pubs.acs.

org/jmc Article

Ispinesib as an Effective Warhead for the Design of

Autophagosome-Tethering Chimeras: Discovery of Potent
Degraders of Nicotinamide Phosphoribosyltransferase (NAMPT)
Guoqiang Dong,*,∥ Ying Wu,∥ Junfei Cheng,∥ Long Chen, Rui Liu, Yu Ding, Shanchao Wu, Junhui Ma,
and Chunquan Sheng*
Cite This: J. Med. Chem. 2022, 65, 7619−7628 Read Online
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sı Supporting Information

ABSTRACT: Autophagosome-tethering compounds (ATTECs) are an emerging new

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technology in targeted protein degradation. However, effective tools and successful

examples for autophagosome-tethering chimeras are still rather limited. Herein, ATTEC
ispinesib was identified for the first time to be an effective warhead to design
autophagosome-tethering chimeras. As a conceptual validation study, the first generation
of autophagic degraders of nicotinamide phosphoribosyltransferase (NAMPT) were
developed by connecting the NAMPT inhibitor and LC3-binding ispinesib through a
flexible linker. In particular, compound A3 significantly induced the degradation of
NAMPT through the autophagy-lysosomal pathway, leading to excellent cellular antitumor
potency. Ispinesib may have broad applications in the design of potent autophagosome-
tethering chimeras.

■ INTRODUCTION
Over the past 2 decades, targeted protein degradation
and engulfs cellular cargos (e.g., proteins and organelles) to form
the double-membrane autophagosome,14 followed by the fusion
exemplified by proteolysis-targeting chimeras (PROTACs) has with lysosomes to generate the autolysosome, resulting in the
aroused great attention and represents an emerging new degradation of cellular cargoes.15,16
technology in the field of drug discovery.1−3 PROTACs are While UPS-dependent PROTACs are limited to degrade the
heterobifunctional chimeras consisting of a warhead binding to soluble intracellular proteins, the ALP pathway can effectively
the protein of interest (POI), a flexible linker, and an E3 ligase remove a broader range of cellular biomolecules (e.g., protein
aggregates and other nonprotein molecules). Therefore,
ligand.4,5 Based on the ubiquitin proteasome system (UPS),
exploiting the power of ALP to degrade target proteins may
PROTACs induce the POI−PROTAC−E3 ternary complex
greatly enrich the targeted protein degradation techniques and
formation by hijacking E3 ligase to recruit POI, leading to its
open a new avenue for drug discovery. Recently, autophagy-
ubiquitination and subsequent proteasomal degradation.4
targeting chimeras (AUTACs)17 and autophagosome-tethering
Recently, a large number of PROTAC molecules have been
compounds (ATTECs)18 have been developed based on
successfully designed to degrade a variety of drug targets.6−8
macroautophagy by hijacking autophagosomes to degrade
More than 10 PROTACs (e.g., ARV-110 and ARV-471) have
bulky cytosolic cargos. Among them, ATTECs act by binding
progressed into clinical trials.9 However, the PROTAC
directly and simultaneously to the mutant HTT protein
technology has several inherent obstacles and limitations,10
(mHTT) and LC3.18 Because the LC3 is attached to the
such as E3 ligase-related drug resistance11 and limited scope of
phagophore membrane and plays a central role in recruiting the
protein targets (only soluble proteins in the cytoplasm).10
specific cargos into the autophagosome (Figure 1A),19 ATTECs
The autophagy-lysosomal pathway (ALP) is another major
have the capacity to drive mHTT directly to the autophagosome
cellular degradation system in eukaryotic cells, which is primarily
for subsequent autophagic degradation. As a proof-of-concept
responsible for the clearance of dispensable proteins, protein
aggregates, droplets, and even defective or surplus organ-
elles.12,13 ALP can be divided into three major subtypes: Received: November 19, 2021
macroautophagy, microautophagy, and chaperone-mediated Published: May 19, 2022
autophagy (CMA).13 Macroautophagy is the most widely
studied form, which is initiated by the formation of a cup-
shaped double-membraned structure known as phagophore
(Figure 1A). During the process, phagophore further expands

© 2022 American Chemical Society https://doi.org/10.1021/acs.jmedchem.1c02001

7619 J. Med. Chem. 2022, 65, 7619−7628
Journal of Medicinal Chemistry
Figure 1. Design of NAMPT degraders via autophagy. (A) Overview of the macroautophagy process. (B) Binding mode of MS2 (green) with NAMPT
and the design strategy of the autophagy-based NAMPT degraders (PDB: 2GVJ). (C) Schematic illustration of the POI degradation via autophagy.
study, Lu et al. identified four ATTECs (i.e., 10O5, ispinesib,
AN1, and AN2, Figure S1 in the Supporting Information). Based
on these molecular glues, they further developed chimeric
autophagy-tethering compounds for the autophagic degradation
of lipid droplets (LDs).20 These molecules were made of LC3-
binding molecules (10O5 and AN2) and LD-binding probes
connected by flexible linkers, which was similar to that of
PROTACs. Despite the progress made in this field, the design
tools for chimeric autophagy-tethering degraders are still rather
limited. Moreover, whether such compounds could effectively
degrade therapeutic targets still remains unknown. To answer
these questions, herein, ispinesib was used for the first time to
design chimeric autophagy-tethering degraders. Ispinesib is a
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potent inhibitor of kinesin spindle protein (KSP) with an IC50
value of 0.5 nM.21,22 KSP is a kinesin motor protein, and it plays
an important role in the formation of a bipolar mitotic spindle
and cell cycle progression through mitosis.22 It has been shown
that abnormal expression of KSP is correlated with a variety of
human cancers. Using ispinesib as a warhead, autophagy-based
degraders of nicotinamide phosphoribosyltransferase
(NAMPT) were successfully identified, which showed excellent
degradation potency as well as antitumor activity. This study
demonstrated that autophagosome-tethering chimeras could be
an effective strategy for targeted protein degradation of drug
targets and discovery of novel therapeutic agents.
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Figure 2. Colocalization experiment of LC3-B protein probes (green) and lysosomes (red). (A) Design of the fluorescent probe for LC3-B by
connecting the FITC group to the amino group of ispinesib. (B) Schematic of the theory for FP assays and the affinity measurement by fluorescence
anisotropy with LC3-B and P1. Error bars represent a standard deviation of the measurements performed in triplicate. (C) Confocal microscopy
images of Hela cells treated by our probe P1 (green) and lysosome probe (red).

■ RESULTS AND DISCUSSION

Design and Synthesis of NAMPT-Degrading ATTECs.
