Structural basis of beneficial design for effective nicotinamide phosphoribosyltransferase inhibitors

Article abstract of DOI:10.3390/molecules25163633

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is an attractive therapeutic strategy for targeting cancer metabolism. So far, many potent NAMPT inhibitors have been developed and shown to bind to two unique tunnel-shaped cavities existing ad

Full text of DOI:10.3390/molecules25163633

molecules
Article
Structural Basis of Beneficial Design for Effective
Nicotinamide Phosphoribosyltransferase Inhibitors
Sei-ichi Tanuma 1,2,*, Kiyotaka Katsuragi 2, Takahiro Oyama 3, Atsushi Yoshimori 4,
Yuri Shibasaki 2, Yasunobu Asawa 5, Hiroaki Yamazaki 3, Kosho Makino 6, Miwa Okazawa 1,
Yoko Ogino 2,7, Yoshimi Sakamoto 8, Miyuki Nomura 8, Akira Sato 2 , Hideaki Abe 3,
Hiroyuki Nakamura 5 , Hideyo Takahashi 6, Nobuhiro Tanuma 8 and Fumiaki Uchiumi 7
1
Department of Genomic Medicinal Science, Research Institute for Science and Technology, Organization for
Research Advancement, Tokyo University of Science, Noda, Chiba 278-8510, Japan;
miwa.okazawa@rs.tus.ac.jp
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda,
2
Chiba 278-8510, Japan; 3b12023@alumni.tus.ac.jp (K.K.); yryr.sbsk040809@gmail.com (Y.S.);
ogino@rs.tus.ac.jp (Y.O.); akirasat@rs.tus.ac.jp (A.S.)
Hinoki Shinyaku Co., Ltd., Chiyoda-ku, Tokyo 102-0084, Japan; takahiro.oyama@hinoki.co.jp (T.O.);
3
hymanami@gmail.com (H.Y.); hideaki.abe@hinoki.co.jp (H.A.)
Institute for Theoretical Medicine Inc., Fujisawa, Kanagawa 251-0012, Japan; yoshimori@itmol.com
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology,
4
5
Yokohama, Kanagawa 226-8503, Japan; asawa.y.aa@m.titech.ac.jp (Y.A.); hiro@res.titech.ac.jp (H.N.)
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda,
6
Chiba 278-8510, Japan; kosho-maki@rs.tus.ac.jp (K.M.); hide-tak@rs.tus.ac.jp (H.T.)
Department of Gene Regulation, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda,
7
Chiba 278-8510, Japan; f_uchiumi@rs.tus.ac.jp
Division of Cancer Chemotherapy, Miyagi Cancer Center Research Institute, Natori, Miyagi 981-1293, Japan;
8
yoshimi.sk.88@gmail.com (Y.S.); nomu.miyu@gmail.com (M.N.); ntanuma@med.tohoku.ac.jp (N.T.)
*
Correspondence: tanuma@rs.tus.ac.jp
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Marialuigia Fantacuzzi and Alessandra Ammazzalorso
Received: 22 July 2020; Accepted: 6 August 2020; Published: 10 August 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is an attractive therapeutic
strategy for targeting cancer metabolism. So far, many potent NAMPT inhibitors have been developed
and shown to bind to two unique tunnel-shaped cavities existing adjacent to each active site
of a NAMPT homodimer. However, cytotoxicities and resistances to NAMPT inhibitors have
become apparent. Therefore, there remains an urgent need to develop effective and safe NAMPT
inhibitors. Thus, we designed and synthesized two close structural analogues of NAMPT inhibitors,
azaindole–piperidine (3a)- and azaindole–piperazine (3b)-motif compounds, which were modified
from the well-known NAMPT inhibitor FK866 (
enzyme inhibitory activity and cellular potency than did 3b and
phenomenon was revealed to be due to apparent electronic repulsion between the replaced nitrogen
atom (N1) of piperazine in 3b and the N atom of His191 in NAMPT by our in silico binding mode
analyses. Indeed, 3b had a lower binding affinity score than did 3a and , although these inhibitors
1). Notably, 3a displayed considerably stronger
1. The main reason for this
δ
1
took similar stable chair conformations in the tunnel region. Taken together, these observations
indicate that the electrostatic enthalpy potential rather than entropy effects inside the tunnel cavity has
a significant impact on the different binding affinity of 3a from that of 3b in the disparate enzymatic
and cellular potencies. Thus, it is better to avoid or minimize interactions with His191 in designing
further effective NAMPT inhibitors.
Keywords: nicotinamide phosphoribosyltransferase; NAD+ biosynthesis; inhibitor; azacyclohexane;
anticancer drug; drug design; enthalpy effect
Molecules 2020, 25, 3633; doi:10.3390/molecules25163633
www.mdpi.com/journal/molecules

