Discovery of potent and efficacious urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors with reduced CYP2C9 inhibition properties

Article abstract of DOI:10.1016/j.bmcl.2013.04.040

Potent, reversible inhibition of the cytochrome P450 CYP2C9 isoform was observed in a series of urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. This unwanted property was successfully removed from the described inhibitors throug

Full text of DOI:10.1016/j.bmcl.2013.04.040

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3531–3538
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent and efficacious urea-containing nicotinamide
phosphoribosyltransferase (NAMPT) inhibitors with reduced CYP2C9
inhibition properties
Janet Gunzner-Toste a, Guiling Zhao a, Paul Bauer b, Timm Baumeister b, Alexandre J. Buckmelter b,
Maureen Caligiuri b, Karl H. Clodfelter b, Bang Fu c, Bingsong Han b, Yen-Ching Ho b, Nikolai Kley b,
Xiaorong Liang a, Bianca M. Liederer a, Jian Lin b, Sophie Mukadam a, Thomas O’Brien a, Angela Oh a,
Dominic J. Reynolds b, Geeta Sharma b, Nicholas Skelton a, Chase C. Smith b, Jasleen Sodhi a,
Weiru Wang a, Zhongguo Wang b, Yang Xiao a, Po-wai Yuen c, Mark Zak a, Lei Zhang c,
Xiaozhang Zheng b, Kenneth W. Bair b, Peter S. Dragovich a,
⇑
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
a r t i c l e i n f o
a b s t r a c t
Article history:
Potent, reversible inhibition of the cytochrome P450 CYP2C9 isoform was observed in a series of urea-
containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. This unwanted property was
successfully removed from the described inhibitors through a combination of structure-based design
and medicinal chemistry activities. An optimized compound which did not inhibit CYP2C9 exhibited
potent anti-NAMPT activity (17; BC NAMPT IC50 = 3 nM; A2780 antiproliferative IC50 = 70 nM), good
mouse PK properties, and was efficacious in an A2780 mouse xenograft model. The crystal structure of
this compound in complex with the NAMPT protein is also described.
Received 16 February 2013
Revised 11 April 2013
Accepted 16 April 2013
Available online 25 April 2013
Keywords:
Nicotinamide phosphoribosyltransferase
NAMPT
Ó 2013 Elsevier Ltd. All rights reserved.
X-ray crystal structure
Cytochrome P450
CYP2C9
Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the
first and rate-limiting step in the conversion of nicotinamide
(NAM) to nicotinamide adenine dinucleotide (NAD), a critical co-
factor for many enzymes involved in maintaining healthy cell
metabolism.1 NAMPT functions as a symmetrical homo-dimeric
protein complex and facilitates the condensation of NAM with
phosphoribosyl pyrophosphate (PRPP; Fig. 1).2 This process is en-
hanced by the ATP-mediated phosphorylation of a key histidine
residue near the NAMPT active site (formed at the dimer interface)
but is not believed to involve covalent bond formation between
NAMPT and its substrates or products.3 The nicotinamide mononu-
cleotide (NMN) produced by NAMPT catalysis is subsequently
transformed in a non-rate-limiting manner to NAD by another en-
zyme (nicotinamide/nicotinate mononucleotide adenyltransferase,
NMNAT; Fig. 1).4 The process described in Figure 1 represents an
efficient means to recycle the NAM produced during NAD
consumption by enzymes such as the sirtuins and PARPs and is
the only known mechanism employed by mammalian cells to
accomplish this important objective.4,5 Inhibition of NAMPT is
therefore anticipated to negatively impact intracellular NAD levels,
and such changes may impair the growth of many cancer cell lines
whose elevated metabolisms and high proliferation rates make
them highly dependent on NAD-consuming enzymes for survival.6
Several classes of NAMPT inhibitors have been previously de-
scribed in the scientific literature, and two specific examples that
progressed to human clinical trials are shown in Figure 2 (com-
pounds 1 and 2).7–10 We recently reported the structure-based
identification of urea-containing NAMPT inhibitors exemplified
by compounds 3 and 4 (Table 1).11–13 While 3 and 4 exhibited
many desirable biological properties, they were also potent (sub-
micromolar), reversible inhibitors of the cytochrome P450 2C9
(CYP2C9) isozyme resulting in their potential to cause unwanted
drug–drug interactions.14 In this work, we describe our efforts to
remove this unfavorable characteristic from this inhibitor series
while retaining the desirable biopharmaceutical properties which
existed in the lead compounds.
