Fragment-based design of 3-aminopyridine-derived amides as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT)

Article abstract of DOI:10.1016/j.bmcl.2013.12.062

The fragment-based identification of two novel and potent biochemical inhibitors of the nicotinamide phosphoribosyltransferase (NAMPT) enzyme is described. These compounds (51 and 63) incorporate an amide moiety derived from 3-aminopyridine, and are thus structurally distinct from other known anti-NAMPT agents. Each exhibits potent inhibition of NAMPT biochemical activity (IC ₅₀ = 19 and 15 nM, respectively) as well as robust antiproliferative properties in A2780 cell culture experiments (IC₅₀ = 121 and 99 nM, respectively). However, additional biological studies indicate that only inhibitor 51 exerts its A2780 cell culture effects via a NAMPT-mediated mechanism. The crystal structures of both 51 and 63 in complex with NAMPT are also independently described.

Full text of DOI:10.1016/j.bmcl.2013.12.062

Bioorganic & Medicinal Chemistry Letters 24 (2014) 954–962
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fragment-based design of 3-aminopyridine-derived amides as potent
inhibitors of human nicotinamide phosphoribosyltransferase
(NAMPT)
a
b
a
a
Peter S. Dragovich a, , Guiling Zhao , Timm Baumeister , Brandon Bravo , Anthony M. Giannetti ,
Yen-Ching Ho b, Rongbao Hua c, Guangkun Li c, Xiaorong Liang a, Xiaolei Ma a, Thomas O’Brien a,
Angela Oh a, Nicholas J. Skelton a, Chengcheng Wang d, Weiru Wang a, Yunli Wang c, Yang Xiao a,
Po-wai Yuen c, Mark Zak a, Qiang Zhao d, Xiaozhang Zheng b
⇑
a Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
b Forma Therapeutics, Inc., 500 Arsenal Street, Watertown, MA 02472, USA
c Pharmaron Beijing, Co. Ltd., 6 Taihe Road, BDA, Beijing 100176, PR China
d Crown Bioscience, Science & Technology Innovation Park, No.6 Beijing West Road, Taicang City, Jiangsu Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The fragment-based identification of two novel and potent biochemical inhibitors of the nicotinamide
phosphoribosyltransferase (NAMPT) enzyme is described. These compounds (51 and 63) incorporate
an amide moiety derived from 3-aminopyridine, and are thus structurally distinct from other known
anti-NAMPT agents. Each exhibits potent inhibition of NAMPT biochemical activity (IC50 = 19 and
15 nM, respectively) as well as robust antiproliferative properties in A2780 cell culture experiments
(IC50 = 121 and 99 nM, respectively). However, additional biological studies indicate that only inhibitor
51 exerts its A2780 cell culture effects via a NAMPT-mediated mechanism. The crystal structures of both
51 and 63 in complex with NAMPT are also independently described.
Received 17 October 2013
Revised 12 December 2013
Accepted 16 December 2013
Available online 21 December 2013
Keywords:
Nicotinamide phosphoribosyltransferase
NAMPT
Fragment-based design
Structure-based design
X-ray crystal structure
Surface plasmon resonance
Ó 2013 Elsevier Ltd. All rights reserved.
Nicotinamide phosphoribosyltransferase (NAMPT, also known
in the literature as pre-B cell colony-enhancing factor (PBEF) as
well as visfatin; EC 2.4.2.12) plays a critical role in cellular metab-
olism.1 The enzyme catalyzes the rate-limiting step in the conver-
sion of nicotinamide (NAM) to the important enzyme co-factor
nicotinamide adenine dinucleotide (NAD).2 This process enables
the efficient intracellular recycling of NAM, which is produced by
the catalytic action of NAD-consuming enzymes such as the PARPs
and Sirtuins, back into NAD (Fig. 1).3 Proper maintenance of NAD
levels is known to be critical to sustaining energetics required for
many cellular functions.4 Inhibition of NAMPT has therefore
emerged as a novel strategy for impairing the proliferation of tu-
mors whose high growth rates may make them more susceptible
to NAMPT disruption relative to non-cancerous cells.5
man clinical trials.8,9 Our own prior discovery efforts identified po-
tent urea and amide-derived NAMPT inhibitors which also
contained terminal biaryl sulfone moieties (compounds 3–6;
Fig. 2).10 In an effort to further diversify these molecules, we also
conducted a surface plasmon resonance (SPR)-based screen to
identify small, structurally novel NAMPT-binding moieties (‘frag-
ments’) which could be combined with our existing compounds.11
In this report, we describe the structure-based elaboration of two
SPR screening hits into potent anti-NAMPT agents that are struc-
turally distinct from other known NAMPT inhibitors.