We choose NAMPT for ATTEC design because it was a highly
challenging target for the development of antitumor agents.
NAMPT is the rate-limiting enzyme of nicotinamide adenine
dinucleotide (NAD), which plays a crucial role in multiple
cellular processes.23−26 However, clinical development of
NAMPT inhibitors (i.e., FK866 and CHS828, Figure S2 in the
Supporting Information) failed due to limited efficacy and dose-
dependent toxicity.27,28 We envisioned that autophagy-based
NAMPT degradation might offer a novel strategy for antitumor
drug discovery.
Similar to the molecular design of PROTAC, the autophagy-
based degrader can also be divided into three parts: a ligand of
POI, a flexible linker, and an LC3 ligand (Figure 1B), which is
supposed to bind to both NAMPT and LC3. As a result, POI
and the LC3 protein could be brought closer, and then, the POI
is labeled, recruited, and fused into autophagosome. The target
protein will be degraded via lysosome-mediated autophagy
(Figure 1C). Previously, our group screened and optimized
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NAMPT inhibitors and identified compound 1 (MS2, Figure
1B) as a potent NAMPT inhibitor (IC50 = 85 nM).29 Docking
studies showed that the terminal piperazine group of compound
1 extended out of the active site of NAMPT without direct
interaction with NAMPT (Figure 1B), offering a favorable site
for the extension of the linker and LC3 ligand.
Then, the key step for the degrader design is to select an
effective LC3 warhead. Among the reported LC3-binding
molecules, indolone LC3 ligands 10O5 and AN1 exist in the
mixture of cis-configuration (active form) and trans-config-
uration (inactive form), which significantly decreased the
degrading potency. Due to its stability and verified config-
uration, herein, we explored the feasibility of ispinesib for the
design of autophagosome-tethering chimeras. To further verify
the direct binding between ispinesib warhead and LC3 protein,
we measured the binding affinity through the isothermal
titration calorimetry (ITC) assay and the KD was 11.8 μM,
indicating that ispinesib could bind to LC3 effectively (Figure S9
in the Supporting Information). Currently, the binding mode of
ispinesib with LC3 is still unknown. Thus, it is important to
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Scheme 1. Synthetic Route of Target Compounds A1−A5a

a
Reagents and conditions: (a) bromide-substituted linkers, DIPEA, DCM, 60 °C, 2 h, yield 60−80%; (b) i. CF3COOH, DCM or LiOH, THF/
MeOH/H2O; ii. compound 2, HATU, DIPEA, DMF, rt, 2 h, yield 36−56%.

determine a suitable site to attach the linker without interfering described previously.31 As shown in Table 1, all the target
the critical interactions with LC3. compounds showed good inhibitory activities with the IC50
To validate the rationality of the structural modification
position of ispinesib and its ability to be engulfed into Table 1. Biological Evaluation of NAMPT Autophagy
autophagosome, the first small-molecule fluorescent probe Degraders
(P1) for LC3 protein was designed and synthesized by
connecting the fluorophore FITC to the amino group of
ispinesib (compound 2) directly through a thiourea linker
(Figure 2A, Scheme S1 in the Supporting Information).
Fluorescence polarization (FP) technology was used to measure
the binding affinity between probe P1 and LC3.30 As shown in
Figure 2B, probe P1 demonstrated nanomolar affinity with a KD
value of 175 nM, indicating that the attachment point for linker
conjugation would improve the binding affinity to LC3 protein.
The colocalization experiment was conducted to verify the
distribution of probe P1 in HeLa cells, which were treated with
the probe for 2 h, followed by staining with red lysosome probes
for another 1.5 h. As shown in Figure 2C, the green fluorescent
probes were colocalized with the red lysosomal probe,
suggesting that the conjugate binding with LC3 could be
engulfed into autophagosome.
On the basis of the above analysis, a series of NAMPT
autophagy degraders were rationally designed and synthesized
by connecting NAMPT inhibitor 1 and ispinesib with different
lengths of alkyl or poly ethylene glycol (PEG) linkers. The target
compounds were synthesized according to Scheme 1. NAMPT
inhibitor 1 was prepared according to the literature protocols.29
Subsequently, intermediates 3a−h were obtained through
substitution reactions between compound 1 and bromide-
substituted linkers under the alkaline condition. After removing
the protective group, the intermediates underwent amide values ranging from 10 to 34 nM, suggesting the good binding
condensation with compound 2 to afford target compounds affinity between the target compounds and NAMPT. Then, we
A1−7. investigated whether target compounds could degrade NAMPT
Evaluation of ATTEC-Induced NAMPT Degradation. in human ovarian cancer A2780 cells, in which NAMPT is
Initially, the NAMPT inhibitory activities of all the designed overexpressed. The results indicated that all the compounds
NAMPT autophagy degraders were determined by the assays were able to trigger NAMPT degradation after treatment for 48
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Figure 3. Characterization of potent NAMPT autophagy degraders. (A) Expression level of NAMPT in A2780 cells treated with target compounds for
48 h. (B) Cell growth inhibition activity of compound A3 and MS2. Error bars represent a standard deviation of the measurements performed in
triplicate.
h (Figure 3A). The degradation efficiency increased gradually
along with the linker length. When the length was 5−7 atoms
(compounds A6, A1 and A2), they showed relatively weak
efficiency with the maximum degradation rates in the range of
1−9% at 0.1 μM. As the length was extended to 8-atom
(compound A3), it showed the best degradation activity.
Compound A3 induced the NAMPT degradation in a
concentration- and time-dependent manner (Figure S3 in the
Supporting Information) with a maximal degradation rate of
91% at 3 μM. When the linker length was further extended to 10-
atoms (compound A5), the degradation activity was obviously
decreased (maximum degradation rate: 33% at about 0.1 μM).
In addition, surface plasmon resonance (SPR) experiments were
performed to further verify the binding affinity between
compound A3 and LC3 protein, with a KD value of 933 nM
(Figure S4 in the Supporting Information). Ispinesib was a well-
known kinesin spindle protein (KSP) inhibitor. Therefore, we
further evaluated the selectivity of compound A3 by monitoring
the degradation of KSP. As shown in Figure S5, compound A3
had no effect on KSP degradation, indicating the selective
NAMPT degrading activity. To evaluate whether the NAMPT
degradation could led to antitumor potency, the cellular
antiproliferative activity of compound A3 was assayed for
several cell lines (Figure 3B), including A2780, MBA-MB-231
(NAMPT overexpression), HCT116 (NAMPT in normal), and
A549 (NAMPT low expression, Figure S6 in the Supporting
Information). Interestingly, the excellent inhibitory activities
were observed in cell lines with overexpressed NAMPT.
