Discovery of 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea as a potent NAMPT (nicotinamide phosphoribosyltransferase) activator with attenuated CYP inhibition

Article abstract of DOI:10.1016/j.bmcl.2021.128048

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the NAD⁺ salvage pathway. Since NAD⁺ plays a pivotal role in many biological processes including metabolism and aging, activation of NAMPT is an attractive therapeutic target for treatment of diverse array of diseases. Herein, we report the continued optimization of novel urea-containing derivatives which were identified as potent NAMPT activators. Early optimization of HTS hits afforded compound 12, with a triazolopyridine core, as a lead compound. CYP direct inhibition (DI) was identified as an issue of concern, and was resolved through modulation of lipophilicity to culminate in 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea (21), which showed potent NAMPT activity accompanied with attenuated CYP DI towards multiple CYP isoforms.

Full text of DOI:10.1016/j.bmcl.2021.128048

Bioorg. Med. Chem. Lett. 43 (2021) 128048
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]
pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea as a potent NAMPT (nicotinamide
phosphoribosyltransferase) activator with attenuated CYP inhibition
,
Mayuko Akiua *, Takashi Tsujia, Yoshitaka Sogawaa, Koji Terayamaa, Mika Yokoyamaa,
Jun Tanakaa, Daigo Asanoa, Ken Sakuraia, Eduard Sergienkob, E. Hampton Sessionsb,
Stephen J. Gardellc, Anthony B. Pinkertonb, Tsuyoshi Nakamuraa
a R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
c Translational Research Institute, AdventHealth, Orlando, FL 32804, USA
A R T I C L E I N F O
A B S T R A C T
Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the NAD+ salvage pathway.
Since NAD+ plays a pivotal role in many biological processes including metabolism and aging, activation of
NAMPT is an attractive therapeutic target for treatment of diverse array of diseases. Herein, we report the
continued optimization of novel urea-containing derivatives which were identified as potent NAMPT activators.
Early optimization of HTS hits afforded compound 12, with a triazolopyridine core, as a lead compound. CYP
direct inhibition (DI) was identified as an issue of concern, and was resolved through modulation of lipophilicity
to culminate in 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea
(21), which showed potent NAMPT activity accompanied with attenuated CYP DI towards multiple CYP
isoforms.
Keywords:
NAMPT activators
NAD+
Triazolopyridines
CYP inhibition
LogD
Introduction
which involves the hydrolysis of linkage between nicotinamide (NAM)
and the ADP-ribosyl moieties. NAD+ is synthesized by two major path-
Nicotinamide adenine dinucleotide (NAD+) is a key cellular factor
which plays an important role in intermediary metabolism.1,2 NAD+
possesses a nicotinamide moiety as the site of a redox reaction, which
makes it an important coenzyme for hydride transfer enzymes essential
for metabolic processes including glycolysis, the TCA cycle and oxida-
tive phosphorylation.3–7 NAD+ is also an important cosubstrate for
certain enzymes, such as sirtuins (SIRTs), poly (ADP-ribose) poly-
merases (PARPs), and cyclic ADP-ribose (cADPR) synthases (CD38 and
CD157).3–7 Based on these multi-faceted functions, several studies sug-
gest that increasing tissue levels of NAD+ mediate beneficial effects to-
ward wide range of diseases, such as metabolic disorders,8
ways; the de novo pathway originating from tryptophan, and via salvage
pathway from NAM, which is the dominant pathway in most
mammals.3–7 In the salvage pathway, NAM is converted to NAM
mononucleotide (NMN) by the enzyme nicotinamide phosphoribosyl-
transferase (NAMPT), the putative rate-limiting step of this salvage
pathway,1–5 which is a homo-dimeric type II phosphoribosyl trans-
ferase.23 NMN is subsequently converted into NAD+ by various nico-
tinamide mononucleotide adenylyltransferases (NMNATs).3–7 In one
study, overexpression of NAMPT in skeletal muscle resulted in an in-
crease in NAD+ levels, leading to improved exercise endurance and
metabolic abnormalities in mice.22 Accordingly, activation of NAMPT
enzyme would elicit elevation of NAD+ level ultimately leading to
modulation or enhancement of metabolism, and thus would offer ther-
apeutic avenues for multiple diseases associated with NAD+ deficiency.