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1. Introduction
Nicotinamide adenine dinucleotide (NAD+) is a critical molecule in control of numerous basic cellular
processes, such as ATP production and maintenance of cellular integrity and genome stability [1–4]
It not only serves as a cofactor for redox bioreactions but can also be a substrate for multiple
NAD+-consuming enzymes, such as poly(ADP-ribose) polymerases, mono-ADP-ribosyltransferases
and sirtuins, participating in the epigenetic regulation of DNA transaction (transcription, replication,
repair and recombination), cellular signaling processes and calcium homeostasis, and thereby in
.
cell proliferation, differentiation and death [3–8]. Given the biological importance of this molecule,
mammalian cells have evolved multiple biosynthetic pathways to produce NAD+; it is synthesized from
tryptophan, nicotinic acid and nicotinamide (NAM)/nicotinamide riboside via de novo, Preiss–Handler
and salvage pathways, respectively [1–4,9–11].
As NAD+ is consumed through many enzymatic processes, accelerated NAD+ depletion is often
characteristic in cancer cells [
the NAM salvage pathway that backs NAM to NAD+ in two steps primarily catalyzed by nicotinamide
phosphoribosyltransferase (NAMPT) [13 17]. Furthermore, in various types of cancer cells, NAMPT
is found to be up-regulated [ 13 16 17], although the molecular mechanisms that dictate the salvage
9–
12]. To rapidly replenish the NAD+ pool, cancer cells rely heavily on
–
8, , ,
pathway choice remain elusive. Thus, NAMPT is considered an attractive target for the development
of new anticancer drugs and therapies [18–31].
NAMPT, which functions as a homodimer with two unique tunnel-shaped cavities existing
adjacent to each active site at the dimer interface, catalyzes the rate-limiting primary step of the salvage
pathway, the transfer of phosphoribosyl residue from 5-phosphoribosyl-1-pyrophosphate (PRPP)
to NAM to produce nicotinamide mononucleotide (NMN) (Figure 1) [
inhibitors, such as FK866 ( ), CSH-828, GNE0617 and STF-11880, which bind to the tunnel cavity
of NAMPT, have been reported to exert anti-tumor suppression effects and have progressed to
clinical trials [18 23 25 30]. However, cytotoxicities dominated by gastrointestinal symptoms and
thrombocytopenia and resistance to NAMPT inhibitors have become apparent [23 35 37]. Therefore,
1–4,32–34]. Many NAMPT
1
,
– ,
,
–
there remains an urgent need to develop effective and safe anticancer NAMPT inhibitors for cancer
chemotherapy. Thus, detailed insights into the tunnel structure of NAMPT are required to generate
effective NAMPT inhibitors with a better therapeutic index and that can overcome resistance.
In the present study, we designed and synthesized two close structural analogues of NAMPT
inhibitors 3a and 3b, which were modified from the best explored NAMPT inhibitor 1 [18
Interestingly, we showed that 3a has a considerably stronger enzyme inhibitory activity and cellular
potency than does 3b or . Furthermore, using these inhibitors as chemical probes, we characterized the
,35].
1
inhibitor-targeting tunnel cavity of NAMPT. Importantly, our in silico binding mode analyses of these
inhibitors with the tunnel cavity of NAMPT revealed that there is apparent electronic repulsion only
between the nitrogen atom of piperazine in 3b and the Nδ atom of His191 in NAMPT. These findings
indicate that the electrostatic enthalpy potential inside the tunnel region has a significant impact on the
different binding affinity of 3a from 3b in the disparate cellular potencies. Thus, these results provide
new insights into the design for further effective NAMPT inhibitors for cancer chemotherapies.

