Identification of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT)

Article abstract of DOI:10.1016/j.bmcl.2013.06.090

Potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors containing 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas were identified using structure-based design techniques. The new compounds displayed improved aqueous solubilities, determined using a high-throughput solubility assessment, relative to previously disclosed urea and amide-containing NAMPT inhibitors. An optimized 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived compound exhibited potent anti-NAMPT activity (18; BC NAMPT IC₅₀ = 11 nM; PC-3 antiproliferative IC₅₀ = 36 nM), satisfactory mouse PK properties, and was efficacious in a PC-3 mouse xenograft model. The crystal structure of another optimized compound (29; NAMPT IC₅₀ = 10 nM; A2780 antiproliferative IC₅₀ = 7 nM) in complex with the NAMPT protein was also determined.

Full text of DOI:10.1016/j.bmcl.2013.06.090

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4875–4885
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived
ureas as potent inhibitors of human nicotinamide
phosphoribosyltransferase (NAMPT)
b
b
b
a
Peter S. Dragovich a, , Kenneth W. Bair , Timm Baumeister , Yen-Ching Ho , Bianca M. Liederer ,
Xiongcai Liu c, Yongbo Liu c, Thomas O’Brien a, Jason Oeh a, Deepak Sampath a, Nicholas Skelton a,
Leslie Wang a, Weiru Wang a, Hongxing Wu c, Yang Xiao a, Po-wai Yuen c, Mark Zak a, Lei Zhang c,
Xiaozhang Zheng b
⇑
a Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA
b Forma Therapeutics Inc., 500 Arsenal Street, Watertown, Massachusetts 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 nicotinamide phosphoribosyltransferase (NAMPT) inhibitors containing 2,3-dihydro-1H-pyrrol-
Received 4 May 2013
Revised 14 June 2013
Accepted 27 June 2013
Available online 6 July 2013
o[3,4-c]pyridine-derived ureas were identified using structure-based design techniques. The new com-
pounds displayed improved aqueous solubilities, determined using
a high-throughput solubility
assessment, relative to previously disclosed urea and amide-containing NAMPT inhibitors. An optimized
2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived compound exhibited potent anti-NAMPT activity (18; BC
NAMPT IC50 = 11 nM; PC-3 antiproliferative IC50 = 36 nM), satisfactory mouse PK properties, and was effi-
cacious in a PC-3 mouse xenograft model. The crystal structure of another optimized compound (29;
NAMPT IC50 = 10 nM; A2780 antiproliferative IC50 = 7 nM) in complex with the NAMPT protein was also
determined.
Keywords:
Nicotinamide phosphoribosyltransferase
NAMPT
Aqueous solubility
X-ray crystal structure
Tumor metabolism
Ó 2013 Elsevier Ltd. All rights reserved.
Nicotinamide phosphoribosyltransferase (NAMPT; also known
in the literature as pre-B cell colony-enhancing factor/PBEF and
visfatin) catalyzes the condensation of nicotinamide (NAM) and
phosphoribosyl pyrophosphate (PRPP) to produce nicotinamide
mononucleotide (NMN; Fig. 1).1 This transformation is dependent
on the phosphorylation of a histidine residue in the NAMPT active
site by ATP, and is the rate-limiting event in the two-step conver-
sion of NAM to the important enzyme co-factor nicotinamide ade-
nine dinucleotide (NAD; Fig. 1).2 The NAM–NAD conversion
represents the only known mechanism for mammalian cells to effi-
ciently produce NAD from NAM that is liberated by NAD-consum-
ing enzymes such as PARPs and Sirtuins.3 Accordingly, NAMPT
plays a key role in maintaining appropriate intracellular NAD lev-
els, and blocking NAMPT activity may thus impair the growth
and/or function of cells which are highly reliant on NAD-depen-
dent processes for survival. Given the elevated metabolisms and
high proliferation rates associated with many tumors and/or
cancer cell lines, NAMPT inhibition is currently viewed as a novel
strategy for the development of new anticancer agents.4,5
We previously described the identification of urea-containing
NAMPT inhibitors exemplified by general structure 1 (Fig. 2) and
compound 5 (Table 1).6–8,32 We also disclosed the preparation of
NAMPT inhibitors bearing amides derived from various bicyclic
heteroaromatic moieties (e.g., structures 2 and 3, Figure 2 and
compounds 6 and 7, Table 1).9 Many examples of both the