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Compound A3 also significantly inhibited the viability of
HCT116 cells, which were highly dependent on the NAD
salvage pathway for survival. In particular, the IC50 value was 46
nM for compound A3 against A2780 cells, which was
significantly more potent than NAMPT inhibitor MS2 (IC50 =
490 nM). Moreover, compound A3 was stable under the
experimental condition and did not degrade into free drugs
(Figure S7 in the Supporting Information).
Investigation of the Mechanism of Action of NAMPT
Autophagic Degrader A3. The underlying mechanism for the
NAMPT-degrading effect of compound A3 was further studied.
First, direct target engagement was performed in the cell lysate
by cellular thermal shift assay (CETSA) to investigate whether
the designed compounds bound to NAMPT directly. As shown
in Figure S8, treatment with compound A3 enhanced the
stability of NAMPT proteins compared to the blank control,
indicating that it was capable of stabilizing NAMPT in cells. In
addition, the CETSA assay also revealed that compound A3 was
able to bind with KSP, which might partly contribute to the
cytotoxicity of compound A3. Thus, as an ATTEC warhead,
ispinesib remained to be further optimized to reduce the binding
activity to KSP. In order to investigate whether the target
compounds caused the NAMPT degradation via lysosomal-
mediated autophagy, different autophagy inhibitors (ammo-
nium chloride, chloroquine, wortmannin, 3-methyladenine, and
LY294002)19,32−35 were added to evaluate their effects on
reversing the expression level of NAMPT. Among them,
ammonium chloride (NH4Cl) and chloroquine can block the
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Figure 4. Compound A3 induced NAMPT degradation via autophagy. (A) A2780 cells were pretreated with MS2, FK866, NH4Cl, chloroquine,
wortmannin, 3-methyladenine or LY294002, followed by treatment with A3 for an additional 48 h. (B) Compound A3 could not reduce NAMPT
levels in Atg7-knockdown cells. Left panel: relative Atg7 levels in A2780 cells and Atg7-knockdown A2780 cells. Right panel: NAMPT levels in A2780
cells and Atg7-knockdown A2780 cells treated with A3 at 500 nM concentration.
function of lysosomes by acidifying the environment of
lysosomes and then impede the fusion process of autophagy
and lysosome. Wortmannin, 3-methyladenine and LY294002
are able to block or interfere with the formation of
autophagosome, thus inhibiting autophagy.19 As shown in
Figure 4A, the total protein level of NAMPT was reversed upon
the addition of autophagy inhibitors, indicating that compound
A3 induced NAMPT degradation through lysosomal-mediated
autophagy. Meanwhile, the coculture of NAMPT inhibitor MS2
or FK866 also led to the recuperation of NAMPT expression,
indicating that the binding mode of compound A3 with
NAMPT was similar to that of FK866 and MS2. LC3 ligand
ispinesib was added to compete with compound A3 for binding
with LC3 protein. However, the A2780 cells were all dead after
pretreating with ispinesib because of high toxicity. As an
alternative, probe P1 was used in the competitive experiment
and the result revealed that the degrading activity was reversed
due to the blockage of LC3 binding (Figure S10 in the
Supporting Information). Atg7 is essential to lysosome-
mediated autophagy.13 When Atg7 was knocked down through
infection via lentivirus in A2780 cells, compound A3 was unable
to induce NAMPT degradation (Figure 4B), indicating that the
degradation relied on the pathway of lysosome-mediated
autophagy.
■ CONCLUSIONS
In summary, ispinesib was identified to be an effective warhead
to design autophagosome-tethering chimeras. Using ispinesib as
the LC3 ligand, a series of novel NAMPT autophagic degraders
were designed and synthesized. Compound A3 effectively
degraded NAMPT, leading to excellent cellular antitumor
potency. Mechanism studies confirmed that compound A3
caused the NAMPT degradation via lysosomal-mediated
autophagy. PROTAC has been emerging as a promising
approach in the field of drug discovery and development.
Recently, using PROTAC technology, we also designed and
identified several NAMPT PROTACs as potent NAMPT
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degraders.36 Although compound A3 did not show better
degradation activity than NAMPT PROTACs, this work proved
that NAMPT could also be effectively degraded by the ALP
system, providing an alternative strategy for targeted degrada-
tion of NAMPT. Moreover, we identified ispinesib as an
effective degradation tag and provided a useful tool for ALP-
based targeted protein degradation. However, it should be noted
that ispinesib is a potent KSP inhibitor and further optimization
is required to improve the affinity and selectivity toward LC3
protein. The application of LC3 binder ispinesib to degrade
more drug targets and structural optimization of ispinesib
warhead is currently in progress.
■
EXPERIMENTAL SECTION
General Methods. All the reagents and solvents were obtained
commercially and used without further purification. 1H NMR and 13C
NMR spectra were collected using Bruker AVANCE300 or
AVANCE600 spectrometers (Leipzig, Bruker Company, Germany)
using tetramethylsilane (TMS) as an interior label and dimethyl
sulfoxide (DMSO)-d6 as solvents. Chemical shifts (δ) are expressed in
parts per million (ppm). The mass spectra were measured on an Esquire
3000 LC-MS mass spectrometer. Thin-layer chromatography analysis
was performed with GF254 silica gel plates to detect reactions (Haiyang
Chemical, Qingdao, China), and visualization of reactants occurred
under 254 nm UV light. Silica gel column chromatography was carried
out with Silica Gel 60 G (Haiyang Chemical, Qingdao, China). High-
performance liquid chromatography analyses were performed on an
Agilent Technologies 1206, instrument to determine purity, and water
and methanol were used as the mobile phase. The final compounds
exhibited the purity greater than 95%.
tert-Butyl 7-(4-((4-(3-(pyridin-3-ylmethyl)thioureido)-
phenyl)sulfonyl)piperazin-1-yl)heptanoate (3a). Compound 1
(500 mg, 1.3 mmol) and tert-butyl bromoacetate (338 mg, 1.3 mmol)
were dissolved in dry dichloromethane (DCM) (20 mL), and then,
N,N-diisopropylethylamine (DIPEA) (335 mg, 2.6 mmol) was slowly
added dropwise. The mixture was stirred at 60 °C for 2 h. After that, the
reaction solvent was evaporated under reduced pressure and the residue
was purified by silica gel column chromatography (DCM/MeOH =
100/1) to obtain a white solid intermediate 3a (512 mg, yield 68%). 1H
NMR (DMSO-d6, 600 MHz) δ: 10.17 (s, 1H), 8.66 (s, 1H), 8.59 (s,
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1H), 8.49 (d, J = 4.7 Hz, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 7.9
Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.40 (dd, J = 7.8, 4.9 Hz, 1H), 4.81 (d,
J = 5.5 Hz, 2H), 2.87 (s, 4H), 2.41 (s, 4H), 2.24 (s, 2H), 1.43−1.47 (m,
2H), 1.38 (s, 9H), 131−1.37 (m, 2H), and 1.17−1.27 (m, 6H).