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Figure 1. Overview of the human (h) nicotinamide phosphoribosyltransferase (NAMPT) as the dimer
form and its enzyme reaction. The homodimer structure of hNAMPT (PDB Code: 2GVJ) [38 42] is
–
shown in the ribbon diagram colored light blue and brown, respectively. The two tunnel-shaped
cavities near the active sites in the hNAMPT molecule are shown in magenta shading. NAMPT
catalyzes the conversion of nicotinamide (NAM) and phosphoribosyl pyrophosphate (PRPP) to produce
nicotinamide mononucleotide (NMN).
2. Results and Discussion
2.1. Synthesis and Biochemical Properties of 3a and 3b
Many NAMPT inhibitors bind to the tunnel-shaped cavity of NAMPT (Figure 1), although the
role of this tunnel in NAMPT function remains unknown [18–31]. These inhibitors have a unique
pharmacophore consisting of three parts (Figure 2): head (a pyridine-like aromatic moiety), linker
(a tunnel-interacting moiety) and tail (a solvent-exposed bulky group). To get more valuable insights into
the tunnel cavity for the development of effective NAMPT inhibitors, we structure-basically designed
and synthesized two close structural analogues of NAMPT inhibitors, namely azaindole–piperidine
(
3a)- and azaindole–piperazine (3b)-motif compounds, which were modified from the head and
linker moieties of (Figure 2, Scheme 1). Since the tail acts as a packing moiety against the solvent
exposed tunnel exit surface, the tail structures of 3a and 3b were fixed as the same benzoyl group
of . The nitrogen-containing aromatic heads present in many NAMPT inhibitors mimic the natural
NAM substrate of the enzyme [18 31]. However, the head moiety of vinylpyridine ring of (Figure 2)
is known to be able to non-specifically interact with proteins [43 44]. Based on this knowledge,
1
1
–
1
,
we first replaced this head to the 5-azaindole heterocyclic ring to improve the specificity to NAMPT
(Figure 2). Secondly, as the linker moiety of azacyclohexane may be considered to become a critical
motif for NAMPT inhibitory activity, we synthesized a close structural set of piperidine (3a)- and
piperazine (3b)-motif NAMPT inhibitors according to the methods of Bair et al. [45] and Vogel et al. [46],
respectively (Scheme 1).

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Figure 2. Chemical structures of 3a, 3b and 1.
O
HO
O
N
O
HN
N
N
O
(i)
(ii)
X
X
N
O
NH2
1a: X=CH
1b:
2a: X=CH
2b:
O
X=N
X=N
N
O
X
N
H
HN
N
3a: X=CH
3b:
X=N
Scheme 1. Synthetic route of compounds 3a and 3b. Reagents and conditions: (i) NH2NH2, EtOH, r.t. to
reflux, 2 h; (ii) 1H-pyrro[3,2-c]pyridine-2-carboxylic acid, EDC-HCl, DIPEA, DMF, r.t., 24 h. The targets
3a and 3b were prepared according to the methods of [45,46], respectively, with slight modifications.
To compare the NAMPT inhibitory activities of 3a
the titration curves from the standard calorimetric enzyme-cycling assay [31
Figure 3a, the inhibitory activity of 3a IC50 = 0.11 µM) was approximately 5-fold stronger than that
of IC50 = 0.52 µM). This result suggests that the replacement of vinylpyridine with an azaindole
,
3b and
1, we determined the IC50 values by
,
36,37]. As shown in
(
1
(
ring could improve anti-NAMPT activity. The binding mode analyses of these inhibitors on the
NAMPT molecule support this observation, as described in the next section; a hydrogen bond formed
between the N1 atom of azaindole and Asp219 of NAMPT (Figure 4b). Surprisingly, the substitution of
piperidine (3a) with piperazine (3b) resulted in a dramatic loss in inhibitory activity against NAMPT;
the inhibitory activity of 3b (IC50 = 5.06
µM) was shown to be about 50-fold weaker than that of
3a. The inhibition curve of 3a was much steeper than that of 3b, suggesting that incubation with 3a

