AND DISCUSSION
■
Inspired by the great success of dual-targeting drugs, especially
bispecific antibodies, we envisage that by combining the
Received: April 9, 2021
Published: May 26, 2021
https://doi.org/10.1021/acs.jmedchem.1c00649
© 2021 American Chemical Society
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Figure 1. Design of novel star-shaped dual PROTACs and the schematic diagram of dual-targeted protein degradation. Dual PROTACs consist of
two ligands binding to two different target proteins and a ligand binding to the E3 ubiquitin ligase. Three ligands are linked through a linker to form
a dual-targeting PROTAC, then recruit the E3 ligase to induce the ubiquitination of both two targets, and subsequent degradation.
concept of PROTAC and dual targeting, a dual PROTAC
molecule with two independent inhibitor units and one E3
ligase ligand can be designed for degrading two targets
simultaneously in completely different pathways (Figure 1).
Dual PROTAC can not only degrade two targets at the same
time, but also imbibe merits of both PROTACs and double
targeting drugs, achieving similar efficacy as bispecific anti-
bodies and still relatively conserving the advantages of small
molecule drugs. To the best of our knowledge, there has been
no report yet on this audacious and innovative idea. Therefore,
it will be very interesting to design and synthesize such a novel
dual PROTAC molecule for proof of concept.
To verify our hypothesis shown above, the rational design of
the novel star-type linker should function as the most pivotal
element for dual PROTACs. Taking biocompatibility into
consideration, natural amino acids will be a good choice. More
importantly, some amino acids, such as tyrosine and serine,
bearing a third reactive site besides normal amine and acid
functional groups, are ideal star-type linkers to connect three
different small molecules (two inhibitors and one E3 ligase
ligand) together (Scheme 1). In addition, the three potential
reactive sites display different reactivities, which makes them
very suitable for sequential organic synthesis manipulation
according to our rational design.
may produce a synergistic effect.3 To verify the concept of dual
PROTAC shown above, we designed the first round of dual
PROTAC molecules by merging EGFR inhibitors and PARP
inhibitors with the E3 ligase ligand in one novel star-shaped
molecule and evaluated their capability to degrade two
independent targets at the cellular level.
As illustrated in Scheme 1, a convergent synthetic strategy
was rationally designed based on our synthetic experience. The
first etherification of the free hydroxyl group of tyrosine and
serine with propargyl bromide provided the protected amino
acid A. Subsequently, a sequential amide condensation reaction
was utilized as a practical operation to induce Gefitinib and
Olaparib to the star-type linker, generating the key
intermediate B. Eventually, the classical copper-promoted
click reaction of azide and alkene was used to connect E3
ligands, CRBN, and VHL, respectively. Based on this concise
synthetic route, a small library of eight dual PROTACs,
including four CRBD ligand-based PROTACs (DP-C 1-4,
Figure S1), four VHL ligand-based PROTACs (DP-V 1-4,
Figure S2), and the corresponding four mono PROTACs in
which one single inhibitor was linked (MP-GC, MP-GV, MP-
OC, MP-OV, Figure S3) were efficiently constructed. Scheme
2 illustrates the detailed synthetic routes for the four mono
PROTACs and eight dual PROTACs.
Drug resistance in advanced cancers has been reported to be
mediated by different factors, such as the epidermal growth
factor receptor (EGFR) over-expression and DNA repair
enzymes. Poly(ADP-ribose) polymerase (PARP) is one such
protein that is known to be a key player in base excision repair
(BER)10 and cellular signaling pathways.11,12 The inhibition of
EGFR leads to the down-regulation of key players in BER and
sensitizes cell response to alkylating agents and ionizing
radiation.6,13 Receptor tyrosine kinase inhibitors (TKIs), just
like EGFR inhibitors (e.g., gefitinib), are widely used in clinics
and have shown excellent therapeutic efficacy.14,15 However,
the emergence of drug resistance, such as that due to T790M
mutation, greatly reduces their efficacy.16 EGFR mutant cancer
cells have been shown to be sensitive to olaparib both in vivo
and in vitro.17 Therefore, inhibition of both EGFR and PARP
Due to drug resistance shown in pancreatic cancer and
nonsmall cell lung cancer, as well as the high expression of
EGFR and PARP,18−23 SW1990 and H1299 cell lines were
used in the experiments. Intracellularly, the degradation of
EGFR in the SW1990 cell line was induced by the CRBN-
based mono PROTAC of Gefitinib (MP-GC) (Figure 2A),
and the CRBN-based mono PROTAC of Olaparib (MP-OC)
also exhibited a PARP degradation effect in the SW1900 cell
line (Figure 2B). However, neither Gefitinib nor Olaparib itself
could induce the degradation of two targets in SW1990
(Figures S4 and S5).
Further experiments revealed that all four CRBN-based dual
PROTACs (DP-C 1-4) had degradation effects on EGFR and
PARP simultaneously in SW1990 cells. As shown in Figure 2C,
compound DP-C-1 showed the best degradation effect on
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Scheme 1. General Synthetic Route Design and the Structures of Dual PROTACs (DP-C 1-4, DP-V 1-4) and Mono PROTACs
a
(MP-GC, MP-GV, MP-OC, MP-OV)
a
Tyrosine and serine were used as star-type linkers to connect two inhibitors and one E3 ligase ligand together. Eight dual PROTACs and four
mono PROTACs were constructed consequently.
both EGFR and PARP. The remaining compounds showed
lower degradation ability on both targets compared to DP-C-1.
These results also indicate that the degradation of EGFR and
PARP by DP-C 1-4 was enhanced by increasing concen-
trations. The simultaneous degradation of EGFR and PARP by
a single dual PROTAC molecule successfully proves the
concept of dual-targeting degradation.
To further demonstrate the degradation kinetics of DP-C-1,
time-dependent experiments were carried out. The results
showed that DP-C-1 began to degrade EGFR and PARP at 6
h. At first, the degradation rate increased with increasing time,
but it started to decrease at 48 h (Figures 2D and S6).
Therefore, DP-C-1 degrades EGFR and PARP in the SW1990
cell line in a time-dependent manner within 48 h and the
optimal degradation rate was around 24 and 36 h.
ubiquitination pathway, the effect of DP-C-1 was assayed by
introducing the proteasome inhibitor MG132. It can be seen
from Figure 2E that due to the inhibitory effect of MG132
itself on the SW1990 cells, the group with MG132 alone had a
slightly lower expression level of both EGFR and PARP
compared to that of the negative control (NC) group.
However, the addition of DP-C-1 in various concentrations
ceases to further decrease the expression levels of both targets,
and they maintain approximately the same level. This shows
that the degradation of DP-C-1 is inhibited by MG132. These
results suggest that the degradation of the target protein
induced by DP-C-1 functions via the proteasome pathway.
To further verify the concept of dual PROTAC, another E3
ligase ligand (VHL-L) was chosen to develop new dual
PROTACs. First, the corresponding mono PROTACs, MP-
GV and MP-OV, were synthesized based on Gefitinib,
Olaparib, and VHL ligands. In the H1299 cell line, MP-GV
and MP-OV could induce EGFR and PARP degradation,
respectively, while exhibiting no effect on the other target
proteins (Figure 3A,B). According to this result, four dual
PROTACs exert their degradation effect on target proteins
through the ubiquitin−proteasome system. Therefore, MG132,
a proteasome inhibitor, is widely used as a tool to verify the
mechanism of such an action.24−26 To further verify that the
degradation of proteins by DP-C-1 functions via the
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Scheme 2. Synthetic Routes for Mono PROTACs (MP-GC, MP-GV, MP-OC, MP-OV) and Dual PROTACs (DP-C 1-4, DP-V
a
1-4)
a
(a) Compound 1 was treated with 1 equiv of compound 2 and 2 equiv of N,N-diisopropylethylamine (DIPEA) in dry dimethylformamide (DMF)
at 85 °C overnight; (b) 1 equiv of acid and amine was treated with 1.2 equiv of 1-ethyl-3(3-dimethylpropylamine) carbodiimide (EDCI), 1-
hydroxybenzotriazole (HOBt), and DIPEA in dry dichloromethane (DCM) at room temperature overnight; (c) compound 7 was treated with 1.1
equiv of compound 8 and 2 equiv of K2CO3 in acetone by refluxing overnight; (d) ester was treated with 5 equiv of NaOH in 1:1 mixture solvent of
methanol and THF at room temperature overnight; (e) 1.0 equiv of alkyne and azide was treated with 1 equiv of CuSO4 and 2 equiv of sodium
ascorbate in 4:1 mixture solvent of THF and water at room temperature for a few hours; (f) phenol or alcohol was treated with 2 equiv of bromide
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Scheme 2. continued
17 and 2 equiv of K2CO3 or NaH in dry DMF for a few hours; and (g) the Boc-protecting group was removed by treatment with HCl ethyl acetate
solution at room temperature overnight.
Figure 2. DP-C-1 degraded EGFR and PARP simultaneously in a dose- and time-dependent manner in SW1990 cells. (A) Changes in associated
proteins 24 h after the addition of MP-GC. (B) Changes in associated proteins 24 h after the addition of MP-OC. (C) Effect of four dual PROTAC
compounds (DP-C 1-4) on the related proteins after 24 h. (D) Effect of 5 μM DP-C-1 on related proteins at different time points. (E) Effect of
DP-C-1 on related proteins after the introduction of 700 nM MG132. EGFR and PARP degradation by DP-C-1 in 24 h could be abrogated by
pretreating with MG132 for 4 h in 700 nM.
PROTACs based on the VHL ligand were further developed
and designated as DP-V 1-4, respectively. To evaluate their
degradation ability of EGFR and PARP, H1299 cells were
treated with these four compounds under six different
concentrations for 36 h. Among the different compounds,
DP-V-4 exhibited the best degradation ability for both EGFR
and PARP (Figures 3C,D and S7) and was selected for further
investigation. Meanwhile, further experiments revealed that
DP-V-4 could also degrade EGFR and PARP in human
epidermal carcinoma A431 cells simultaneously (Figure S8).
In H1299 cells, DP-V-4 degraded EGFR and PARP
simultaneously in a dose-dependent manner (Figure 3D). At
a concentration of 0.47 μM, DP-V-4 could induce about 50%
of PARP degradation, and as the concentration increased, it
induced PARP degradation to a greater level. For EGFR, DP-
V-4 also induced degradation at higher levels when the
compound concentration gradually increased. Moreover, DP-
V-4 could significantly induce EGFR and PARP degradation at
15 μM. Apart from EGFR and PARP, p-EGFR concentrations
decreased visibly when treated with DP-V-4, which could be
the result of its degradation by DP-V-4, or reduced supply
from EGFR.