Preparation methods of target compounds 3b−g were the same as
that of target compound 3a.
tert-Butyl 8-(4-((4-(3-(pyridin-3-ylmethyl)thioureido)-
phenyl)sulfonyl)piperazin-1-yl)octanoate (3b). White solid,
yield 53%. 1H NMR (DMSO-d6, 600 MHz) δ: 10.14 (s, 1H), 8.64 (s,
1H), 8.58 (d, J = 2.0 Hz, 1H), 8.48 (dd, J = 4.8, 1.4 Hz, 1H), 7.76−7.83
(m, 3H), 7.66 (d, J = 8.74 Hz, 2H), 7.39 (dd, J = 7.9, 4.8 Hz, 1H), 4.80
(d, J = 5.6 Hz, 2H), 2.86 (s, 4H), 2.40 (s, 4H), 2.23 (s, 2H), 2.14 (t, J =
7.3 Hz, 2H), 1.41−1.48 (m, 2H), 1.38 (s, 9H), 1.30−1.36 (m, 2H), and
1.18−1.23 (m, 6H). High-resolution mass spectrometry (HRMS) (ESI,
negative) m/z calcd for C29H43N5O4S2 [M − H]+, 588.2684; found,
588.2701.
Ethyl 9-(4-((4-(3-(pyridin-3-ylmethyl)thioureido)phenyl)-
sulfonyl)piperazin-1-yl)nonanoate (3c). White solid, yield 55%.
1
H NMR (DMSO-d6, 600 MHz) δ: 10.11 (s, 1H), 8.59−8.66 (m, 1H),
8.57 (d, J = 1.78 Hz, 1H), 8.47 (dd, J = 1.4, 15.4 Hz, 1H), 7.72−7.83 (m,
3H), 7.60−7.69 (m, 2H), 7.38 (dd, J = 7.8, 4.8 Hz, 1H), 4.79 (d, J = 5.5
Hz, 2H), 3.98−4.11 (m, 2H), 3.17 (d, J = 5.3 Hz, 1H), 2.85 (s, 4H),
2.39 (s, 4H), 2.16−2.23 (m, 4H), 1.27−1.51 (m, 4H), and 1.08−1.27
(m, 10H).
Ethyl 10-(4-((4-(3-(pyridin-3-ylmethyl)thioureido)phenyl)-
sulfonyl)piperazin-1-yl)decanoate (3d). White solid, yield 58%.
1
H NMR (DMSO-d6, 600 MHz) δ: 8.89 (s, 1H), 7.77 (d, J = 8.2 Hz,
1H), 7.71 (d, J = 7.8 Hz, 1H), 7.42−7.47 (m, 5H), 7.33−7.39 (m, 2H),
5.02 (q, J = 6.75.5 Hz, 1H), 4.71 (t, J = 9.2 Hz, 1H), 4.50 (s, 1H), 4.07
(s, 1H), 3.84 (d, J = 11.4 Hz, 1H), 3.68 (dd, J = 11.4, 3.6 Hz, 1H), 2.49
(s, 4H), 2.27−2.34 (m, 1H), 1.95−2.00 (m, 1H), 1.53 (d, J = 7.0 Hz,
3H), and 1.13−1.18 (m, 11H). HRMS (ESI, negative) m/z calcd for
C29H43N5O4S2 [M − H]+, 588.2684; found, 588.2673.
tert-Butyl 11-(4-((4-(3-(pyridin-3-ylmethyl)thioureido)-
phenyl)sulfonyl)piperazin-1-yl)undecanoate (3e). White solid,
yield 65%. 1H NMR (DMSO-d6, 600 MHz) δ: 10.13 (s, 1H), 8.62 (s,
1H), 8.57 (d, J = 1.7 Hz, 1H), 8.47 (dd, J = 4.8, 1.6 Hz, 1H), 7.79 (d, J =
8.8 Hz, 2H), 7.74−7.78 (m, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.36−7.39
(m, 1H), 4.79 (d, J = 5.4 Hz, 2H), 3.56 (s, 3H), 2.85 (s, 4H), 2.39 (s,
4H), 2.26 (t, J = 7.4 Hz, 2H), 2.22 (t, J = 7.2 Hz, 2H), 1.44−1.52 (m,
2H), and 1.29−1.35 (m, 2H).
tert-Butyl 3-(2-(4-((4-(3-(pyridin-3-ylmethyl)thioureido)-
phenyl)sulfonyl)piperazin-1-yl)ethoxy)propanoate (3f). White
solid, yield 70%. 1H NMR (DMSO-d6, 600 MHz) δ: 10.12 (s, 1H), 8.62
(t, J = 5.7 Hz, 1H), 8.55 (d, J = 1.2 Hz, 1H), 8.46 (d, J = 3.5 Hz, 1H),
7.56−7.82 (m, 5H), 7.36 (dd, J = 7.9, 5.0 Hz, 1H), 4.77 (d, J = 5.7 Hz,
2H), 3.48 (t, J = 6.4 Hz, 2H), 3.39 (t, J = 5.3 Hz, 2H), 2.81 (s, 4H),
2.38−2.47 (m, 6H), 2.34 (t, J = 5.9 Hz, 2H), and 1.33 (s, 9H). HRMS
(ESI, positive) m/z calcd for C26H37N5O5S2 [M + H]+, 564.2309;
found, 564.2329.
tert-Butyl 3-(2-(2-(2-(4-((4-(3-(pyridin-3-ylmethyl)-
thioureido)phenyl)sulfonyl)piperazin-1-yl)ethoxy)ethoxy)-
ethoxy)propanoate (3g). White solid, yield 65%. 1H NMR (DMSO-
d6, 600 MHz) δ: 10.19 (s, 1H), 8.69 (s, 1H), 8.57 (s, 1H), 8.47 (d, J =
4.1 Hz, 1H), 7.72−7.89 (m, 3H), 7.66 (d, J = 8.8 Hz, 2H), 7.37 (dd, J =
7.6, 4.9 Hz, 1H), 4.79 (d, J = 5.3 Hz, 2H), 3.56 (t, J = 6.1 Hz, 2H), 3.42−
3.50 (m, 10H), 2.73−3.01 (m, 4H), 2.55−2.72 (m, 4H), 2.40 (t, J = 6.4
Hz, 2H), 1.38 (s, 9H), and 1.22−1.26 (m, 2H).