Compound 7 was identified from our SPR-based screening
methods as a moderately potent NAMPT binder with high ligand
efficiency (Table 1).11,12 The compound also demonstrated the abil-
ity to inhibit NAMPT in biochemical assessments, although its po-
tency was somewhat attenuated relative to its binding properties
(Table 1).13 A co-crystal structure of the molecule in complex with
NAMPT was subsequently determined and revealed that the mole-
cule occupied the nicotinamide-binding region of the protein’s
active site (Fig. 3).14 Not unexpectedly, the pyridine portion of 7
formed face-to-face pi-stacking interactions with the side
Multiple examples of NAMPT inhibitors are known in the scien-
tific and patent literature, and the most advanced of these agents
[GMX-1778 (1)6 and APO-866 (2)7; Fig. 2] have progressed to hu-
⇑
Corresponding author.
E-mail address: dragovich.peter@gene.com (P.S. Dragovich).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.062

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 24 (2014) 954–962
955
O
P
O
O
P
O
-
-
O
O
-
O
-
O
N
NH2
O
O
P
O
O
O
O
NAMPT
-
NH2
O
-
+
O
P
O
O
-
N
O
HO
OH
HO
OH
3-
ATP ADP + PO4
NAM
PRPP
NMN
NMNAT
NH2
O
NH2
N
N
N
N
PARPs
Sirtuins
N
O
OH
O
HO
HO
OH
O
P
O
O
-
O
O P
O
O
-
NAD
Figure 1. NAD recycling and NAMPT biochemical mechanism.
O
N
N
N
O
N
O
N
H
N
H
N
N
H
Cl
1
2
O
O
O
S
O
N
N
H
N
N
N
H
N
S
H
N
O
O
H2N
3
4
6
O
F
O
N
N
N
N
H
H
N
N
H
S
F
N
S
CF3
O
O
O
O
5
Figure 2. Examples of known NAMPT inhibitors.
chains of Phe-193 and Tyr-180 in a manner that was similar to that
observed for many other NAMPT inhibitors.10 A hydrogen bond
was also observed between the amide NH of 7 and the NAMPT
Asp-219 residue. However, in contrast to the binding of previ-
ously-studied amide-containing NAMPT inhibitors such as 5,10c
the amide moiety of 7 was positioned much deeper in the nicotin-
amide-binding region of the protein (Fig. 3). This location oriented
the amide carbonyl of 7 directly toward the Arg-311 side chain
instead of toward a water-filled region of the active site that was
typically occupied by the carbonyl group present in other known
NAMPT inhibitors (e.g., 4–6, Fig. 2).10c–e Importantly, the observed
binding of 7 positioned the compound’s pyridine nitrogen in a
location that was similar to that observed for the N-atoms present
in many other heterocycles which bound to the same NAMPT
region (e.g., compare the location of the imidazopyridine moiety
present in 5 with that of the pyridine contained in 7; Fig. 3). Such
positioning was consistent with 7 functioning as a NAMPT sub-
strate and undergoing enzyme-catalyzed condensation with PRPP
in the NAMPT active site, although this possibility was not exam-
ined experimentally (c.f., Fig. 1).15
Our first attempts to improve the inhibitory activity of 7 in-
volved replacing the N-methyl-pyrazole moiety with other isoster-
ic methyl-containing heterocycles. However, as shown in Table 1,
none of these modifications afforded significant improvements in
SPR-binding affinity to NAMPT or biochemical inhibitory activity
(compare 7 with compounds 8–16).16 Attempts to extend various
methyl-containing heterocycles into the NAMPT tunnel region
occupied by the benzyl portion of 5 (c.f., Fig. 3) were also not

956
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 24 (2014) 954–962
Table 1
Initial SAR of 3-aminopyridine-amide NAMPT inhibitors
N
O
N
H
R
a
b
Compd
R
NAMPT IC50 (uM)
NAMPT Kd (uM)
NAMPT SPR LEc
0.47
N
7
91
5.2
N
N
H
N
8
>100
>100
34
176
129
0.36
0.35
0.40
0.38
0.44
0.38
N
9
N
N
10
11
12
13
36
55
12
61
129
20
13
S
S
>100
38
O
O
>100
N
S
S
O
14
15
16
50
0.35
0.42
0.44
>100
>100
N
All biochemical and SPR results are reported as the arithmetic mean of at least 2 separate runs.
a
NAMPT biochemical inhibition.
NAMPT SPR binding.
Ligand efficiency based on SPR Kd value.
b
c
successful. This outcome was obtained regardless of whether the
extending moieties were appended to a position adjacent to (com-
pounds 17–20) or once-removed from (compounds 21–23) the
heterocyclic methyl group (Table 2). These results collectively indi-
cated that replacement of the methyl-pyrazole present in 7 with
non-isosteric and/or structurally diverse moieties would likely be
required to properly optimize the potency of the 3-aminopyri-
dine-amide inhibitor series.
