Discovery of trans-3-(pyridin-3-yl)acrylamide-derived sulfamides as potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors for the potential treatment of cancer

Article abstract of DOI:10.1016/j.bmcl.2019.04.013

Nicotinamide phosphoribosyltransferase (NAMPT) has emerged as a promising target for the discovery of anticancer drugs. Based on NAMPT inhibitor FK866 that has been advanced into phase II trial, we identified a trans-3-(pyridin-3-yl)acrylamide compound 13 incorporating with a biarylsulfanilamide moiety as a new NAMPT inhibitor. Further structure-activity relationship (SAR) exploration led to additional biarylsulfanilamide-derived compounds with high in vitro NAMPT inhibitory potency and antiproliferative activity. In particular, compound 23, the most potent NAMPT inhibitor (IC₅₀ = 5.08 nM), showed single-digit nanomolar antiproliferative activity against DU145, Hela, and H1975 cells with IC₅₀ values of 2.90 nM, 2.34 nM, and 2.24 nM, respectively, and even subnanomolar level against K562, MCF-7, and HUH7 cells with IC₅₀ values of 0.46 nM, 0.23 nM and 0.53 nM, respectively. Our findings provided promising lead compounds for the discovery of more potent NAMPT inhibitors as anticancer drugs.

Full text of DOI:10.1016/j.bmcl.2019.04.013

Accepted Manuscript
Discovery of trans-3-(pyridin-3-yl)acrylamide-derived sulfamides as potent
nicotinamide phosphoribosyltransferase (NAMPT) inhibitors for the potential
treatment of cancer
Kuojun Zhang, Yong Ni, Jiaxuan Chen, Zhengchao Tu, Xiaoxing Wu, Dong
Chen, Hequan Yao, Sheng Jiang
PII:
S0960-894X(19)30222-7
https://doi.org/10.1016/j.bmcl.2019.04.013
BMCL 26380
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 February 2019
31 March 2019
6 April 2019
Please cite this article as: Zhang, K., Ni, Y., Chen, J., Tu, Z., Wu, X., Chen, D., Yao, H., Jiang, S., Discovery of
trans-3-(pyridin-3-yl)acrylamide-derived sulfamides as potent nicotinamide phosphoribosyltransferase (NAMPT)
inhibitors for the potential treatment of cancer, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://
doi.org/10.1016/j.bmcl.2019.04.013
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Discovery of trans-3-(pyridin-3-
yl)acrylamide-derived sulfamides as potent
nicotinamide phosphoribosyltransferase
(NAMPT) inhibitors for the potential
treatment of cancer
Leave this area blank for abstract info.
Kuojun Zhang, Yong Ni, Jiaxuan Chen, Zhengchao Tu, Xiaoxing Wu, Hequan Yao, Dong Chen, Sheng Jiang*
O
NAMPT IC50 = 5.08 nM
O
O
K562
IC50 = 0.46 nM
IC50 = 0.23 nM
IC50 = 0.53 nM
S
N
N
MCF-7
HUH-7
H
H
N
23

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery of trans-3-(pyridin-3-yl)acrylamide-derived sulfamides as potent
nicotinamide phosphoribosyltransferase (NAMPT) inhibitors for the potential
treatment of cancer
Kuojun Zhanga,b,c,§, Yong Nia,b,§, Jiaxuan Chenc,§, Zhengchao Tua, Xiaoxing Wuc, Dong Chenc, Hequan
Yaoc, Sheng Jiangc,*
a Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; bUniversity of Chinese Academy of Sciences,
Beijing 100049, China ; cState Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical
University, Nanjing, 210009, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Nicotinamide phosphoribosyltransferase (NAMPT) has emerged as a promising target for the
discovery of anticancer drugs. Based on NAMPT inhibitor FK866 that has been advanced into
phase II trial, we identified a trans-3-(pyridin-3-yl)acrylamide compound 13 incorporating with
a biarylsulfanilamide moiety as a new NAMPT inhibitor. Further structure-activity relationship
(SAR) exploration led to additional biarylsulfanilamide-derived compounds with high in vitro
NAMPT inhibitory potency and antiproliferative activity. In particular, compound 23, the most
potent NAMPT inhibitor (IC50 = 5.08 nM), showed single-digit nanomolar antiproliferative
activity against DU145, Hela, and H1975 cells with IC50 values of 2.90 nM, 2.34 nM, and 2.24
nM, respectively, and even subnanomolar level against K562, MCF-7, and HUH7 cells with IC50
values of 0.46 nM, 0.23 nM and 0.53 nM, respectively. Our findings provided promising lead
compounds for the discovery of more potent NAMPT inhibitors as anticancer drugs.
