Discovery of potent and efficacious cyanoguanidine-containing nicotinamide phosphoribosyltransferase (Nampt) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.11.006

A co-crystal structure of amide-containing compound (4) in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein and molecular modeling were utilized to design and discover a potent novel cyanoguanidine-containing inhibitor bearing a sulfone moiety (5, Nampt Biochemical IC₅₀ = 2.5 nM, A2780 cell proliferation IC₅₀ = 9.7 nM). Further SAR exploration identified several additional cyanoguanidine-containing compounds with high potency and good microsomal stability. Among these, compound 15 was selected for in vivo profiling and demonstrated good oral exposure in mice. It also exhibited excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model. The co-crystal structure of this compound in complex with the NAMPT protein was also determined.

Full text of DOI:10.1016/j.bmcl.2013.11.006

Bioorganic & Medicinal Chemistry Letters 24 (2014) 337–343
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent and efficacious cyanoguanidine-containing
nicotinamide phosphoribosyltransferase (Nampt) inhibitors
a
a
a
Xiaozhang Zheng a, , Timm Baumeister , Alexandre J. Buckmelter , Maureen Caligiuri ,
Karl H. Clodfelter a, Bingsong Han a, Yen-Ching Ho a, Nikolai Kley a, Jian Lin a, Dominic J. Reynolds a,
Geeta Sharma a, Chase C. Smith a, Zhongguo Wang a, Peter S. Dragovich b, Angela Oh b, Weiru Wang b,
Mark Zak b, Yunli Wang c, Po-wai Yuen c, Kenneth W. Bair a
⇑
a Forma Therapeutics, Inc., 500 Arsenal Street, Watertown, MA 02472, USA
b Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
c Pharmaron Beijing, Co. Ltd, 6 Tai He Road, BDA, Beijing 100176, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A co-crystal structure of amide-containing compound (4) in complex with the nicotinamide phosphori-
bosyltransferase (Nampt) protein and molecular modeling were utilized to design and discover a potent
novel cyanoguanidine-containing inhibitor bearing a sulfone moiety (5, Nampt Biochemical IC50 = 2.5 nM,
A2780 cell proliferation IC50 = 9.7 nM). Further SAR exploration identified several additional cyanoguani-
dine-containing compounds with high potency and good microsomal stability. Among these, compound
15 was selected for in vivo profiling and demonstrated good oral exposure in mice. It also exhibited
excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model.
The co-crystal structure of this compound in complex with the NAMPT protein was also determined.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 6 September 2013
Revised 1 November 2013
Accepted 5 November 2013
Available online 14 November 2013
Keywords:
Nicotinamide phosphoribosyltransferase
Nampt
cyanoguanidine
X-ray crystal structure
Nicotinamide adenine dinucleotide (NAD) is an abundant and
essential biomolecule used as a co-factor and/or substrate for many
biological processes.1 Mammals have multiple biosynthetic path-
ways to produce NAD; a de novo pathway using tryptophan and
three pathways using exogenous biomolecules as the precursors—
nicotinamide (NAM), nicotinic acid (NA), and nicotinamide ribose
(NR).2 NAM can be converted to NAD via a two-step pathway that
uses nicotinamide phosphoribosyltransferase (Nampt) as the rate
limiting enzyme (Fig. 1a). In an ATP hydrolysis coupled reaction,
Nampt catalyzes the formation of NAD through the condensation
of NAM with 5-phosphoribosyl-1-pyrophosphate (PRPP) to form
nicotinamide mononucleotide (NMN), the immediate precursor to
NAD (Fig. 1b).3 NAD can be consumed in a manner that releases
nicotinamide (NAM) by enzymes which catalyze mono- and
poly-(ADP)-ribosylations, NAD dependent-protein deacetylations
(Sirutins), and cyclic-ADP-ribose production. The primary mecha-
nism of maintaining intracellular NAD levels is its synthesis from
released NAM via the Nampt-dependent salvage pathway.4 In can-
cers, elevated poly-ADP-ribose polymerase (PARP) activity has been
well documented, which leads to an elevated rate of cellular NAD
mitochondrial oxidative phosphorylation, glycolysis, and the citric
acid cycle. These processes are used to produce ATP and assorted
essential biosynthetic precursors upon whose levels cancer cells
are exquisitely dependent. Additionally, NAD and NADH have
essential functions in maintaining the reductive environment that
protects cells from reactive oxygen species which are elevated in
cancers.5 Cancer cells that consume more NAD and are more depen-
dent upon NAD driven processes should be extremely sensitive to
inhibition of the NAD salvage pathway. Therefore, Nampt is an
inviting target for potential development of novel cancer therapies.