Accordingly, crystal structures of several other NAMPT frag-
ment screening hits were determined to identify those with good
potential for combination with lead compound 7.11 The nitro-
substituted benzimidazole 24 (Fig. 4), which exhibited moderate
SPR-based NAMPT affinity and good ligand efficiency, emerged
from this exploration as a particularly attractive possibility. As
shown in Fig. 5, the compound bound to NAMPT in the tunnel re-
gion with the nitro group located near where the amide moiety of 7
resided in the 7-NAMPT crystal structure. A hydrogen bond was
observed between one nitro group oxygen atom of 24 and the side
chain of Arg-311 which was shifted from its location in the
7-NAMPT crystal structure in order to facilitate this interaction.
The benzimidazole portion of 24 formed many favorable van der
Waals contacts with hydrophobic portions of the NAMPT tunnel
region (Ile-309, Ile-351, and the face of His-191) but did not inter-
act with any observed water molecules. Somewhat unexpectedly,
the benzimidazole of 24 did not bind to NAMPT in a manner that
was co-planar with the methyl-pyrazole contained in 7. However,
analysis of the two overlaid structures suggested that replacement
of the latter moiety with a simple phenyl ring would be tolerated
and would offer alternate vectors for further elaboration into the
NAMPT tunnel region (Fig. 5).
As shown in Table 3, effecting the described substitution affor-
ded a molecule (25) with significantly improved biochemical
NAMPT inhibitory activity relative to lead compound 7. Impor-
tantly, extension of 25 via modification of the 4-position on the
phenyl ring was typically tolerated and often improved biochemi-
cal potency as compared to the unsubstituted inhibitor (26–35;
Table 3). Two notable exceptions to this trend were the amino-
containing molecules 29 and 32 which, based on the 24-NAMPT
co-crystal structure, unfavorably positioned their polar amino
groups in the center of the hydrophobic NAMPT tunnel region.
Encouragingly, however, transformation of the amine moieties
present in either compound to the corresponding carbamates

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 24 (2014) 954–962
957
Table 2
Elaboration of 3-aminopyridine-amide NAMPT inhibitors
a
b
Compd
R
NAMPT IC50
(uM)
NAMPT Kd
M)
NAMPT
SPR LEc
(
l
N
N
17
>100
>100
234
0.31
NA
18
>500
N
N
19
20
>100
>100
177
0.24
NA
N
N
N
>500
21
22
N
N
>100
>100
59
0.36
0.29
N
Figure 3. Crystal structure of compound
7 in complex with NAMPT (resolu-
tion = 2.86 Å; PDB accession code 4N9B). Protein side chains from the two NAMPT
monomers which form the dimeric active site cleft are depicted in white and grey,
respectively. The van der Waals surface of the ligand binding pocket is also shown
in grey. The inhibitor is presented as orange tubes with corresponding crystallo-
graphic water molecules and hydrogen bonds indicated by orange spheres and
dashed orange lines, respectively. The thin green lines depict the location of
233
N
N
compound
5 in the binding site (PDB accession code 4KFO). Hydrogen bond
N
23
>100
481
0.26
interactions between 5 and NAMPT are indicated by the dashed green lines.
and/or sulfonamides afforded molecules with more promising
NAMPT inhibition properties (30–31 and 33–35). This observation
prompted us to systematically explore appending phenyl-contain-
ing acyl and sulfonyl moieties to the amines present in 29 and 32.
We anticipated that some of the resulting compounds would suc-
cessfully traverse the NAMPT tunnel region and interact with a
post-tunnel (solvent-exposed) area of the protein in a manner sim-
ilar to that noted for the terminal portions of larger known NAMPT
inhibitors (e.g., compound 1–6, Fig. 2; see also Figs. 3 and 6–8).
Table 4 depicts the outcome of this systematic exploration. Mol-
ecules incorporating benzoyl-derived amides were moderately po-
tent NAMPT inhibitors when the amides were directly attached to
the compounds’ central phenyl rings (36, 40, 44, and 48). However,
inclusion of a methylene moiety between the amides and the cen-
tral phenyl rings significantly impaired the potencies of the result-
ing compounds (37, 41, 45, and 49). In contrast, direct central-ring
attachment of phenyl-containing sulfonamides afforded ineffective
NAMPT inhibitors (38, 42, 46, and 50) while extension of the same
sulfonamide moieties by a methylene group provided much more
potent inhibitors (39, 43, 47, 51). The ability of several compounds
contained in Table 4 to inhibit the proliferation of A2780 cells was
also tested. Most molecules did not exhibit meaningful activity in
this assessment, likely due to insufficient NAMPT inhibition po-
tency. However, compounds 47 and 51 displayed measurable anti-
proliferation activity and the latter (more potent) molecule was
selected for additional characterization studies.