To further explore the mode of action, different time points
were set in H1299 cells after being treated with 4 μM of DP-V-
4. At 6 h, DP-V-4 showed the best degradation effect, and
EGFR and PARP recovered as time went on (Figure 3E),
which might be explained by the accumulation of newly
synthesized proteins in cells.
To further verify that the degradation of proteins in cells by
DP-V-4 functions via the ubiquitination pathway, the effect of
DP-V-4 was assayed by introducing the proteasome inhibitor
MG132. In H1299 cells, when pretreated with 1 μM MG132
for 4 h, the degradation of EGFR and PARP by DP-V-4
completely terminated (Figure 3F). These results proved that
DP-V-4 degrades EGFR and PARP through the ubiquitin−
proteasome system.
Besides degradation ability, we attempted to identify the
antiproliferative activity of dual PROTACs in H1299 tumor
cells. The CCK-8 assay determined Gefitinib, Olaparib, and
DP-V-4 with IC50s of 6.56 1.07, 35.93 1.05, and 19.92
1.08 μM, respectively (Figure 3G). The relatively weaker
antiproliferative activity of DP-V-4 might be explained by its
poorer solubility and cell penetrability, owing to its higher
molecular weight.
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Figure 3. DP-V-4 degraded EGFR and PARP simultaneously in a dose- and time-dependent manner in H1299 cells. (A) Changes in associated
proteins 36 h after the addition of MP-GV. (B) Changes in associated proteins 36 h after the addition of MP-OV. (C) Effect of three dual
PROTAC compounds (DP-V 1-3) on related proteins after 36 h. (D) Effect of DP-V-4 on related proteins with different concentrations at 36 h.
(E) Effect of 3.75 μM DP-V-4 on related proteins at different times. (F) Effect of DP-V-4 on related proteins after the introduction of 1 μM
MG132. EGFR and PARP degradation by DP-V-4 in 6 h could be abrogated by pretreating with MG132 for 4 h in 1 μM. (G) The antiproliferative
activity of Gefitinib, Olaparib, and DP-V-4 at 72 h was identified by a cell counting kit-8 (CCK-8) assay. Half-maximal inhibitory concentration
(IC50) was expressed as mean standard deviation (SD).
To further confirm the target engagement of these dual
PROTACs, microscale thermophoresis (MST) was employed
to assay the binding affinity between the two targets and two
representative dual PROTACs, DP-C-1 (CRBN based) and
DP-V-4 (VHL based). As shown in Figure 4, although a little
weaker binding was observed for both DP-C-1 and DP-V-4
compared to Gefitinib with EGFR (Kd 0.47 0.05 μM) and
Olaparib with PARP (Kd 5.31 1.11 μM), they still retained
strong binding with EGFR and PARP, with the disassociation
constants for DP-C-1 at 2.74 0.13 and 7.89 0.96 μM with
EGFR and PARP, respectively, and for DP-V-4 at 5.47 0.67
and 12.80 2.16 μM with EGFR and PARP, respectively.
In this study, we proposed the new concept of dual-targeting
PROTAC. Inspired by bispecific antibodies,5 we creatively
proposed the idea of designing a dual-targeting PROTAC
molecule with two different warheads, in which an E3 ligand
was linked to two inhibitors with different types/pathways of
targets, and was capable of simultaneously degrading two
completely different target proteins in tumor cells. To verify
this concept, we used Gefitinib (an existing EGFR inhibitor)
and Olaparib (a PARP inhibitor)3 as substrates to synthesize
dual-targeting degradation chimeric molecules (dual PRO-
TAC) of EGFR and PARP proteins with different linker
lengths and different E3 ligands (CRBN or VHL), which
successfully degraded EGFR and PARP simultaneously in
cancer cells. This is the first successful example of dual
PROTACs.
study can be easily adapted for the synthesis of more dual-
targeting PROTAC molecules just like building blocks, and it
will greatly expand the application of PROTAC technology
while opening up a new avenue for drug discovery. In the
present clinical practice, the combination therapy of two
different inhibitors, or even the treatment of some bispecific
antibodies, could be substituted by the strategy shown in this
study through designing a corresponding single dual PROTAC
molecule for double targeting degradation, with the same or
even better therapeutic effects.
It is easy to recognize that in the field of combination
therapy of cancer, dual PROTACs can be quickly applied to
replace many current combinations. For example, we could
design and synthesize the dual PROTACs targeting two
kinases, which are synthetically lethal, or targeting a tumor
immune target plus an adjuvant immune target, kinase, or
energy metabolism target, or targeting an epigenetic target plus
an antiapoptotic target, etc. Of course, in addition to the
obvious advantages listed above, the increased molecular
weight of dual PROTACs will give rise to some issues in the
aspect of drug properties and pharmacokinetics. The approach
to solving these problems lies in two directions. One is to
employ a nanodrug delivery system to improve drug
absorption, and the other is to simplify the inhibitor part
while retaining the minimum pharmacophore. We are
currently working on realizing these approaches.
CONCLUSIONS
In addition, we used biocompatible natural amino acids as a
linker to achieve the rapid synthesis of star-type dual-targeting
PROTACs. The technique and method established in this
■
In conclusion, we have designed and synthesized the first dual-
targeting PROTAC molecules with two different warheads,
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Figure 4. Binding affinity of DP-C-1 and DP-V-4 with EGFR and PARP. (A−C) Measurement of affinity of DP-C-1, DP-V-4, and Gefitinib to
EGFR by MST with Kd values of 2.74 0.13, 5.47 0.67, and 0.47 0.05 μM, respectively. (D−F) Measurement of affinity of DP-C-1, DP-V-4,
and Olaparib to PARP by MST with Kd values of 7.89 0.96, 12.80 2.16, and 5.31 1.11 μM, respectively.
compounds for the specific time, the cells were harvested and washed
once with phosphate-buffered saline (PBS) and then lysed in
radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime,
#P0013C) according to the directions. The suspension was
centrifuged at 13 000 rpm for 15 min to remove insoluble material,
and a bicinchoninic acid (BCA) assay was employed to determine the
concentration of soluble proteins (Beyotime, #P0011). The lysate
protein (20−40 μg) was subjected to 10% sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoreti-
cally transferred to poly(vinylidene difluoride) (PVDF) membranes
(Millipore, #IPVH00010). The membranes were blocked with 5%
nonfat milk or 5% bovine serum albumin (BSA) for 1 h and then
incubated with the specific primary antibodies with a 1:1000 dilution,
respectively, at 4 °C overnight. The primary antibodies and secondary
antibodies were purchased from Abclonal and Biosharp, respectively.
Membranes were imaged by the Tanon-5200 Chemiluminescent
Imaging System with ECL luminescence reagents (Meilunbio,
#MA0186).
and realized the simultaneous degradation of two completely
different types of target proteins by one small molecule in
tumor cells. We believe that this technique will greatly expand
the application of PROTAC technology and open up a new
field of drug discovery.
EXPERIMENTAL SECTION
■
Cell Culture. Cells were purchased from the Chinese Typical
Culture Collection Center. The cells were cultured in Dulbecco’s
modified Eagle’s medium containing 25 mM glucose (high DMEM,
GIBCO) supplemented with 10% (v/v) fetal bovine serum in a
saturated humidified atmosphere of 5% CO2 at 37 °C. All cell lines
were authenticated by short tandem repeat (STR) analysis.
Immunoblotting. The cells were seeded in six-well plates at an
individual density for 24 h and then treated with dimethyl sulfoxide
(DMSO) or compounds at the indicated concentrations for various
times. After being treated with the indicated concentrations of
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Cell Proliferation Inhibition Assay. About 4000 cells per well
were seeded in 96-well plates in 100 μL of medium and incubated
overnight. After that, the cells were treated with a series of
concentrations of specific compounds for a further 72 h. Cell
counting kit-8 (CCK-8) (Targetmol, #C0005) was used to identify
the antiproliferative activity of compounds, and plates were read at
450 nm using a Bio-Tek Synergy 2 microplate reader. Data were
analyzed using GraphPad Prism 7.0 Software.
ionization (ESI) source in the positive-ion mode. The purity of
compounds was determined by high-performance liquid chromatog-
raphy (HPLC) analyses, and the purity was >95% for all tested
compounds.
General Procedure for the Preparation of Compound 3. A 25 mL
reaction tube was charged with compound 1 (0.27 g, 1.0 mmol, 1.0
equiv) and 1 equiv of compound 2. Then, 5 mL of dry DMF and 2
equiv of DIPEA were added to the mixture and the resulting mixture
was stirred at 85 °C overnight. The reaction was monitored by TLC.
When the reaction was completed, 50 mL of ethyl acetate was added
and the mixture was washed with brine several times and then dried
over Na2SO4. The solvent was removed and the residue was purified
with column chromatography on silica gel, eluting with petroleum
ether and ethyl acetate (gradient elution from 2:1 to 1:1) to afford the
corresponding product 3 as a yellow solid.
Clone, Expression, and Purification of PARP. Human PARP-1
ART domain Asp788-Trp1014 (NCBI RefSeq: NP_001609.2)
containing a GST tag followed by the HRV-3C cleavage site was
cloned into a PGEX-6p-1 and heterologously expressed in Escherichia
coli strain BL21 (DE3).27 The cells were cultured at 37 °C for 3−4 h
and supplemented with 0.4 mM isopropyl β-D-1-thiogalactopyrano-
side (IPTG) and 0.1 mM ZnSO4 to boost protein expression. The
cells were collected by centrifugation at 3000g for 10 min after
overnight growth at 16 °C. The cell pellet was resuspended in lysis
buffer (50 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid
(HEPES), pH 8.0, 200 mM NaCl, 5 mM 2-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride (PMSF)) and homogenized using a
homogenizer on ice, followed by centrifugation at 40 000g for 1 h.
After centrifugation, the supernatant was collected and incubated with
3−4 mL of GST agarose (ABclonal) in a gravity column (Bio-Rad),
washed with 30 mL of buffer A (50 mM HEPES, pH 8.0, 200 mM
NaCl, 1 mM dithiothreitol (DTT)). The washed agarose was left on-
column in buffer A and digested with HRV-3C protease overnight.