Several classes of Nampt inhibitors have been reported in
the scientific literature6,7 and the most advanced compounds
GMX-17788 (1), its prodrug GMX-17779 (not shown), and
APO-86610 (2) progressed to clinical trials during the past decade
(Fig. 2). We recently reported the structure-based identification
of urea- and amide-containing Nampt inhibitors, exemplified by
compounds 3 and 4 (Fig. 2).11,12 In the current work, we describe
the continuation of our Nampt-related research leading to the dis-
covery of novel and highly potent cyanoguanidine-containing
Nampt inhibitors.
consumption. As
NADH are used in multiple biological redox reactions such as
a
co-factor, NAD and its reduced form
Our previous exploration of urea- and amide-containing Nampt
inhibitors indicated that changes within the linker region of the
compounds were tolerated as long as the positioning of the left
hand side (LHS) aromatic ring and the right hand side (RHS) moiety
remained unaffected (Fig. 3). In particular, crystallographic water
⇑
Corresponding author.
E-mail address: xzheng@formatherapeutics.com (X. Zheng).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.006

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X. Zheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 337–343
a
NAMPT
NMN
Exogenous
NR
Exogenous
NAM
NAD Salvage
NAM
NMNAT
Pathway
Exogenous
Trp, NA
ADP-ribose,
NAD
PARPs,
ADP-ribosylated
Sirtuins
,
proteins,
etc.
etc.
b
2-O3PO
PPi
O-
P
O
O-
P
O
O
O
O
O
N
O
NAMPT
2-O3PO
+
N
O-
O
+
NH2
OH
HO
HO
OH
NMN
NH2
NAM
PRPP
3-
ATP
ADP + PO4
Figure 1. (a) Schematic representation of NAD biosynthetic pathways found in mammalian cells. Nampt, the rate limiting step in the biosynthesis of NAD from NAM,
catalyzes the conversion of NAM to NMN. NMN is further converted to NAD by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT). NAD can also be
produced through alternative pathways using Trp (tryptophan), NA (nicotinic acid), and NR (nicotinamide ribose) as the precursors. (b) The phosphoribosyltransferase
reaction catalyzed by Nampt.
O
N
N
N
O
N
O
N
H
N
H
N
N
H
Cl
1 (GMX-1778)
2 (APO-866)
O
O
N
O
S
O
N
H
O
N
N
H
N
H
NH
N
S
O
O
4
3
Figure 2. Examples of Nampt inhibitors.
molecules in the linker region shifted to accommodate variation in
the linker hydrogen bonding network between Nampt residues
Ser275 and Asp219.11,12 To further examine the effects of different
linker groups, we used molecular modeling to explore whether the
cyanoguanidine linker in compound 1 could replace the amide
moiety present in compound 4, for which a co-crystal structure
in complex with NAMPT was obtained (Fig. 3). The docked pose
of the resulting hybrid molecule 5 overlapped well with both the
pyridine and cyanoguanidine portion of docked 1 as well as with
the RHS of compound 4 (Fig. 4). Compound 5 was therefore synthe-
sized, and it demonstrated very good biochemical and cellular
anti-Nampt potency (Table 1).13 The molecule thus provided an
excellent starting point for further inhibitor optimization.
reduced cell potencies of 7 and 8 relative to 5 and 6 may be
due to improper positioning of the LHS pyridine atom which pre-
vents facile formation of the corresponding inhibitor-PRPP ribose
adducts in the Nampt active site.15 The sulfone containing com-
pounds (5 and 7) displayed better mouse liver microsomal stabil-
ity than the sulfonamide-containing inhibitors (6 and 8), and this
trend was also observed with other Nampt inhibitors we previ-
ously studied.11,12
With this information, our research then focused on 4-pyridine-
cyanoguanidine sulfones by exploring various substituents on the
RHS of the molecules. Our previous work with related urea- and
amide-containing compounds indicated that alterations to the
biarylsulfone portion of the inhibitor could greatly improve the
potency and biopharmaceutical properties.11,12 Similar modifica-
tions were expected to be tolerated in the cyanoguanidine-
containing Nampt inhibitor series since the biarylsulfone terminus
was anticipated to be highly solvent-exposed. As shown in Table 2,
inserting a methylene group between the sulfone and phenyl ring
led to compound 9 which maintained good biochemical activity
As shown in Table 1, a compound containing a sulfonamide
RHS moiety was also a potent Nampt inhibitor in biochemical
and cell-based assays. Interestingly, moving the pyridine nitrogen
atom from the 4-position to 3-position (compounds 7 and 8)
resulted in equal biochemical potency but also produced
a
several-fold loss of cellular activity (compare to 5 and 6). The

X. Zheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 337–343
339
N
N
N
O
N
H
N
H
N
Cl
N
N
LHS
Linker
RHS
N
H
N
H
1
S
O
O
O
N
H
5
NH
N
S
O
O
Linker
LHS
RHS
4
Figure 3. Discovery of novel cyanoguanidine-containing Nampt inhibitors.