Keywords:
NAMPT inhibitor
Anticancer
Acrylamide
Biarylsulfanilamide
Antiproliferative activity
2009 Elsevier Ltd. All rights reserved.
Metabolic reprogramming has been considered as one of prototypical cancer hallmarks, which has sparked much interest in discovering
2
novel effective anticancer drugs targeting cancer metabolism.1, Among these targets, nicotinamide adenine dinucleotide (NAD)
metabolism has attracted much attention due to its indispensable role in sustaining cancer cell growth and malignant transformation.3
Nicotinamide adenine dinucleotide (NAD) served as an essential cofactor for multiple redox reactions as well as a consumable substrate
for sirtuins and poly(ADP-ribose) polymerase 1 (PARP1), which are intimately associated with energy metabolism and signaling
transduction processes.4-6 In mammals, NAD can be produced by de novo biosynthesis starting with tryptophan (Trp), and more
commonly by salvage pathways from various precursors, including nicotinic acid (NA), nicotinamide (NAM), and nicotinamide
riboside (NR).7 Considering that NAD-consuming enzymes, such as sirtuins and PARP1, consumes NAD while liberating nicotinamide
NAM as a byproduct, the NAM-dependent salvage pathway is the main and efficient source of NAD. In this biosynthetic process,
nicotinamide phosphoribosyltransferase (NAMPT) is the first rate-limiting enzyme, which facilitates the condensation reaction between
NAM and phosphoribosyl pyrophosphate (PRPP) to generate nicotinamide mononucleotide (NMN). Subsequently NMN is converted
into NAD catalyzed by nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT). Cancer cells have accelerated growth
rates and an elevated energy demand than normal cells and thus are more dependent on NAMPT to maintain required NAD levels. In
addition, NAD and its reduced form NADH play a critical role in maintaining the redox balance of the tumor microenvironment,
protecting cells from reactive oxygen species (ROS) that are increased in cancers. Due to the great importance of NAD in cancer
biological processes, various cancer cells are highly dependent on NAMPT activity for survival, proliferation, and malignant
transformation. Importantly, normal cells can undergo an alternative nicotinic acid phosphoribosyltransferase (NAPRT)-mediated
pathway for NAD synthesis from nicotinic acid (NA), therefore protecting themselves from NAMPT inhibition,8, 9 while most cancer
cells lack the NAPRT activity.10, 11As such, NAMPT has emerged as a promising target for the development of selective and effective
anticancer drugs.
To date, a number of NAMPT inhibitors with diverse structures have been reported (Figure 1). Among these compounds, FK866
(also known as APO866, 1)12, 13 and CHS828 (also known as GMX1778, 2),14 the most advanced NMAPT inhibitors, have entered into
phase
II
trials.
However,
their
* Corresponding author E-mail address: jiangsh9@gmail.com (S. Jiang); § These authors contributed equally to this work.