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resulted in the formation of an entity capable of extremely tight binding to the tunnel cavity of NAMPT.
From these observations, we suspect that unfavorable interactions between the piperazine moiety of
3b and the tunnel cavity of NAMPT were responsible for the considerably weak anti-NAMPT activity.
Figure 3. Biochemical properties of 3a and 3b. (a) The inhibitory activities of 3a (open circle), 3b (open
triangle) and (open square) against NAMPT enzyme were examined, as described previously [31
1
,
36,37].
NAMPT activity was measured using a coupled-enzyme reaction system (CycLex NAMPT colorimetric
assay kit) in 96-well plate format using the one-step method. Data are the averages of three independent
experiments, and the bars indicate the standard error (SE) values. (
(circle) and 3b (triangle) on HCT116 cells were investigated as described previously [31
cells were treated with the indicated concentrations of each inhibitor with (closed symbol) or without
(open symbol) 100 M NMN for 72 h. The cell activity (% of control) was measured by use of the WST-8
b) The anti-proliferative effects of 3a
,
36,37]. HCT116
µ
assay. Data are presented as the means of three independent experiments
the SE values.
±
SE, and the bars indicate
To confirm the marked differences of anti-NAMPT activities between 3a and 3b, the anti-proliferation
effects of these inhibitors on the human colon cancer cell line HCT116 were examined after continuous
exposure for 72 h [31,36,37]. As shown in Figure 3b, compound 3b displayed significantly weaker
cell activity; the 50% effective concentration (EC50) value was calculated to be 87.5 nM, which was
approximately 200-fold higher than that of 3a (EC50 = 0.48 nM). These results of the different inhibition
degrees in cell-based assay from those in enzyme assay (Figure 3) paralleled observations by others
that the degrees of potency of cell-based assessments were not always identical with those of NAMPT
enzymatic inhibition [26,28,47]. In addition, there was little cytotoxicity on non-cancer cell lines, such
as TIG-120, WI38 and MRC-5 cells. Both 3a and 3b caused more than 90% reduction of NAD+ levels in
the cancer cells after 24 h-treatment and thereby induced cell death. Furthermore, supplementation of
the product of NAMPT, NMN, to the cell culture medium could significantly rescue HCT116 cells from
cell death by treatment with 3a or 3b (Figure 3b). Additionally, the dose–response curves of 1 were
similar to those of 3a. These results confirmed that NAMPT is a target of both inhibitors. Collectively,
these results indicate that the substitution of one carbon (C) atom of the piperidine moiety in 3a for a
nitrogen (N) atom (piperazine moiety in 3b) exerts a critical effect on the anti-NAMPT activity.
2.2. Binding Modes of 3a and 3b to the Tunnel Cavity of NAMPT
To understand the different NAMPT inhibitory activities of 3a and 3b, we analyzed their binding
modes to NAMPT (PDB Code: 2GVJ [38
and LigandScout [49]. The superimpositions of these inhibitors in the tunnel cavity revealed that they
adopted almost identical chair binding forms as with the case in (Figure 4a). The NAMPT residues
that contacted these inhibitors were observed in very similar locations in the structure. These results
–42]) by our in silico binding mode analyses using RDKit [48]
1

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indicate that the binding modes of 3a and 3b in the tunnel cavity of NAMPT do not significantly alter
as compared with that of 1.
When observed closely, the position of the head regions of 3a and 3b in the tunnel cavity were
nearly identical to that of
may have been introduced by their slightly different orientations of the head-azaindole bicyclic ring
from the head-pyridine ring of inside the tunnel cavity of NAMPT. The azaindole rings of 3a and 3b
were sandwiched between Phe193A and Tyr18B, forming tight aromatic stacks like the pyridine
ring of . Additionally, the nitrogen atoms (N1) in the azaindole rings of 3a and 3b could interact with
1 (Figure 4b). The subtle shifts observed in 3a and 3b from the position of 1
1
π–π
1
Asp219 of NAMPT by a hydrogen bond (Figure 4b), thus suggesting that they might be involved in the
tighter specific binding of 3a and 3b to NAMPT than that of 1.
However, this specific contacts of the head-azaindole moiety with NAMPT could not explain
the discrepancy in the considerably weaker inhibitory activity of 3b than that of 3a or 1. Therefore,
to better understand this phenomenon, we focused on the importance of linker-motifs for the potencies
of NAMPT inhibitors. Thus, we analyzed closely the binding forms of the linker-azacyclohexane
motifs of 3a and 3b in the tunnel cavity of NAMPT. The carbon atom (C4) of piperidine in 3a and
nitrogen atom (N1) of piperazine in 3b were revealed to protrude approximately 1Å deeper into
the NMA binding active site, as compared to the corresponding carbon atom of piperidine in
Importantly, the electronic repulsion of the replaced nucleophilic N1 atom of piperazine in 3b with the
atom of the imidazole side chain of His191, located 3.82 Å away from the N1 atom, was revealed to
occur (Figure 4c). In contrast, there was no electronic repulsion of the corresponding carbon atom of
piperidine in 3a with the N atom as well as that of . These results clearly show that the electronic
1.
N
δ
δ
1
repulsion interferes with the proper binding of 3b to the tunnel cavity of NAMPT.
It is noteworthy that Zheng and colleagues have reported that the imidazole ring of His191 interacts
with the linker aromatic group of GNE series inhibitors through a herringbone stacking [44
Furthermore, NAMPT mutations, including H191R, D93del, Q388R, S165Y and G217V, have been
reported to confer resistance to NAMPT inhibitors [50 52]. In addition, we have recently shown that
an NAMPT mutation variant of HCT116 cells generated by continuously exposing cells to increasing
concentrations of , which confers cross-resistance to diverse NAMPT inhibitors, such as CHS-828,
GNE-617 and STF-118804, was mapped to only H191R [36 37]. Thus, this His191 residue should
,45,47].
–
1
,
be considered with higher priority in designing further effective NAMPT inhibitors. That is, it is
preferable to avoid interactions with His191 in designing novel NAMPT inhibitors.