N-(1-(3-Benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-
2-methylpropyl)-4-methyl-N-(3-(7-(4-((4-(3-(pyridin-3-
ylmethyl)thioureido)phenyl)sulfonyl)piperazin-1-yl)-
heptanamido)propyl)benzamide (A1). Intermediate 3a (519 mg,
1.0 mmol), ispinesib (516 mg, 1.0 mmol), HATU (570 mg, 1.5 mmol),
and DIPEA (194 mg, 1.5 mmol) were dissolved in dry dimethylforma-
mide (DMF) (10 mL), and then, the mixture was stirred at room
temperature for 1 h. After that, the mixture was poured into iced water
(30 mL). A large amount of white solid was precipitated and then
filtrated under vacuum. The precipitant was dried at 50 °C to constant
weight and then purified via column chromatography to obtain the
target compound A1 (360 mg, yield 36%). 1H NMR (DMSO-d6, 600
MHz) δ: 10.14 (s, 1H), 8.63 (s, 1H), 8.57 (d, J = 1.7 Hz, 1H), 8.47 (dd,
7625
pubs.acs.org/jmc Article
J = 4.8, 1.3 Hz, 1H), 8.22 (d, J = 8.6 Hz, 1H), 7.81 (d, J = 8.6 Hz, 2H),
7.75−7.78 (m, 2H), 7.63−7.67 (m, 3H), 7.34−7.38 (m, 3H), 7.28−
7.33 (m, 2H), 7.18−7.26 (m, 6H), 8.87 (d, J = 16.4 Hz, 1H), 5.53 (d, J =
10.4 Hz, 1H), 4.80 (d, J = 5.5 Hz, 2H), 3.17−3.26 (m, 2H), 2.66−2.75
(m, 1H), 2.34−2.50 (m, 4H), 2.31 (s, 3H), 2.16−2.27 (m, 2H), 1.68−
1.79 (m, 2H), 1.21−1.35 (m, 9H), 1.05−1.17 (m, 4H), 0.89 (d, J = 6.8
Hz, 3H), 0.79−0.86 (m, 2H), and 0.47 (d, J = 6.2 Hz, 3H); 13C NMR
(DMSO-d6, 150 MHz) δ: 185.98, 177.24, 176.85, 166.33, 160.45,
154.11, 153.40, 152.37, 149.25, 144.70, 143.88, 141.87, 140.51, 139.37,
138.96, 134.09, 133.87, 133.62, 133.22, 132.65, 131.86, 131.60, 131.07,
128.66, 126.76, 124.27, 99.99, 64.21, 66.75, 50.95, 50.39, 49.92, 47.67,
40.92, 40.43, 35.39, 34.16, 33.71, 33.57, 31.65, 30.24, 26.11, 24.71, and
23.36; HRMS (ESI, positive) m/z calcd for C54H65ClN9O5S2 [M +
H]+: 1018.4233; found, 1018.4260.
Preparation methods of target compounds A2-A7 were the same as
that of target compound A1.
N-(1-(3-Benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-
2-methylpropyl)-4-methyl-N-(3-(8-(4-((4-(3-(pyridin-3-
ylmethyl)thioureido)phenyl)sulfonyl)piperazin-1-yl)-
octanamido)propyl)benzamide (A2). White solid, yield 52%. 1H
NMR (DMSO-d6, 600 MHz) δ: 10.13 (s, 1H), 8.39−8.72 (m, 3H),
8.23 (d, J = 8.2 Hz, 1H), 7.72−7.88 (m, 4H), 7.59−7.70 (m, 3H),
7.14−7.43 (m, 11H), 5.88 (d, J = 16.2 Hz, 1H), 5.54 (d, J = 9.8 Hz, 1H),
5.06 (d, J = 16.5 Hz, 1H), 4.80 (s, 2H), 3.19−3.28 (m, 2H), 2.86 (s,
4H), 2.67−2.76 (m, 1H), 2.39 (s, 4H), 2.32 (s, 3H), 2.22 (s, 2H),
1.67−1.81 (m, 2H), 1.37−1.20 (m, 7H), 1.15 (s, 4H), 1.09 (s, 2H),
0.82−0.94 (m, 4H), and 0.47 (d, J = 4.6 Hz, 3H); HRMS (ESI,
positive) m/z calcd for C55H67ClN9O5S2 [M + H]+, 1032.4390; found,
1032.4368.
N-(1-(3-Benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-
2-methylpropyl)-4-methyl-N-(3-(9-(4-((4-(3-(pyridin-3-
ylmethyl)thioureido)phenyl)sulfonyl)piperazin-1-yl)-
nonanamido)propyl)benzamide (A3). White solid, yield 43%. 1H
NMR (DMSO-d6, 600 MHz) δ: 10.17 (s, 1H), 8.38−8.77 (m, 3H),
8.23 (s, 1H), 7.72−7.95 (m, 4H), 7.66 (s, 3H), 7.06−7.52 (m, 12H),
5.88 (d, J = 12.0 Hz, 1H), 5.55 (s, 1H), 5.06 (d, J = 14.0 Hz, 1H), 4.80
(s, 2H), 3.24 (s, 4H), 2.58−3.05 (m, 6H), 2.07−2.47 (m, 10H), 1.75 (s,
2H), 1.01−1.50 (m, 15H), 0.90 (s, 5H), and 0.48 (s, 3H); 13C NMR
(DMSO-d6, 150 MHz) δ: 181.23, 172.45, 172.02, 161.56, 155.72,
149.42, 148.67, 147.64, 144.46, 139.92, 139.09, 137.16, 135.72, 134.61,
134.26, 129.32, 129.11, 128.85, 128.44, 127.87, 127.12, 126.88, 126.33,
123.86, 121.93, 119.55, 59.45, 57.72, 52.12, 46.38, 45.64, 45.19, 42.90,
40.93, 36.17, 35.72, 30.69, 29.24, 29.15, 29.07, 28.81, 27.22, 26.59,
25.53, 21.36, 19.95, and 18.63; HRMS (ESI, positive) m/z calcd for
C56H67ClN9O5S2 [M − H]−, 1044.4401; found, 1045.4424.