Figure 4. Overlay of compound 5 (green carbons) with: (A) compound 4 from PDB code 4LWW and (B) compound 1 (each in magenta) in complex with Nampt (grey). The
structure of compounds 5 and 1 represent minimum energy conformations obtained from docking with GlideXP.14
Table 1
Initial SAR of cyanoguanidine-containing Nampt inhibitors
N
X
Y
N
N
H
N
H
R
O
S
O
a,c
b,c
Entry
X
Y
R
Nampt IC50 (nM)
29
A2780 IC50 (nM)
18
MLM % remainingd
54
HLM % remaininge
75
4f
5
6
7
8
N
CH
CH
N
2.5
2.4
2.2
4.4
9.7
50
57
12
14
5.5
96
60
83
52
O
O
N
N
N
CH
CH
30
N
175
a
b
c
d
e
f
Nampt biochemical inhibition.
A2780 cell proliferation inhibition. This inhibition can be reversed by addition of 0.33 mM of NMN.
Unless otherwise noted, all biochemical and cell-based assay results are reported as the arithmetic mean of at least 2 separate runs (n = 2).
Mouse liver microsomal stability—% remaining at 30 min.
Human liver microsomal stability—% remaining at 30 min.
For structure see Figure 2.

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X. Zheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 337–343
Table 2
RHS SAR of cyanoguanidine-containing Nampt inhibitors
N
N
N
N
H
N
H
R
O
S
O
MLM % remainingd
HLM % remaininge
27
Solubilityf (
lM)
a,c
b,c
Entry
R
Nampt IC50 (nM)
A2780 IC50 (nM)
9
7.5
264
27
30
10
11
5.4
2.0
14.8
0.3
49
56
89
ALQg
NAh
N
>2000
O
N
N
12
13
1.7
0.6
3.7
1.8
67
71
85
83
67
F
1.3
F
CF3
14
15
16
17
2.4
4.4
1.9
3.6
2.9
2.2
3.4
14
74
55
58
11
62
45
55
10
28
16
51
18
OCF3
OMe
N
a
b
c
Nampt biochemical inhibition.
A2780 cell proliferation inhibition. This inhibition can be reversed by addition of 0.33 mM of NMN.
Unless otherwise noted, all biochemical and cell-based assay results are reported as the arithmetic mean of at least 2 separate runs (n = 2).
Mouse liver microsomal stability—% remaining at 30 min.
Human liver microsomal stability—% remaining at 30 min.
Aqueous solubility.
d
e
f
g
h
Above Limit of Quantification (>100
Not Available.
lM).
but displayed greatly reduced cellular potency. The conversion of
the terminal phenyl ring of compound 5 to a cyclohexyl moiety
(compound 10), did not impact potency but resulted in a signifi-
cant loss of mouse liver microsome stability. Incorporating a nitro-
gen atom into the terminal ring (compound 11) maintained
biochemical potency but led to a dramatic loss of cellular activity.
However, introducing a morpholino group attached to a terminal
pyridine ring (compound 12) greatly improved the cellular poten-
cies. Modification of the terminal phenyl ring by introducing sub-
stituents such as 3,5-di-F, 3-CF3, 3-OCF3, and 3-OMe afforded
very potent Nampt inhibitors in both biochemical and cell-based
assessments (13–16). A fused bicyclic aryl terminal system (17)
maintained good biological activities but also exhibited impaired
microsomal stabilities. In summary, RHS SAR exploration led to
the discovery of several potent Nampt inhibitors with good micro-
somal stability as well as acceptable aqueous solubility.