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Figure 4. Binding modes of 3a
and in the tunnel cavity of NAMPT were analyzed by our in silico binding mode analysis using
RDKit [48] and LigandScout [49]. ( ) Close-up view of head regions of these three NAMPT inhibitors in
the tunnel cavity of NAMPT using Open3DALIGN [53]. ( ) Close-up view of the binding interactions
of azacyclohexane-linker moieties of these NAMPT inhibitors with His191A located in the tunnel
, 3b and 1 in the tunnel cavity of NAMPT. (a) Binding modes of 3a, 3b
1
b
c
cavity of NAMPT. Carbons are colored green (3a), magenta (3b) and gray (1). Nitrogen and oxygen are
colored blue and red, respectively.

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2.3. Influence of Enthalpic Effects of 3a and 3b on the Interactions with Tunnel Cavity of NAMPT
The C-to-N conversion in the azacylohexane-linker moiety was considered to be possible to
introduce some entropic differences in the compounds’ abilities to interact with the tunnel cavity of
NAMPT. Although piperazine and piperidine rings are known to form various conformations from
chairs to half-chair and skewed boat-to-boat forms in aqueous solution [54], piperazine-containing 3b
and piperidine-containing 3a took nearly identical positions as a chair form to that occupied by
1 in
the tunnel cavity analyzed by in silico binding modes and crystal structure analyses [38 42] (Figure 4).
–
In addition, both C4 in 3a (Figure 5a) and tertiary amine-type nitrogen (N1) containing 3b (Figure 5b)
might prefer equatorial conformations to axial ones by 1,3-diaxial hydrogen interaction [54], taking a
stable chair conformation in the tunnel cavity of NAMPT. Therefore, such entropic effects in 3a and 3b
in the tunnel space of NAMPT may not be so different from each other. Thus, it is possible to consider
that the dramatic differences in enzymatic and cellular potencies between 3a and 3b may be due to
enthalpic effects rather than entropic effects inside the tunnel cavity of NAMPT.
To prove this hypothesis, we attempted to analyze the binding affinity scores of 3a and 3b
to NAMPT (PDB Code: 2GVJ) by our in silico binding mode analyses using RDKit [48] and
LigandScout [49], as described in Materials and Methods. Remarkably, the binding affinity scores of 3a
and 3b (non-protonation form) (Figure 5b) were calculated to be
−
29.07 and 22.13 kJ/mol, respectively,
−
while that of 28.05 kJ/mol) was near to the value of 3b (Table 1). Thus, the piperidine-motif was
1
(
−
revealed to be beneficial for 3a potency and has valuable influences on the interaction with the tunnel
cavity of NAMPT.
The N1 and N4 atoms in 3a and 3b, respectively, would take an sp2-like bond, due to the effect
of the proximity of the benzoyl group (R2) on the lone pair (Figure 5a,b). In contrast, although it is
hard to know whether or not the N1 atom of piperazine in 3b, which takes an sp3 bond like the C4
atom in 3a, would be protonated in the tunnel cavity of NAMPT, it is possible to be protonated in
physiological conditions. Thus, the binding affinity score of 3b in the protonation state was measured
by LigandScout. Interestingly, it was calculated to be
the non-protonated form (Table 1). In the protonated N1 atom, the electrostatic interaction becomes
attractive to the N of His 191 but repulsive against the protonated N . However, as the imidazole ring
of His has aromaticity, the positive charge introduced by protonation does not tend to be localized on
the N . Thus, the repulsion may not be so strong. In addition, it seems that a hydrogen bond between
the protonated N of His191 and the N1 atom in 3b could not be formed, because the angle that
H-N1 (piperazine) takes is 106.4◦. Furthermore, of course, the influence of the hydrophobic amino
−
23.85 kJ/mol and was not as good as that of
δ
δ
δ
δ
N
δ
acids in the tunnel region on the protonated N1 in 3b cannot be ruled out, and they may negatively
influence this interaction. Accordingly, the electrostatic enthalpy effects were suggested to interfere
with the proper binding of 3b to the tunnel cavity of NAMPT. For this reason, both the non-protonated
and protonated forms of 3b are suggested to have lower binding affinities to the tunnel cavity of
NAMPT than does 3a. These outcomes indicate that the electrostatic potential (enthalpy term) inside
the tunnel region of NAMPT has a significant impact on the different binding affinity of 3b from that
of 3a.