urea-containing inhibitors and the heteroaromatic compounds
exhibited desirable biological properties including potent NAMPT
inhibition activity, acceptable oral bioavailability, and efficacy in
mice bearing human tumor xenografts. However, compounds from
both of these inhibitor classes often displayed poor aqueous solu-
bilities which complicated their further development. We there-
fore hypothesized that the introduction of a urea derived from a
cyclic, aliphatic amine into the inhibitor design (structure 4,
Fig. 2) might favorably alter intra/inter hydrogen bonding charac-
teristics relative to ureas such as 1 and thereby improve aqueous
solubility properties. This design modification also involved the
saturation of an aromatic ring present in structures 2 and 3, and
such alterations were also anticipated to beneficially impact
⇑
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.2013.06.090

4876
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4875–4885
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
O
and 13, Table 1) was not tolerated, likely due to tight steric con-
straints in the NAMPT active site. Similarly, replacing the urea moi-
ety present in 8 with the corresponding sulfonyl urea functional
group led to complete loss of biological activity (compound 14, Ta-
ble 1). The above SAR studies identified the 2,3-dihydro-1H-pyrrol-
o[3,4-c]pyridine-derived urea as optimal for obtaining potent
biochemical and cell-based NAMPT inhibition properties, and this
functionality was therefore incorporated into all molecules de-
scribed in the remainder of this work.
?
R
R
N
N
N
H
N
N
H
N
H
1
4
O
O
R
R
O
N
N
We next explored variation of the biaryl sulfone moiety present
in compound 8. As shown in Table 2, numerous substituted aro-
matic and heteroaromatic groups were tolerated at the inhibitor
terminus (compounds 15–21). These results paralleled those from
our previous SAR studies of related NAMPT inhibitors and were
consistent with this portion of the molecules residing in a sol-
vent-exposed region in the NAMPT binding site.9 We also explored
the inclusion of various aliphatic sulfones and sulfonamides into
the inhibitor design. As shown in Table 2, saturation of the termi-
nal phenyl ring present in 8 afforded a compound (22) with
encouraging NAMPT inhibition properties but unacceptably low
stability in human liver microsomes. The poor in vitro stability of
22 could be improved by blocking metabolism on the cyclohexane
ring (23) or by reducing compound lipophilicity through the incor-
poration of various polar functional groups in this region of the
molecule (24–31). However, many of these modifications were also
detrimental to anti-NAMPT potency in the biochemical and/or cell-
based assessments. Compounds containing terminal sulfonamide
moieties (32–40) were generally weaker NAMPT inhibitors than
the related sulfones, but several exceptions to this trend were
identified (38–40). With the exception of compound 22, in vitro
human liver microsomal stabilities for all compounds depicted in
Table 2 were in the stable to moderate range. Measured aqueous
solubilities were also generally favorable with many compounds
H
H
NH
N
N
2
3
Figure 2. Design of ureas derived from cyclic, aliphatic amines as NAMPT
inhibitors.
solubility parameters.10 In this work, we describe the identification
of NAMPT inhibitors bearing such aliphatic urea moieties as well as
the subsequent transformation of the initial lead molecules into
more optimized compounds.11
As shown in Table 1, an inhibitor containing a urea moiety de-
rived from 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine exhibited potent
anti-NAMPT properties in both biochemical and cell culture assess-
ments (compound 8).12 The compound’s antiproliferation effects
were ameliorated by the addition of 0.33 mM of NMN (the product
of NAMPT catalysis; c.f., Fig. 1) to the cell assay, strongly implicat-
ing NAMPT inhibition as the causative mechanism. Importantly,
the molecule also displayed improved aqueous solubility relative
to our previously characterized NAMPT inhibitors bearing unsub-
stituted ureas or heteroaromatic amides (compare to compounds
5, 6, and 7; Table 1).13 Introduction of an amino substituent adja-
cent to the pyridine nitrogen atom present in 8 significantly wors-
ened NAMPT inhibition and solubility properties (compound 9).