O
Cl
H
H
N
O
N
N
O
N
O
O
S
N
H
N
N
N
NH
N
O
1
(FK866/APO866)
2
3
(CHS828/GMX1778)
F
O
O
O
H
N
N
H
NH
S
O
N
N
H
N
N
N
N
O
F
O
N
N
N
4
6
5
O
O
O
S
HN
N
N
N
NH
N
O
H
N
O
H
N
O
O
N
S
N
7
N
N
O
N
9
8
O
O
N
Cl
NH
N
O
O
O
O
S
NH
N
N
NH
N
N
N
H
O
S
H
H
N
H
N
O
CF3
NH
O
10
11
12
Figure 1．Examples of reported NAMPT inhibitors.
Figure 2. Discovery of novel trans-3-(pyridin-3-yl)acrylamide-containing NAMPT inhibitors and overlay of the binding pose of FK866 (yellow) and compound
13 (green carbons) in complex with NAMPT (2GVJ).
clinical applications have been limited owning to short half-life in circulation and dose-limiting thrombocytopenia and gastrointestinal
toxicity.15 Previous efforts by our group have identified highly potent trans-3-(pyridin-3-yl)acrylamide-derived NAMPT inhibitors
which also contained terminal indole groups (compound 12) endowed with outstanding efficiency in tumor xenograft model and
desirable in vivo preclinical pharmacokinetic properties.16 In the current work, we describe our continuous efforts leading to the
discovery of novel and highly potent trans-3-(pyridin-3-yl)acrylamide-containing NAMPT inhibitors.
Generally, the pharmacophore model of the NAMPT inhibitors is composed of four modules: a cap group mimicking NAM, a
connecting unite, a linker and a tail group.17 The cap group combined with the connecting unit constitutes the core which is
indispensable for NAMPT enzyme inhibition. The linker should have an appropriate length and geometry, allowing the tail group to
protrude toward the solvent-exposed surface and to project into the hydrophobic cleft in the rim of the enzyme. The tail group plays a
critical role in the enzyme inhibition due to its capability of making hydrophobic interactions with the hydrophobic cleft. Moreover,
diversified structures can fit into this cleft well. From the cocrystal structure of FK866 (1) complexed with NAMPT (PDB ID: 2GVJ18),
we found that the hydrophobic cleft can accommodate a larger group than the benzoylpiperidine ring. Accordingly, we retained the core
group and linker of 1 while attempting to replace the benzoylpiperidine
Table 1. In vitro NAMPT inhibitory activity of target compounds with different linker.

O
N
H
n
H
N
S
N
O
O
13 17
-
N
H
H
N
N
S
O
n
O
O
18 21
-
Compd
13
14
15
16
17
18
19
20
n
2
1
3
4
5
1
3
4
5
/
NAMPT IC50 (nM)
17.77±2.13
5920±532
28.39±1.99
14.42±0.72
16.41±1.32
234.50±23.50
16.54±1.35
10.53±0.97
31.32±3.47
6.80±0.70
21
FK866
aFK866 was used as the positive control. Values are means of three experiments.
ring with biarylsulfanilamide motif to design compound 13 (Figure 2A). As shown in Figure 2B, the docked pose of the resulting
molecule 13 overlapped well with the pyridine portion of docked FK866. And the biarylsulfanilamide motif can fit well into the tail
hydrophobic pocket as depicted in Figure 2C. Compound 13 was therefore synthesized, and demonstrated very good biochemical anti-
NAMPT potency (Table 1). Thus, this molecule represents a good hit compound for further medicinal chemistry optimization.
As mentioned above, the length and position of the linker installed is very important for the appropriate positioning of cap group and
tail group. Given that biarylsulfanilamide motif can fit into the cleft of enzyme, we next explored whether other length linker are
allowed, leading to the synthesis of compounds 14-17. Further, we examined the position where the linker was connected to the tail
benzene group by synthesizing compounds 18-21. Compounds 14-21 were tested for in vitro NAMPT enzyme inhibitory activity with
FK866 as the positive control, and all these compounds showed weaker NAMPT inhibition versus FK866. As expected, the length of
the linker is very important for NAMPT enzyme inhibition, and the six-methylene spacer was the best for inhibitory potency
(compounds 16 and 20). As shown in Table 1, the increase of the linker length has little influence on potency, while shortening the
distance to three-methylene spacer significantly reduced NAMPT inhibition (compounds 14 and 18). Generally, better inhibitory
potency was observed for the compounds linked to the meta-position of sulfanilamide group than those attached to the para-position of
sulfanilamide.