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Figure 5. Stable conformers of 3a and 3b in the tunnel cavity of NAMPT. The conformational states of
3a ) and 3b with its protonation form ( ) in the tunnel cavity of NAMPT were analyzed by in silico
binding mode analysis and crystal structure of (PDB code: 2GVJ) [38 42]. The hydrogen interactions
(
a
b
1
–
of axial-methylene with 1,3-diaxial hydrogens are represented by dashed lines.
Table 1. Biochemical properties and binding affinity scores of 3a and 3b NAMPT inhibitors.
Binding Affinity Score b (kJ/mol)
a
Compound
IC50 (µM)
Non-Protonation
Protonation
3a
(piperidine)
3b
(piperazine)
1
0.11
5.06
0.52
−29.07
-
−23.85
-
−22.13
−28.05
(piperidine)
a
b
The IC50 values of these three NAMPT inhibitors were determined by the titration curves in Figure 3a. The
binding affinity scores of these three inhibitors in the tunnel cavity of NAMPT were calculated by LigandScout [49].

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3. Materials and Methods
3.1. Enzymatic NAMPT Assay
For the inhibitory effects of 3a and 3b on NAMPT activity, recombinant NAMPT activity was
measured using a coupled-enzyme reaction system (CycLex NAMPT Colorimetric Assay Kit: MEDICAL
& BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Japan) according to the manufacturer’s
instructions, as described previously [31,36,37]. Briefly, NAMPT enzyme and tested compounds
were put on the 96-well transparent plate. The mixture of the rest of contents (ATP, NAM, PRPP,
nicotinamide nucleotide adenylyltransferase 1 (NMNAT1), water-soluble tetrazolium salts (WST-1),
alcohol dehydrogenase, diaphorase and ethanol (EtOH)) were then added onto the wells. After a
brief incubation, the absorbance of the samples was detected at 450 nm on every 10 min until 60 min
using a SYNERGY HTX (BioTek Instruments, Inc.; Winooski, VT, USA). The inhibition rates were
calculated by the initial slope of the absorbance of the tested samples divided by that of the control
wells. The IC50 values for the samples were calculated by 4-parameter logistic regression using Image J
1.48v (https://imagej.nih.gov/ij/).
3.2. Cells and Cell Culture
HCT116 cell was obtained from American Type Culture Collection (ATCC) (Manassas, US) and
cultured in Dulbecco’s modified Eagle medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation)
supplemented with 10% of fetal bovine serum (Biosera Europe; Nuaillé, France), 100 Units/mL of
penicillin G and 100 µg/mL of Streptomycin (FUJIFILM Wako Pure Chemical Corporation) [36,37].
3.3. Cell Activity Assay
One thousand HCT116 cells were inoculated into the 96-well transparent cell culture plate and
cultured for 24 h. The medium was changed to new medium that contained the tested compounds
and was further cultivated for 72 h. Following the culture, 1/10 volume of the WST-8 reagent
(FUJIFILM Wako Pure Chemical Corporation) was added and incubated for 1 h. After the incubation,
the absorbance of the samples was detected at 450 nm using a SYNERGY HTX (BioTek Instruments,
Inc.). Cell activity was expressed as percentage of control cells treated with vehicle alone [31,36,37].
3.4. In Silico Binding Mode and Binding Affinity Score Analyses
The crystal structure of NAMPT was downloaded from the Protein Data Bank (PDB code:
2GVJ) for molecular binding mode analyses. In silico binding mode analyses were done using
RDKit [48] and LigandScout [49]. First, 1000 conformations of each inhibitor were generated using the
EmbedMultipleConfs and MMFFGetMoleculeForceField functions of RDKit. Second, the conformations
of each inhibitor were aligned to the crystal structure of
1 using Open3DALIGN [53] implemented in
RDKit. The conformation of each inhibitor, which has the highest alignment score, was selected. Third,
energy minimization of the selected conformation of each inhibitor with NAMPT was performed,
and then the binding affinity score of each inhibitor was calculated using LigandScout [49].
3.5. Synthetic Procedures of the Target Compounds
3.5.1. General Experimental Methods
1H-NMR spectra were recorded at 400 MHz using a JEOL ECZ400 operating (JEOL Ltd.; Tokyo,
Japan) at the indicated frequencies. Chemical shifts were expressed in ppm relative to the internal
standard tetramethylsilane (ppm = 0.00). All reagents and solvents were purchased from FUJIFILM
Wako Pure Chemical Corporation. The progress of all reactions was monitored by TLC using ethyl
acetate/hexane, acetone/hexane, dichloromethane (CH2Cl2)/MeOH and CH2Cl2/MeOH/triethylamine
(TEA) as the solvent system, and spots were visualized by irradiation with ultraviolet light (254 nm) or
ninhydrin reaction. Column chromatography was performed using silica gel (200–300 mesh).