Herein, we report the further optimization of series of compounds pos-
sessing a (pyridin-4-ylmethyl) urea moiety.
cardiovascular and kidney disease,9–11 mitochondria disease,12 neuro-
degeneration,13–15 stroke,
wasting disorder.20–22
retinal degeneration,18,19 or muscle
16,17
Ongoing NAD+ biosynthesis is necessary to preserve the NAD+ level,
given that NAD+ as a cosubstrate contributes as a donor of ADP-ribose,
* Corresponding author.
E-mail address: akiu.mayuko.bs@daiichisankyo.co.jp (M. Akiu).
https://doi.org/10.1016/j.bmcl.2021.128048
Received 8 February 2021; Received in revised form 9 April 2021; Accepted 14 April 2021
Available online 19 April 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

M. Akiu et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128048
Table 2
Exploration of 6–5 bicyclic cores.
O
CO2Et
O
O
N
H
N
N
N
N
H
H
N
HTS hit 2
HTS hit 1
EC50 (Emax) = 0.53~1.4 M (33%)
μ
EC50 (Emax) = 3.3~3.7 M (75%)
μ
Cpd
R
NAMPT Enzyme
Cell-based assaya
Hybridize the two
structural features
This Work
Derivatization
assay (EC50
Emax)
/
(HEK293A) (%@1, 3, 10
μM)
O
S
O
R2
R
O
N
9
0.51
μM/ 103%
73, 107, 131
R1
X
N
H
N
H
O
N
N
Y
N
H
N
H
3
10
11
12
0.040
0.012
0.083
μM/ 155%
μM/ 135%
μM/ 131%
121, 150, 176
115, 148, 145
130, 139, 161
N
O
O
S
O
Ar
N
H
N
H
N
4
Potent NAMPT activators
(previously reported)
Fig. 1. Design principles of novel NAMPT activators.
a
Percentage indicates the amount of NAD+ increase achieved with 1, 3, 10
μM
of the compound, compared to that achieved with 30 μM of compound 1.
Table 1
Initial SAR exploration.
Table 3
Physicochemical and in vitro ADME properties of selected 6–5 bicyclic analogs.
Cpd
logDa
PAMPA
Solubility
Metabolic
Stabilityb (h,
mou) (%)
CYP DIc
(1A2/
permeability (pH
5.0, pH 7.4) (nm/
s)
(pH 1.2, 6.8)
(μ
g/mL)
2C9/
2D6/
Cpd
R
X
Y
NAMPT
Cell-based assaya
(HEK293A) (%
3A4) (%)
Enzyme assay
(EC50
/
@1, 3, 10
μ
M)
10
11
12
3.2
3.5
3.0
46.0, >50
>50, >50
14.2, 27.6
Insolubled
640, 0.5
87, 83
83, 91
95, 86
88/ 74/
50/ 75
81/ 78/
70/ 53
63/ 79/
21/ 37
Emax)
5
N
CH
63%@100
NTc
μ
Mb
>680, 92
a
The distribution coefficients (logD) were measured between 1-octanol and
6
7
N
CH
0.48
91%
μ
μ
M/
M/
25, 45, 62
phosphate buffered saline (pH 7.4).
b
The % remaining value at 1 μM concentration of compounds reacted with
human or mouse microsomes for 30 min.
c
The % inhibition value at 10 μM concentration of compounds reacted with
corresponding CYP isoforms for 10 min.
d
N
CH
N
0.22
55%
44, 76, 89
NTc
Solubility could not be determined because the compound was insoluble to
DMSO.
Table 4
8
CH
IC50 0.032
Exploration of left-hand region of triazolopyridines.
μM
a
Percentage indicates the amount of NAD+ increase achieved with 1, 3, 10
μM
of the compound, compared to that achieved with 30
μ
M of compound 1.
Cpd
R
NAMPT Enzyme
Cell-based assaya
CYP DIb (1A2/
2C9/2D6/
b
Percentage indicates the amount of NAMPT activation achieved with 100
assay (EC50
Emax)
/
(HEK293A) (%@1, 3,
μ
M of the compound, compared to that achieved with 30
μ
M of compound 1.
10
μM)
3A4) (%)
c
Not tested.