Among the potent and stable compounds, 15 was selected to
progress to in vivo mouse PK and xenograft efficacy studies. As
shown in Table 3, compound 15 exhibited good exposures in mice
following oral administration using a solution formulation. As
depicted in Figure 5, compound 15 also displayed robust efficacy
in an A2780 human ovarian carcinoma xenograft model with rela-
tively minor changes in body weights. Encouragingly, oral admin-
istration of the compound for 5 days (75 mg/kg BID; maximum
tolerated dose) produced rapid tumor regression. The anti-tumor
effects exhibited by compound 15 were durable, with the mean
tumor volume of the treated group failing to return to baseline
levels by the end of the study (day 13). The described xenograft

X. Zheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 337–343
341
Table 3
thiourea inhibitor whose interactions with Asp219 were mediated
by a crystallographic water molecule.11a For both 4 and 15, the sul-
fone oxygens formed water mediated hydrogen bonds with the
backbone carbonyls of Val350 and Tyr188. The trifluoromethoxy
group of 15 was positioned in a pocket surrounded by the residues
of His191, Val242, Ser241, Tyr240, and Tyr188. This orientation left
considerable space for ligand expansion in the vicinity of the
terminal phenyl ring and explained the wide tolerance of various
terminal sulfones noted in the SAR.
Compound 15 in vivo mouse PK parameters after oral administration (75 mg/kg)
PK parameters
Cmax M)
Tmax (h)
AUCinf
t1/2(h)
Mean (n = 3) SD
(
l
47.9 17.5
2.00
286.8 194.1
2.16
(
lM h)
The inhibitors described in this work were prepared by the
general synthetic method depicted in Scheme 1. Commercially
available dimethyl cyanocarbonimidodithioate (18) was reacted
with 3 or 4-aminopyridines (19) under basic conditions to afford
cyanocarbonimidothioate intermediates (20) which were subse-
quently condensed with a variety of benzylamines (21) to provide
the desired products 5–17.
The synthesis of benzylamine 21a is shown in Scheme 2. Sulfo-
nyl chloride (22) was coupled with bridged morpholine (23) to
provide the corresponding sulfonamide (24). This intermediate
was then reduced to the benzylamine (21a) by hydrogenation.
The preparation of benzylamine 21b is depicted in Scheme 3.
Sulfinate (25)16 was coupled with benzyl bromide (26) to provide
the corresponding sulfone intermediate (27). Removal of the acetyl
group from 27 then led to the benzyl amine 21b.
results confirmed that a cyanoguanidine-containing Nampt inhib-
itor with appropriate biological and PK properties can function as a
potent in vivo anti-cancer agent.
To enable further optimization of the described cyanoguani-
dine-containing Nampt inhibitors, we obtained a co-crystal struc-
ture of compound 15 in complex with the protein (Fig. 6B). The
right hand side of 15 closely overlapped with the co-crystal of 4
and with structures of our other previously disclosed urea-,
thiourea-, and amide-containing inhibitors.11,12 The cyano nitrogen
of 15 was located at a position close to that occupied by the sulfur
atom of our related thiourea inhibitor (PDB code 4JRS)11a and
formed a direct hydrogen bond with Ser275 while replacing the
and displaced a crystallographic water that mediated a similar
interaction in the structure of 4. The two NH groups of 15 formed
direct hydrogen bonds with Asp219 in contrast to a related
1400
1300
Vehicle control
Compound 15 (75 mg/kg PO BIDX5)
22
20
18
1200
1100
1000
900
800
700
600
500
400
300
200
100
dosing period
dosing period
16
0
0
2
4
6
8
10
12
14
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
days
days
Figure 5. Left panel: efficacy of compound 15 (dosed 75 mg/kg BID) in A2780 mouse xenograft model. Right panel: body weight loss as a result of compound 15
administration in xenograft experiment. Eight (8) mice were employed per group. Vehicle = 60% PEG400/10% EtOH/30% D5W. Data reflect mean values ( standard deviation).
Figure 6. (A) Co-crystal structure of 4 (green carbons) in complex with NAMPT (PDB code 4LWW, resolution 1.64 Å). (B) Co-crystal structure of 15 (green carbons) in complex
with Nampt (PDB code 4LTS, resolution 1.70 Å). Important crystallographic waters present in each structure are shown as red spheres, and possible hydrogen bonds are
depicted as dashed yellow lines. For comparison, each picture shows the overlay of the ligand of the other structure in magenta.

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X. Zheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 337–343
N
H2N
N
X
Y
N
X
Y
R
O
N
S
X
Y
N
O
NH2
N
N
H
N
H
21
(ii)
19
N
H
SMe
R
O
MeS
SMe
S
(i)
O
5 - 17
20
18
X = N, Y = CH
or
X = CH, Y = N
Scheme 1. Synthesis of cyanoguanidine-containing compounds. Reagents and conditions: (i) NaH, DMAP, DMF, 0 to 25 °C, 18 h, 39–84%; (ii) Et3N, DMAP, pyridine, 60 °C, 12 h,
36–73%.