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3.5.2. General Procedures for Synthesizing the Target Compounds 3a and 3b
Compounds 1-benzoylpiperidin-4-yl)butyl)isoindoline-1,3-dione (1a) and 2-(4-(4-benzoylpiperazin-
1-yl)butyl)isoindoline-1,3-dione (1b) weresynthesized accordingto Gilig etal. [55] withslight modifications.
3.5.3. (4-(4-aminobutyl)piperidin-1-yl)(phenyl)methanone (2a)
Hydrazine hydrate (0.3 mL, 6.17 mmol) was added under Ar2 atmosphere to a solution of 1a
(1.0 g, 2.56 mmol) in EtOH at 20 ◦C. After stirring at 20 ◦C for 10 min, the mixture was heated under
reflux for 2 h. After cooling to 20 ◦C, the white precipitate was filtered off and washed with EtOH
(10 mL). The EtOH solutions were combined, and the solvent was evaporated in vacuo. The residue
was taken in CH2Cl2 (20 mL) and a saturated aqueous solution of K2CO3 (20 mL). Vigorous stirring for
10 min produced two clear phases. The aqueous phase was extracted with CH2Cl2 (10 mL, 3 times).
The combined organic phases were washed with brine (30 mL) and dried over Na2SO4. After solvent
evaporation in vacuo, 2a was obtained (0.530 g) as a pale-yellow oil.
3.5.4. N-(4-(1-benzoylpiperidin-4-yl)butyl)-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (3a)
First, 1H-pyrro[3,2-c]pyridine-2-carboxylic acid (0.149 g, 0.918 mmol) was added to a solution
of 2a obtained above (0.239 g, 0.918 mmol) in dry DMF (8.0 mL) in a 25 mL round-bottomed flask.
HOBt (0.186 g, 1.38 mmol), EDC-HCl (0.352 g, 1.84 mmol) and N, N-diisopropylethylamine (0.462 mL,
2.75 mmol) were added sequentially, and the suspension was allowed to stir at room temperature
overnight. The reaction mixture was then poured into a separatory funnel containing water and ethyl
acetate. The aqueous phase was separated and extracted three times with EtOAc. The combined
organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The resulting residue was
purified by column chromatography (CH2Cl2/MeOH = 9/1) to afford the target product 3a (0.215 g,
1
yield 58%, more than 95%, as judged by ODS(C18) HPLC) as white crystals. H-NMR (DMSO-d6)
d 11.94 (s, 1H), 8.88 (s, 1H), 8.59 (t, 1H), 8.19 (d, 1H), 7.39 (m, 3H), 7.31 (m, 3H), 7.20 (s, 1H), 4.43 (m,
1H), 3.51 (m, 1H), 3.26 (m, 2H), 2.96 (m, 1H), 2.70 (m, 1H), 1.70 (m, 2H), 1.51 (m, 3H), 1.29 (m, 4H),
1.05 (m, 2H).
3.5.5. 4-(4-Aminobutyl)piperazin-1-yl)(phenyl)methanone (2b)
Compound 2b was obtained from 1b by similar synthetic processes as described in Section 3.5.3.
3.5.6. N-(4-(4-Benzoylpiperazin-1-yl)butyl)-1H-pyroro[3,2-c]pyridine-2-carboxamide (3b)
The procedure was the same as described above for the synthesis of 3a. Compound 3b was
obtained as pale-yellow powder (0.104 g, yield 28%, more than 95%, as judged by ODS(C18) HPLC).
1H-NMR (DMSO-d6)
δ 11.97 (s, broad, 1H), 8.90 (s, 1H), 8.61 (t, 1H), 8.19 (d, 1H), 7.33–7.43 (m, 5H),
7.23 (s, 1H), 3.26–3.58 (m, 9H), 2.30–2.38 (m, 5H), 1.48–1.55 (m, 3H).
3.6. Statistical Analysis
All data were obtained from at least three independent experiments and were expressed as
mean ± standard error (SE).
4. Conclusions
In this study, to understand the structural basis of beneficial design for effective NAMPT
inhibitors, we designed and synthesized a close structural analogue set of potent and specific
azacyclohexane-motif NAMPT inhibitors, namely 3a (piperidine-motif) and 3b (piperazine-motif)
modified from
1. In addition, these two compounds have 5-azaindole ring as the head structure, which
is replaced by the vinylpyridine ring of
1. Through biochemical experiments and in silico binding mode
analyses using these inhibitors, we clearly showed that 3a is the most potent NAMPT inhibitor among
these inhibitors, and that the azaindole-head and piperidine-linker of NAMPT inhibitors are promising

