Identification of benzothiophene amides as potent inhibitors of human nicotinamide phosphoribosyltransferase

Article abstract of DOI:10.1016/j.bmcl.2015.12.101

Nicotinamide phosphoribosyltransferase (Nampt) is an attractive therapeutic target for cancer. A Nampt inhibitor with novel benzothiophene scaffold was discovered by high throughput screening. Herein the structure-activity relationship of the benzothiophe

Full text of DOI:10.1016/j.bmcl.2015.12.101

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of benzothiophene amides as potent inhibitors
of human nicotinamide phosphoribosyltransferase
Wei Chen y, Guoqiang Dong y, Shipeng He y, Tianying Xu, Xia Wang, Na Liu, Wannian Zhang,
⇑
⇑
Chaoyu Miao , Chunquan Sheng
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nicotinamide phosphoribosyltransferase (Nampt) is an attractive therapeutic target for cancer. A Nampt
inhibitor with novel benzothiophene scaffold was discovered by high throughput screening. Herein the
structure–activity relationship of the benzothiophene Nampt inhibitor was investigated. Several new
inhibitors demonstrated potent activity in both biochemical and cell-based assays. In particular, com-
Received 27 September 2015
Revised 17 December 2015
Accepted 29 December 2015
Available online xxxx
pound 16b showed good Nampt inhibitory activity (IC50 = 0.17
lM) and in vitro antitumor activity
(IC50 = 3.9 M, HepG2 cancer cell line). Further investigation indicated that compound 16b could effi-
ciently induce cancer cell apoptosis. Our findings provided a good starting point for the discovery of novel
antitumor agents.
l
Keywords:
Nampt inhibitors
Antiproliferative activity
Structure–activity relationship
Ó 2015 Published by Elsevier Ltd.
Cancer is a disease of genetic heterogeneity, but the metabolic
alternation is the common future of malignant cells.1,2 Although
Warburg suggested that energy generation in cancer cells switch
form the Krebs (tricarboxylic acid) cycle to glycolysis to sustain
robust proliferation as early as in 1924,3,4 it is only recently that
targeting cancer metabolism has emerged as a therapeutic strategy
for the development of antineoplastic agents.5 Among the meta-
bolic targets, considerable attention has been given to the biosyn-
thetic pathways leading to nicotinamide adenine dinucleotide
(NAD).6,7 NAD is an important cofactor in redox reactions and
serves as the substrate for poly (ADP-ribose) polymerases (PARPs),8
mono (ADP-ribose) transferases (ARTs),2 and sirtuins,9 which con-
vert NAD to nicotinamide (NAM). In cancer cells, NAD is rapidly
consumed because of increased demand for ATP and high activity
of NAD-consuming enzymes such as PARPs and Sirtuins.9–11 Thus,
malignant cells are more sensitive to NAD availability as compared
with normal cells. Rapidly proliferating cancers requires constant
re-synthesis of NAD in order to maintain sufficient levels for cell
survival. Biochemically, there are three main pathways to generate
NAD in eukaryotic cells: a de novo pathway using tryptophan (Trp)
as the precursor, the primary salvage pathway in which NAD is
recycled from NAM, and the alternative salvage pathway using
nicotinic acid. The main source of cellular NAD is from the primary
salvage pathway using NAM as the precursor.12 Nicotinamide
phosphoribosyltransferase (Nampt) catalyzes the rate-limiting
step.13,14 Given that Nampt plays a key role in the replenishment
of NAD, inhibition of Nampt can dramatically impact NAD metabo-
lism and cancer proliferation. Thus, Nampt is considered as an
attractive target for the development of new cancer therapies.
To date, several classes of Nampt inhibitors have been reported
in the scientific and patent literatures.2,12,15 The most advanced
compounds FK866, CHS828 and its prodrug GMX1777 (Fig. 1) have
entered the human clinical trials.16–18 FK866 is in phase 2 clinical
trials for the treatment of cutaneous T-cell lymphoma (CTCL),
while CHS828 is in phase 1 trials against metastatic melanoma.
However, the clinical development of FK866 has been hampered
by dose-limiting thrombocytopenia.19 CHS828 was also reported
with thrombocytopenia and various gastrointestinal symptoms.20
With regard to limited classes of small-molecule Nampt inhibitors,
it is highly desirable to discover new inhibitors with novel
chemical scaffolds and potent antiproliferative activity (e.g.
compound 1).15,21,22
The pharmacophoric model (Fig. 1) of the Nampt inhibitors con-
sists of a cap group (typically a meta or para substituted pyridine),
a connecting unit, a linker and a tail group.2 Previously, we
described the identification of compound 2 (Fig. 1) as a novel
Nampt inhibitor by high throughput screening.15 It showed submi-
cromolar activity against Nampt (IC50 = 0.15 lM). Interestingly, the
⇑
Corresponding authors. Tel.: +86 21 81871271 (C.M.), +86 21 81871239 (C.S.).