Relocation or removal of the pyridine nitrogen atom contained
within 8 afforded molecules (10 and 11) which retained potent
NAMPT biochemical inhibition properties but which were devoid
of anti-NAMPT cell activity. This phenomenon likely arises from
the inability of these compounds to form PRPP-derived phosphor-
ibosylated adducts in the NAMPT active site.14 Incorporation of lar-
ger aliphatic rings into the urea inhibitor design (compounds 12
displaying values >100 l
M in the high-throughput assessment.13
In anticipation of conducting in vivo experiments with the de-
scribed NAMPT inhibitors, we profiled selected examples in a vari-
ety of additional in vitro DMPK assessments. We focused these
efforts on molecules which exhibited potent anti-NAMPT activity
(A2780 IC50 6 0.020
liver microsomes (HLM CL 6 10 mL/min/kg), and appropriate
aqueous solubility (>50 M in the high throughput assessment).
As shown in Table 3, the majority of these compounds were not
lM), good in vitro stability toward human
l

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4875–4885
4877
Table 1
Initial structure–activity relationships
X
R
N
H
S
O
O
Compd
R
X
NAMPT IC50a
(lM)
A2780 IC50b
0.61
(
lM)
Solubilityc
10
(lM)
5d
C@O
0.019
N
N
H
6
7
8
C@O
C@O
C@O
0.020
0.009
0.014
0.018
0.009
0.015
3
NH
N
N
N
O
21
115
N
N
N
9
C@O
0.075
0.38
5.5
H2N
N
N
10
11
C@O
C@O
0.060
0.012
>2.0
>2.0
144
1
N
N
N
12
13
C@O
C@O
0.31
>2.0
>2.0
>2.0
115
102
N
N
N
N
14
SO2
>2.0
>2.0
3
All biochemical and cell-based assay results are reported as the arithmetic mean of at least two separate runs. Solubility determinations are n = 1. See supplementary data for
experimental details associated with each assessment.
a
NAMPT biochemical inhibition.
b
Antiproliferation activity determined in cell culture experiments using A2780 cell line. This inhibition can be reversed by addition of 0.33 mM of NMN.
Aqueous solubility.
Contains piperidine derived sulfonamide in place of phenyl sulfone.
c
d
potent inhibitors of the cytochrome P450 (CYP) 3A4 isoform in
both time-dependent and non-time-dependent assessments (two
substrates examined). They also exhibited minimal inhibition of
several other CYP isoforms (2D6, 1A2, 2C19) but were strong inhib-
itors of CYP2C9. Similar CYP2C9 inhibition properties were noted
previously for related urea-containing NAMPT inhibitors.6b In con-
trast to their favorable stability toward human liver microsomes
(Table 2), the optimized 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-
derived ureas were relatively labile when tested in the presence
of mouse liver microsomes (Table 4). However, all such compounds
displayed improved stability properties when assessed using
mouse hepatocytes (Table 4). The molecules also exhibited a range
of in vitro permeability properties in MDCK cells, with those bear-
ing polar substituents displaying the lowest (poorest) values (19,
28, 29; Table 4). Mouse plasma protein binding for the compounds
was typically moderate (ca. 90% bound), although several mole-
cules with significantly higher unbound fractions were also identi-
fied (19, 29; Table 4). Collectively, the above assessments indicated
that compound 18 possessed an ideal combination of potent
NAMPT inhibition activity and attractive in vitro DMPK properties.
The molecule also exhibited antiproliferative effects against a
number of additional human tumor cell lines (Table 5), although
in some instances such activity was slightly attenuated relative
to potency against the A2780 line. Based on these favorable char-
acteristics, compound 18 was therefore progressed to in vivo
mouse PK and xenograft efficacy studies.