N-(1-(3-Benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-
2-methylpropyl)-4-methyl-N-(3-(10-(4-((4-(3-(pyridin-3-
ylmethyl)thioureido)phenyl)sulfonyl)piperazin-1-yl)-
decanamido)propyl)benzamide (A4). White solid, yield 42%. 1H
NMR (DMSO-d6, 600 MHz) δ: 10.15 (s, 1H), 8.61−8.71 (m, 1H),
8.56 (s, 1H), 8.46 (dd, J = 4.7 Hz, 1H), 8.22 (d, J = 8.5 Hz, 1H), 7.71−
7.84 (m, 3H), 7.56−7.70 (m, 3H), 7.11−7.45 (m, 11H), 5.87 (d, J =
16.1 Hz, 1H), 5.53 (d, J = 10.0 Hz, 1H), 5.05 (d, J = 16.7 Hz, 1H), 4.79
(s, 2H), 3.10−3.27 (m, 4H), 2.84 (s, 3H), 2.54 (s, 2H), 2.34−2.43 (m,
4H), 2.32 (s, 3H), 2.14−2.24 (m, 2H), 1.95−2.09 (m, 1H), 1.66−1.83
(m, 2H), 1.00−1.40 (m, 14H), 0.73−0.97 (m, 5H), and 0.46 (d, J = 5.5
Hz, 3H); 13C NMR (DMSO-d6, 150 MHz) δ: 181.23, 172.46, 172.07,
161.56, 155.71, 149.38, 148.64, 147.63, 144.43, 139.92, 139.10, 137.13,
135.73, 134.61, 134.23, 130.08, 129.51, 129.32, 129.11, 128.83, 128.44,
127.87, 127.11, 126.85, 126.32, 123.87, 121.97, 119.53, 59.45, 57.72,
52.11, 46.36, 45.63, 45.17, 42.89, 40.88, 36.17, 35.72, 31.71, 30.67,
29.45, 29.29, 29.14, 29.05, 28.80, 27.21, 27.04, 26.56, 25.53, 22.51,
21.34, 19.945, 18.61, and 14.37; HRMS (ESI, positive) m/z calcd for
C57H69ClN9O5S2 [M − H]+, 1058.4557; found, 1058.4539.
N-(1-(3-Benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-
2-methylpropyl)-4-methyl-N-(3-(11-(4-((4-(3-(pyridin-3-
ylmethyl)thioureido)phenyl)sulfonyl)piperazin-1-yl)-
undecanamido)propyl)benzamide (A5). White solid, yield 39%.
1
H NMR (DMSO-d6, 600 MHz) δ: 10.21 (s, 1H), 8.69 (s, 1H), 8.58 (d,
J = 1.8 Hz, 1H), 8.49 (dd, J = 4.6, 1.4 Hz, 1H), 8.23 (d, J = 8.6 Hz, 1H),
7.82 (d, J = 8.7 Hz, 2H), 7.75−7.79 (m, 2H), 7.63−7.67 (m, 3H),
https://doi.org/10.1021/acs.jmedchem.1c02001
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Journal of Medicinal Chemistry
7.20−7.40 (m, 11H), 5.88 (d, J = 16.2 Hz, 1H), 5.54 (d, J = 10.5 Hz,
1H), 5.06 (d, J = 16.2 Hz, 1H), 4.80 (d, J = 5.6 Hz, 2H), 3.24 (t, J = 8.1
Hz, 2H), 2.86 (s, 4H), 2.67−2.76 (m, 1H), 2.39 (s, 4H), 2.33 (s, 3H),
2.20−2.24 (m, 2H), 1.96−2.01 (m, 1H), 1.72−1.81 (m, 3H), 1.46−
1.55 (m, 2H), 1.27−1.34 (m, 6H), 1.23−1.26 (m, 6H), 1.09−1.13 (m,
2H), 0.90 (d, J = 6.8 Hz, 3H), and 0.47 (d, J = 6.2 Hz, 3H); 13C NMR
(DMSO-d6, 150 MHz) δ: 181.23, 172.45, 172.03, 161.56, 155.71,
149.39, 148.65, 147.63, 144.48, 139.91, 139.09, 137.14, 135.71, 134.59,
134.25, 130.09, 129.31, 129.09, 128.83, 128.43, 127.87, 127.12, 126.86,
126.32, 123.86, 121.88, 119.54, 59.44, 57.67, 52.07, 46.34, 45.63, 45.14,
42.89, 36.17, 35.72, 31.71, 30.84, 30.68, 29.52, 29.36, 29.30, 29.15,
29.07, 28.80, 27.20, 27.00, 26.56, 25.54, 22.52, 21.34, 19.93, 18.61, and
14.37; HRMS (ESI, positive) m/z calcd for C58H73ClN9O5S2 [M + H]+,
1074.4859; found, 1074.4846.
N-(1-(3-Benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-
2-methylpropyl)-4-methyl-N-(3-(3-(2-(4-((4-(3-(pyridin-3-
ylmethyl)thioureido)phenyl)sulfonyl)piperazin-1-yl)ethoxy)-
propanamido)propyl)benzamide (A6). White solid, yield 40%. 1H
NMR (DMSO-d6, 600 MHz) δ: 10.15 (s, 1H), 8.60−8.72 (m, 1H),
8.57 (d, J = 1.6 Hz, 1H), 8.47 (dd, J = 3.4, 1.4 Hz, 1H), 8.22 (d, J = 8.6
Hz, 1H), 7.74−7.83 (m, 4H), 7.59−7.66 (m, 3H), 7.19−7.40 (m,
12H), 5.87 (d, J = 16.3 Hz, 1H), 5.52 (d, J = 10.3 Hz, 1H), 5.04 (d, J =
16.3 Hz, 1H), 4.79 (d, J = 5.3 Hz, 2H), 3.19−3.27 (m, 2H), 2.80 (m,
4H), 2.35−2.47 (m, 8H), 2.31 (s, 4H), 1.93−2.01 (m, 2H), 1.25−1.33
(m, 2H), 0.88 (d, J = 6.5 Hz, 3H), 0.75−0.87 (m, 2H), and 0.46 (d, J =
6.2 Hz, 3H); HRMS (ESI, positive) m/z calcd for C52H59ClN9O6S2 [M
− H]−, 1004.3724; found, 1004.3693.