Molecules 2020, 25, 3633
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motifs that result in enthalpically higher potent inhibitory activity against NAMPT molecule and
cancer cells. The main reason for this phenomenon was revealed to be due to the electronic repulsion
of the piperazine-linker motif of 3b with His191 of NAMPT. Importantly, this H191 is known to be a
drug-resistance sensitive amino acid residue [36,37,50–52]. Thus, it is better to avoid interactions with
His191 in designing effective NAMPT inhibitors. Further studies on the tunnel cavity of NAMPT using
rigid analogues of azaindole-piperidine-motif inhibitors will allow a more definitive understanding
of the inhibitor pharmacophore. Although additional confirmatory studies, such as the synthesis of
novel azaindole-piperidine-motif NAMPT inhibitors and details of in vivo efficacy and resistibility are
warranted, our findings provide valuable insights into the design for effective NAMPT inhibitors that
offer improved therapeutic potential by making high specificity and avoiding resistance to NAMPT.
Furthermore, on the basis of the characteristics of NAMPT functions and the mechanisms of action
of NAMPT inhibitors, we are now trying to design additional azaindole–piperidine-motif NAMPT
inhibitors available in vivo, such as the translation of a potent one into a payload for antibody–drug
conjugates, which are an important therapeutic modality enabling targeted drug delivery to cancer
cells [56].
Author Contributions: Conceptualization, S.-i.T. and N.T.; software, A.Y.; validation, T.O.; formal analysis, T.O.
and A.Y.; investigation, K.K., Y.S. (Yuri Shibasaki), M.O., Y.O., Y.S. (Yoshimi Sakamoto) and M.N.; resources,
Y.A., K.M., H.N. and H.T.; writing—original draft preparation, S.-i.T.; writing—review and editing, S.-i.T., A.Y.,
Y.A., H.Y., A.S., H.N., M.O., H.T., N.T. and F.U.; visualization, T.O., A.Y. and H.A.; supervision, S.-i.T.; project
administration, S.-i.T.; funding acquisition, S.-i.T. and N.T. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported in part by P-CREATE for division of Pharmaceutical Research and Development
from the Japan Agency for Medical Research and Development, AMED grant (18cm0106133h0001) and the
Hinoki Foundation.
Acknowledgments: The authors would like to thank staffs for Science and Technology, Organization for Research
Advancement, Tokyo University of Science for their assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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