As shown in Figure 3 and Table 6, inhibitor 18 exhibited accept-
able bioavailability and plasma exposure following oral adminis-
tration to female NCR nude mice.15 Single-dose IV administration
of the compound determined that the molecule’s in vivo mouse
CL value resided somewhere in between those predicted from
the in vitro mouse microsome and hepatocyte stability assess-
ments (c.f., Tables 4 and 6). Importantly, BID administration of
18 at 40 mg/kg/dose afforded free mouse plasma levels which ex-
ceeded the compound’s A2780 and PC-3 antiproliferation cell cul-
ture IC50 values for at least 12 h following the first dose (Fig. 3).
Accordingly, compound 18 displayed significant efficacy in the
NCI-PC3 human prostate cancer xenograft model with relatively

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P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4875–4885
Table 2
Structure–activity relationships of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas
O
N
N
H
R
S
N
O
O
Compd
R
NAMPT IC50a
(
lM)
A2780 IC50b
(lM)
HLM CLhepc (mL/min/kg)
7.5
Solubilityd
115
(lM)
8
0.014
0.008
0.015
15
16
0.0027
14
13
10
83
CF3
CF3
N
F
0.015
0.010
17
0.0071
0.0063
8
16
F
18
19
20
21
22
0.011
0.017
0.017
0.020
0.002
0.011
0.020
0.0073
0.011
0.013
3.5
6
183
124
105
192
145
N
N
NH2
N
N
11
4
N
N
16
F
F
23
0.078
0.013
6
35
O
24
0.026
0.044
0.035
0.032
6
7
206
208
25e
O
N
O
O
26
0.083
0.11
6
187
N
N
27
28
29
0.027
0.031
0.010
0.054
11
10
5
216
121
214
O
0.0074
0.0070
O
N

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4875–4885
4879
M)
Table 2 (continued)
Compd
R
NAMPT IC50a
0.048
(
lM)
A2780 IC50b
0.099
(l
M)
HLM CLhepc (mL/min/kg)
11
Solubilityd
240
(l
N
N
30
31
0.058
0.026
9
142
O
32
33
0.071
0.044
0.068
0.098
9
6
59
N
OH
174
N
N
N
34f
35f
0.028
0.77
0.070
0.15
ND
8
216
215
OH
OH
N
36
37
0.038
0.059
0.071
0.046
9
9
160
196
N
N
O
N
O
38
39
40
N
0.022
0.020
0.0074
0.0095
0.010
13
13
6
194
272
6.2
N
O
N
N
0.0056
N
See supplementary data for experimental details associated with each assessment. All biochemical and cell-based assay results are reported as the arithmetic mean of at least
two separate runs. Solubility and HLM determinations are n = 1.
a
NAMPT biochemical inhibition.
b
Antiproliferation activity determined in cell culture experiments using A2780 cell line. This inhibition can be reversed by addition of 0.33 mM of NMN.
Human hepatic clearance predicted from human liver microsomes (stable, moderate, labile = <6, 6–15, >15 mL/min/kg, respectively).
Aqueous solubility.
Racemic mixture.
c
d
e
f
Single enantiomer (unknown absolute configuration). ND = not determined.
Table 3
CYP inhibition properties of selected NAMPT inhibitors
c
Compd
Time-dependent CYP Inhib
3A4 TDI (T) (% AUC shift)a
CYP Inhib IC50
3A4 (T)
(lM)
3A4 TDI (M) (% AUC shift)b
3A4 (M)
2D6
1A2
2C19
2C9
8
<15
23
<15
<15
<15
<15
<15
<15
<15
<15
<15
<15
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
5–10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3.7
>10
6.2
6.2
>10
>10
0.054
0.077
0.065
0.073
0.30
18
19
21
28
29
0.27
See supplementary data for experimental details associated with each assessment. All assay results are reported as the arithmetic mean of at least two separate runs. ND = not
determined.
a
Time-dependent inhibition of 3A4 cytochrome P450 isoform (substrate = testosterone); >15% AUC shift considered possible TDI risk.