⇑
Corresponding author. Tel.: +1 650 467 6854.
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.2013.04.040

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J. Gunzner-Toste et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3531–3538
2-O3PO
N
2-
O
P
PO3
N
O
H247
2-
O
OPO3
+
O-
N
O
HO
OH
NAMPT
ADP
ATP
NH2
PRPP
NAM
N
2-O3PO
HN
O
H247
+
N
O
+
PPi
NH2
3-
NAMPT
HO
OH
NMN
PO4
NH2
O
N
N
N
NH2
N
N
O
NMNAT
OH
OH
O
HO
HO
O
O
-
O
P O P
O
O
-
O
NAD
Figure 1. NAD recycling and NAMPT biochemical mechanism.
O
N
N
N
N
O
O
N
H
N
H
N
N
H
Cl
7,8
9
1
2
(GMX-1778)
(APO-866)
Figure 2. NAMPT inhibitors which progressed to clinical development.
Table 1
Methyl-scan of urea-containing NAMPT inhibitors
O
O
R2
S
O
N
B =
A =
R1
O
N
H
N
H
R1
R2
NAMPT IC50 (nM)
A2780 IC50 (nM)
CYP2C9 IC50 (nM)
a
b
c
Entry
3
4
A
B
3
28
144
990
68
200
N
5
6
A
B
146
>2000
>2000
>2000
4600
>10,000
N
7
8
A
B
59
1400
>2000
>2000
>10,000
>10,000
N
9
10
A
B
7
40
144
1100
125
644
N
11
12
A
B
12
1500
>2000
>2000
1960
>10,000
N
N
13d
14d
A
B
930
1600
>2000
>2000
139
1300
See Supplementary data for experimental details associated with each assessment. All biochemical, cell-based, and CYP assay results are reported as the arithmetic mean of
two separate runs (n = 2).
a
NAMPT biochemical inhibition.
b
Antiproliferation activity determined in cell culture experiments using A2780 cell line. All antiproliferation effects were reversed by addition of 330 lM NMN.
Cytochrome P450 inhibition activity (CYP2C9 isoform).
Racemic mixture.
c
d

J. Gunzner-Toste et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3531–3538
3533
Table 2
Pyridine alteration of urea-containing NAMPT inhibitors
O
O
S
O
R
N
H
N
H
a
b
c
d
Entry
R
NAMPT IC50 (nM)
A2780 IC50 (nM)
CYP2C9 IC50 (nM)
HLM CLhep (mL/min/kg)
N
3
3
144
68
8
N
15
16
680
13
>2000
>2000
>10,000
12
6
O
N
1770
Cl
N
17
18
3
70
>10,000
5
H2N
N
>2000
>2000
ND
74
ND
N
N
19
13
739
8
F
N
N
20
21
12
846
515
460
3
5
N
1300
>2000
N
N
N
22
23
600
14
>2000
>2000
149
6
3
N
>10,000
H2N
N
ND = not determined. See Supplementary data for experimental details, associated with each assessment. All biochemical, cell-based, CYP, and HLM assay results are reported
as the arithmetic mean of two separate runs (n = 2).
a
NAMPT biochemical inhibition.
b
Antiproliferation activity determined in cell culture experiments using A2780 cell line. All antiproliferation effects were reversed by addition of 330
Cytochrome P450 inhibition activity (CYP2C9 isoform).
Metabolic stability toward human liver microsomes (stable, moderate, labile = <6, 6–15, >15 mL/min/kg, respectively).
lM NMN.
c
d
We identified the exposed pyridine moiety contained in 3 and 4
pare 3 and 4 with 13 and 14). Based on the above results, we aban-
as one possible source of the undesirable CYP2C9 inhibition activ-
ity.15 We therefore began our studies by introducing a methyl
group at various locations on the pyridine ring in an attempt to
block interactions with the CYP2C9 protein. As expected, addition
of a methyl group adjacent to the pyridine N-atom dramatically re-
duced CYP2C9 inhibition activity (compounds 5–8, Table 1). Less
impact on anti-CYP2C9 properties was unsurprisingly seen when
the methyl group was located at the 3-position relative to the pyr-
idine N-atom (inhibitors 9 and 10). However, compounds bearing
methyl groups at the 4-position relative to the pyridine N-atom
also exhibited relatively weak CYP2C9 inhibition activity (11 and
12; Table 1). This outcome may result from an altered conforma-
tional preference of the pyridine ring relative to inhibitors 5–10
that precludes efficient binding to the cytochrome P450 enzyme.