13
0.022
μ
M/ 217%
118, 154, 174
86/ 59/ 23/ 0
Our HTS campaign, which utilized a fluorescence thermal shift assay
14
0.14 μM/ 87%
103, 123, 138
69/ 59/ 13/ 0
followed by an enzymatic assay using purified NAMPT, led to the
identification of urea compound 1 as well as proline compound 2 as hit
compounds that led to a moderate enhancement of NAMPT activity.24
The NAMPT activation activity of the compounds were determined by
measuring the EC50 and Emax (maximal NAMPT activation) as
described previously.24,25 While the urea compound 1 exhibited a fairly
high Emax of 75%, the proline compound 2 was more potent, with an
a
Percentage indicates the amount of NAD+ increase achieved with 1, 3, 10
μM
of the compound, compared to that achieved with 30
μ
M of compound 1.
b
The % inhibition value at 10 μM concentration of compounds reacted with
corresponding CYP isoforms for 10 min.
2

M. Akiu et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128048
Table 5
Exploration of right-hand region of triazolopyridines.
Cpd
R
NAMPT Enzyme assay (EC50 / Emax)
0.070 M/ 120%
Cell-based assay a (HEK293A) (%@1, 3, 10
108, 115, 99
μ
M)
CYP DIb (1A2/ 2C9/ 2D6/ 3A4) (%)
43/ 78/ 50/ 65
logDc
15
μ
3.4
16
0.35
μM/ 124%
61, 103, 127
28/ 90/ 36/ 0
4.3
17
18
>100
μ
M
NTd
8/ 81/ 24/ 0
NTd
4.4
5.8
2.4
μ
M/ 101%
M/ 75%
0, 32, 55
NTd
19
20
μ
16, 29, 69
NTd
NTd
2.6
0.12
0.18
0.19
μ
μ
μ
M/ 153%
M/ 132%
M/ 117%
88, 115, 150
51/ 75/ 27/ 40
21
22
71, 107, 150
102, 135, 133
15/ 42/ 16/ 25
12/ 79/ 17/ 4
1.8
2.8
23
0.083
μ
M/ 105%
73, 108, 88
6/ 44/ 13/ 18
1.5
a
Percentage indicates the amount of NAD+ increase achieved with 1, 3, 10
μM of the compound, compared to that achieved with 30 μM of compound 1.
b
c
The % inhibition value at 10 μM concentration of compounds reacted with corresponding CYP isoforms for 10 min.
The distribution coefficients (logD) were measured between 1-octanol and phosphate buffered saline (pH 7.4).
d
Not tested.
EC50 range (0.53–1.4
μ
M) but a lower Emax. We previously reported
phenyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-3-(pyridin-4-ylmethyl)urea
(12) and 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyr-
idin-6-yl]-3-(pyridin-4-ylmethyl)urea (21) with a triazolopyridine core
structure, which displayed potent activities in NAMPT enzyme and cell-
based assays (Fig. 1).
intensive derivatization around 1, which led to the discovery of potent
NAMPT activators such as sulfonamides 3 and sulfones 4.24–28 Concur-
rently, we conducted an initial structure activity relationship (SAR)
study around the HTS hit 2, but the SAR was narrow, with only minor
structural variations resulting in a loss of activity (data not shown). We
therefore attempted to hybridize the two structural features of 1 and 2,
in anticipation of acquiring a potent novel scaffold. Herein, we report
the results of those efforts, which culminated in the discovery of 1-(2-
The test compounds in Tables 1–5 were synthesized following the
general procedures described in Schemes 1-6.
The general procedure for the synthesis of triazolopyridine de-
rivatives is depicted in Scheme 1. The triazolopyridine core structure of
N
Scheme 1. Synthesis of tiazolopyridine derivatives.
O
N
f
R
R
N
Reagents and conditions: (a) Ethyl O-mesitylsulfo-
N
N
N
N
H
N
H
H2
N
nylacetohydroxamate, pyridine, perchloric acid,
dichloromethane, 91%; (b) RCOCl, pyridine, 80 ◦C,
36–71%; or RCO2H, EDCI, HOAt, triethylamine,
dichloromethane, 50–89%; (c) 2 M NaOHaq, meth-
anol, THF, 65%-quant.; (d) DPPA, triethylamine,
dioxane, 80 ◦C, then 4-picolylamine, 14–39%; (e)
DPPA, triethylamine, DMF, then H2O, 80 ◦C,
23–29%; (f) 4-Nitrophenyl [(pyridin-4-yl)methyl]
carbamate,30 DIPEA, 1,4-dioxane, 80 ◦C, 38–61%;
(g) DPPA, triethylamine, CH2Cl2, then t-butylalco-
hol, toluene, 100 ◦C, 74%; (h) 4 M HCl in dioxane,
CH2Cl2; (i) 4-Nitrophenyl [(pyridin-4-yl)methyl]
carbamate, DIPEA, 1,4-dioxane, 80 ◦C, 67% over
two steps.