O
HN
N
H2N
O
N
O
23
Cl
O
N
O
N
O
S
(i)
S
S
(ii)
O
O
O
24
22
21a
Scheme 2. Synthesis of compound 21a. Reagents and conditions: (i) (i-Pr)2NEt, CH2Cl2, 0 to 25 °C, 2 h, 78%; (ii) Raney Ni, H2, 45 psi, MeOH, 100 °C, 16 h, 58%.
O
O
Br
N
H
N
H
H2N
26
ONa
S
O
(ii)
(i)
S
S
O
O
O
O
25
27
21b
Scheme 3. Synthesis of compound 21b. Reagents and conditions: (i) (n-Bu)4N+IÀ, H2O, 70 °C, 2 h, 54%; (ii) 12 M HCl-i-PrOH, 100 °C, 16 h, 80%.
The synthesis of compound 21c is shown in Scheme 4. Treat-
ment of 4-fluorobenzonitrile (28) with cyclohexanethiol (29) gave
the thioether intermediate (30). Oxidation of 30 with mCPBA pro-
vided the corresponding sulfone (31) which was then reduced to
compound 21c.
The preparation of benzylamines (21d–21k) is described in
Scheme 5. Sulfinate (28) underwent Chan–Lam oxidative coupling
with aryl boronic acids (32) to provide the corresponding biaryl
sulfone intermediates (33). Removal of the acetyl group from 33
led to the benzyl amines 21d–21k.
SH
H2N
N
N
N
29
S
S
F
(i)
(ii)
(iii)
S
O
O
O
O
28
31
30
21c
Scheme 4. Synthesis of compound 21c. Reagents and conditions: (i) K2CO3, DMF, 120 °C, 20 h, 84%; (ii) mCPBA, CH2Cl2, 25 °C, 4 h, 70%; (iii) 10% Pd/C, H2, 45 psi, MeOH, 25 °C,
1 h, 43%.
O
O
(HO)2B R
H2N
N
H
N
H
32
R
O
ONa
R
O
S
S
O
(ii)
(i)
S
O
O
25
33
21
Entry
21d
21e
21f
R
phenyl
3-pyridyl
3-(6-morpholino)-3-pyridiyl
3,5-di-F-phrnyl
21g
21h
21i
3-CF -phenyl
3
3-OCF -phenyl
3
21j
3-OMe-phenyl
8-quinolinyl
21k
Scheme 5. Synthesis of compounds 21d–21k. Reagents and conditions: (i) Cu(OCOCH3)2, K2CO3, DMSO, 25 °C, 16 h, 15–45%; (ii) 12 M HCl-i-PrOH, 100 °C, 16 h, 80–100%.

X. Zheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 337–343
7. Burgos, E. S. Curr. Med. Chem. 2011, 18, 1947.
343
As described above, we identified novel and potent Nampt
inhibitors which contain the cyanoguanidine moiety. Several of
these compounds exhibit nanomolar antiproliferative activity
against human tumor lines in in vitro cell culture experiments,
and a representative example (compound 15) demonstrated good
mouse PK properties and was efficacious in an A2780 mouse xeno-
graft model. A co-crystal structure of compound 15 obtained in
complex with Nampt suggested additional possibilities for future
inhibitor modification.
8. (a) Watson, M.; Roulston, A.; Bélec, L.; Billot, X.; Marcellus, R.; Bédard, D.;
Bernier, C.; Branchaud, S.; Chan, H.; Dairi, K.; Gilbert, K.; Goulet, D.; Gratton,
M.-O.; Isakau, H.; Jang, A.; Khadir, A.; Koch, E.; Lavoie, M.; Lawless, M.; Nguyen,
M.; Paquette, D.; Turcotte, E.; Berger, A.; Mitchell, M.; Shore, G. C.; Beauparlant,
P. Mol. Cell. Biol. 2009, 29, 5872; (b) Ravaud, A.; Cerny, T.; Terret, C.; Wanders, J.;
Bui, B. N.; Hess, D.; Droz, J.-P.; Fumoleau, P.; Twelves, C. Eur. J. Cancer 2005, 41,
702; (c) Hovstadius, P.; Larsson, R.; Jonsson, E.; Skov, T.; Kissmeyer, A.-M.;
Krasilnikoff, K.; Bergh, J.; Karlsson, M. O.; Lönnebo, A.; Ahlgren, J. Clin. Cancer
Res. 2002, 8, 2843. Compound 1 is also known in the literature as CHS828.