All biochemical and SPR results are reported as the arithmetic mean of at least 2
separate runs.
a
NAMPT biochemical inhibition.
NAMPT SPR binding.
Ligand efficiency based on SPR Kd value (NA = not applicable).
b
c
O2N
N
NAMPT SPR KD = 126
µM
N
H
NAMPT SPR LE = 0.44
24
Figure 4. NAMPT fragment screening hit.
positioned similarly to both the methyl-pyrazole present in 7 and
the benzimidazole contained in 24. However, the phenyl group of
51 was not precisely co-planar with either of these entities and in-
stead filled a space in the tunnel region that resided between them.
This orientation was drastically different from the binding mode
observed previously for the central benzyl group of compound 5
in the same area of the protein (Fig. 6). In contrast, the terminal
phenyl-sulfonamide portion of 51 interacted with NAMPT in a
manner that was very similar to that noted for the terminal sulfone
present in 5 with water-mediated hydrogen bonds being formed
with both sulfonamide oxygen atoms (Fig. 6). The pyridine N-atom
of 51 was situated very close to the exposed imidazopyridine nitro-
gen of 5 in the NAMPT nicotinamide binding region, and this posi-
tioning was consistent with 51 possibly functioning as a NAMPT
substrate.15 The collective analysis of the 51-NAMPT co-crystal
structure demonstrated how a compound that was structurally
distinct from other known NAMPT inhibitors such as compounds
3–6 could effectively and uniquely occupy the protein’s active
site.17
The co-crystal structure of inhibitor 51 in complex with NAMPT
is shown in Figure 6. The molecule’s amino-pyridine fragment
bound to the protein in a manner that was very similar to, although
not quite co-planar with, the binding mode described above for the
identical portion of compound 7. Accordingly, the pyridine ring of
51 was situated between the side chains of Phe-193 and Tyr-180
and a hydrogen bond was observed between the compound’s
amide NH moiety and the Asp-219 residue. The central phenyl ring
of inhibitor 51 occupied the NAMPT tunnel region and was
Further analysis of the 24-NAMPT co crystal structure described
above (Fig. 5) suggested that 6,5-bicyclic aromatic moieties could

958
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 24 (2014) 954–962
Figure 6. Crystal structure of compound 51 in complex with NAMPT (resolu-
tion = 1.70 Å; PDB accession code 4N9D). Protein side chains from the two NAMPT
monomers which form the dimeric active site cleft are depicted in white and grey,
respectively. The van der Waals surface of the ligand binding pocket is also shown
in grey. The inhibitor is presented as blue tubes with corresponding crystallo-
graphic water molecules and hydrogen bonds indicated by blue spheres and dashed
blue lines, respectively. The thin green lines depict the location of compound 5 in
the binding site (PDB accession code 4KFO). Hydrogen bond interactions between 5
and NAMPT are indicated by the dashed green lines.
Figure 5. Crystal structure of compound 24 in complex with NAMPT (resolu-
tion = 1.75 Å; PDB accession code 4N9C). Protein side chains from the two NAMPT
monomers which form the dimeric active site cleft are depicted in white and grey,
respectively. The van der Waals surface of the ligand binding pocket is also shown
in grey. The inhibitor is presented as cyan tubes with corresponding crystallo-
graphic water molecules and hydrogen bonds indicated by cyan spheres and cyan
lines, respectively. The thin orange lines depict the location of compound 7 in the
binding site (PDB accession code 4N9B). Hydrogen bond interactions between 7 and
NAMPT are indicated by the dashed orange lines.
Table 3
Additional elaboration of 3-aminopyridine-amide NAMPT inhibitors
N
O
N
H
R
a
Compd
R
NAMPT IC50
(
lM)
25
26
27
28
29
30
31
32
33
34
35
H
CH3
Ph
13
13
1.0
1.0
>100
0.42
10.3
>100
13
Cyclohexyl
NH2
NHBoc
NHSO2CH3
CH2NH2
CH2NHBoc
CH2NHCbz
CH2NHSO2CH3
3.3
5.0
All biochemical results are reported as the arithmetic mean of at least 2 separate
runs.