E-mail addresses: cymiao@smmu.edu.cn (C. Miao), shengcq@hotmail.com
benzothiophene inhibitor 2 did not contain a pyridine or nitrogen-
containing heterocycle moiety in the cap group. This molecule
demonstrated that a non-nitrogen heterocycle NAM mimetic can
(C. Sheng).
y
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2015.12.101
0960-894X/Ó 2015 Published by Elsevier Ltd.
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2
W. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Nampt inhibitor pharmacophoric model
Cap group
Connecting
unit
Linker
Tail group
(NAM mimic)
Cl
O
N
H
N
H
N
O
O
N
R
N
N
H
CN
N
CHS828 R = no substituent
FK866
O
GMX1777 R =
O
O
O
Cl
4
O
O
O
F
S
S
N
H
N
O
O
N
N
N
H
S
N
H
N
H
O
S
F
O
O
N
2
MS0
1
Figure 1. Pharmacophoric model and chemical structures of the Nampt inhibitors.
Table 1
be tolerated in the Nampt binding site. However, the in vitro
antitumor potency of inhibitor 2 was poor (Table 1) and its struc-
ture–activity relationships (SAR) is still unknown. Thus, a series of
novel benzothiophene Nampt inhibitors were designed, synthe-
sized and assayed.
Nampt inhibition and in vitro antitumor activity of target compounds
Compd
IC50 (lM)
Nampt
HCT116
MDA-MB-231
HepG2
7
8a
8b
8c
1.4 0.11
5.3 0.32
0.86 0.05
>10
>10
>10
0.95 0.08
1.1 0.12
1.0 0.09
>10
0.17 0.01
>10
159 11
169 21
>307
86 9.2
>401
196 15
>401
25 1.9
61 5.7
198 16
>320
>371
191 14
>307
>328
>401
>328
>401
36 2.8
10 1.2
>276
137 14
99 8.7
>307
80 9.0
>401
The synthetic routes of the target compounds were shown in
Schemes 1–3. As depicted in Scheme 1, commercially available
compound 3 was reacted with the substituted carboxylic acids in
the present of HBTU and Et3N to give target compounds 7, 8a–c,
and 9. Compounds 13a–b were synthesized according to the proce-
dures outlined in Scheme 2. Treatment of compound 3 and (tert-
butoxycarbonyl)glycine (10) via condensation reaction afforded
the key intermediate 11. After the removal of the Boc protecting
group of 11, amide 12 was coupled with carboxylic acids (5a–b)
to give the target compounds 13a–b. Finally, the compounds bear-
ing the imidazole tails (15a–b, 16a–c, 17) were synthesized using
the condition similar to that of compound 7 (Scheme 3). Condensa-
tion of 3-(1H-imidazol-1-yl)propan-1-amine (14) with carboxylic
acids afforded the target compounds 15a–b, 16a–c,and 17.
Our previously described fluorometric assay was used to evalu-
ate the Nampt inhibitory activity.15 As shown in Table 1, most of
the benzothiophene derivatives exhibited moderate to good anti-
Nampt activity. The replacement of the benzothiophene scaffold
with benzofuran (compound 7) led to significant decrease of the
Nampt inhibitory activity. The removal of the benzene ring of the
benzothiophene scaffold afforded the substituted thiophene
derivatives. Introduction of methyl group at the thiophene C-5
position yielded several good Nampt inhibitors. Compounds 8b,
9
13a
13b
15a
15b
16a
16b
16c
17
47 4.2
>401
5.2 0.45
11 1.3
102 12
3.9 0.33
60 5.5
>276
>320
>390
>276
>293
>390
>276
281 20
>10
0.15 0.02
2
82 7.9
Table 1, these compounds showed better activity against HepG2
than the other two cell lines. Moreover, most target compounds
exhibited better antiproliferative potency than lead compound 2.
Among them, compound 16b exhibited the best antiproliferative
activity against the HepG2 cell line (IC50 = 3.9 lM). Despite good
in vitro Nampt inhibitory potency, the 5-methylthiophenes 8b
and 13b lost activity for cancer cell lines. In consistent with the
Nampt inhibitory activities, compounds containing the pyridine
in cap group (9 and 17) were completely inactive in the antitumor
assays.
13b and 16b had an IC50 value of 0.86
respectively. Compound 16b showed comparable Nampt inhibi-
tory activity to lead compound 2 (IC50 = 0.15 M). However, com-
l
M, 0.95
l
M and 0.17
l
M,
Molecular docking studies were performed using GOLD 5.024 to
investigate the binding mode of compounds 2 and 16b with
Nampt. The crystal structure of Nampt in complex with FK866
was obtained from protein database bank (PDB ID: 2GVJ)25 and
prepared for molecular docking analysis in Discovery Studio 3.026
by removing the ligand from the binding pocket, merging nonpolar
hydrogens, adding polar hydrogens, and rendering Gasteiger
charges to each atom. Conformations were generated by genetic
algorithm and scored using GoldScore as fitness function. The best
conformation was chosen to analyze the ligand–protein interac-
tion. As depicted in Figure 2, the benzothiophene scaffold of com-
pound 2 was sandwiched between the side chains of Phe193 and
l
pounds with a nitro group at the C-5 position (8c and 16c) lost
their inhibitory potency in both biochemical and cell-based assays.