After HRV-3C protease digestion, the flow-through containing an
untagged PARP-1 ART domain was collected, concentrated, and
purified by size-exclusion chromatography on a Superdex 200 10/300
GL column (GE Healthcare) pre-equilibrated with buffer C (20 mM
HEPES, pH 8.0, 150 mM NaCl, 1 mM DTT). The protein peak was
collected for further use.
Microscale Thermophoresis (MST) Assay. The MST assay was
performed according to the supplied labeling protocol. The EFGR
kinase domain (purchased from Sino Biological: Cat 10001-H20B2)
and the PARP-1 ART domain were diluted with assay buffer (20 mM
HEPES, pH 7.5, 150 mM NaCl) to 10 μM and labeled with the
Monolith NTTM Protein Labeling Kit RED (Cat # L001).28 After
protein purification, the labeled EFGR and PARP concentration were
adjusted to 200 nM. The high concentration of compounds was used
as the initial concentration for subsequent gradient dilution. Mixed
the different concentrations of compounds with EFGR or PARP and
incubated for 10 min at room temperature. The samples were loaded
into Monolith standard-treated capillaries, and the thermophoresis
was measured at room temperature after 30 min incubation on a
Monolith NT.115 instrument (Germany). Furthermore, the laser
power was set to 40% using 15 s on-time. The light-emitting diode
(LED) power was set to 100%. The dissociation constant Kd values
were fitted using NT-Analysis software.29
4-((2-(2-Azidoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)-
1
isoindoline-1,3-dione (3a). 0.11 g, 29% yield. H NMR (400 MHz,
CDCl3) δ 8.31 (s, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.11 (d, J = 7.2 Hz,
1H), 6.94 (d, J = 8.4 Hz, 1H), 6.50 (t, J = 5.6 Hz, 1H), 4.92 (dd, J =
12.0, 5.2 Hz, 1H), 3.73 (t, J = 5.2 Hz, 2H), 3.68 (t, J = 5.2 Hz, 2H),
3.50 (q, J = 5.6 Hz, 2H), 3.41 (t, J = 5.2 Hz, 2H), 2.87−2.72 (m, 3H),
2.14−2.10 (m, 1H).
4-((2-(2-(2-Azidoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperi-
din-3-yl)isoindoline-1,3-dione (3b). 0.14 g, 33% yield. 1H NMR (400
MHz, CDCl3) δ 9.06 (brs, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.09 (dd, J =
7.2, 2.0 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.50 (t, J = 5.6 Hz, 1H),
4.96−4.92 (m, 1H), 3.74 (t, J = 5.2 Hz, 2H), 3.70−3.67 (m, 6H),
3.48 (q, J = 5.6 Hz, 2H), 3.38 (t, J = 4.4 Hz, 2H), 2.80−2.73 (m, 3H),
2.13−2.10 (m, 1H).
General Procedure for Amide Condensation. One equiv of acid
and amine was added to 50 mL of dry dichloromethane at 0 °C, then
1.2 equiv of EDCI, HOBt, and DIPEA was added in a sequence.
Then, the reaction mixture was warmed to room temperature and
stirred overnight. The reaction was monitored by TLC. When the
reaction was completed, 50 mL of dichloromethane and 50 mL of
saturated aqueous NaHCO3 were added. The organic phase was
separated and washed with 50 mL of brine and then dried over
Na2SO4. The solvent was removed with a rotary evaporator. The
residue was purified with column chromatography on silica gel,
eluting with dichloromethane and methanol to afford the correspond-
ing amide product.
(2S,4R)-1-((S)-2-(4-Azidobutanamido)-3,3-dimethylbutanoyl)-4-
hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxa-
mide (6). 6 was prepared according to the general procedure for the
amide condensation, starting from compound 4 (0.86 g, 2.0 mmol, 1.0
equiv) and 1 equiv of compound 5. Chromatography purification
used dichloromethane and methanol as elution solvents (gradient
elution from 50:1 to 20:1) to afford the corresponding product as a
white solid, 0.71 g, 65% yield. 1H NMR (400 MHz, CDCl3) δ 8.70 (s,
1H), 7.37−7.32 (m, 5H), 6.36 (d, J = 8.4 Hz, 1H), 4.70 (t, J = 8.0 Hz,
1H), 4.58−4.52 (m, 3H), 4.34 (dd, J = 15.2, 5.6 Hz, 1H), 4.06 (d, J =
11.6 Hz, 1H), 3.63 (dd, J = 11.2, 3.6 Hz, 1H), 3.32 (td, J = 6.4, 1.6
Hz, 2H), 2.50 (s, 3H), 2.48−2.45 (m, 1H), 2.36−2.34 (m, 2H),
2.17−2.11 (m, 1H), 1.87 (dtd, J = 14.2, 6.8, 3.2 Hz, 2H), 0.94 (s,
9H).
Synthesis of Compounds and Their Characterization.
General Information. Compound 7 (CAS: 184475-71-6) and
compound 13 (CAS: 763111-47-3) were both purchased from Bide
Pharmatech Ltd. Unless otherwise noted, reagents and solvents were
obtained from commercial suppliers and were used directly without
further purification. Reactions were monitored by thin-layer
chromatography (TLC) analysis, which was performed on aluminum
plates precoated with silica gel (GF-254), and visualized by UV
fluorescence (λmax = 254 nm) and/or by staining with 1% w/v
KMnO4 in 0.5 M aqueous K2CO3. Products were purified by flash
column chromatography, which was performed using silica gel (230-
General Procedure for the Preparation of Compound 9. A 50 mL
reaction tube was charged with compound 7 (0.64 g, 2.0 mmol, 1.0
equiv), 1.1 equiv of compound 8, and 2 equiv of K2CO3. Then, 10 mL
of acetone was added. The reaction tube was sealed and the resulting
mixture was heated to refluxing and stirred overnight. The reaction
was monitored by TLC. When the reaction was completed, the
mixture was cooled down to room temperature and filtered. The
filtrate was removed using a rotary evaporator. The residue was
purified with column chromatography on silica gel, eluting with
petroleum ether and ethyl acetate (1:1) to afford the corresponding
product 9 as a white solid.
1
400 mesh). H NMR spectra were recorded on a 400 MHz Bruker
1
Avance III spectrometer. Chemical shifts for H NMR spectra are
reported in parts per million (ppm) from tetramethylsilane with the
solvent resonance as the internal standard (CDCl3, 7.26 ppm;
DMSO-d6, 2.50 ppm). Data are reported as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p =
quintet, m = multiplet, br = broad), coupling constants (Hz), and
integration. A high-resolution mass spectrum (HRMS) was measured
on a FLEX-PC (ultraflex TOF/TOF) instrument equipped with an
atmospheric pressure chemical ionization (APCI) or electrospray
Ethyl 2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquina-
zolin-6-yl)oxy)acetate (9a). 0.34 g, 42% yield. 1H NMR (400
MHz, DMSO-d6) δ 9.66 (brs, 1H), 8.48 (d, J = 4.4 Hz, 1H), 8.10 (d, J
= 6.8 Hz, 1H), 7.87 (s, 1H), 7.74 (d, J = 5.0 Hz, 1H), 7.15 (td, J =
7846
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13.2, 2.8 Hz, 1H), 7.22 (d, J = 3.2 Hz, 1H), 4.94 (s, 2H), 4.21 (q, J =
7.2 Hz, 2H), 3.96 (s, 3H), 1.24 (t, J = 7.2 Hz, 3H).
3.80 (brs, 3H), 3.59 (brs, 2H), 3.46 (brs, 1H), 3.30 (brs, 2H), 2.62−
2.55 (m, 4H), 1.99−1.97 (m, 1H).
General Procedure for the Alkylation of the Hydroxyl Group.
Phenol or alcohol was treated with 2 equiv of bromide 17 and 2 equiv
of K2CO3 or NaH in dry DMF at room temperature or at 0 °C for a
few hours. The reaction was monitored by TLC. When the reaction
was completed, 200 mL of ethyl acetate and 200 mL of brine were
added. The organic phase was separated and washed with 50 mL of
brine, and then dried over Na2SO4. The solvent was removed using a
rotary evaporator. The residue was purified with column chromatog-
raphy on silica gel, eluting with petroleum ether and ethyl acetate to
afford the corresponding product.
Methyl 4-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyqui-
1
nazolin-6-yl)oxy)butanoate (9b). 0.43 g, 51% yield. H NMR (400
MHz, CDCl3) δ 8.68 (s, 1H), 8.17 (brs, 1H), 8.05−8.02 (m, 2H),
7.87 (s, 1H), 7.26 (s, 1H), 7.16 (t, J = 8.8 Hz, 1H), 4.31 (t, J = 8.0 Hz,
2H), 4.02 (s, 3H), 3.86 (s, 3H), 2.59 (t, J = 6.0 Hz, 2H), 2.29−2.22
(m, 2H).
Methyl 6-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyqui-
1
nazolin-6-yl)oxy)hexanoate (9c). 0.54 g, 60% yield. H NMR (400
MHz, CDCl3) δ 8.64 (s, 1H), 7.90 (dd, J = 6.4, 2.4 Hz, 1H), 7.83
(brs, 1H), 7.63−7.58 (m, 1H), 7.24 (d, J = 5.2 Hz, 2H), 7.15 (t, J =
8.4 Hz, 1H), 4.11 (t, J = 8.4 Hz, 2H), 3.98 (s, 3H), 3.68 (s, 3H), 2.39
(t, J = 7.2 Hz, 2H), 1.91 (p, J = 7.2 Hz, 2H), 1.74 (p, J = 7.2 Hz, 2H),
1.55 (p, J = 7.2 Hz, 2H).
General Procedure for Saponification. One equiv of ester and 5
equiv of NaOH were added to the 1:1 mixture solvent of methanol
and THF. The resulting mixture was stirred at room temperature
overnight. The reaction was monitored by TLC. When the reaction
was completed, 2 M HCl ethyl acetate solution was added to the
mixture until the pH reached 2. Then, the solvent was removed to
afford the corresponding acid product, which was used directly for the
next step.
2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)acetic Acid (10a). 10a was prepared according to the general
procedure for saponification, starting from compound 9a (0.41 g, 1.0
mmol, 1.0 equiv). 0.34 g, 90% yield, white solid. 1H NMR (400 MHz,
DMSO-d6) δ 13.15 (brs, 1H), 12.06 (s, 1H), 8.85 (s, 1H), 8.78 (s,
1H), 8.12 (dd, J = 6.8, 2.4 Hz, 1H), 7.86 (ddd, J = 9.0, 4.4, 2.6 Hz,
1H), 7.60 (s, 1H), 7.53 (t, J = 13.2 Hz, 1H), 5.09 (s, 2H), 4.00 (s,
3H).