N
27
15, 19
e
O
N
N
N
N
O
N
N
c
a, b
d
R
R
OMe
R
N
N
N
N
H
N
H
MeO2C
HO2C
H2
N
N
N
26
25
24
12, 16-18, 22, 23
g
N
N
O
N
N
O
h, i
N
N
N
H
N
H
N
N
O
N
H
N
20
28
3

M. Akiu et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128048
Scheme 2. Synthesis of compound 21. Re-
O
O
agents and conditions: (a) Ethoxycarbonyl
N
N
N
N
MeO
S
O
OMe
a
b
c
Br
NH2
isothiocyanate, dioxane, 50 ◦C, 66%; (b)
N
N
N
N
H
N
H
OEt
MeO2C
H2N
N
MeO2C
Hydroxylammonium chloride, DIPEA, meth-
anol, ethanol, 87%; (c) t-Butyl nitrite, copper
(II) bromide, acetonitrile, 50 ◦C, 58%; (d) 1-
31
30
24
29
Methyl-1H-pyrazole-5-boronic acid pinacol
N
N
N
O
N
N
N
N
N
[1,1′-bis(diphenylphosphino)ferro-
N
N
N
N
O
ester,
g, h
N
e, f
d
N
H
N
H
N
N
cene]palladium(II)
dichloride
dichloro-
O
N
H
MeO2C
N
methane adduct, potassium carbonate,
dioxane, H2O, 60 ◦C, 45%; (e) 2 N NaOHaq,
methanol, THF, 60%; (f) DPPA, triethyl-
amine, CH2Cl2, then t-butylalcohol, toluene,
21
32
33
100 ◦C, 76%; (g) 4 M HCl in dioxane, CH2Cl2; (h) Triphosgene, triethylamine, 4-picolylamine, dichloromethane, 29% over two steps.
O2N
N
N
N
N
N
N
O
a
b
O
N
N
N
H2N
N
H
N
O
N
N
N
H
H
H
27a
13
34
N
N
c
O
N
N
H
N
H
N
O
14
Scheme 3. Synthesis of compounds 13 and 14. Reagents and conditions: (a) 4-Nitrophenyl chloroformate, toluene, 80 ◦C; (b) 1H-Pyrazol-4-ylmethylamine dihy-
drochloride, DIPEA, methanol, 23% over two steps; (c) Triphosgene, DIPEA, dichloromethane, then oxazol-5-ylmethylamine, 28%.
CHO
NO2
O
a
c
b
N
N
N
N
N
N
H
N
H
N
Br
Br
H2
N
N
36
37
10
35
Scheme 4. Synthesis of compound 10. Reagents and conditions: (a) Tri-n-butylphosphine, aniline, 2-propanol, 80 ◦C, 76%; (b) Benzophenone imine, palladium
acetate, 4,5- bis(diphenylphosphino)-9,9-dimethylxanthene, cesium carbonate, 1,4-dioxane, 100 ◦C then 2 M HCl, THF, 40%; (c) 4-Nitrophenyl [(pyridin-4-yl)
methyl]carbamate, triethylamine, 1,4-dioxane, 80 ◦C, 64%.
Scheme 5. Synthesis of compound 11. Reagents
Br
Br
and conditions: (a) LiHMDS, THF, ꢀ 78 ◦C, then
methyl benzoate, 43%; (b) Hydroxylammonium
chloride, sodium hydroxide, methanol, 60 ◦C, 47%;
(c) Trifluoroacetic anhydride, triethylamine,
dichloromethane, 85%; (d) Iron (II) chloride,
dimethoxyethane, 90 ◦C, 75%; (e) Benzophenone
Br
Br
OH
O
N
b
a
c
d
N
N
N
N
N
38
41
39
40
imine,
palladium
acetate,
4,5-
bis
O
e
f
N
N
N
(diphenylphosphino)-9,9-dimethylxanthene, cesium
carbonate, 1,4-dioxane, 100 ◦C then 2 M HCl, THF,
58%; (f) 4-Nitrophenyl [(pyridin-4-yl)methyl]
carbamate, triethylamine, 1,4-dioxane, 80 ◦C, 60%.