Time-dependent inhibition of 3A4 cytochrome P450 isoform (substrate = midazolam); >15% AUC shift considered possible TDI risk.
Cytochrome P450 inhibition activity (various isoforms; T = testosterone substrate, M = midazolam substrate).
b
c

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P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4875–4885
Table 4
In vitro DMPK properties of selected NAMPT inhibitors
Compd
MLM CLhepa(mL/min/kg)
MH CLhepb(mL/min/kg)
MDCK Papp,A–B ꢀ 10ꢁ6 cm/sc
Mouse PPB (fu)d
8
75
71
63
71
56
65
43
36
26
45
19
25
8.3
2.5
0.50
4.1
0.10
0.10
0.093
0.14
0.20
0.12
0.078
0.36
18
19
21
28
29
See supplementary data for experimental details associated with each assessment. All assay results are reported as the arithmetic mean of at least two separate runs.
a
Mouse hepatic clearance predicted from mouse liver microsomes (stable, moderate, labile = <27, 27–63, >63 mL/min/kg, respectively).
Mouse hepatic clearance predicted from 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
To enable further optimization of the described urea-containing
series of NAMPT inhibitors, we obtained a crystal structure of com-
Antiproliferation effects determined for compound 18 in various human tumor cell
Table 5
pound 29 in complex with the NAMPT enzyme.16 As shown in Fig-
ure 5, the compound made numerous favorable contacts with the
protein. For example, the pyridine ring of the 2,3-dihydro-1H-pyr-
rolo[3,4-c]pyridine-derived urea moiety participated in an offset
face-to-face pi-stack with the side chains of NAMPT residues
Tyr180 and Phe193. This binding location was previously observed
to be occupied by the pyridine ring of the nicotinamide NAMPT
substrate as well as by heterocycles associated with several other
classes of NAMPT inhibitors.6,9,17 The electron density of the
NAMPT-29 structure did not allow unambiguous orientation of
the pyridine nitrogen atom. The depicted urea rotamer placed this
atom within hydrogen bonding distance of a water molecule and
linesa,b
HT1080
25
PC3
MiaPaCa2
70
HCT116
22
IC50 (nM)
1
36 19
9
4
a
CyQuant endpoint; arithmetic mean of three separate runs (n = 3, standard
deviations are also shown).
b
All antiproliferation effects were reversed by addition of 330 lM NMN, strongly
implicating NAMPT inhibition as the causative MOA. See Supplementary data for
experimental details.
minor decreases in body weights (Fig. 4; see supplementary data
for additional details).15
Figure 3. Mean ( standard deviation) plasma concentration-time profiles of compound 18 in female NCR nude mice following intravenous and oral administration.
Table 6
In vivo PK properties of compound 18 in nude mice.
b
c
f
Route
Dosea (mg/kg)
Cmax
(lM)
Tmax (h)
Vssd (L/kg)
CLe(mL/min/kg)
T1/2 (h)
Fg (%)
IV
PO
5
5
—
0.33
—
0.25
2.04
—
57.7
—
1.12
1.85
—
20.3
a
b
c
d
e
f
Formulation = 35:10:15:40 PEG400:EtOH:PG:H2O (solution).
Maximum plasma concentration.
Time at which maximum plasma concentration observed.
Volume of distribution (steady-state).
Clearance.
Half-life.
g
Bioavailibility. See supplementary data for experimental descriptions.