With these in hand, our study investigated various substituents on the tail benzene ring of molecule 20, the most potent compound
among this series. As shown in Table 2, the substituents have little effect on NAMPT inhibition. The benzene ring substituted by
methyl group at the ortho-position resulted in the most active compound 23 (IC50 = 5.08 nM), which was selected as the lead compound
because it showed better activity than the positive control FK866 (IC50 = 6.80 nM).
Table 2. Biarylsulfanilamide SAR of NAMPT inhibitors.
H
O
N
S
O
NH
R
O
N
Compd
22
23
24
25
26
27
28
29
R
4-CH3
2-CH3
3-CH3
2,5-CH3
4-CH3CH2
4-C(CH3)3
4-Cl
NAMPT IC50 (nM)
9.78±0.89
5.08±0.52
12.28±1.13
11.63±1.13
31.54±1.67
9.47±0.75
10.75±0.86
9.20±0.83
4-F
30
31
32
FK866
3-Cl-4-F
4-CF3
4-OPh
/
9.70±0.95
24.53±2.06
13.09±0.86
6.80±0.70
aFK866 was used as the positive control. Values are means of three experiments.

All the newly-prepared compounds were assessed on the antiprolifrative activities against the K562 (human myelogenous leukemia),
MCF-7 (human breast cancer cells), DU145 (human prostate cancer), Hela (human cervical carcinoma), H1975 (human non-small lung
cancer), and HUH7 (human liver cancer) using the CCK8 (Cell Counting Kit-8) assay,19 with FK866 as positive control. As shown in
Table 3, these compounds are most sensitive to K562 and MCF-7 cells, but least sensitive to A431 and A549 cells. Although these
compounds exhibited poor efficiency against A549 cells, they seemed to be sensitive to H1975 cells that is a type of human non-small
lung cancer cells harboring the EGFRL858R/T790M mutation. Hence, they may represent promising lead compounds for the discovery of
novel alternative therapeutic agents for the third-generation EGFR inhibitors in the treatment of EGFR-gene-mutated non-small lung
cancer.
Importantly,
compound
23,
the
most
potent
compound
Table 3. Antiproliferative activities of all the target compounds.a,b
IC50 (nM)
Compd
K562
9.56±0.78
4902±90
MCF-7
5.03±0.45
4120±68
DU145
47.90±4.82
>10000
Hela
184.00±16.60
>10000
H1975
63.60±5.28
>10000
HUH7
24.20±2.36
>10000
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
145.90±13.3
48.50±4.85
56.50±5.10
86.80±8.10
16.25±1.56
0.88±0.07
61.10±5.49
2.98±0.25
0.46±0.03
4.78 ±0.39
0.84±0.07
3.89±0.31
24.7±1.98
6.41±0.53
0.93±0.08
5.31±0.45
44.9±2.79
5.12±0.42
0.96±0.08
95.70±8.9
36.00±3.80
37.60±3.37
142.0±13.7
12.49±1.09
0.63±0.07
42.60±3.83
1.97±0.17