4-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)butanoic Acid (10b). 10b was prepared according to the
general procedure for saponification, starting from compound 9b
(0.42 g, 1.0 mmol, 1.0 equiv). 0.37 g, 92% yield, white solid. 1H NMR
(400 MHz, DMSO-d6) δ 12.03 (s, 2H), 8.84 (s, 1H), 8.69 (s, 1H),
8.10 (dd, J = 6.8, 2.4 Hz, 1H), 7.87−7.84 (m, 1H), 7.56 (s, 1H), 7.53
(t, J = 9.2 Hz, 1H), 4.32 (t, J = 6.4 Hz, 2H), 3.99 (s, 3H), 2.46 (t, J =
7.2 Hz, 2H), 2.03 (p, J = 6.8 Hz, 2H).
Methyl 2-((tert-Butoxycarbonyl)amino)-3-(4-(prop-2-yn-1-
yloxy)phenyl)propanoate (18). 18 was prepared according to the
general procedure for the alkylation of the hydroxyl group, starting
from compound 16 (5.90 g, 20 mmol, 1.0 equiv), 2 equiv of
compound 17, and 2 equiv of K2CO3 at room temperature for 6 h.
Chromatography purification used petroleum ether and ethyl acetate
as elution solvents (8:1) to afford the corresponding product as a
yellow oil, 6.50 g, 98% yield. 1H NMR (400 MHz, CDCl3) δ 7.06 (d, J
= 8.4 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 4.98 (d, J = 8.0 Hz, 1H), 4.67
(d, J = 2.4 Hz, 2H), 4.55 (dd, J = 14.0, 6.0 Hz, 1H), 3.72 (s, 3H), 3.03
(qd, J = 14.0, 6.0 Hz, 2H), 2.53 (t, J = 2.4 Hz, 1H), 1.42 (s, 9H).
2-((tert-Butoxycarbonyl)amino)-3-(4-(prop-2-yn-1-yloxy)-
phenyl)propanoic Acid (19). 19 was prepared according to the
general procedure for saponification, starting from compound 18
(0.66 g, 2.0 mmol, 1.0 equiv). 0.54 g, 85% yield, yellow oil, which was
used directly for the next step.
tert-Butyl(1-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-1-oxo-3-(4-(prop-2-yn-1-yloxy)-
phenyl)propan-2-yl)carbamate (20). 20 was prepared according to
the general procedure for the amide condensation, starting from
compound 13 (0.73 g, 2.0 mmol, 1.0 equiv) and 1 equiv of compound
19. Chromatography purification used dichloromethane and meth-
anol as elution solvents (gradient elution from 80:1 to 30:1) to afford
1
the corresponding product as a white solid, 0.60 g, 45% yield. H
NMR (400 MHz, CDCl3) δ 10.76 (brs, 1H), 8.49−8.46 (m, 1H),
7.80−7.69 (m, 3H), 7.30−7.28 (m, 2H), 7.13 (t, J = 8.8 Hz, 2H),
7.02 (dd, J = 15.2, 8.8 Hz, 1H), 6.99 (dd, J = 15.6, 8.4 Hz, 2H), 5.44
(t, J = 8.0 Hz, 1H), 4.84−4.71 (m, 1H), 4.67 (dd, J = 10.8, 6.0 Hz,
2H), 4.28 (s, 2H), 3.83 (brs, 1H), 3.60−3.35 (m, 3H), 3.27−3.21 (m,
1H), 3.08−3.01 (m, 2H), 2.97 (dd, J = 13.6, 5.6 Hz, 1H), 2.89 (dd, J
= 12.8, 5.6 Hz, 1H), 2.53 (t, J = 2.4 Hz, 1H), 1.42 (s, 9H).
General Procedure for the Deprotection of the Boc Group. Boc-
protected amine was treated with HCl ethyl acetate solution at room
temperature overnight to remove the Boc group. The reaction was
monitored by TLC. When the reaction was completed, the solvent
was removed to afford the corresponding product as a white solid,
which was used directly for the next step.
6-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)hexanoic Acid (10c). 10c was prepared according to the
general procedure for saponification, starting from compound 9c
(0.45 g, 1.0 mmol, 1.0 equiv). 0.42 g, 96% yield, white solid. 1H NMR
(400 MHz, DMSO-d6) δ 12.13 (s, 2H), 8.84 (s, 1H), 8.73 (s, 1H),
8.12 (dd, J = 6.8, 2.4 Hz, 1H), 7.89−7.86 (m, 1H), 7.56 (s, 1H), 7.52
(t, J = 8.8 Hz, 1H), 4.29 (t, J = 6.4 Hz, 2H), 3.98 (s, 3H), 2.26 (t, J =
7.2 Hz, 2H), 1.81 (p, J = 7.2 Hz, 2H), 1.60 (p, J = 7.2 Hz, 2H), 1.48
(p, J = 7.2 Hz, 2H).
4-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(prop-2-yn-1-yl)butanamide (12). 12 was prepared
according to the general procedure for the amide condensation,
starting from compound 10b (81 mg, 0.2 mmol, 1.0 equiv) and 1
equiv of compound 11. Chromatography purification used dichloro-
methane and methanol as elution solvents (gradient elution from 80:1
to 30:1) to afford the corresponding product as a white solid, 55 mg,
63% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.54 (brs, 1H), 8.51 (s,
1H), 8.39 (t, J = 5.2 Hz, 1H), 8.13 (dd, J = 6.8, 2.4 Hz, 1H), 7.82−
7.79 (m, 2H), 7.45 (t, J = 8.8 Hz, 1H), 7.21 (s, 1H), 4.15 (t, J = 6.0
Hz, 2H), 3.95 (s, 3H), 3.88 (dd, J = 5.2, 2.4 Hz, 2H), 3.11 (t, J = 2.4
Hz, 1H), 2.36 (t, J = 7.2 Hz, 2H), 2.07 (p, J = 6.8 Hz, 2H).
4-(4-Fluoro-3-(4-(pent-4-ynoyl)piperazine-1-carbonyl)benzyl)-
phthalazin-1(2H)-one (15). 15 was prepared according to the general
procedure for the amide condensation, starting from compound 13
(0.73 g, 2.0 mmol, 1.0 equiv) and 1 equiv of compound 14.
Chromatography purification used dichloromethane and methanol as
elution solvents (gradient elution from 100:1 to 50:1) to afford the
4-(3-(4-(2-Amino-3-(4-(prop-2-yn-1-yloxy)phenyl)propanoyl)-
piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (21).
21 was prepared according to the general procedure for the
deprotection of the Boc group, starting from compound 20 (0.2
mmol, 1.0 equiv) to afford the corresponding product quantitatively
as a white solid, which was used directly for the next step.
2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(1-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-1-oxo-3-(4-(prop-2-yn-1-yloxy)-
phenyl)propan-2-yl)acetamide (22). 22 was prepared according to
the general procedure for the amide condensation, starting from
compound 10a (75 mg, 0.2 mmol, 1.0 equiv) and 1 equiv of
compound 21. Chromatography purification used dichloromethane
and methanol as elution solvents (gradient elution from 40:1 to 10:1)
to afford the corresponding product as a white solid, 81 mg, 44%
1
yield. H NMR (400 MHz, CDCl3) δ 12.58 (s, 1H), 9.50 (d, J = 2.4
Hz, 1H), 8.53 (s, 1H), 8.25 (d, J = 6.4 Hz, 1H), 8.22 (t, J = 6.8 Hz,
1H), 8.11 (dd, J = 5.2, 2.0 Hz, 1H), 7.96−7.93 (m, 1H), 7.90−7.86
(m, 1H), 7.84−7.77 (m, 3H), 7.47−7.42 (m, 2H), 7.33 (d, J = 4.0 Hz,
1H), 7.22 (t, J = 7.2 Hz, 1H), 7.11 (dd, J = 11.2, 6.4 Hz, 2H), 6.81
(dd, J = 11.2, 6.4 Hz, 2H), 5.01 (ddd, J = 44.8, 13.8, 6.6 Hz, 1H),
1
corresponding product as a white solid, 0.69 g, 78% yield. H NMR
(400 MHz, CDCl3) δ 10.86 (brs, 1H), 8.49−8.47 (m, 1H), 7.79−7.72
(m, 3H), 7.38−7.27 (m, 2H), 7.05 (t, J = 9.2 Hz, 1H), 4.30 (s, 2H),
7847
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4.71−4.69 (m, 4H), 4.32 (s, 2H), 3.94 (s, 3H), 3.56−3.44 (m, 7H),
3.11−3.08 (m, 2H), 2.97−2.84 (m, 3H).
(m, 2H), 2.42 (t, J = 6.8 Hz, 1H), 2.35 (t, J = 6.8 Hz, 1H), 1.88−1.81
(m, 2H), 1.44 (s, 9H).
2-((tert-Butoxycarbonyl)amino)-3-(prop-2-yn-1-yloxy)propanoic
Acid (24). 24 was prepared according to the general procedure for the
alkylation of the hydroxyl group, starting from compound 23 (4.10 g,
20 mmol, 1.0 equiv), 2 equiv of compound 17, and 2 equiv of NaH at
0 °C, then the mixture was warmed to room temperature, and stirred
overnight. Chromatography purification used petroleum ether and
ethyl acetate as elution solvents (1:1) to afford the corresponding
product as a yellow oil, 3.61 g, 74% yield. 1H NMR (400 MHz,
CDCl3) δ 9.51 (brs, 1H), 5.42 (d, J = 8.4 Hz, 1H), 4.50 (dd, J = 5.2,
3.2 Hz, 1H), 4.19−4.18 (m, 2H), 4.00 (dd, J = 9.2, 3.2 Hz, 1H), 3.81
(dd, J = 9.2, 3.2 Hz, 1H), 2.48 (t, J = 2.4 Hz, 1H), 1.46 (s, 9H).
tert-Butyl(1-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-1-oxo-3-(prop-2-yn-1-yloxy)-
propan-2-yl)carbamate (25). 25 was prepared according to the
general procedure for the amide condensation, starting from
compound 13 (0.73 g, 2.0 mmol, 1.0 equiv) and 1 equiv of
compound 24. Chromatography purification used dichloromethane
and methanol as elution solvents (gradient elution from 100:1 to
50:1) to afford the corresponding product as a white solid, 0.59 g,
tert-Butyl(6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-6-oxohexyl)carbamate (29c). 29c
was prepared according to the general procedure for the amide
condensation, starting from compound 13 (0.73 g, 2.0 mmol, 1.0
equiv) and 1 equiv of compound 28c. Chromatography purification
used dichloromethane and methanol as elution solvents (gradient
elution from 40:1 to 20:1) to afford the corresponding product as a
1
white solid, 0.98 g, 85% yield. H NMR (400 MHz, CDCl3) δ 10.80
(brs, 1H), 8.49−8.47 (m, 1H), 7.78−7.72 (m, 3H), 7.33 (brs, 2H),
7.05 (t, J = 8.8 Hz, 1H), 4.60 (brs, 1H), 4.30 (s, 2H), 3.78 (brs, 3H),
3.57 (brs, 2H), 3.43 (brs, 1H), 3.29 (brs, 2H), 3.11 (brs, 2H), 2.37−
2.30 (m, 2H), 1.66−1.65 (m, 2H), 1.51−1.50 (m, 2H), 1.44 (s, 9H),
1.37−1.36 (m, 2H).