N
Br
N
N
N
H
N
H
H2N
42
N
43
11
methyl ester 25 was constructed by amination of the pyridine nitrogen
atom of compound 24,29 which was followed by condensation with
appropriate acyl chlorides, either purchased or generated by reacting
the corresponding carboxylic acid with thionyl chloride, or by conden-
sation with the corresponding carboxylic acid utilizing EDCI as a
coupling reagent. Compound 25 was then hydrolysed to carboxylic acid
26. Subsequent Curtius rearrangement of 26, followed by in situ reaction
of the corresponding isocyanates with 4-picolylamine afforded urea
compounds 12, 16–18, 22, and 23. As an alternative pathway, carbox-
ylic acid 26 was transformed to either aniline 27 or Boc-protected ani-
line 28 via a Curtius rearrangement, which was further reacted with 4-
nitrophenyl [(pyridin-4-yl) methyl]carbamate30 to give the corre-
sponding urea compounds 15, 19, and 20.
thiourea 29, which was subsequently condensed with hydrox-
ylammonium chloride to give 30.29 In order to install the right-hand
pyrazole ring, the amino group of 30 was converted to the bromine by
a Sandmeyer reaction, followed by Suzuki coupling with 1-methyl-1H-
pyrazole-5-boronic acid pinacol ester. Subsequent basic hydrolysis and
a Curtius rearrangement yielded Boc-protected aniline 32. Deprotection
of the Boc group, followed by urea formation by triphosgene and 4-pico-
lylamine afforded the final compound 21.
Scheme 3 depicts the synthesis of compounds 13 and 14. As for
compound 13 with a pyrazole group, aniline 27a was reacted with
commercially available 4-nitrophenyl chloroformate to afford the
carbamate intermediate 34, which was coupled with 1H-pyrazol-4-
ylmethylamine to afford the final product 13. Synthesis of compound
14 with an oxazole group was accomplished by treating aniline 27a with
triphosgene followed by coupling with oxazol-5-ylmethyl amine.
Scheme 2 illustrates the synthesis of pyrazole analog 21. The tri-
azolopyridine core structure was constructed from pyridine 24 via
4

M. Akiu et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128048
Scheme 6. Synthesis of compounds 5–9. Reagents and conditions: (a)
4-Methylbenzyl bromide, sodium hydride, DMF, ice bath to rt; (b) Iron
powder, 2 M HCl, ethanol, THF, 80 ◦C, 29–44% over two steps; (c)
Triphosgene, triethylamine, 4-picolylamine or 3-picolylamine,
dichloromethane, 61–73%; (d) p-Toluoyl chloride, triethylamine,
dichloromethane; (e) H2, Pd/C, EtOH, THF, 50 ◦C, 37% over two
steps; (f) 4-Nitrophenyl [(pyridin-4-yl)methyl]carbamate, triethyl-
amine, 1,4-dioxane, 60 ◦C, 45–96%.
H
N
N
O
a, b
N
c
N
H
N
O2N
H2N
H
N
45
44
5
O
O
N
d, e
c
O
N
N
H
N
H
H2N
X
46
Y
6 (X=N, Y=CH)
8 (X=CH, Y=N)
O
O
H
N
N
d, b
O
f
N
O2N
N
H
N
H
H2N
N
47
7
48
H
N
H
N
O
f
N
H
N
H
H2N
N
49
9
Scheme 4 and 5 delineates the synthesis of indazole and pyr-
azolopyridine derivatives. The indazole core structure of bromide 36
was generated by reductive cyclization of 4-bromo-2-nitrobenzaldehyde
35 as reported by Genung et al (Scheme 4).31 Pd-catalyzed amination of
36 with benzophenone imine followed by acid cleavage provided aniline
37, which was converted to the final urea compound 10 by coupling
with 4-nitrophenyl [(pyridin-4-yl)methyl]carbamate. As for the pyr-
azolopyridine derivative 11, its core structure was constructed following
the synthetic route reported by Stevens et al (Scheme 5).32 Starting
material 38 was converted to ketone 39 which was reacted with
hydroxylammonium chloride to afford oxime 40, then subsequently
converted to pyrazolopyridine 42 via azirine intermediate 41. The ob-
tained bromide 42 was transformed to the final urea compound 11
following the procedure analogous to those depicted in Scheme 4.
Synthesis of indoline and indole derivatives 5–9 are illustrated in
Scheme 6. Starting from commercially available 5-nitroindoline 44 or 5-
nitroindole 47, its nitrogen was either alkylated or acylated, subse-
quently followed by reduction of nitro group by either iron powder (45
and 48) or hydrogenation (46). Aniline intermediates were converted to
final urea compounds utilizing either the triphosgene method (5, 6 and
8) or carbamate coupling (7 and 9). The 2-phenylindole derivative 9 was
prepared from the corresponding aniline 49 which was commercially
available.