N-(1-(3-Benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-
2-methylpropyl)-4-methyl-N-(12-oxo-1-(4-((4-(3-(pyridin-3-
ylmethyl)thioureido)phenyl)sulfonyl)piperazin-1-yl)-3,6,9-tri-
oxa-13-azahexadecan-16-yl)benzamide (A7). White solid, yield
51%. 1H NMR (600 MHz, DMSO-d6) δ: 10.16 (s, 1H), 8.66 (s, 1H),
8.58 (s, 1H), 8.48 (d, J = 4.5 Hz, 1H), 8.23 (d, J = 8.7 Hz, 1H), 7.74−
7.86 (m, 4H), 7.60−7.71 (m, 3H), 7.18−7.43 (m, 11H), 5.89 (d, J =
16.1 Hz, 1H), 5.54 (d, J = 10.0 Hz, 1H), 5.06 (d, J = 15.6 Hz, 1H), 4.79
(s, 2H), 3.37−3.48 (m, 11H), 3.21−3.28 (m, 2H), 2.85 (s, 4H), 2.70−
2.77 (m, 1H), 2.41−2.49 (m, 6H), 2.34 (s, 3H), 1.98−2.06 (m, 2H),
1.24−1.31 (m, 3H), 0.90 (d, J = 6.5 Hz, 3H), 0.81−0.88 (m, 2H), and
0.48 (d, J = 6.3 Hz, 3H); 13C NMR (150 MHz, DMSO-d6) δ: 185.98,
177.20, 174.66, 166.34, 160.45, 154.18, 153.43, 152.41, 149.20, 144.72,
143.91, 141.92, 140.48, 139.37, 138.99, 134.12, 133.88, 133.63, 133.24,
132.64, 131.94, 131.64, 131.11, 128.63, 126.70, 124.31, 74.87, 74.82,
74.69, 73.46, 74.84, 64.20, 61.76, 57.13, 51.13, 49.96, 47.53, 41.13,
41.01, 33.57, 26.11, 24.70, and 23.38; HRMS (ESI, positive) m/z calcd
for C56H69ClN9O8S2 [M + H]+, 1094.4394; found, 1094.4362.
(R)-N-(1-(3-Benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-
yl)-2-methylpropyl)-N-(3-(3-(3′,6′-dihydroxy-3-oxo-3H-spiro-
[isobenzofuran-1,9′-xanthen]-5-yl)thioureido)propyl)-4-
methylbenzamide (P1). The intermediate 4 (0.1 g, 0.2 mmol) and 5
(75 mg, 0.2 mmol) were dissolved in anhydrous DMF (10 mL). Then,
tetraethylammonium (40 mg, 0.4 mmol) was added and the mixture
was reacted at room temperature. After 2 h, the reaction solution was
poured into water (100 mL) and extracted with EA (40 mL × 3). The
organic phases were collected and concentrated under reduced pressure
to give the crude product. Purification was applied with C18 column
[H2O (0.1% TFA): MeOH = 20: 80] to afford the target compound P1
as a yellow solid (74 mg, yield 41%). 1H NMR (600 MHz, DMSO-d6) δ
10.11 (s, 2H), 9.68 (s, 1H), 8.21 (d, 1H, J = 8.5 Hz), 8.05 (s, 1H), 7.78
(s, 1H), 7.70 (s, 1H), 7.57−7.60 (m, 2H), 7.35−7.37 (m, 2H), 7.20−
7.31 (m, 7H), 7.13 (d, 1H, J = 8.0 Hz), 6.67 (d, 2H, J = 2.4 Hz), 6.55−
6.59 (m, 4H), 5.89 (d, 1H, J = 16.0 Hz), 5.57 (d, 1H, J = 10.4 Hz), 5.09
(d, 1H, J = 16.5 Hz), 3.36−3.37 (m, 2H), 3.00 (s, 2H), 2.76−2.80 (m,
1H), 2.24 (s, 3H), 1.51−1.53 (m, 1H), 1.04−1.11 (m, 1H), 0.92 (d,
3H, J = 6.6 Hz), and 0.49 (d, 3H, J = 6.6 Hz). 13C NMR (150 MHz,
DMSO-d6) δ 180.63, 172.45, 168.95, 161.61, 159.95, 155.67, 152.35,
147.70, 141.63, 139.94, 139.19, 137.18, 134.19, 129.39, 129.11, 128.46,
127.87, 127.23, 126.88, 126.40, 124.39, 119.64, 112.99, 110.22, 102.72,
59.546, 45.676, 42.602, 41.417, 29.725, 28.884, 21.321, 20.001, and
18.64. HRMS (ESI): m/z calcd for C51H44ClN5O7S, 906.4421; found,
906.2742 [M + H]+.
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NAMPT Enzymatic Inhibition Assay. All of the enzymatic
reactions were conducted at 30 °C for 90 min in a 50 μL mixture
containing 50 mM Tris HCl (pH 8.0), 12.5 mM MgCl2, 2 mM ATP, 1
mM DTT, 0.02% bovine serum albumin (BSA), 0.4 mM phosphor-
ibosyl pyrophosphate, 20 μM nicotinamide, 30 μg/mL of alcohol
dehydrogenase, 1.5% alcohol, 0.01% Tween 20, 10 μg/mL NAMPT,
and the test compounds. Fluorescence intensity was measured at an
excitation of 360 nm and an emission of 460 nm, and then, the
fluorescent intensity data were analyzed using Graphpad Prism.
Fluorescence Polarization Measurements. KD values were
measured using the fluorescence anisotropy assay with fluorescein-
labeled ispinesib as the ligand in black flat-bottom half-area 96-well
plates (Corning #3650). The fluorescein-labeled ispinesib was diluted
to 50 nM with N-(2-hydroxyethyl)piperazine-N’-ethanesulfonic acid
(HEPES) buffer. A twofold serial dilution of LC3 protein in HEPES
buffer (100 μL) was added to a 200 μL of solution of fluorescent probe
(final concentration at 25 nM). The fluorescence anisotropy was
measured on BioTek Synergy H2 with the 485 nm excitation and 535
nm emission filters after 30 min incubation at room temperature. The
KD values were determined by fitting the displacement curves to the
following equation using Mathamatica 9 (Wolfram Research Inc.)
Q × F × AB + (1 − F ) × AF
A=
1 − (1 − Q ) × F
F=
(KD + LST + RT − (KD + LST + RT)2 − 4 × LST × RT )
2 × LST
In the equations, A is the measured fluorescence polarization value,
Q is the ratio of the fluorescence intensity of the probe in the bound and
free status, AB and F represent the anisotropies of bound and free probe,
respectively, KD is the equilibrium dissociation constant of the
fluorescent probe, LST is the concentration of the probe, and RT is
the receptor protein concentration.
Western Blot Analysis. A2780 cells were seeded in six-well plates
at a density of 1 × 106/well. After incubation at 37 °C with 5% CO2
overnight, compounds at different concentrations were added. After 48
h, the medium was discarded. Cells were washed with phosphate-
buffered saline (PBS) three times and then lysed on ice with RIPA cell
lysis buffer for 30 min. Cell lysates were collected and centrifuged at 1.2
× 105 g at 4 °C for 15 min. The supernatants were quantified via the
bicinconinic acid assay kit and then degenerated at 100 °C. Then, they
were analyzed at an equal amount of protein via sodium dodecyl
sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). Subse-
quently, they were transferred to the polyvinylidene fluoride membrane
and then blocked with 5% BSA in Tris-buffered saline and Tween 20 at
room temperature for 2 h. Then, they were incubated with primary
antibodies at 4 °C overnight separately. After being washed with PBS
three times every 10 min, they were incubated with secondary
antibodies at room temperature for 1 h. After being washed with PBS
another three times every 10 min, the membranes were read by Odyssey
Infrared Imaging.