0.23±0.01
4.00±0.29
0.60±0.05
3.64±0.29
26.3±2.38
5.94±0.49
0.91±0.09
6.30±0.51
67.8±6.01
3.84±0.31
0.41±0.04
581±52
333±28
778±39
239.00±17.30
330.00±33.80 1225.00±102.50
408±86
112.00±9.70
328.00±29.80
438.00±40.10
165.00±14.20
140.00±9.70
6.73±0.54
429.00±25.70
34.40±3.10
2.90±0.17
377.00±30.70
223.00±20.60
115.00±10.20
105.00±8.37
30.40±2.82
598.00±33.9
38.80±2.82
2.34±0.22
65.80±5.62
22.60±1.13
132.00±11.90
1520.00±61.80 157.30±10.80
170.00±13.90
99.90±6.99
261.00±28.70
146.00±11.20
6.37±0.59
316.00±18.5
17.10±1.26
2.24±0.18
40.70±3.26
4.88±0.43
33.40±2.69
384.00±35.60
41.10±3.86
2.82±0.21
138.00±10.60
6.92±0.56
0.53±0.03
13.20±1.19
2.58±0.21
34.60±2.77
163.00±9.88
24.10±2.17
4.28±0.38
39.40±2.56
164.00±14.10
32.70±1.98
1.05±0.09
41.70±2.91
7.48±0.56
40.10±2.81
281.00±19.70
84.40±5.98
10.60±0.86
150.00±11.70
1410.00±75.70 1450.00±71.10 174.60±12.60
50.90±4.70
5.12±0.48
33.30±2.76
1.34±0.12
25.00±1.57
385.00±30.10
31
32
FK866
262.00±19.30
3.75±0.27
25.60±2.18
4.76±0.32
aFK866 was used as the positive control. bInhibition of cell growth by the listed compounds was determined by using CCK-8 assay.
b
c
H
H
CH2OH
CHO
a
H
COOH
COOCH3
N
N
N
S
S
S
R
R
NaO3S
O
O
O
O
R
O
O
35
36
33
34
OEt
OH
OEt
H
N
H
n
O
d
H
e
f
n
N
n
N
S
S
S
O
R
R
O
O
O
O
O
R
O
O
O
O
N
S
OEt
N
n
39
37
38
N
N
42
Ph
N
NH2
NH
H
H
n
n
N
g
N
h
S
S
O
R
R
O
O
O
O
40
13 32
-
Scheme 1. Synthesis of compounds 13-32. Reagents and conditions: (a) (i) CH3OH, sulfoxide chloride, 0 °C to reflux; (ii) sulfoxide chloride, DMF, toluene, 0 to
80 °C; (iii) anilines 41, toluene, rt, 67% in three steps; (b) KBH4/LiCl, anhydrous THF, reflux, 89% ; (c) silica gel, PCC, DCM, 73%; (d) 42, NaHMDS, THF, -20
°C, 30%; (e) Pd/C, H2, CH3CH2OH, 83%; (f) KBH4/KCl, anhydrous THF, reflux, 65%; (g) (i) MsCl, triethylamine, DCM, 0 °C, (ii) NaN3, DMF, 80 °C, (iii)
Pd/C, H2, CH3CH2OH, 50% in three steps; (h) (E)-3-(pyridin-3-yl)acrylic acid (43), EDCI, HOBt, triethylamine, DMF, rt, 80%.
in the aspect of enzyme inhibition, showed outstanding anti-prolifrative activities against most cell lines with IC50 values in the
nanomolar range.
An in silico study of synthesized compounds that possessed potent NAMPT inhibition was performed for prediction of
physicochemical properties exploring Accelrys Discovery Studio 3.0 (Table S1). From the predicted results, we can see that some of
them exhibited comparable or improved physicochemical properties versus FK866.