Unfortunately, as shown in Table 1, all methyl modifications which
favorably influenced CYP2C9 inhibition activity also had negative
impacts on NAMPT biochemical and cell-based inhibition proper-
ties (compounds 5–8 and 11–12). Similarly, incorporation of a
methyl substituent at the benzylic position of the inhibitor design
resulted in loss of NAMPT inhibition potency (respectively com-
doned our attempts to introduce a methyl moiety on the pyridine
ring of the inhibitors under study and instead explored alternate
approaches to reducing the ability of the pyridine N-atom to favor-
able interact with CYP2C9.16
As shown in Table 2, incorporation of a methoxy substituent
adjacent to the pyridine N-atom present in compound 3 afforded
a molecule which exhibited weaker CYP2C9 and NAMPT inhibition
properties (compound 15). A more favorable result was observed
when a Cl substituent was utilized in place of the methoxy group
with the resulting compound retaining potent attenuation of
NAMPT biochemical activity while exhibiting only moderate inhi-
bition of CYP2C9 (16, Table 2). Unfortunately, this molecule lacked
meaningful cell-proliferation activity, suggesting that it may not
readily combine with PRPP in the NAMPT active site.17,18 Encourag-
ingly, incorporation of a 2-amino-pyridine substituent into the
inhibitor design afforded a compound with potent anti-NAMPT
activity in both biochemical and cellular assays and minimal
CYP2C9 inhibitory properties (17, Table 2). However, dimethyla-
tion of the 2-amino-pyridine moiety drastically impaired NAMPT
inhibition at both the biochemical and cellular level (compare 18

3534
J. Gunzner-Toste et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3531–3538
Table 3
Sulfonamide SAR of urea-containing NAMPT inhibitors
O
O
R
S
O
N
N
H
N
H
a
b
c
d
Entry
R
NAMPT IC50 (nM)
A2780 IC50 (nM)
CYP2C9 IC50 (nM)
HLM CLhep (mL/min/kg)
N
N
O
O
4
28
990
200
750
6
24
4
15
17
25e
N
N
O
52
745
601
17
26
27
O
O
10
33
251
1050
554
8
3
N
N
1600
28
47
>2000
1140
8
O
N
N
O
29
103
27
>2000
188
>10,000
>10,000
7
7
30f
N
H
NH
31f
32f
N
6
5
76
>10,000
>10,000
4
2
NH
136
N
See Supplementary data for experimental details associated with each assessment. All biochemical, cell-based, CYP, and HLM assay results are reported as the arithmetic
mean of two separate runs (n = 2).
a
NAMPT biochemical inhibition.
b
Antiproliferation activity determined in cell culture experiments using A2780 cell line. All antiproliferation effects were reversed by addition of 330 lM NMN.
Cytochrome P450 inhibition activity (CYP2C9 isoform).
Metabolic stability toward human liver microsomes (stable, moderate, labile = <6, 6–15, >15 mL/min/kg, respectively).
Racemic mixture of trans-isomers.
c
d
e
f
Racemic mixture.
with 17, Table 2). Introduction of a fluorine substituent at the 3-po-
inhibitor design that were not assessed during our initial explora-
sition relative to the pyridine N-atom present in compound 3 had
minimal effects on CYP2C9 inhibition activity (compare 19, Table 2
with 3, Table 1). However, incorporation of pyridazine, pyrimidine,
and pyrazine moieties into the inhibitor design ameliorated
CYP2C9 inhibition properties to varying degrees (compounds 20–
23, Table 2). Unfortunately, all of these modifications also nega-
tively impacted anti-NAMPT activity in either biochemical and/or
cell-based assays. Encouragingly, all compounds depicted in Table 2
displayed good to excellent in vitro stability toward human liver
microsomes suggesting their potential to exhibit low clearance val-
ues in vivo.