30 was prepared according to the general procedure for the
deprotection of the Boc group, starting from compound 29 (0.2
mmol, 1.0 equiv) to afford the corresponding product quantitatively
as a white solid, which was used directly for the next step.
tert-Butyl(1-((2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-
yl)methyl)benzoyl)piperazin-1-yl)-2-oxoethyl)amino)-1-oxo-3-
(prop-2-yn-1-yloxy)propan-2-yl)carbamate (31a). 31a was prepared
according to the general procedure for amide condensation, starting
from compound 24 (0.24 g, 1.0 mmol, 1.0 equiv) and 1 equiv of
compound 30a. Chromatography purification used dichloromethane
and methanol as elution solvents (gradient elution from 30:1 to 15:1)
to afford the corresponding product as a white solid, 0.27 g, 50%
1
50% yield. H NMR (400 MHz, CDCl3) δ 10.86 (brs, 1H), 8.49−
8.47 (m, 1H), 7.79−7.71 (m, 3H), 7.35−7.32 (m, 2H), 7.05 (t, J =
8.8 Hz, 2H), 5.51 (d, J = 8.0 Hz, 1H), 4.83 (d, J = 28.8 Hz, 1H), 4.30
(s, 2H), 4.15−4.11 (m, 2H), 3.98 (brs, 1H), 3.81−3.36 (m, 9H), 2.44
(d, J = 11.2 Hz, 1H), 1.43 (s, 9H).
4-(3-(4-(2-Amino-3-(prop-2-yn-1-yloxy)propanoyl)piperazine-1-
carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (26). 26 was pre-
pared according to the general procedure for the deprotection of the
Boc group, starting from compound 25 (0.2 mmol, 1.0 equiv) to
afford the corresponding product quantitatively as a white solid, which
was used directly for the next step.
1
yield. H NMR (400 MHz, CDCl3) δ 11.27 (d, J = 12.0 Hz, 1H),
8.49−8.46 (m, 1H), 7.78−7.77 (m, 2H), 7.75−7.73 (m, 1H), 7.44 (d,
J = 12.8 Hz, 1H), 7.36−7.28 (m, 2H), 7.05 (t, J = 9.2 Hz, 1H), 5.50
(d, J = 6.8 Hz, 1H), 4.39 (brs, 1H), 4.31 (s, 2H), 4.17−4.08 (m, 4H),
3.98 (brs, 1H), 3.79 (brs, 2H), 3.72−3.68 (m, 1H), 3.61 (brs, 1H),
3.53 (brs, 1H), 3.41 (brs, 1H), 3.33 (brs, 2H), 2.46 (q, J = 2.4 Hz,
1H), 1.46 (s, 9H).
4-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(1-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-1-oxo-3-(prop-2-yn-1-yloxy)-
propan-2-yl)butanamide (27). 27 was prepared according to the
general procedure for the amide condensation, starting from
compound 10b (81 mg, 0.2 mmol, 1.0 equiv) and 1 equiv of
compound 26. Chromatography purification used dichloromethane
and methanol as elution solvents (gradient elution from 40:1 to 20:1)
to afford the corresponding product as a white solid, 110 mg, 64%
tert-Butyl(1-((4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-
yl)methyl)benzoyl)piperazin-1-yl)-4-oxobutyl)amino)-1-oxo-3-
(prop-2-yn-1-yloxy)propan-2-yl)carbamate (31b). 31b was pre-
pared according to the general procedure for the amide condensation,
starting from compound 24 (0.24 g, 1.0 mmol, 1.0 equiv) and 1 equiv
of compound 30b. Chromatography purification used dichloro-
methane and methanol as elution solvents (gradient elution from
30:1 to 15:1) to afford the corresponding product as a white solid,
1
yield. H NMR (400 MHz, CDCl3) δ 12.60 (s, 1H), 9.57 (brs, 1H),
8.51 (s, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.28−8.25 (m, 2H), 8.13 (dd, J
= 6.8, 2.4 Hz, 1H), 7.98−7.91 (m, 3H), 7.87−7.78 (m, 3H), 7.40−
7.36 (m, 1H), 7.24 (t, J = 9.2 Hz, 1H), 5.00−4.90 (m, 1H), 4.32 (s,
2H), 4.16 (s, 4H), 3.95 (s, 3H), 3.63−3.53 (m, 8H), 3.47 (s, 2H),
3.16−3.13 (m, 2H), 2.39 (q, J = 6.8 Hz, 2H), 2.05 (q, J = 6.0 Hz,
2H).
1
0.35 g, 61% yield. H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H),
8.26 (d, J = 7.6 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.90 (t, J = 7.6 Hz,
2H), 7.84 (t, J = 7.6 Hz, 1H), 7.44 (brs, 1H), 7.37 (t, J = 6.4 Hz, 1H),
7.24 (t, J = 9.2 Hz, 1H), 6.81 (t, J = 6.4 Hz, 1H), 4.33 (s, 2H), 4.12
(d, J = 12.4 Hz, 2H), 4.06−4.02 (m, 1H), 3.64 (brs, 1H), 3.58−3.46
(m, 5H), 3.41−3.38 (m, 1H), 3.19 (brs, 1H), 3.14 (brs, 1H), 3.08
(brs, 2H), 2.33 (t, J = 6.8 Hz, 1H), 2.26 (t, J = 6.8 Hz, 1H), 1.63 (q, J
= 5.6 Hz, 1H), 1.35 (d, J = 24.8 Hz, 9H), 1.24 (s, 2H).
tert-Butyl(2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-2-oxoethyl)carbamate (29a). 29a
was prepared according to the general procedure for the amide
condensation, starting from compound 13 (0.73 g, 2.0 mmol, 1.0
equiv) and 1 equiv of compound 28a. Chromatography purification
used dichloromethane and methanol as elution solvents (gradient
elution from 40:1 to 20:1) to afford the corresponding product as a
tert-Butyl(1-((6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-
yl)methyl)benzoyl)piperazin-1-yl)-6-oxohexyl)amino)-1-oxo-3-
(prop-2-yn-1-yloxy)propan-2-yl)carbamate (31c). 31c was prepared
according to the general procedure for the amide condensation,
starting from compound 24 (0.24 g, 1.0 mmol, 1.0 equiv) and 1 equiv
of compound 30c. Chromatography purification used dichloro-
methane and methanol as elution solvents (gradient elution from
30:1 to 15:1) to afford the corresponding product as a white solid,
1
white solid, 0.86 g, 82% yield. H NMR (400 MHz, CDCl3) δ 10.91
(brs, 1H), 8.49−8.47 (m, 1H), 7.79−7.73 (m, 3H), 7.36−7.33 (m,
2H), 7.05 (t, J = 8.8 Hz, 1H), 5.51 (d, J = 16.0 Hz, 1H), 4.30 (s, 2H),
4.03−3.94 (m, 2H), 3.79 (brs, 3H), 3.60 (brs, 1H), 3.50 (brs, 1H),
3.37−3.33 (m, 3H), 1.45 (s, 9H).
1
0.46 g, 76% yield. H NMR (400 MHz, CDCl3) δ 11.06 (d, J = 46.8
Hz, 1H), 8.49−8.46 (m, 1H), 7.79−7.70 (m, 3H), 7.34−7.32 (m,
2H), 7.05 (t, J = 9.2 Hz, 1H), 6.57 (brs, 1H), 5.51−5.46 (m, 1H),
4.30 (s, 2H), 4.28−4.23 (m, 1H), 4.19−4.11 (m, 2H), 3.93−3.89 (m,
1H), 3.70−3.66 (m, 4H), 3.55 (brs, 2H), 3.43 (brs, 1H), 3.28 (brs,
4H), 2.48 (brs, 1H), 2.37 (t, J = 7.2 Hz, 1H), 2.29 (t, J = 7.2 Hz, 1H),
1.66 (p, J = 7.2 Hz, 2H), 1.55 (p, J = 7.2 Hz, 2H), 1.45 (s, 9H), 1.38
(p, J = 7.2 Hz, 2H).