Table 6
Effects on hERG current of compound 12 and 21.
Cpd
hERG inhibitiona (%)
3
μM
10
μM
30
μM
12
21
20
11
51
14
69
25
a
% inhibition at given compound concentration.
scaffold hopping investigation seeking an alternative structure for pyr-
rolidine ring. Introduction of a fused bicyclic structure forming an
indoline ring resulted in weak NAMPT activation activity (5). Further-
more, we were delighted to see that incorporation of amide linker to the
side chain of the indoline moiety (6) led to stronger activity in NAMPT
enzyme assay as well as moderate cellular activity. Converting the
indoline ring to indole (7) led to stronger potency, though its Emax was
lower than that of indoline analog. Moreover, the 3-pyridyl analog of
compound 7 elicited a switch in mode of action from NAMPT activation
to NAMPT inhibition (8).35 Although compound 7 showed highest ac-
tivity in NAMPT enzyme assay among these series, the cellular activity
was suboptimal, so we decided to further explore the central core region
aiming enhancement of activity.
Our initial attempt focused on the 2-phenyl-1H-indole 9 which
showed increased activity in NAMPT enzyme and cell-based assays
compared to its N-substituted analog 7 (Table 2). Encouraged by this
result, we screened nitrogen-containing 6–5 fused bicyclic aromatic
rings with a phenyl substituent in a similar position. Among several ring
systems we have attempted, indazole (10), pyrazolopyridine (11) and
triazolopyridine (12) exhibited potent NAMPT activation activity with
increased potency and efficacy. Compounds 10, 11 and 12 also showed
potent activity in the cell-based assay, with the NAD+ increase
exceeding 100% at all the tested concentrations.
Our initial SAR exploration is summarized in Table 1. For our pri-
mary SAR driving assay we utilized human NAMPT enzyme, with ac-
tivity evaluated by chemically converting the NMN produced by NAMPT
to a fluorescent substance, and using the fluorescence intensity as an
index of amount of NMN production.33 The Emax of the synthesized
compounds were calculated as percentage of the maximal activity ach-
ieved with 30
μ
M of compound 1.34 We also employed a cell-based assay
measuring the level of NAD+ increase in HEK293A cells across a range of
concentrations utilizing a NAD/NADH-Glo assay kit. Unfortunately, our
initial attempt to simply merge the two structural features of 1 and 2, the
(pyridin-4-ylmethyl) urea moiety and the pyrrolidine ring, resulted in
loss of activity (data not shown); therefore, we conducted a further
The compounds which showed potent activities in NAMPT enzyme
and cell-based assays were further profiled to assess their
5

M. Akiu et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128048
commonly known that there is a correlation between CYP inhibition and
lipophilicity.37 Although introduction of an N-methylpyrrole (20) was
tolerated in terms of NAMPT activation activity and reduced the com-
pound’s logD to 2.6, high CYP2C9 DI remained. Somewhat surprisingly,
installing pyrazole ring to this region had a profound effect in reduction
of CYP DI; N-methylpyrazole analog 21 was an equally potent NAMPT
activator, while its lowered logD of 1.8 led to a significant reduction of
CYP DI to the desired level. A bulkier substituent on the pyrazole ring
(22) improved cellular activity but was accompanied with higher
CYP2C9 DI presumably caused by the increased lipophilicity. Of note,
low lipophilicity of compound 23, which possess a pyrazole ring with a
methoxyethyl substituent, also translated to reduced level of CYP DI,
which was successfully accompanied by equipotent NAMPT activation
activity.
Table 7
Physicochemical and in vitro ADME properties of compound 21.
ADME properties
Compound 21
Solubility (pH = 1.2, 6.8) (
μ
g/mL)
>680, 18
97, 83
30
Metabolic stabilitya (h, mou) (%)
Plasma protein bindingb (mouse) (%)
PAMPA permeability (pH 5.0, pH 7.4) (nm/s)
0.4, 0.8
a
The % remaining value at 1 μM concentration of compounds reacted with
human or mouse microsomes for 30 min.
b
The % unbound in mouse plasma.
Table 8
Pharmacokinetic parameters of compound 12 and 21 (C57BL/6N mice, po
administrationa, 10 mg/kg).