In addition to altering the NAMPT inhibitors in the vicinity of
the pyridine moiety, we also explored whether modifications else-
where in the compounds’ structures could favorably impact
CYP2C9 inhibition properties. We noted that, as depicted in Table 1,
replacement of the terminal phenyl sulfone moiety present in a
variety of molecules with a morpholine-derived sulfonamide con-
sistently afforded compounds with more favorable (i.e., weaker)
CYP2C9 inhibition profiles. We therefore examined the introduc-
tion of additional terminal sulfonamides into the urea-containing
tions.11 As shown in Table 3, compounds which terminated in
substituted or bicyclic morpholine-derived sulfonamides exhibited
relatively weak CYP2C9 inhibition properties (24–27). Further
attenuation of the unwanted cytochrome P450 inhibition activity
was achieved by employing terminal sulfonamides containing po-
lar spirocyclic moieties (compounds 28–32, Table 3). Similarly, use
of sulfonamides derived from various 1,4-diazepanes, 4-amino-
piperidines, and amino-cyclohexanes typically afforded potent
NAMPT inhibitors with relatively benign CYP2C9 inhibitory prop-
erties (compounds 33–41, Table 4). Several of the described sulfon-
amide-containing compounds failed to exhibit sub-micromolar
potency in cell based assessments in spite of their attractive
NAMPT biochemical inhibition activities (27–29, Table 3). As men-
tioned above, this phenomenon may result from the inability of
these compounds to productively form PRPP adducts in the NAMPT
active site, although alternate explanations are also possible.17,18 In
general, the sulfonamides depicted in Tables 3 and 4 displayed
good to moderate stability toward human liver microsomes,
although several highly lipophilic molecules were determined to
be metabolically labile (compounds 24, 25, and 36).

J. Gunzner-Toste et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3531–3538
3535
Table 4
Sulfonamide SAR of urea-containing NAMPT inhibitors
O
R
O
S
O
N
H
N
H
N
a
b
c
d
Entry
R
NAMPT IC50 (nM)
A2780 IC50 (nM)
CYP2C9 IC50 (nM)
HLM CLhep (mL/min/kg)
N
N
NH
NH
33
45
594
>10,000
2180
6
34e
100
99
8
N
N
NH
NH
35e
36e
11
3
109
80
930
110
5
18
CF3
F
F
37
38
8
410
351
>10,000
>10,000
3
6
HN
NH
142
HN
N
39
40
4
8
52
>10,000
>10,000
5
2
HN
HN
HN
NH2
OH
369
41
4
42
>10,000
4
OH
See Supplementary data for experimental details associated with each assessment. All biochemical, cell-based, CYP, and HLM assay results are reported as the arithmetic
mean of two separate runs (n = 2).
a
NAMPT biochemical inhibition.
b
Antiproliferation activity determined in cell culture experiments using A2780 cell line. All antiproliferation effects were reversed by addition of 330
Cytochrome P450 inhibition activity (CYP2C9 isoform).
Metabolic stability toward human liver microsomes (stable, moderate, labile = <6, 6–15, >15 mL/min/kg, respectively).
Racemic mixture.
lM NMN.
c
d
e
Table 5
In vitro stability, permeability, plasma protein binding and solubility of selected NAMPT inhibitors
MDCK Papp,A–B (ꢀ10ꢁ6 cm/s)
Mouse PPBd (fu)
Sole
(lM)
a
b
c
Compd
MLM CLhep (mL/min/kg)
MH CLhep (mL/min/kg)
17
24
31
34
39
41
42
81
44
48
27
61
12
55
9.2
16
6.3
19
2.9
3.1
0.07
0.10
0.20
0.10
0.027
0.011
0.43
0.39
0.55
0.35
109
112
75
107
161
136
All assay results except solubility determinations are reported as the arithmetic mean of two separate runs (n = 2; solubility: n = 1). See Supplementary data for experimental
details associated with each assessment.
a
Stability toward mouse liver microsomes (stable, moderate, labile = <27, 27–63, >63 mL/min/kg, respectively).
Metabolic stability toward mouse hepatocytes (stable, moderate, labile = <27, 27–63, >63 mL/min/kg, respectively).
Apparent permeability coefficients (Papp,A–B) across MDCK cell monolayers.
Mouse plasma protein binding; fraction unbound.
b
c
d
e
Aqueous solubility measured in high-throughput assessment.