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4875–4885
4881
O2
N
1250
1000
750
500
250
0
O
Y
Vehicle
X
Z
H2N
i
ii
O
N
H
NH
Y
8-40
+
R
R
A
40 mg/kg 18
S
S
O
O
O
O
*p<.05
X
N
N
Z
A
41
42
43
44
CH
CH
CH2
iii, iv
CH CNH2 CH2
45 CH
N
CH
CH CH2CH2
CH2CH2
CH2
*
46
47
N
CH
CH
14
CH
N
Scheme 1. General synthesis of urea-containing compounds. Reagents and condi-
tions: (i) (4-NO2)PhC(O)Cl, toluene, 110 °C, 2 h; (ii) EtOH, reflux, 2 h, 45–82%; (iii)
OCNSO2Cl, 2-bromoethanol, CH2Cl2, 0 °C, 30 min; then 41, iPrNEt2 CH2Cl2, 0 °C,
40 min, 47%; (iv) 43, CH3CN, 70 °C, 14 h, 74%.
solvent-exposed and made relatively few specific protein interac-
tions. For example, Van der Waals contacts were noted only be-
tween the piperidine ring of 29 and Ile309 as well as between
the ligand’s tetrahydropyran ring and Pro307. A water molecule
was also observed within hydrogen bonding distance of the piper-
idine nitrogen atom, although this entity was not present in the
other ligand binding site contained in the NAMPT dimer.16
0
3
7
10
14
18
Rx
Day
Figure 4. Efficacy of compound 18 (dosed 40 mg/kg BID) in the NCI-PC3 human
prostate cancer xenograft model. Eight (8) mice were tested per group. Vehi-
cle = 60% PEG400/10% EtOH/30% D5W. Data reflect mean tumor volumes
standard error of mean).
(
The inhibitors described in this work were prepared by the
general method depicted in Scheme 1.18 In this approach, various
benzylic amines (41) were converted to their corresponding 4-ni-
tro-phenyl carbamates (42) and these entities were subsequently
condensed with bicyclic amines 43–47 to give the desired ureas.19
With the exception of 44 (see below), the required bicyclic amines
were commercially available and the preparations of 43,20 45,21
and 4622 are also described in the literature. The sulfonyl urea 14
was prepared from amine 41 (R = Ph) and 2,3-dihydro-1H-pyrrol-
o[3,4-c]pyridine (43) by sequentially reacting the amine compo-
nents with 2-oxo-1,3-oxazolidine-3-sulfonyl chloride generated
in situ from 2-bromoethanol and chlorosulfonyl isocyanate.23
The synthesis of bicyclic amine 44 (required for the preparation
of compound 9) is depicted in Scheme 2.24 6-Chloronicotinic acid
(48) was converted to the corresponding acyl chloride and this
properly positioned it for reaction with the PRPP co-factor in the
NAMPT active site.14 The urea linkage of 29 extended into a polar
portion of the substrate tunnel region and the urea NH formed a
hydrogen bond with the side chain of Asp219. As shown in Figure 5,
a water-mediated hydrogen bond was observed to bridge between
the urea carbonyl oxygen of 29 and the side chain of Ser275. The
benzylic portion of 29 formed favorable Van der Waals contacts
with the more hydrophobic regions of the NAMPT substrate tunnel
including the side chains of Val242 and Ile351. The central phenyl
ring of the inhibitor phenyl ring also participated in a favorable
edge-to-face pi interaction with the His191 side chain. The sulfone
moiety of 29 made one hydrogen bond to a structural water
molecule while the substituted piperidine fragment was highly
Figure 5. Co-crystal structure of inhibitor 29 (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. Hydrogen bonds are indicated with dashed yellow lines, and the van der Waals surface of the
protein is shown in grey. Two phosphates (orange), likely originating from the crystallization buffer, are also depicted. The resolution of the structure is 1.84 Å.

4882
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4875–4885
Cl
R
Cl
Cl
Cl
iv
OH
OH
iii
v
O
N
N(i-Pr)2
N
R
N
N
O
O
O
53
54
51
52
48
49
R = CHO
R = CH2OH
R = OH
R = Cl
50 R = N(i-Pr)2
iv
i
ii
vi
H
N
Cl
vii
R
N
DMB
N
R
OCH3
N
N
DMB =
55
56
R = DMB
R = H
viii
OCH3
44
Scheme 2. Synthesis of amine 44. Reagents and conditions: (i) SOCl2, DMF, toluene, 85 °C, 5 h, 95%; (ii) HN(i-Pr)2, DIEA, CH2Cl2, 10 °C, 2 h, 85%; (iii) LDA, HCOOEt, Et2O,
ꢁ75 °C, 1 h, 65%; (iv) NaBH4, EtOH, 25 °C, 1 h, 78%; (v) HCl (6 M, aq), 120 °C, 2 h, 46%; (vi) SOCl2, H2N–DMB, CH2Cl2, 25 °C, overnight, 37%; (vii) Pd2(dba)3, dppf, H2N–DMB,
toluene, 90 °C, overnight, 58%; (viii) TFA, CH2Cl2, reflux, overnight, 52%.