Figure 7. Crystal structure of compound 24 in complex with NAMPT (resolu-
tion = 1.75 Å; PDB accession code 4N9C). Protein side chains from the two NAMPT
monomers which form the dimeric active site cleft are depicted in white and grey,
respectively. The van der Waals surface of the ligand binding pocket is also shown
in grey. The inhibitor is presented as cyan tubes with corresponding crystallo-
graphic water molecules and hydrogen bonds indicated by cyan spheres and dashed
cyan lines, respectively. The thin green lines depict the location of compound 2 in
the binding site (PDB accession code 2GVJ). Hydrogen bond interactions between 2
and NAMPT are indicated by the dashed green lines.
a
NAMPT biochemical inhibition.
also be productively utilized as replacements for the methyl-
pyrazole present in fragment lead 7. Accordingly, a benzimidazole
containing an appropriately-positioned 3-amino-pyridine-derived
amide displayed encouraging biochemical NAMPT inhibitory activ-
ity (compound 52, Table 5). Replacement of the benzimidazole
present in 52 with several other 6,5-bicyclic aromatic systems also
afforded biochemically active NAMPT inhibitors (53–58, Table 5),
although several compound potencies were somewhat attenuated
relative to that exhibited by 52. Unfortunately, all of these
molecules failed to demonstrate anti-NAMPT activity in cell cul-
ture assessments. These outcomes were not entirely surprising
since the compound concentrations tested in these cell-based
experiments were somewhat low relative to the corresponding
biochemical IC50 values. Attempts to further elaborate the benz-
imidazole contained in 52 by derivatizing the nitrogen atom meta
to the carboxamide substituent were unsuccessful (compounds 59
and 60, Table 5). However, analysis of the bound conformations of
fragment 24 and the very potent NAMPT inhibitor 2 suggested that

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 24 (2014) 954–962
959
Table 5
3-Aminopyridine-amide NAMPT inhibitors containing 6,5-bicyclic moieties
N
O
N
H
R
a
b
Compd
52
R
NAMPT IC50
M)
A2780 IC50
M)
(
l
(l
N
1.5
>2.0
N
H
O
53
54
55
56
57
58
9.1
3.4
26
>2.0
>2.0
>2.0
>2.0
>2.0
ND
N
N
O
S
Figure 8. Crystal structure of compound 63 in complex with NAMPT (resolu-
tion = 1.72 Å; PDB accession code 4N9E). Protein side chains from the two NAMPT
monomers which form the dimeric active site cleft are depicted in white and grey,
respectively. The van der Waals surface of the ligand binding pocket is also shown
in grey. The inhibitor is presented as salmon tubes with corresponding crystallo-
graphic water molecules and hydrogen bonds indicated by salmon spheres and
dashed salmon lines, respectively. The thin green lines depict the location of
N
N
4.1
21
S
compound
2 in the binding site (PDB accession code 2GVJ). Hydrogen bond
interactions between 2 and NAMPT are indicated by the dashed green lines.
O
Table 4
Systematic exploration of amino-containing 3-aminopyridine-amide NAMPT inhibi-
tors
21
S
N
N
N
O
59
60
>100
>100
ND
ND
N
H
N
H
R
X
n
a
b
Compd
R
X
n
NAMPT IC50
M)
A2780 IC50
M)
N
N
(
l
(l
36
37
C@O
C@O
0
1
0.61
25
>2.0
ND
All biochemical and cell-based results are reported as the arithmetic mean of at
least 2 separate runs.
NAMPT biochemical inhibition.
Antiproliferation activity determined in cell culture experiments using A2780
cell line. ND = not determined.
38
39
SO2
SO2
0
1
>100
0.21
>2.0
ND
a
40
41
C@O
C@O
0
1
1.5
40
>2.0
ND
b
42
43
SO2
SO2
0
1
83
0.014
>2.0
ND
elaboration with molecular fragments derived directly from the
structure of inhibitor 2.
44
45
46
47
C@O
C@O
SO2
SO2
0
1
0
1
0.64
3.1
62
>2.0
>2.0
>2.0
0.88
Cl
The described methyl-containing derivative of 52 was prepared
(compound 61) and it displayed biochemical NAMPT inhibition
properties that were equivalent to those exhibited by the unme-
thylated parent molecule (Table 6). Further elaboration of the
methyl group present in 61 with several moieties inspired by the
structure of inhibitor 2 was either tolerated (62) or dramatically
improved biochemical NAMPT inhibitory activity (63). However,
related derivatization of the 61 benzimidazole 2-position
weakened anti-NAMPT potency with the larger appended groups
having the most deleterious effects (compounds 64–67, Table 6).
Cell-culture antiproliferation effects were assessed with several
of the compounds depicted in Table 6, but only inhibitor 63
exhibited significant potency in these experiments. Somewhat
surprisingly, however, the antiproliferative effects associated with
63 were not completely reversed/eliminated by the addition of
NMN (the biochemical product of NAMPT catalysis; c.f. Fig. 1) to
the cell culture media. This result suggested that the cell effects
0.019
48
49
50
51
C@O
C@O
SO2
SO2
0
1
0
1
0.29
80
36
>2.0
ND
>2.0
0.12
0.019
All biochemical and cell-based results are reported as the arithmetic mean of at
least 2 separate runs.