The explorations described above identified multiple com-
pounds which exhibited potent anti-NAMPT properties in both bio-
chemical and cellular assays as well as minimal CYP2C9 inhibition
properties. In anticipation of conducting in vivo animal experi-
ments with such molecules, we profiled them in various in vitro
DMPK assays designed to assess their in vivo potential. With the
exception of inhibitor 24, the compounds examined displayed
moderate stability when exposed to mouse liver microsomes
(Table 5). In addition, all such molecules exhibited improved sta-
bility profiles when mouse hepatocytes were utilized in lieu of
microsomes. Inhibitors 17 and 24 displayed acceptable permeabil-
ity in MDCK assessments, but compounds bearing highly polar sul-

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J. Gunzner-Toste et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3531–3538
1200
Vehicle
Compound 17
1000
800
600
400
200
0
Figure 3. Mean ( standard deviation) plasma concentration–time profiles of
compound 17 in BALB/c mice (n = 3) following intravenous and oral administration.
0 1012
14
16
18
20
22
24
26
Days
Table 6
Dosing
Mean pharmacokinetic parameters of compound 17 in BALB/c mice (n = 3)
Route Dosea
(mg/kg)
Cmax
M)
Tmax
(h)
Vss (L/ CLe (mL/
T1/2
(h)
Fg
(%)
b
c
d
f
Figure 4. Efficacy of compound 17 (dosed 50 mg/kg BID) in A2780 mouse xenograft
model. Eight (8) mice were employed per group. Vehicle = 60% PEG400/10% EtOH/
30% D5W. Data reflect mean values ( standard deviation).
(
l
kg)
min/kg)
iv
1
—
2.9
—
0.83
0.51
—
4.9
—
2.6
2.7
—
31
po
5
See Supplementary data for experimental descriptions
Table 7
a
Formulation = 10:60:30 EtOH/PEG400/D5W (v/v/v, solution).
Highest observed plasma concentration.
Time at which Cmax occured.
Volume of distribution at steady-state.
Clearance.
Half-life.
Antiproliferation effects determined for compound 17 in various cell linesa,b
b
c
HT1080
104 17
PC3
MiaPaCa2
>1000
HCT116
87 13
d
IC50 (nM)
149 76
e
f
a
CyQuant endpoint; arithmetic mean of three separate runs (n = 3, standard
g
Bioavailibility.
deviations are also shown).
b
All antiproliferation effects were reversed by addition of 330 lM NMN. See
Supplementary data for experimental details.
fonamide moieties did not (31, 34, 39, and 41, Table 5). Mouse
plasma protein binding properties varied considerably among the
compounds depicted in Table 5 with the more polar analogs exhib-
iting higher free fractions. Encouragingly, all compounds contained
in Table 5 displayed good aqueous solubility when examined in a
high-throughput assay which measured this property. Of the mol-
ecules profiled in the in vitro DMPK assessments, inhibitor 17
exhibited the most optimal combination of in vitro metabolic sta-
bility, MDCK permeability, protein binding, and aqueous solubility.
The compound was therefore progressed to in vivo mouse PK and
xenograft efficacy studies.
I309
S275
P237
R311
I351
Y18’
As shown in Figure 3, compound 17 exhibited good exposures
in mice following oral administration using a solution formulation.
The molecule’s in vivo clearance (uncorrected for protein binding
effects) observed after IV dosing in mice was low and was some-
what lower than that predicted by in vitro microsome and hepato-
cyte stability experiments (Table 6). As depicted in Figure 4,
compound 17 displayed good efficacy in an A2780 human ovarian
carcinoma xenograft model with relatively minor changes in body
weights (see Supplementary data for additional details). Consistent
with its expected NAMPT inhibition mechanism, the molecule po-
tently reduced NAD levels in A2780 cells with an IC50 value be-
V242
H191
D219
Y188
F193
Figure 5. Co-crystal structure of inhibitor 17 (green) in complex with NAMPT. The
ligand binds to a site formed by the interface of two NAMPT protein monomers
(depicted as white and cyan, respectively). Water molecules are depicted as red
spheres and a bound phosphate is shown in orange. Hydrogen bonds are indicated
with dashed yellow lines, and the van der Waals surface of the protein is shown in
grey. The resolution of the structure is 2.4 Å.
tween 30 and 75 nM in
a 48 h in vitro assessment (see
Supplementary data for additional details). Compound 17 also
exhibited antiproliferative effects against a number of additional
cell lines (Table 7), although in some instances such activity was
attenuated relative to potency against the A2780 line. In addition,
compound 17 did not inhibit non-2C9 CYP isoforms against which
it was tested (IC50 >10 lM vs CYPs 3A4, 2D6, 1A2, and 2C19) sug-
gesting a low risk of causing unwanted drug–drug interactions via
inhibition of these enzymes.