As for compound 21 and lead compound 12, their human ether-a-go-
go related gene (hERG) inhibition was also evaluated (Table 6). Com-
pound 21 exhibited lower tendency of hERG inhibition compared to
compound 12, of which the decrease in lipophilicity may also be a
factor.
Cpd
Structure
T1/2
(h)
Cmax
M)
AUC0-24
M∙h)
(
μ
(μ
12
0.9
9.2
25
Physicochemical and in vitro ADME properties of compound 21 are
shown in Table 7. Compound 21 was highly soluble in acidic medium
and exhibited excellent metabolic stability in human and mouse plasma.
Compound 21 also exhibited substantial level of free fraction (30%) in
mouse plasma. Unfortunately, the low lipophilicity of compound 21
abrogated membrane permeability, which can potentially impact oral
bioavailability. Indeed, compound 21 showed lower exposure compared
to compound 12 when orally administered to mice (Table 8), indicating
additional optimization will be required in order to acquire an optimal
compound with a well-balanced overall preclinical profile.
21
1.9
0.1
0.3
a
Compounds were administered with 0.5% methylcellulose as a solvent.
physicochemical and in vitro ADME properties (Table 3). In general,
these compounds exhibited high membrane permeability and metabolic
stability in human and mouse microsomes. However, most compounds
were associated with CYP direct inhibition (DI) which was presumably
caused by the exposed pyridine ring incorporated at the left-hand side,
as such heterocycles are typically thought to cause CYP inhibition by
binding to the heme center of the CYP enzymes.36 CYP inhibition was
prevalently observed with several NAMPT inhibitors containing an
exposed pyridine ring36 as well as our previously described NAMPT
activators. Notably, triazolopyridine analog 12 showed acceptable sol-
ubility in the neutral pH, along with attenuated CYP DI for CYP2D6 and
CYP3A4. Concomitantly considering the ADME properties and NAMPT
activation activity, compound 12 became our lead compound for further
SAR around the triazolopyridine template with the aim to see whether
attenuating CYP DI while possessing desired levels of NAMPT potency
was feasible.
In summary, optimization of HTS hits led to the discovery of (pyr-
idin-4-ylmethyl) urea derivatives with bicyclic core structures that are
potent NAMPT activators in both biochemical and cell-based assays. In
particular, triazolopyridine 12 was identified as a lead compound, dis-
playing potent NAMPT activation activity and favorable ADME prop-
erties albeit with high levels of CYP direct inhibition. Through
additional SAR investigation, lowering logD was shown to be beneficial
for the reduction of CYP inhibition, which yielded compound 21 that
had attenuated CYP inhibition along with hERG inhibition. Unfortu-
nately, the low lipophilicity of compound 21 led to low membrane
permeability, which translated into poor oral exposure. Further opti-
mization to give compounds with high oral exposure while maintaining
low risk of CYP inhibition will be necessary.
Initially, we explored the left-hand region of our compound, the
exposed pyridine ring (Table 4). As this moiety is hypothesized to be
crucial for the compound’s interaction with NAMPT enzyme, an inten-
sive exploration of this left-hand region previously identified 4-pyrazole
and 5-oxazole as heterocycles showing NAMPT activation other than 4-
pyridine.28 4-Pyrazole and 5-oxazole were also tolerated with this
template, displaying optimal activities in NAMPT enzyme and cell-based
assays (13 and 14). Unfortunately, although decreasing tendency for DI
of CYP2C9 and CYP3A4 was observed, CYP1A2 DI could not be atten-
uated to a desired level.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128048.