NAMPT biochemical inhibition.
Antiproliferation activity determined in cell culture experiments using A2780
cell line (ND = not determined). This inhibition can be reversed by addition of
0.33 mM of NMN.
a
b
functionalization of the 52 nitrogen atom para to the carboxamide
substituent might be more fruitful (Fig. 7). Specifically, it was
envisioned that introduction of a methyl group at this location in
52 would be tolerated and would thereby enable additional

960
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 24 (2014) 954–962
Table 6
Optimization of benzimidazole-containing 3-aminopyridine-amide NAMPT inhibitors
N
O
N
N
R1
N
H
R2
R1
R2
NAMPT IC50
(
lM)
A2780 IC50 (lM)
a
b
Compd
52
61
H
CH3
H
H
1.5
1.4
>2.0
ND
62
H
3.7
>2.0
O
N
63
H
0.015
0.099c
64
65
CH3
CH3
CH3
Ph
6.1
14
ND
ND
66
CH3
>100
ND
N
N
Boc
O
67
CH3
>100
ND
All biochemical and cell-based results are reported as the arithmetic mean of at least 2 separate runs.
a
NAMPT biochemical inhibition.
Antiproliferation activity determined in cell culture experiments using A2780 cell line (ND = not determined).
Antiproliferative effects were only partially reversed by addition of 0.33 mM of NMN.
b
c
Table 7
N
O
Antiproliferation effects determined for compounds 51 and 63 in various human
i
8 11 13 23 25 28 30
- , - , - , ,
tumor cell linesa,b
+
33, 34, 52-59, 62, 64-66
R
R
HO
NH2
CyQuant IC50 values in nM
HT1080
213
>1000
PC3
MiaPaCa2c
HCT116
ii
51
63
9
217 67
>1000
855 77
>1000
271 31
>1000
61
a
CyQuant endpoint; arithmetic mean of 3 separate runs (n = 3, standard devia-
tions are also shown).
O
iii
b
60
All antiproliferation effects were reversed by addition of 330 lM NMN, strongly
+
implicating NAMPT inhibition as the causative MOA.
H3CO
c
n = 2. See Supplementary data for experimental details.
Scheme 1. Synthesis of compounds containing 3-aminopyridine-derived amides.
Reagents and conditions: (i) HATU, (i-Pr)2NEt, DMF, 25–50 °C, 3–18 h, 4–76%; (ii)
HATU, (i-Pr)2NEt, DMF, 45 °C, 3 h (forms HOAt adduct), then 3-aminopyridine,
microwave, DMF, 95 °C, 1 h, 25%; (iii) AlCl3, 1,2-dichloroethane, 70 °C, 15 h, 35%.
observed for compound 63 might result from inhibition of biolog-
ical targets in addition to and/or other than NAMPT. More detailed
characterization studies of compound 63 were therefore under-
taken to better explore this possibility.
oriented directly toward the Arg-311 side chain. As also expected
from analysis of many inhibitor-NAMPT co-crystal structures, the
pyridine nitrogen atoms of compounds 63 and 2 were located in
similar positions in the NAM-binding region of the protein that
were consistent with each molecule possibly functioning as a
NAMPT substrate.15
In addition to the various studies described above, more exten-
sive cell-culture experiments were conducted with both com-
pounds 51 and 63 to characterize their anti-NAMPT properties in
greater detail. Compound 51 potently reduced NAD levels in
A2780 cells with an IC50 value between 24 and 58 nM (Figs. S1
and S2). In contrast, compound 63 exhibited negligible effects on
A2780 NAD levels when tested under similar experimental condi-
tions (IC50 >1000nM; Fig. S3). The latter observation was consis-
tent with the inability of NMN to reverse the A2780
antiproliferation effects exhibited by compound 63 and suggested
that, in spite of the compound’s ability to biophysically associate
A co-crystal structure of compound 63 in complex with NAMPT
was determined and it confirmed that the compound could bio-
physically associate with the protein. The terminal portion of the
molecule, which bound in the post-tunnel, solvent-exposed
NAMPT region, closely mimicked the bound conformation of the
corresponding portion of inhibitor 2 (Fig. 8). Such binding enabled
many favorable van der Waals contacts between the benzoyl moi-
ety present in 63 and aliphatic portions of the side chains of
NAMPT residues Arg-349 and Ala-379. Similar favorable van der
Waals interactions were also noted between the piperidine frag-
ment of 63 and the hydrophobic Val-242 and Ile-309 NAMPT tun-
nel-region residues. As was observed for other 3-amino-pyridine
amides described in this work, the pyridine portion of 63 formed
face-to-face pi-stacking interactions with the side chains of Phe-
193 and Tyr-180, a hydrogen bond existed between the compound’s
amide NH and Asp-219, and the molecule’s amide carbonyl was

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 24 (2014) 954–962
961
O
O
O
NO2
Br
R
NH
i
HO
iii
HO
N
N
HO
68
69
70
R = NO2
R = NH2
ii
71
Scheme 2. Synthesis of acid required to prepare compound 62. Reagents and conditions: (i) Cyclohexylmethanamine, K2CO3, DMF, 110 °C, 14 h, 19%; (ii) H2, 10% Pd on C,
MeOH, 25 °C, 2 h, 91%; (iii) HC(OEt)3, EtOH, 70 °C, 14 h, 87%.