To enable further optimization of the described urea-containing
series of NAMPT inhibitors, we obtained a crystal structure of
compound 17 in complex with the NAMPT protein.19 As shown
in Figure 5, the amino pyridine ring of 17 forms a pi-stack between

J. Gunzner-Toste et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3531–3538
3537
O
O
O
O
O
O
S
S
R2
S
O
Cl
O
N
(i)
(ii)
Cl
R1
R1
R3
O
C
N
H
N
H
N
H
N
H
N
42
43
Scheme 1. Synthesis of urea-containing compounds. Reagents and conditions: (i) R1NH2, THF, rt, 18 h; (ii) R2NHR3, Et3N, DCM, rt, 1 h, 53%.
O
S
O2N
H2N
Na
(i)
(ii)
O
S
S
O
O
O
O
O
44
45
O
O
O
S
S
(iii)
(iv)
O
O
R1
O
N
H
N
H
N
H
46
47
Scheme 2. Alternate synthesis of urea-containing compounds. Reagents and conditions: (i) 1-fluoro-4-nitrobenzene, DMSO, 130 °C, 2 days, 95%; (ii) MeOH/THF, H2, 10% Pd
on C, rt, 18 h, 81%; (iii) K2CO3, PhOC(O)Cl, THF, rt, 1 h, 86%; (iv) R1NH2, Et3N, 1,4-dioxane, 60 °C, 1 h, 82%.
the side chains of NAMPT residues Phe193 and Tyr18’, with the
amino group extending into the active site that is ordinarily occu-
pied by phosphorylated ribose. The inhibitor is positioned with the
pyridine nitrogen atom appropriately oriented to form an adduct
with bound PRPP in the active site.20 The urea NH groups partici-
pate in two hydrogen bonds with a bound water molecule, which
in turn makes hydrogen bonds to the side chain of Asp219 and
the backbone carbonyl oxygen of Val242. The urea carbonyl oxy-
gen atom of 17 is positioned near the side chain hydroxyl group
of Ser275 (3.1 Å). However, the C@Oꢂ ꢂ ꢂOH angle is close to 90° sug-
gesting that an optimal hydrogen bond interaction may not be
formed between the two moieties. The aniline-phenylsulfone por-
tion of 17 extends through a hydrophobic and aromatic tunnel of
the NAMPT dimer formed by the side chains of His191, Val242,
Ile309 and Ile351, while the terminal phenyl moiety contacts the
solvent exposed surface of the protein. The inhibitor sulfone oxy-
gen atoms are both within hydrogen bonding distance of bound
water molecules, and one of them is close to the backbone carbonyl
oxygen of Tyr188 (3.1 Å)—likely an unfavorable interaction. The
ring of the terminal phenyl sulfone packs against the side chain
of the NAMPT Pro237 residue, but otherwise does not make signif-
icant contact with the protein. This orientation left considerable
space for ligand expansion in the vicinity of the terminal phenyl
ring and explains the wide tolerance of various terminal sulfona-
mides noted in the SAR results described above.
The inhibitors described in this work were prepared by the two
general synthetic methods depicted in Schemes 1 and 2.21 In the
first approach, reaction of commercially available 4-(chlorosulfo-
nyl)phenyl isocyanate with various amines under neutral condi-
tions afforded sulfonyl chloride-containing intermediates of
general structure 42. These entities were separately condensed
with additional amines in the presence of a tertiary base to give
the desired products (general structure 43, Scheme 1). In the sec-
ond method, sodium benzenesulfinate was transformed to the ani-
line-containing biphenyl sufone 45 (via the nitro-intermediate 44)
using known chemistries.22 Sulfone 45 was then converted to car-
bamate 46 which was subsequently condensed with a variety of
amines to provide the desired products (general structure 47).
The amines required for the preparation of compounds 37 and 38
were prepared using literature methods.23
As described above, unwanted reversible CYP2C9 inhibition
activity was successfully removed from a series of potent urea-
containing NAMPT inhibitors through a combination of structure-
based design and medicinal chemistry activities. An optimized
compound (17) with potent anti-NAMPT properties was thereby
identified which did not reversibly inhibit CYP2C9 or any other
CYP isoform against which it was tested. The compound also dis-
played good mouse PK properties and was efficacious in an
A2780 mouse xenograft model. A crystal structure of compound
17 obtained in complex with NAMPT suggested additional possibil-
ities for inhibitor modification, and these activities will be de-
scribed in subsequent disclosures.