Having concluded that CYP DI could not be attenuated through
conversion of the exposed 4-pyridine ring, we turned our attention to the
right-hand region of our compound, where we explored additional
structural modifications (Table 5). As for the substituent on the phenyl
ring, the ortho position (15) was favorable in terms of NAMPT activation
activity, whereas meta substitution (16) led to decrease of activity, and
para substitution (17) was detrimental. CYP2C9 DI was consistently
observed among these phenyl analogs. As Zak et al. illustrated in their
work that increasing the fraction of sp3 carbon atoms (Fsp3) was
favorable for reducing CYP inhibition,36 we attempted to introduce
aliphatic groups to this region (18 and 19), but this resulted in an
impairment of NAMPT activation. We subsequently explored installa-
tion of heterocycles in the aim of decreasing lipophilicity, for it is
References
1 Xiao W, Wang R-S, Handy DE, Loscalzo J. Antioxd Redox Signal. 2018;28:251–272.
2 Imai S, Gurante L. Trends Cell Biol. 2014;24:464–471.
3 Canto C, Menzies K, Auwerx J. Cell Metab. 2015;22:31–53.
4 Rajman L, Chewalek K, Sinclari DA. Cell Metab. 2018;27:529–547.
5 Yang Y, Sauve AA. Biochim Biophys Acta. 1864;2016:1787–1800.
6 Verdin E. Science. 2015;350:1208–1213.
7 Katsuyaba E, Auwerx J. EMBO J. 2017;36:2670–2683.
8 Yoshino J, Mills KF, Yoon MJ, Imai S. Cell Metab. 2011;14:528–536.
9 Hershberger KA, Martin AS, Hirschey MD. Nat Rev Nephrol. 2017;13:213–225.
10 Lee CF, Chaves JD, Garcia-Menendez L, et al. Circulation. 2016;134:883–894.
11 Tran MT, Zsengeller ZK, Berg AH, et al. Nature. 2016;531:528–532.
12 Khan NA, Auranen M, Paetau I, et al. EMBO Mol Med. 2014;6:721–731.
13 Araki T, Sasaki Y, Milbrandt J. Science. 2004;305:1010–1013.
6

M. Akiu et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128048
14 Gong B, Pan Y, Vempati P, et al. Neurobiol Aging. 2013;34:1581–1588.
15 Jia H, Li X, Gao H, et al. J Neurosci Res. 2008;86:2083–2090.
16 Ayoub IA, Lee EJ, Ogilvy CS, Beal MF, Maynard KI. Neurosci Lett. 1999;259:21–114.
17 Wang SN, Miao CY. Stroke Vasc Neurol. 2019;4:83–139.
27 Burgos ES, Ho M, Almo SC, Schramm VL. Proc Natl Acad Sci USA. 2009;106:
13748–13753.
28 Pinkerton AB, Sessions EH, Hershberger P, et al. Bioorg Med Chem Lett. 2021. https://
doi.org/10.1016/j.bmcl.2021.128007.
18 Lin JB, Kubota S, Ban N, et al. Cell Rep. 2016;17:69–85.
29 Hamadouchi C, Maiti P, Warshawsky AM, et al. J Med Chem. 2018;61:934–945.
¨
19 Williams PA, Harder JM, Cardozo BH, Foxworth NE, John SWM. Commun Integr Biol.
2018;11, e1356956. https://doi.org/10.1080/19420889.2017.1356956.
20 Gomes AP, Price NL, Ling AJY, et al. Cell. 2013;155:1624–1638.
21 Zhang H, Ryu D, Gariani K, et al. Science. 2016;352:1436–1443.
22 Costford SR, Brouwers B, Hopf ME, et al. Mol Metab. 2018;7:1–11.
23 Wang T, Zhang X, Bheda P, Revollo JR, Imai S, Wolberger C. Nat Struct Mol Biol.
2006;13:661–662.
30 Christensen MK, Bjorkling F. WO2010/023307.
31 Genung NE, Wei L, Aspnes GE. Org Lett. 2014;16:3114–3117.
32 Stevens KL, Jung DK, Alberti MJ, et al. Org Lett. 2005;7:4753–4756.
33 Formentini L, Moroni F, Chiarugi A. Biochem Pharmacol. 2009;77:1612–1620.
34 NAMPT activation elicited by 1 reached an Emax plateau at a compound
concentration of 30 μM and showed highly reproducible NAMPT activation; thus, we
considered it suitable as a standard for calculation of Emax of the synthesized test
compounds.
24 Gardell SJ, Hopf M, Khan A, et al. Nat Commun. 2019;10:3241.
25 The maximal NAMPT activation for individual compounds (Emax) in the enzyme
assay was determined as a percentage of the maximal activation by 2.5 mM ATP,
since ATP phosphorylation of NAMPT H247 causes activation of NAMPT enzyme
(see Ref. 26 and 27).
35 This switch in mode of action was also observed with our previous reported NAMPT
activators, see Ref. 24 and 28.
36 Zak M, Yuen P, Liu X, et al. J Med Chem. 2016;59:8345–8368.
37 Yang Y, Engkivst O, Llinas A, Chen H. J Med Chem. 2012;55:3667–3677.
26 Burgos ES, Schramm VL. Biochemistry. 2008;47:11086–11096.
7