The synthetic routes for the target compounds were depicted in Scheme 1. Commercially available sodium benzenesulfonate 33 was
methylated to give corresponding benzoic ester, which was condensed with various anilines 41 via sulfuryl chloride intermediate to
provide the corresponding sulfonamides 34. Reduction of 34 with potassium borohydride (KBH4) in the presence of lithium chloride
(LiCl) provided alcohols 35, which was followed by PCC oxidation to afford aldehydes 36. Employing a modified-Julia procedure,

aldehydes 36 were coupled with 1-phenyl-1H-tetrazole-phenyl sulfone (42) in the presence of sodium bis(trimethylsilyl)amide
(NaHMDS) to yield the key intermediate 37. Compounds 39 were obtained through two-step reductive reactions, i.e., hydrogen
reduction of double bond under the Pd/C catalysis and further reduction of ester group using the KBH4/KCl system. Subsequent
transformation of alcohols 39 int amines 40 involved a three-step sequence: (i) mesylation of alcohol 39, (ii) installation of the azide
groud, and (iii) hydrogen reduction via Pd/C catalyst. Coupling amines 40 with (E)-3-(pyridin-3-yl)acrylic acid (43) in the presence of
1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDCI) and 1-hydroxybenzotriazole (HOBt) under basic condition afforded target
compounds 13-32.
In conclusion, we identified novel and potent trans-3-(pyridin-3-yl)acrylamide-derived biarylsulfanilamide containing NAMPT
inhibitors. Several of these compounds exhibited nanomolar antiproliferative activity against human tumor lines. In particularly,
compound 23 that was the most potent NAMPT inhibitor (IC50 = 5.08 nM) among all the newly synthesized compounds showed single-
digit nanomolar antiproliferative activity against DU145, Hela, and H1975 cells with IC50 values of 2.90 nM, 2.34 nM, and 2.24 nM,
respectively. More importantly, 23 exhibited outstanding activities against K562, MCF-7, and HUH7 cells within subnanomolar range
(IC50 = 0.46 nM, 0.23 nM and 0.53 nM, respectively). Taken together, this study provides a good starting point for the development of
novel NAMPT inhibitors, and further structural optimization and biological assays are in progress.
Acknowledgments
This work was supported by the National Natural Science Foundation (81773559, 21472191, 21672050), the Double First-Class
University Project (CPU2018GY03), the International Cooperation Special Grant (2016A050502036) from the Science and Technology
Development Project of Guangdong Province, the International Cooperation Grant (201704030099) of Guangzhou, the Science and
Technology Planning Project of Guangdong Province (2013A022100019), the Project of State Key Laboratory of Natural Medicines,
China Pharmaceutical University (SKLNMZZRC201810), Chinese Pharmaceutical Association-Yiling Biopharmaceutical Innovation
Foundation.
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19. Cell lines, K562, MCF-7, DU145, Hela, A549, H1975, HUH7 and A431 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells
were routinely grown and maintained in RPMI or DMEM media with 10% FBS and 1% penicillin/streptomycin. All cell lines were incubated in a
Thermo/Forma Scientific CO2 water jacketed incubator with 5% CO2 in air at 37 °C. Cell viability assay was determined by the CCK8 (DOjinDo, Japan)
assay. Cells were seeded at a density of 400-800 cells/well in 96-well plates and treated with various concentration of tested compounds or vehicle. After 72
h incubation, CCK8 reagent was added and absorbance was measured at 450 nm using Envision 2104 multilabel reader (Perkin Elmer, USA). Dose response
curves were plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc. USA).
Supplementary Material
Supplementary data (experimental protocols and structural characterization of target compounds) associated with this article can be
found, in the online version.

Graphical Abstract
.
Discovery of trans-3-(pyridin-3-
yl)acrylamide-derived sulfamides as potent
nicotinamide phosphoribosyltransferase
(NAMPT) inhibitors for the potential
treatment of cancer
Leave this area blank for abstract info.
Kuojun Zhang, Yong Ni, Jiaxuan Chen, Zhengchao Tu, Xiaoxing Wu, Hequan Yao, Dong Chen, Sheng Jiang*
O
NAMPT IC50 = 5.08 nM
O
O
K562
IC50 = 0.456 nM
IC50 = 0.226 nM
IC50 = 0.525 nM
S
N
N
MCF-7
HUH-7
H
H
N
23