32 was prepared according to the general procedure for the
deprotection of the Boc group, starting from compound 31 (0.2
mmol, 1.0 equiv) to afford the corresponding product quantitatively
as a white solid, which was used directly for the next step.
tert-Butyl(4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-4-oxobutyl)carbamate (29b). 29b
was prepared according to the general procedure for the amide
condensation, starting from compound 13 (0.73 g, 2.0 mmol, 1.0
equiv) and 1 equiv of compound 28b. Chromatography purification
used dichloromethane and methanol as elution solvents (gradient
elution from 40:1 to 20:1) to afford the corresponding product as a
1
white solid, 0.84 g, 76% yield. H NMR (400 MHz, CDCl3) δ 10.94
(brs, 1H), 8.49−8.46 (m, 1H), 7.78−7.73 (m, 3H), 7.34−7.27 (m,
2H), 7.05 (t, J = 8.8 Hz, 1H), 4.81 (brs, 1H), 4.30 (s, 2H), 4.38 (brs,
3H), 3.57−3.56 (m, 2H), 3.43 (brs, 1H), 3.30 (brs, 2H), 3.20−3.17
7848
https://doi.org/10.1021/acs.jmedchem.1c00649
J. Med. Chem. 2021, 64, 7839−7852

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
4-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(1-((4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-4-oxobutyl)amino)-1-oxo-3-(prop-
2-yn-1-yloxy)propan-2-yl)butanamide (33a). 33a was prepared
according to the general procedure for the amide condensation,
starting from compound 10b (40 mg, 0.1 mmol, 1.0 equiv) and 1
equiv of compound 32b. Chromatography purification used dichloro-
methane and methanol as elution solvents (gradient elution from 30:1
to 10:1) to afford the corresponding product as a white solid, 37 mg,
38% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 9.56 (s,
1H), 8.51 (s, 1H), 8.26 (dd, J = 7.6, 1.2 Hz, 1H), 8.15 (t, J = 7.6 Hz,
1H), 8.13 (dd, J = 7.2, 2.4 Hz, 1H), 8.04−7.99 (m, 1H), 7.95 (t, J =
8.4 Hz, 1H), 7.88 (t, J = 7.6 Hz, 1H), 7.85−7.77 (m, 3H), 7.46−7.42
(m, 2H), 7.36 (brs, 1H), 7.25−7.21 (m, 2H), 4.43 (p, J = 6.8 Hz,
1H), 4.32 (s, 2H), 4.15−4.11 (m, 4H), 3.94 (s, 3H), 3.60−3.57 (m,
4H), 3.50−3.99 (m, 4H), 3.30 (brs, 1H), 3.19 (brs, 1H), 3.13 (brs,
1H), 3.09−3.03 (m, 2H), 2.43−2.41 (m, 2H), 2.31 (t, J = 7.2 Hz,
1H), 2.24 (t, J = 7.2 Hz, 1H), 2.05 (p, J = 7.2 Hz, 2H), 1.61 (brs,
2H).
6-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(1-((2-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-2-oxoethyl)amino)-1-oxo-3-(prop-
2-yn-1-yloxy)propan-2-yl)hexanamide (33b). 33b was prepared
according to the general procedure for the amide condensation,
starting from compound 10c (43 mg, 0.1 mmol, 1.0 equiv) and 1
equiv of compound 32a. Chromatography purification used dichloro-
methane and methanol as elution solvents (gradient elution from 30:1
to 10:1) to afford the corresponding product as a white solid, 43 mg,
45% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 9.77 (s,
1H), 8.54 (s, 1H), 8.26 (d, J = 7.6 Hz, 1H), 8.14−8.10 (m, 1H), 8.02
(t, J = 4.8 Hz, 1H), 7.97−7.95 (m, 1H), 7.90−7.78 (m, 3H), 7.45 (t, J
= 8.8 Hz, 2H), 7.36 (brs, 1H), 7.24 (brs, 1H), 7.21 (s, 1H), 4.56 (brs,
1H), 4.33 (s, 2H), 4.16−4.13 (m, 3H), 3.99 (brs, 1H), 3.95 (s, 3H),
3.63−3.44 (m, 6H), 3.19 (brs, 1H), 3.13 (brs, 1H), 3.05−2.99 (m,
1H), 2.75 (d, J = 4.4 Hz, 1H), 2.22 (t, J = 6.0 Hz, 2H), 1.86−1.82 (m,
2H), 1.61 (t, J = 6.4 Hz, 2H), 1.49 (p, J = 7.6 Hz, 2H).
6-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(1-((6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-6-oxohexyl)amino)-1-oxo-3-(prop-
2-yn-1-yloxy)propan-2-yl)hexanamide (33c). 33c was prepared
according to the general procedure for the amide condensation,
starting from compound 10c (43 mg, 0.1 mmol, 1.0 equiv) and 1
equiv of compound 32c. Chromatography purification used dichloro-
methane and methanol as elution solvents (gradient elution from 30:1
to 10:1) to afford the corresponding product as a white solid, 53 mg,
52% yield. 1H NMR (400 MHz, CDCl3) δ 11.91 (d, J = 53.2 Hz, 1H),
9.15 (s, 1H), 8.58 (s, 1H), 8.40 (d, J = 7.2, 1H), 7.88−7.86 (m, 1H),
7.74−7.67 (m, 4H), 7.64−7.62 (m, 2H), 7.30−7.28 (m, 2H), 7.23−
7.19 (m, 2H), 7.09−6.99 (m, 2H), 4.70−4.64 (m, 1H), 4.25 (s, 2H),
4.15−4.00 (m, 2H), 3.99 (brs, 2H), 3.94 (s, 3H), 3.82−3.65 (m, 6H),
3.51 (brs, 3H), 3.38−3.21 (m, 6H), 2.45 (s, 1H), 2.35−2.23 (m, 4H),
1.81 (t, J = 6.8 Hz, 2H), 1.66 (t, J = 6.8 Hz, 2H), 1.57−1.43 (m, 5H).
General Procedure for Copper-Promoted Click Reaction to
Access the Corresponding Mono and Dual PROTACs. A 5 mL
reaction tube was charged with 1.0 equiv of alkyne and 1 equiv of
azide. Then, 2 mL of THF was added. The reaction mixture was
stirred at room temperature. One equiv of CuSO4 and 2 equiv of
sodium ascorbate were dissolved in 0.5 mL of water and the solution
color became brown. The brown aqueous solution was added
dropwise to the THF solution. Then, the resulting mixture was stirred
at room temperature for 0.5−3 h. The reaction was monitored by
TLC. When the reaction was completed, 10 mL of ethyl acetate and
10 mL of brine were added. The organic phase was separated and
dried over Na2SO4. The solvent was removed using a rotary
evaporator. The residue was purified with column chromatography
on silica gel, eluting with dichloromethane and methanol (gradient
elution from 30:1 to 8:1) to afford the corresponding PROTAC
products as a yellow or white solid. In the case of Mono PROTACs
MP-GC, MP-GV, MP-PC, MP-PV, the click reaction was performed
on a 0.05 mmol scale, while the reaction for dual PROTACs DP-C 1-
4 and DP-V 1-4 was performed on a 0.02 mmol scale.
4-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-((1-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindo-
lin-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-
1
butanamide (MP-GC). Yellow solid, 13 mg, 32% yield. H NMR
(400 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.59 (s, 1H), 8.51 (s, 1H),
8.42 (t, J = 5.6 Hz, 1H), 8.13 (dd, J = 6.8, 2.4 Hz, 1H), 7.89 (s, 1H),
7.82 (s, 1H), 7.80−7.77 (m, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.44 (t, J =
9.2 Hz, 1H), 7.20 (s, 1H), 7.07 (d, J = 8.8 Hz, 1H), 7.02 (d, J = 7.2
Hz, 1H), 6.56 (t, J = 6.0 Hz, 1H), 5.06 (dd, J = 12.8, 5.6 Hz, 1H),
4.49 (t, J = 4.8 Hz, 2H), 4.31 (d, J = 5.6 Hz, 2H), 4.15 (t, J = 6.0 Hz,
2H), 3.94 (s, 3H), 3.80 (t, J = 5.2 Hz, 2H), 3.58 (t, J = 5.2 Hz, 2H),
3.41 (dd, J = 10.8, 5.2 Hz, 2H), 2.94−2.84 (m, 1H), 2.61−2.54 (m,
2H), 2.36 (t, J = 7.2 Hz, 2H), 2.10−2.02 (m, 3H). HRMS (pos. ESI):
m/z [M + H]+ for C39H38ClFN10O8 calcd: 829.2619, found:
829.2631.
2-(2,6-Dioxopiperidin-3-yl)-4-((2-(2-(4-(3-(4-(2-fluoro-5-((4-oxo-
3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-3-oxo-
propyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)isoindoline-1,3-
dione (MP-OC). Yellow solid, 16 mg, 39% yield. 1H NMR (400 MHz,
DMSO-d6) δ 12.61 (s, 1H), 11.11 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H),
7.96 (d, J = 8.0 Hz, 1H), 7.89 (td, J = 8.0, 1.6 Hz, 1H), 7.85−7.80 (m,
2H), 7.56 (dd, J = 12.8, 7.6 Hz, 1H), 7.46−7.43 (m, 1H), 7.37 (d, J =
6.4 Hz, 1H), 7.24 (t, J = 8.8 Hz, 1H), 7.11 (dd, J = 8.4, 5.6 Hz, 1H),
7.03 (dd, J = 6.8, 4.4 Hz, 1H), 6.58 (d, J = 4.4 Hz, 1H), 5.07 (dd, J =
12.8, 5.6 Hz, 1H), 4.49 (t, J = 4.4 Hz, 2H), 4.34 (s, 2H), 3.82 (d, J =
3.2 Hz, 2H), 3.62−3.61 (m, 4H), 3.52 (brs, 2H), 3.45 (brs, 2H), 3.37
(brs, 1H), 3.30 (brs, 1H), 3.16 (brs, 2H), 2.90−2.82 (m, 3H), 2.72−
2.54 (m, 4H), 2.06−2.03 (m, 1H). HRMS (pos. ESI): m/z [M + H]+
for C42H42FN10O8 calcd: 833.3166, found: 833.3161.
(2S,4R)-1-((S)-2-(4-(4-((4-((4-((3-Chloro-4-fluorophenyl)amino)-
7-methoxyquinazolin-6-yl)oxy)butanamido)methyl)-1H-1,2,3-tria-
zol-1-yl)butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-
methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (MP-GV).
White solid, 17 mg, 35% yield. 1H NMR (400 MHz, CDCl3) δ
8.95 (brs, 1H), 8.67 (s, 1H), 8.61 (s, 1H), 8.04 (d, J = 4.4 Hz, 1H),
7.96 (brs, 1H), 7.88 (s, 1H), 7.46 (s, 1H), 7.36−7.29 (m, 5H), 7.20
(s, 1H), 7.14 (t, J = 8.8 Hz, 1H), 7.07 (d, J = 5.6 Hz, 1H), 6.53 (d, J =
8.0 Hz, 1H), 4.74 (t, J = 4.8 Hz, 1H), 4.59−4.47 (m, 4H), 4.44 (d, J =
8.8 Hz, 1H), 4.38−4.34 (m, 2H), 4.25−4.22 (m, 1H), 4.19−4.15 (m,
2H), 4.07 (d, J = 12.0 Hz, 1H), 3.97 (s, 3H), 3.62 (dd, J = 11.2, 3.2
Hz, 1H), 2.50 (s, 3H), 2.45 (t, J = 4.8 Hz, 3H), 2.22−2.01 (m, 7H),
0.93 (s, 9H). HRMS (pos. ESI): m/z [M + H]+ for C48H56ClFN11O7S
calcd: 984.3752, found: 984.3759.