with the protein and potently inhibit its biochemical activity, the
A2780 cell culture outcomes likely result from non-NAMPT-related
biological activity. Accordingly, compound 63 did not exhibit
meaningful antiproliferative effects against four other cancer cell
lines against which it was tested (Table 7). Encouragingly, how-
ever, compound 51, which strongly reduced NAD levels in A2780
cells, exhibited potent to moderate antiproliferative activity
against all of these same lines (Table 7). Taken together, the above
results indicate that careful biological characterization should be
conducted with novel NAMPT inhibitors to confirm that any ob-
served antiproliferative effects truly result from inhibition of the
targeted NAMPT enzyme.
H
Boc
N
O
N
O
Boc
NH2
i
N
N
RO
+
O
EtO
N
H
72
73
R = Et
R = H
ii
Scheme 3. Synthesis of of acid required to prepare compound 66. Reagents and
conditions: (i) 10% Pd/C, EtOH, reflux, 48 h, 89%; (ii) 1:5 1 N NaOH:EtOH, 85 °C, 18 h,
94%.
The compounds described in this work were either purchased
from Sigma–Aldrich (7, 12, and 24) or were prepared by the meth-
ods shown in Schemes 1–5.18 Many were synthesized by HATU-
mediated coupling of commercially available carboxylic acids with
3-aminopyridine (8–11, 13–23, 25–28, 30, 33, 34, 52–58, 62, 64–
66; Scheme 1). The acid required for the preparation of compound
59 was obtained by hydrolysis of the corresponding (commercially
available) methyl ester while those needed to make 62 and 66
were prepared as described in Schemes 2 and 3, respectively. In
the case of inhibitor 60, the desired amide linkage was formed di-
rectly from 3-aminopyridine and the appropriate methyl ester
using an AlCl3-mediated coupling technique (Scheme 1). Removal
of the Boc groups present in compounds 30 and 33 using various
acidic conditions respectively afforded the amino-containing mol-
ecules 29 and 32 in good yield. These entities were subsequently
transformed into the corresponding amides and sulfonamides by
reaction with various acid- and sulfonyl-chlorides (compounds
31, 35–51; Scheme 4). The preparation of compound 63 was
ii
36 40 44 48
,
,
,
i
30
33
29
32
31 38 42 46 50
,
,
,
,
iii
ii
37 41 45 49
,
,
,
iv
35 39 43 47 51
,
,
,
,
iii
Scheme 4. Alternate synthesis of amide and sulfonamide-containing compounds.
Reagents and conditions: (i) 4.0 M HCl, 1,4-dioxane, 25 °C, 6 h, 85%; (ii) RC(O)Cl,
Et3N, CH2Cl2, 25 °C, 18 h, 11–26%; (iii) RSO2Cl, (i-Pr)2NEt, CH2Cl2, 25 °C, 18 h, 4–45%;
(iv) TFA, CH2Cl2, 25 °C, 3 h, 90%.
O
O
O
NO2
Br
R
ii
RO
i
iv
H3CO
N
N
RO
NH
N
Boc
68
74
R = H
R = CH3
75
76
R = NO2
R = NH2
iii
N
Boc
77
78
R = CH3
R = H
v
vi
N
O
N
N
viii
N
H
63
N
R
79
80
R = Boc
R = H
vii
Scheme 5. Synthesis of compound 63. Reagents and conditions: (i) EDCꢀHCl, (i-Pr)2NEt, HOBt, MeOH, 25 °C, 1 h, 87%; (ii) tert-butyl-4-(aminomethyl)piperidine-1-
carboxylate, K2CO3, DMF, 100 °C, 20 h, 65%; (iii) Raney Ni, H2, MeOH, 25 °C, 0.5 h, 45%; (iv) PPTS, HC(OEt)3, CH2Cl2, 25 °C, 20 h, 72%; (v) KOH, EtOH/H2O, 25 °C, 20 h, 82%; (vi) 3-
aminopyridine, HATU, (i-Pr)2NEt, DMF, 25 °C, 20 h, 55%; (vii) TFA, CH2Cl2, 25 °C, 0.5 h, 82%; (viii) PhC(O)Cl, Et3N, CH2Cl2, 25 °C, 0.5 h, 14%.