9. Beauparlant, P.; Bédard, D.; Bernier, C.; Chan, H.; Gilbert, K.; Goulet, D.; Gratton,
M.-O.; Lavoie, M.; Roulston, A.; Turcotte, E.; Watson, M. Anti-Cancer Drugs 2009,
20, 346.
10. (a) Olesen, U. H.; Thougaard, A. V.; Jensen, P. B.; Sehested, M. Mol. Cancer Ther.
2010, 9, 1609; (b) Holen, K.; Saltz, L. B.; Hollywood, E.; Burk, K.; Hanauske, A.-R.
Invest. New Drugs 2008, 26, 45. Compound 2 is also known in the literature as
FK866.
Acknowledgments
We acknowledge Forma’s high speed synthesis group for their
synthetic contributions and the ADME group for generating the
microsomal and solubility data, Shohini Ganguly and Rashida Gar-
cia-Dancey for helping to generate the cellular data, Lakshmanan
Manikandan, Saradhi Vijay, and Danilal C. Sharma for generating
the in vivo PK and efficacy data, Agilent Technologies (Woburn,
MA) for generating the Rapidfire LCMS data, and Professor Yigong
Shi’s group (Tsinghua University, Beijing, China) for providing a
Nampt DNA construct and Nampt protein.
11. (a) Zheng, X.; Bauer, P.; Baumeister, T.; Buckmelter, A. J.; Caligiuri, M.;
Clodfelter, K. H.; Han, B.; Ho, Y.-C.; Kley, N.; Lin, J.; Reynolds, D. J.; Sharma, G.;
Smith, C. C.; Wang, Z.; Dragovich, P. S.; Oh, A.; Wang, W.; Zak, M.; Gunzner-
Toste, J.; Zhao, G.; Yuen, P.-w.; Bair, K. W. J. Med. Chem. 2013, 56, 4921; (b)
Gunzner-Toste, J.; Zhao, G.; Bauer, P.; Baumeister, T.; Buckmelter, A. J.;
Caligiuri, M.; Clodfelter, K. H.; Fu, B.; Han, B.; Ho, Y.-C.; Kley, N.; Liang, X.;
Liederer, B. M.; Lin, J.; Mukadam, S.; O’Brien, T.; Oh, A.; Reynolds, D. J.; Sharma,
G.; Skelton, N.; Smith, C. C.; Sodhi, J.; Wang, W.; Wang, Z.; Xiao, Y.; Yuen, P.-w.;
Zak, M.; Zhang, L.; Zheng, X.; Bair, K. W.; Dragovich, P. S. Bioorg. Med. Chem. Lett.
2013, 23, 3531.
12. Zheng, X.; Bauer, P.; Baumeister, T.; Buckmelter, A. J.; Caligiuri, M.; Clodfelter,
K. H.; Han, B.; Ho, Y.-C.; Kley, N.; Lin, J.; Reynolds, D. J.; Sharma, G.; Smith, C. C.;
Wang, Z.; Dragovich, P. S.; Gunzner-Toste, J.; Liederer, B. M.; Ly, J.; O’Brien, T.;
Oh, A.; Wang, L.; Wang, W.; Xiao, Y.; Zak, M.; Zhao, G.; Yuen, P.-w.; Bair, K. W. J.
Med. Chem. 2013, 56, 6413.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.11.
006.
13. The antiproliferative effects exhibited in cell culture by 5 were completely
reversed when the compound was tested in the presence of 0.33 mM NMN (the
product of the Nampt-catalyzed condensation of nicotinamide and PRPP cf.,
Fig. 1). This result strongly suggests that the observed cell-based effects result
from Nampt inhibition. Related NMN experiments were conducted in cell-
based assessments with all inhibitors reported in this work and all displayed
similar reversals of their antiproliferative effects.
References and notes
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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 experiments (Refs.
11,12). Once formed, the PRPP-adducts may accumulate intracellularly and
thereby enhance cell culture antiproliferation effects (see Ref. 8 for additional
information and discussion). However, there are many other factors that also
likely influence NAMPT inhibitor cell potency including: biochemical potency,
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. Bair, K.W. Baumeister, T. Buckmelter, A.J. Clodfelter, K.H. Dragovich, P.S.
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