(2S,4R)-1-((S)-2-(4-(4-(3-(4-(2-Fluoro-5-((4-oxo-3,4-dihydroph-
thalazin-1-yl)methyl)benzoyl)piperazin-1-yl)-3-oxopropyl)-1H-
1,2,3-triazol-1-yl)butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (MP-
1
OV). White solid, 14 mg, 36% yield. H NMR (400 MHz, CDCl3) δ
11.33 (brs, 1H), 8.69 (s, 1H), 8.43 (s, 1H), 7.78−7.71 (m, 4H),
7.44−7.29 (m, 6H), 7.24−7.19 (m, 2H), 7.07−7.00 (m, 1H), 4.78−
4.72 (m, 1H), 4.64−4.55 (m, 2H), 4.34−4.22 (m, 5H), 4.15 (t, J =
11.2 Hz, 1H), 3.70−3.67 (m, 2H), 3.48 (brs, 2H), 3.31 (brs, 1H),
3.17 (brs, 1H), 3.02 (brs, 2H), 2.76 (brs, 2H), 2.48 (s, 3H), 2.43−
2.07 (m, 10H), 0.99 (s, 9H). HRMS (pos. ESI): m/z [M + Na]+ for
C51H58FN11NaO7S calcd: 1010.4118, found: 1010.4128.
2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(3-(4-((1-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoi-
soindolin-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-
phenyl)-1-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-1-oxopropan-2-yl)acetamide (DP-
1
C-1). Yellow solid, 11 mg, 42% yield. H NMR (400 MHz, DMSO-
d6) δ 12.58 (s, 1H), 11.08 (s, 1H), 9.49 (s, 1H), 8.53 (s, 1H), 8.25 (d,
J = 5.2 Hz, 1H), 8.21 (t, J = 6.8 Hz, 1H), 8.11−8.10 (m, 2H), 7.94 (t,
J = 7.2 Hz, 1H), 7.87 (t, J = 7.2 Hz, 1H), 7.84−7.82 (m, 1H), 7.80−
7.76 (m, 1H), 7.54 (t, J = 6.0 Hz, 1H), 7.44−7.42 (m, 2H), 7.35 (brs,
1H), 7.22 (t, J = 6.8 Hz, 1H), 7.13−7.07 (m, 3H), 7.01 (d, J = 6.0 Hz,
1H), 6.84 (t, J = 7.6 Hz, 2H), 6.57 (t, J = 4.8 Hz, 1H), 5.04−4.99 (m,
4H), 4.70 (brs, 2H), 4.54 (brs, 2H), 4.32 (brs, 2H), 4.06−4.02 (m,
1H), 3.96 (s, 3H), 3.84 (t, J = 4.0 Hz, 2H), 3.60 (t, J = 4.0 Hz, 2H),
3.51 (brs, 2H), 3.43 (q, J = 4.0 Hz, 4H), 3.10 (brs, 2H), 2.94−2.81
(m, 4H), 1.99−1.97 (m, 2H), 1.76−1.71 (m, 1H). HRMS (pos. ESI):
7849
https://doi.org/10.1021/acs.jmedchem.1c00649
J. Med. Chem. 2021, 64, 7839−7852

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
m/z [M + H]+ for C66H60ClF2N14O12 calcd: 1313.4166,
found:1313.4152.
(2S,4R)-1-((2S)-2-(4-(4-((2-(4-((4-((3-Chloro-4-fluorophenyl)-
amino)-7-methoxyquinazolin-6-yl)oxy)butanamido)-3-(4-(2-fluo-
ro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-
1-yl)-3-oxopropoxy)methyl)-1H-1,2,3-triazol-1-yl)butanamido)-3,3-
dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-
pyrrolidine-2-carboxamide (DP-V-2). White solid, 10 mg, 36% yield.
1H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 9.57 (s, 1H), 8.98
4-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(3-((1-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoin-
dolin-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-1-
(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-
piperazin-1-yl)-1-oxopropan-2-yl)butanamide (DP-C-2). Yellow
1
solid, 12 mg, 48% yield. H NMR (400 MHz, DMSO-d6) δ 12.59
(s, 1H), 8.57 (t, J = 6.0 Hz, 1H), 8.51 (s, 1H), 8.34 (d, J = 8.0 Hz,
1H), 8.25 (d, J = 7.6 Hz, 1H), 8.11 (dd, J = 6.8, 2.4 Hz, 1H), 8.05 (d,
J = 13.6 Hz, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.97−7.93 (m, 1H), 7.88
(td, J = 8.0, 1.2 Hz, 1H), 7.84−7.76 (m, 3H), 7.46−7.35 (m, 7H),
7.21 (s, 2H), 5.13 (d, J = 3.6 Hz, 1H), 4.97−4.92 (m, 1H), 4.55−4.50
(m, 3H), 4.46−4.41 (m, 2H), 4.32 (brs, 5H), 4.22 (dd, J = 16.0, 5.6
Hz, 1H), 4.14 (d, J = 5.6 Hz, 2H), 3.94 (s, 3H), 3.65−3.41 (m, 10H),
3.13 (brs, 2H), 2.44 (s, 3H), 2.38 (d, J = 6.4 Hz, 2H), 2.28−2.15 (m,
2H), 2.03−2.01 (m, 5H), 0.92 (s, 9H). HRMS (pos. ESI): m/z [M +
H]+ for C71H77ClF2N15O11S calcd: 1420.5299, found: 1420.5281.
(2S,4R)-1-((2S)-2-(4-(4-((2-(6-((4-((3-Chloro-4-fluorophenyl)-
amino)-7-methoxyquinazolin-6-yl)oxy)hexanamido)-3-((2-(4-(2-
fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-
piperazin-1-yl)-2-oxoethyl)amino)-3-oxopropoxy)methyl)-1H-
1,2,3-triazol-1-yl)butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (DP-
V-3). White solid, 12 mg, 41% yield. 1H NMR (400 MHz, DMSO-d6)
δ 12.60 (s, 1H), 10.05 (brs, 1H), 8.98 (s, 1H), 8.59 (brs, 2H), 8.25
(d, J = 7.6 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 8.11−8.08 (m, 2H),
8.03−7.99 (m, 2H), 7.97−7.93 (m, 2H), 7.89 (brs, 1H), 7.83 (d, J =
7.6 Hz, 1H), 7.79−7.76 (m, 1H), 7.45−7.36 (m, 6H), 7.22 (s, 2H),
5.15 (brs, 1H), 4.55−4.52 (m, 4H), 4.45−4.50 (m, 2H), 4.34−4.30
(m, 5H), 4.21 (dd, J = 16.0, 5.6 Hz, 1H), 4.14 (t, J = 6.4 Hz, 2H),
3.99 (brs, 2H), 3.95 (s, 3H), 3.64−3.57 (m, 6H), 3.51−3.46 (m, 2H),
3.19 (brs, 1H), 3.13 (brs, 1H), 2.43 (s, 3H), 2.28−2.15 (m, 5H),
2.06−2.01 (m, 3H), 1.83−1.81 (m, 3H), 1.60 (brs, 2H), 1.46 (brs,
2H), 0.92 (s, 9H). HRMS (pos. ESI): m/z [M + H]+ for
C75H84ClF2N16O12S calcd: 1505.5826, found: 1505.5829.
(s, 1H), 11.09 (s, 1H), 9.52 (s, 1H), 8.50 (s, 1H), 8.32 (d, J = 8.0 Hz,
1H), 8.26−8.24 (m, 1H), 8.11 (dd, J = 6.8, 2.4 Hz, 1H), 8.01−7.99
(m, 1H), 7.94−7.92 (m, 1H), 7.88 (td, J = 7.2, 1.2 Hz, 1H), 7.82−
7.76 (m, 2H), 7.53 (t, J = 8.0 Hz, 1H), 7.45−7.41 (m, 2H), 7.35 (brs,
1H), 7.20 (brs, 2H), 7.08−7.02 (m, 2H), 7.01 (d, J = 7.2 Hz, 1H),
6.56 (brs, 1H), 5.05 (dd, J = 12.8, 5.2 Hz, 1H), 4.97−4.92 (m, 1H),
4.50 (brs, 4H), 4.32 (brs, 2H), 4.14−4.13 (m, 2H), 3.94 (s, 3H), 3.83
(brs, 2H), 3.58−3.42 (m, 12H), 3.12 (brs, 2H), 2.93−2.85 (m, 1H),
2.51−2.50 (m, 2H), 2.49−2.36 (m, 2H), 2.04 (brs, 2H), 1.83−1.81
(m, 1H). HRMS (pos. ESI): m/z [M + H]+ for C62H60ClF2N14O12
calcd: 1265.4166, found: 1265.4172.
4-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(3-((1-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoin-
dolin-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-1-
((4-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-
benzoyl)piperazin-1-yl)-4-oxobutyl)amino)-1-oxopropan-2-yl)-
1
butanamide (DP-C-3). Yellow solid, 12 mg, 45% yield. H NMR
(400 MHz, DMSO-d6) δ 12.59 (s, 1H), 11.10 (s, 1H), 9.56 (s, 1H),
8.50 (s, 1H), 8.25 (d, J = 7.6 Hz, 1H), 8.12 (dd, J = 6.8, 2.4 Hz, 2H),
8.00−7.76 (m, 6H), 7.53 (t, J = 8.0 Hz, 1H), 7.43 (t, J = 8.8 Hz, 2H),
7.35 (brs, 1H), 7.24−7.19 (m, 2H), 7.07 (d, J = 8.8 Hz, 1H), 7.01 (d,
J = 7.2 Hz, 1H), 6.56 (t, J = 5.6 Hz, 1H), 5.05 (dd, J = 12.8, 5.2 Hz,
1H), 4.50−4.42 (m, 4H), 4.31 (d, J = 7.6 Hz, 2H), 4.12 (t, J = 5.2 Hz,
2H), 3.94 (s, 3H), 3.83 (brs, 2H), 3.58−3.57 (m, 5H), 3.49−3.42 (m,
5H), 3.29 (brs, 1H), 3.17 (brs, 1H), 3.12 (brs, 1H), 3.05−3.04 (m,
2H), 2.93−2.85 (m, 1H), 2.51−2.50 (m, 2H), 2.42 (t, J = 8.0 Hz,
2H), 2.28 (t, J = 7.2 Hz, 1H), 2.21 (t, J = 7.2 Hz, 1H), 2.03 (brs, 3H),
1.59 (brs, 2H), 1.23 (brs, 2H). HRMS (pos. ESI): m/z [M + H]+ for
C66H67ClF2N15O13 calcd: 1350.4694, found: 1350.4681.