Acknowledgments
We thank Drs. Krista Bowman and Jiansheng Wu and their
respective Genentech research groups for performing many pro-
tein expression and purification activities, the DMPK group at
Genentech, especially Leslie Wang, Jonathan Cheong, Jason Halla-
day, and Ning Liu, for their contributions to the generation of the
in vitro DMPK data, Rashida Garcia-Dancey and Shohini Ganguly
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 generat-
ing the Rapidfire LCMS data, and professor Yigong Shi’s group
(Tsinghua University, Beijing, China) for providing the NAMPT
DNA construct. We also thank Dr. Peter Jackson for many helpful
discussions regarding NAMPT.
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.
04.040.

3538
J. Gunzner-Toste et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3531–3538
D. M. WO 2011/109441.; (c) Arigon, J.; Bernhart, C.; Bouaboula, M.; Dimalta, A.;
References and notes
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1. (a) He, J.; Tu, C.; Li, M.; Wang, S.; Guan, X.; Lin, J.; Li, Z. Curr. Pharm. Des.
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13. The antiproliferative effects exhibited in cell culture by
completely eliminated (‘reversed’) when the compounds were tested in the
presence of 330 M NMN (the product of the NAMPT-catalyzed condensation
3 and 4 were
l
of nicotinamide and PRPP; cf, Figure 1). This result strongly suggests that the
observed cell-based effects result from NAMPT inhibition. Related NMN
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17. As described previously (Ref. 11),
a cell-potent, urea-containing NAMPT
inhibitor was observed to function as a substrate for the NAMPT-catalyzed
phosphoribosylation reaction and thereby form the corresponding PRPP-
derived phosphoribosylated adduct (cf, Figure 1).
biochemically potent) compound which could not form
A
a
related (and
similar ribose
adduct was inactive in cell-based experiments, suggesting that adduct
formation contributed to the cell culture activity of this inhibitor class.
However, as described in Ref. 11, PRPP-derived ribose adduct formation was
also observed for several biochemically potent urea-containing NAMPT
inhibitors that were poorly active in cell culture experiments. In addition,
the ability of a given inhibitor and/or its corresponding PRPP-derived ribose
adduct to effectively compete with NAM also contributed to the resulting cell
potency. Accordingly, additional experiments are required to better
understand the relationships between NAMPT enzyme inhibition,
phosphoribosylated adduct formation, and associated cellular activity. Other
phosphoribosylated NAMPT inhibitor adducts have been reported (Ref. 7a) and
a non-phosphorylated inhibitor-ribose entity has also been described (Kang, G.
B.; Bae, M.-H.; Kim, M.-K.; Im, I.; Kim, Y.-C.; Eom, S. H. Mol. Cells 2009, 27, 667).
18. Other explanations for poor cell culture activity exhibited by biochemically
potent NAMPT inhibitors include poor cell penetration and/or high protein
binding.
8.
A prodrug of compound 1 was also progressed to human clinical trials:
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the scientific and patent literature. Curtin, M. L.; Sorensen, B. K.; Heyman, H. R.;
Clark, R. F.; Woller, K. R.; Shah, O. J.; Michaelides, M.; Tse, C.; Vasudevan, A.;
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K. H.; Han, B.; Ho, Y.-C.; Kley, N.; Lin, J.; Reynolds, D. J.; Sharma, G.; Smith, C. C.;
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19. NAMPT is believed to function as a symmetrical homodimer with two active
sites formed at opposite ends of the dimer interface. Accordingly, the protein
crystalized with such
a dimer present in the asymmetric unit. The two
monomer chains were highly similar (RMSD 0.17 Å for 436 C-alpha atoms) and
both active sites contained a molecule of compound 17 in a similar orientation.
In the crystallography discussion, 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. The crystal structure has
been deposited in the PDB (code 4JNM).
20. The described positioning is assumed as the resolution of the crystal structure
cannot distinguish the pyridine N-atom from the alternate CH possibility.
21. Chemical purities were >95% for all final compounds as assessed by LCMS
analysis with UV detection at 220 nm.
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