Pyridine is a classical cap group in the known Nampt inhibitors.
However, when benzothiophene was replaced by the meta
substituted pyridine, compounds 9 and 17 were inactive in the
anti-Nampt and antiproliferative assay. Thus, the SAR of the
benzothiophenes might be different from that of classical Nampt
inhibitors.
The synthesized compounds were also evaluated for antiprolif-
erative activities against the HCT116 (colon cancer cells), MDA-
MB-231 (breast cancer cells) and HepG2 (liver cancer cells) human
cancer cell lines using the standard MTT assay.23 As shown in
Tyr18 and formed
p–p interactions. The oxygen atom of amide
formed a hydrogen bond with the hydroxyl group of Ser275. The
phenyl and alkyl group interacted with Val242, Ala244 and
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W. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
COOH
O
H
N
4b
O
O
O
O
7
R
COOH
S
5a-c
H2N
O
O
a
R
H
N
8a R = 3-CH3
8b R = 5-CH3
8c R = 5-NO2
O
O
S
O
3
8a-c
COOH
O
O
N
6
N
H
9
O
N
Scheme 1. Reagents and conditions: (a) HBTU, Et3N, DMF, rt, 2 h, 64–83%.
O
O
O
O
O
H
N
a
H
N
Boc
OH
O
N
H
Boc
R
H2N
3
10
11
R
OH
O
O
S
O
O
O
O
H
N
b
5a-b
O
N
H
S
H2N
c
N
H
O
13a-b
12
13a R = 3-CH3
13b R = 5-CH3
Scheme 2. Reagents and conditions: (a) HBTU, Et3N, DMF, rt, 2 h, 85%; (b) CF3COOH, DCM, rt, 1 h, 70%; (c) HBTU, DIPEA, DMF, 2 h, 67–71%.
COOH
X
4a-b
15a X = S
15b X = O
H
N
N
N
X
O
15a-b
R
COOH
S
a
16a R = 3-CH3
R = 5-CH3
16c R = 5-NO2
R
H
5a-c
H2N
N
N
16b
N
N
N
S
14
O
16a-c
O
COOH
N
6
N
H
N
N
N
17
Scheme 3. Reagents and conditions: (a) HBTU, Et3N, DMF, rt, 2 h, 59–78%.
Ile309 mainly through hydrophobic and van der Waals interac-
tions. Compound 16b had similar binding mode to lead
compound 2. The thiophene intercalated into Phe193 and Tyr18
and formed face-to-face stacking interaction. Besides the
hydrogen bonding interaction between the amide oxygen atom
and Ser275, an additional hydrogen bond was observed between
the nitrogen atom of imidazole and His191. The alkyl group
attached to the imidazole formed hydrophobic and van der Waals
interactions with Ala244 and Ile309. The terminal imidazole group
was located at the outside of active site and faced to the solvent.
To investigate whether the target compounds can lead to cell
death by apoptosis, an annexin VFITC/propidium iodide (PI)
binding assay was performed. HepG2 cells were treated with
vehicle alone or compounds 15a and 16b at 5 M for 48 h, stained
a
l
with Annexin V-FITC and PI, and analyzed by flow cytometry. As
shown in Figure 3, both 15a and 16b caused significant induction
of apoptosis in the Hep2 cell line. The percentage of apoptotic cells
for 15a and 16b was 26.30% and 48.54% (Q2 + Q4), respectively.
The results revealed that compounds 15a and 16b could efficiently
induce apoptosis of HepG2 cells.
In summary, SARs of the novel benzothiophene Nampt inhibitor
was obtained. Several derivatives demonstrated good Nampt inhi-
bitory potency and antiproliferative activity. Especially, compound
16b was proven to a potent Nampt inhibitor, which also showed
p–p
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4
W. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Figure 2. Binding mode of compound 2 (A) and 16b (B) in the active site of Nampt (PDB ID: 2GVJ). The figure was generated using PyMol (http://www.pymol.org/).
Figure 3. Cell apoptosis induced by compound 15a and 16b. HepG2 cells were treated with DMSO and 5
cytometer (n = 3).
lM of compounds for 48 h. Apoptosis was examined by flow
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the HepG2 cell line. Taken together, the present study provides a
good starting point for the development of novel Nampt inhibitors.
Further structural optimization is in progress.
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Commission of Shanghai Municipality (Grant 14YF1405400).
Supplementary data
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characterization of target compounds) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/j.
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