(2S,4R)-1-((2S)-2-(4-(4-((2-(6-((4-((3-Chloro-4-fluorophenyl)-
amino)-7-methoxyquinazolin-6-yl)oxy)hexanamido)-3-((6-(4-(2-
fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)-
piperazin-1-yl)-6-oxohexyl)amino)-3-oxopropoxy)methyl)-1H-
1,2,3-triazol-1-yl)butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (DP-
V-4). White solid, 11 mg, 35% yield. 1H NMR (400 MHz, DMSO-d6)
δ 12.60 (s, 1H), 10.29 (brs, 2H), 8.98 (s, 1H), 8.65 (s, 1H), 8.59 (t, J
= 6.0 Hz, 1H), 8.25 (d, J = 7.6 Hz, 1H), 8.08 (dd, J = 6.8, 2.0 Hz,
1H), 8.05 (brs, 1H), 8.02−7.94 (m, 4H), 7.88 (t, J = 7.6 Hz, 1H),
7.82 (t, J = 7.6 Hz, 1H), 7.78−7.76 (m, 1H), 7.49 (t, J = 8,8 Hz, 1H),
7.42−7.36 (m, 5H), 7.21−7.21 (m, 2H), 5.15 (brs, 1H), 4.55−4.40
(m, 5H), 4.32 (brs, 5H), 4.21 (dd, J = 15.6, 5.6 Hz, 1H), 4.14 (t, J =
5.2 Hz, 2H), 3.96 (s, 3H), 3.65−3.62 (m, 3H), 3.55−3.49 (m, 5H),
3.15 (d, J = 16.4 Hz, 3H), 3.02 (brs, 3H), 2.43 (s, 3H), 2.28−2.19 (m,
7H), 2.06−2.00 (m, 3H), 1.89−1.83 (m, 1H), 1.82 (brs, 2H), 1.58
(brs, 2H), 1.44−1.34 (m, 7H), 0.92 (s, 9H). HRMS (pos. ESI): m/z
[M + H]+ for C79H92ClF2N16O12S calcd: 1561.6452, found:
1561.6462.
6-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(3-((1-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoi-
soindolin-4-yl)amino)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-
methoxy)-1-((6-(4-(2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)benzoyl)piperazin-1-yl)-6-oxohexyl)amino)-1-oxopropan-
2-yl)hexanamide (DP-C-4). Yellow solid, 12 mg, 41% yield. 1H NMR
(400 MHz, CDCl3) δ 11.06 (d, J = 16.08 Hz, 1H), 10.01 (s, 1H), 8.99
(s, 1H), 8.59 (s, 1H), 8.41 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 6.4 Hz,
1H), 7.74−7.70 (m, 4H), 7.59−7.57 (m, 2H), 7.46 (t, J = 8.0 Hz,
1H), 7.33−7.28 (m, 3H), 7.08−7.04 (m, 4H), 6.86 (dd, J = 8.8, 4.4
Hz, 1H), 6.50 (d, J = 4.4 Hz, 1H), 4.94−4.89 (m, 1H), 4.54−4.49 (m,
5H), 4.25 (s, 2H), 4.02 (t, J = 7.2 Hz, 2H), 3.98 (s, 3H), 3.84 (brs,
3H), 3.68−3.64 (m, 5H), 3.60 (s, 4H), 3.52 (brs, 2H), 3.36 (brs, 2H),
3.28 (brs, 2H), 3.21 (brs, 3H), 2.80−2.70 (m, 2H), 2.32−2.29 (m,
3H), 2.22−2.21 (m, 1H), 1.84 (brs, 2H), 1.69 (brs, 2H), 1.57−1.43
(m, 7H). HRMS (pos. ESI): m/z [M + H]+ for C72H79ClF2N15O14
calcd: 1450.5582, found: 1450.5536.
(2S,4R)-1-((2S)-2-(4-(4-((4-(2-(2-((4-((3-Chloro-4-fluorophenyl)-
amino)-7-methoxyquinazolin-6-yl)oxy)acetamido)-3-(4-(2-fluoro-
5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoyl)piperazin-1-
yl)-3-oxopropyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-
butanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-
thiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (DP-V-1). White
ASSOCIATED CONTENT
■
1
sı
solid, 14 mg, 48% yield. H NMR (400 MHz, DMSO-d6) δ 12.60
* Supporting Information
(s, 1H), 9.54 (s, 1H), 8.98 (s, 1H), 8.57 (t, J = 6.0 Hz, 1H), 8.54 (s,
1H), 8.25 (d, J = 7.6 Hz, 2H), 8.18 (d, J = 7.6 Hz, 1H), 8.11 (dd, J =
6.8, 2.4 Hz, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.94 (t, J = 7.2 Hz, 1H),
7.89−7.78 (m, 4H), 7.47−7.37 (m, 6H), 7.26 (brs, 1H), 7.23 (t, J =
8.8 Hz, 1H), 7.11 (t, J = 8.8 Hz, 2H), 6.86 (t, J = 8.4 Hz, 2H), 5.14
(d, J = 3.2 Hz, 1H), 5.06−4.98 (m, 3H), 4.71 (brs, 2H), 4.54 (d, J =
9.2 Hz, 1H), 4.46−4.41 (m, 2H), 4.35−4.33 (m, 5H), 4.21 (dd, J =
16.0, 5.2 Hz, 1H), 3.96 (s, 3H), 3.66−3.43 (m, 7H), 3.11 (brs, 2H),
2.93−2.85 (m, 3H), 2.44 (s, 3H), 2.30−2.15 (m, 2H), 2.04−2.02 (m,
3H), 0.94 (s, 9H). HRMS (pos. ESI): m/z [M + H]+ for
C75H77ClF2N15O11S calcd: 1468.5299, found: 1468.5287.
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00649.
1H NMR spectra of compounds in the text, HRMS
spectra of MP-GC, MP-OC, MP-GV, MP-OV, DP-C 1-
4 and DP-V 1-4 and HPLC for purity determination of
DP-C 1-4, DP-V 1-4, MP-GC, MP-OC, MP-GV, and
MP-OV (PDF)
Molecular formula strings (CSV)
7850
https://doi.org/10.1021/acs.jmedchem.1c00649
J. Med. Chem. 2021, 64, 7839−7852

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Tongji-Rongcheng Center for Biomedicine, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan 430030, China
Xuechen Zhou − Hubei Key Laboratory of Natural Medicinal
Chemistry and Resource Evaluation, School of Pharmacy,
Tongji-Rongcheng Center for Biomedicine, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan 430030, China
AUTHOR INFORMATION
■
Corresponding Authors
Lixia Chen − Wuya College of Innovation, Key Laboratory of
Structure-Based Drug Design & Discovery, Ministry of
Education, Shenyang Pharmaceutical University, Shenyang
110016, China; orcid.org/0000-0003-2196-1428;
Email: syzyclx@163.com
Yirong Zhou − Hubei Key Laboratory of Natural Medicinal
Chemistry and Resource Evaluation, School of Pharmacy,
Tongji-Rongcheng Center for Biomedicine, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan 430030, China; orcid.org/0000-0002-3470-
4562; Email: zhouyirong@hust.edu.cn
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00649
Author Contributions
§M.Z, J.H., and X.G. contributed equally to this work.
Hua Li − Hubei Key Laboratory of Natural Medicinal
Chemistry and Resource Evaluation, School of Pharmacy,
Tongji-Rongcheng Center for Biomedicine, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan 430030, China; Wuya College of Innovation, Key
Laboratory of Structure-Based Drug Design & Discovery,
Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, China; orcid.org/0000-0003-1903-
836X; Email: li_hua@hust.edu.cn
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (NSFC) (grant numbers 81773637,
U1803122, and 81903863), the National Mega-project for
Innovative Drugs (grant number 2019ZX09721001-004-007,
China), the Chunhui Program-Cooperative Research Project
of the Ministry of Education, the Natural Science Foundation
of Hubei Province (no. 2020CFB642), the 100 Talents
Program of the Hubei Provincial Government, the Liaoning
Revitalization Talents Program (no. XLYC1807182), and the
Liaoning Province Natural Science Foundation (no. 2020-
MZLH-31). The Center of Analysis and Testing of Huazhong
University of Science and Technology is gratefully acknowl-
edged for the characterization of the new compounds. We
thank Kexing Li from the School of Life Sciences, Tsinghua
University, for English editing help.
Authors
Mengzhu Zheng − Hubei Key Laboratory of Natural
Medicinal Chemistry and Resource Evaluation, School of
Pharmacy, Tongji-Rongcheng Center for Biomedicine, Tongji
Medical College, Huazhong University of Science and
Technology, Wuhan 430030, China
Junfeng Huo − Hubei Key Laboratory of Natural Medicinal
Chemistry and Resource Evaluation, School of Pharmacy,
Tongji-Rongcheng Center for Biomedicine, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan 430030, China
Xiaoxia Gu − Hubei Key Laboratory of Natural Medicinal
Chemistry and Resource Evaluation, School of Pharmacy,
Tongji-Rongcheng Center for Biomedicine, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan 430030, China
Yali Wang − Wuya College of Innovation, Key Laboratory of
Structure-Based Drug Design & Discovery, Ministry of
Education, Shenyang Pharmaceutical University, Shenyang
110016, China
Canrong Wu − Hubei Key Laboratory of Natural Medicinal
Chemistry and Resource Evaluation, School of Pharmacy,
Tongji-Rongcheng Center for Biomedicine, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan 430030, China
Qingzhe Zhang − Hubei Key Laboratory of Natural
Medicinal Chemistry and Resource Evaluation, School of
Pharmacy, Tongji-Rongcheng Center for Biomedicine, Tongji
Medical College, Huazhong University of Science and
Technology, Wuhan 430030, China
Wang Wang − Wuya College of Innovation, Key Laboratory of
Structure-Based Drug Design & Discovery, Ministry of
Education, Shenyang Pharmaceutical University, Shenyang
110016, China
Yang Liu − Wuya College of Innovation, Key Laboratory of
Structure-Based Drug Design & Discovery, Ministry of
Education, Shenyang Pharmaceutical University, Shenyang
110016, China
ABBREVIATIONS
■
EDCI, 1-ethyl-3(3-dimethylpropylamine)carbodiimide; HOBt,
1-hydroxybenzotriazole; DCM, dichloromethane; DIPEA,
N,N-diisopropylethylamine; DMSO, dimethyl sulfoxide
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