Cell Proliferation Assay. Cells were seeded at a density of 5 × 103
cells per well. After being incubated overnight, they were treated with
DMSO, MS2, or A3 as indicated for 72 h. The culture medium was
discarded, and 10% CCK8 containing culture medium was added. After
being incubated at 37 °C, the OD values at a wavelength of 450 nm were
read. The cell proliferation rates versus concentration of compounds
was analyzed using Graphpad Prism.
Cell Thermal Shift Assay. A2780 cells were seeded at a density of 1
× 107/well and then incubated at 37 °C with 5% CO2 overnight. Then,
compounds at the indicated concentration were added. After 4 h,
culture medium was discarded and the cells were collected and lysed
under treatment in liquid nitrogen for 5 min (three times). After being
centrifuged at 1.2 × 105 g at 4 °C for 15 min, the supernatants were
divided into four parts and heated at 48 , 52, 55, and 58 °C, respectively.
Subsequently, they were centrifuged at 1.2 × 105 g at 4 °C for 15 min.
https://doi.org/10.1021/acs.jmedchem.1c02001
J. Med. Chem. 2022, 65, 7619−7628
Journal of Medicinal Chemistry
The supernatants were degenerated at 100 °C and then analyzed via
SDS-PAGE to detect the expression level of NAMPT protein.
Knockdown of Atg7 in A2780 Cells. Cells were seeded into 24-
well plates at a density of 1.5×105/well. After being incubated at 37 °C
with 5% CO2 overnight, 250 μL of medium containing shRNA and 2
μg/mL polybrene was replaced for culture medium. After 4 h, another
250 μL of culture medium was added. After 24 h, the medium was
discarded and replaced by fresh culture medium. Cells were collected
after another 48 h for analysis of the expression level of Atg7.
Molecular Docking. The crystal structure of NAMPT in complex
with its inhibitor was obtained from the Protein Data Bank (PDB:
2GVJ37) and prepared for docking using the protein preparation tool in
Discovery Studio 3.0.38 During this process, the ligands and waters were
removed and hydrogens were added to the structure. Staged
minimization was performed with default setting. The docking studies
were carried out using GOLD 5.0.39 The binding site was defined as
whole residues within a 15 Å radius subset encompassing the ligand.
Conformations were generated by genetic algorithm and scored using
GoldScore as fitness function. The best conformation was chosen to
analyze the ligand−protein interaction. The image representing the
best pose was prepared using PyMOL.
Isothermal Titration Calorimetry. The thermodynamic param-
eters of the binding of ispinesib to recombinant LC3B were determined
by ITC using VP-ITC or ITC200 calorimetry (MicroCal VP-ITC,
Malvern). Since the solubility of ispinesib in the ITC buffer is relatively
low, we performed the titration by injecting a 12 μl of aliquot of LC3B
protein sample (1500 μM) into the cell containing 100 μM ispinesib
(the ITC buffer includes 20 mM HEPES-HCl, pH 7.3, 1% DMSO, and
100 mM NaCl) at a time interval of 120 s to ensure that the titration
peak returned to baseline. Altogether, 13 aliquots were titrated in each
individual experiment. The dissociation constant KD, stoichiometry of
binding (n), and the binding enthalpy ΔH were calculated using
MicroCal Origin 7.0 software with a one-site binding model.
■sı
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c02001.
Chemical structures of ATTECs; chemical structures of
NAMPT inhibitors; NAMPT levels in A2780 cells treated
with compound A3 at 1 μM concentration for the
indicated time points; affinity measurement by SPR with
LC3-B and A3; KSP levels in A2780 cells treated with
compound A3; cell growth inhibition of compound A3;
stability of A3; CETSA of NAMPT with compound A3;
validation of the interaction between LC3-B and ispinesib
by ITC at 25 °C; A2780 cells pretreated with ispinesib,
followed by treatment with A3 for an additional 48 h;
synthetic route of probe P1; and 1H- and 13C NMR
spectra of target compounds (PDF)
SMILES molecular formula strings (CSV)
Molecular docking of compound MS2 with NAMPT
(PDB)
■
AUTHOR INFORMATION
Corresponding Authors
Guoqiang Dong − School of Pharmacy, Second Military
Medical University, Shanghai 200433, China; orcid.org/
0000-0003-1708-7803; Email: gdong@smmu.edu.cn
Chunquan Sheng − School of Pharmacy, Second Military
Medical University, Shanghai 200433, China; orcid.org/
0000-0001-9489-804X; Email: shengcq@smmu.edu.cn
Authors
Ying Wu − School of Pharmacy, Second Military Medical
University, Shanghai 200433, China; Department of
7627
pubs.acs.org/jmc Article
Pharmacy, 920th Hospital of Joint Logistics Support Force,
Kunming 650032, China
Junfei Cheng − School of Pharmacy, Second Military Medical
University, Shanghai 200433, China
Long Chen − School of Pharmacy, Second Military Medical
University, Shanghai 200433, China
Rui Liu − School of Life Sciences, Fudan University, Shanghai
200433, China
Yu Ding − School of Life Sciences, Fudan University, Shanghai
200433, China
Shanchao Wu − School of Pharmacy, Second Military Medical
University, Shanghai 200433, China
Junhui Ma − School of Pharmacy, Second Military Medical
University, Shanghai 200433, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c02001
Author Contributions
∥
G.D., Y.W. and J.C. made equal contributions to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (grant 82030105 to C.S.), the National
Key Research and Development Program of China (grant
2020YFA0509100 to C.S.), and Shanghai Rising-Star Program
(20QA1411700 to G.D.).
■ ABBREVIATIONS
ALP, autophagy-lysosomal pathway; ATTEC, autophagosome-
tethering compound; AUTACs, autophagy-targeting chimeras;
CMA, chaperone-mediated autophagy; DIPEA, N,N-diisopro-
pylethylamine; LDs, lipid droplets; Mhtt, mutant HTT protein;
NAD, nicotinamide adenine dinucleotide; NAMPT, nicotina-
mide phosphoribosyltransferase; PEG, poly(ethylene glycol);
PROTACs, proteolysis targeting chimeras
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