962
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 24 (2014) 954–962
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somewhat more complicated and was accomplished by the synthe-
sis depicted in Scheme 5.
In this report, we describe the fragment-based identification of
two potent biochemical inhibitors of the nicotinamide phosphori-
bosyltransferase enzyme that are structurally distinct from other
known anti-NAMPT agents. Each of these new compounds (51
and 63) incorporates an amide moiety derived from 3-amino-
pyridine, and this entity enables them to bind to NAMPT in a
manner that is unique relative to how other NAMPT inhibitors
associate with the protein. Although both compounds exhibit
antiproliferative activity in A2780 cell culture experiments, only
inhibitor 51 appears to exert these effects via a NAMPT-mediated
mechanism. This molecule also displays antiproliferative proper-
ties in several other cancer cell lines. Collectively, our results
provide new chemical options for the design of potent NAMPT
inhibitors, and also caution regarding the need for careful biologi-
cal characterization of any novel molecules so identified.
Acknowledgments
We thank Drs. Krista Bowman and Jiansheng Wu and their
respective Genentech research groups for performing many pro-
tein expression and purification activities. We also thank Shohini
Ganguly for generating all the biochemical and cell IC50 results. Fi-
nally, we thank Dr. Peter Jackson for many helpful discussions
regarding NAMPT and Dr. Jeff Blaney for many useful suggestions
associated with fragment-based ligand design.
Supplementary data
11. Giannetti, A. M.; Zheng, X.; Skelton, N. J.; Wang, W.; Bravo, B.; Bair, K. W.;
Baumeister, T.; Cheng, E.; Crocker, L.; Feng, Y.; Gunzner-Toste, J.; Ho, Y.-C.; Hua,
R.; Liederer, B. M.; Liu, Y.; Ma, X.; O’Brien, T.; Oeh, J.; Sampath, D.; Shen, Y.;
Wang, C.; Wang, L.; Wu, H.; Xiao, Y.; Yuen, P.-W.; Zak, M.; Zhao, G.; Zhao, Q.;
Dragovich, P. S. submitted to J. Med. Chem.
12. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
13. The SPR assessments omitted the PRPP co-substrate and ATP additive that
were included in the biochemical inhibition assays (c.f., Fig. 1). The latter entity
is known in the literature to improve NAMPT catalytic efficiency (see: Burgos,
E. S.; Schramm, V. L. Biochemistry 2008, 47, 11086). Thus, it was not surprising
that that SPR Kd values did not perfectly correspond to biochemical IC50
measurements. SPR techniques were utilized primarily to identify the novel
NAMPT binding fragments and to optimize their affinity in the absence of
meaningful biochemical inhibitory activity. Once biochemical IC50 values of
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
12.062.
References and notes
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<20 lM were achieved for a given inhibitor series, biochemical methods were
preferentially used in subsequent optimization activities.
14. NAMPT is believed to function as a symmetrical homodimer with two active
sites formed at opposite ends of the dimer interface. Accordingly, the protein
3. The de novo NAD synthesis pathway requires eight enzyme-catalyzed steps to
produce the co-factor from tryptophan. See Ref. 2 for additional details.
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crystalized with such
a dimer present in the asymmetric unit. The two
monomer chains in the dimers of the co-crystal structures described in this
work were very similar, and both active sites contained co-crystallized
molecules in similar orientations. In the crystallography discussions, the
NAMPT residues are designated with prime and non-prime notation (e.g.,
Tyr180, Phe193) to distinguish the monomer chain in which a given residue
resides.
15. We currently believe that many cell-potent NAMPT inhibitors form PRPP-
derived phosphoribosylated adducts in the protein’s active site which block the
function of the enzyme. This belief is consistent with the repeated observation
of these adducts by mass spectrometry in biochemical and/or crystallographic
experiments (e.g., compounds 5 and 6; see Ref. 10a,c,e). Once formed, the
PRPP-adducts may accumulate intracellularly and thereby enhance cell culture
antiproliferation effects (see Ref. 6a for additional information and discussion).
However, there are many other factors that also likely influence NAMPT
inhibitor cell potency including: biochemical inhibition activity, the ability of a
given inhibitor and/or its corresponding PRPP-derived ribose adduct to
effectively compete with the NAM substrate, cell membrane permeability,
and/or protein binding.
16. The improvements in biochemical inhibition exhibited by compounds 10, 12,
and 14 relative to 7 were deemed too incremental to warrant additional
follow-up activities.
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17. Compound 51 was previously described in the literature as STF-31, an inhibitor
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as determined by as determined by LCMS analysis with UV detection at
220 nm.
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