NC
NC
Br
v
NH
Ar3
S
N
F
60
vi
iv
iii
NC
NC
i
ii
vii
Ar1 SO2Li
Ar1 Br
Ar
41
+
S
Br
57
58
O
O
59
F
F
Ar1
=
Ar2
=
Ar3
=
NHBoc
N
n-Pr
N
N
N
Scheme 3. Synthesis of amines 41 required to prepare compounds 8 and 17–20. Reagents and conditions: (i) n-BuLi, SO2, THF, ꢁ78 °C, 1 h, 93%; (ii) DMF, 120 °C, 16 h, 15%,
(iii) n-PrBr, KOH, EtOH, 80 °C, 12 h; (iv) Ar2SO2Na, DMSO, 130 °C, 16 h, 94%; (v) Ar3SH, K2CO3, DMF, 145 °C, 12 h, 89%; (vi) Oxone, CH3CN/H2O, 25 °C, 2 h, 81%; (vii) Raney Ni,
2 N NH3 in CH3OH, H2, 25 °C, 1 h, 42–68%.
Boc
Boc
N
H
Br
CF3
N
H
i
ii
Ar1
=
=
Ar
S
N
SO2Na
SO2Na
O
O
O
61
63
62
64
41
CF3
Ac
Ac
N
H
R2
N
H
iii
iv
R2
S
N
O
N
Scheme 4. Synthesis of amines 41 required to prepare compounds 15, 16, 21, and 22. Reagents and conditions: (i) Ar1, DMSO, 120 °C, overnight, 8%; (ii) TFA, CH2Cl2, 25 °C,
40 min; (iii) R2-B(OH)2, Cu(OAc)2, K2CO3 or Et3N, DMSO, 60 °C, 16 h; (iv) 3.0 M HCl, i-PrOH, reflux, 16–48 h, 31–63%.
entity was subsequently condensed with N,N-diisopropylamine to
afford amide 50. Directed deprotonation of 50 with lithium diiso-
propylamide followed by trapping with ethyl formate provided
aldehyde 51 which was then reduced to give the corresponding
alcohol (52). Intramolecular cyclization of 52 was then accom-
plished under acidic conditions at elevated temperature and the
resulting lactone (53) was subsequently reduced to afford diol 54.
After exposure of 54 to thionyl chloride, the resulting dichloride
(not shown) was condensed with 2,4-dimethoxybenzylamine to
provide amine 55. A second 2,4-dimethoxybenzylamino substituent
was then introduced using a palladium-catalyzed process to give
intermediate 56. Removal of both 2,4-dimethoxybenzyl moieties
present in 56 under strongly acidic conditions afforded amine 44.
The syntheses of the benzylic amines (41) required to prepare
ureas 822 and 17–20 are depicted in Scheme 3. Various aryl bro-
mides (57) were converted to their corresponding lithium sulfi-
nates (58) by n-butyllithium-mediated metal–halogen exchange
followed by trapping with sulfur dioxide gas.25 The sulfinates were
subsequently coupled with 4-bromobenzonitrile at elevated tem-
perature to give various cyano-containing sulfones (59).26 Reduc-
tion of the cyano groups present in these entities with hydrogen/
Raney nickel27 afforded the desired amines (41). The aryl bromide
57 required for the synthesis of inhibitor 20 was prepared by alkly-
ation of 4-bromo-1H-pyrazole.28 Two alternate syntheses of
sulfones 59 are also depicted in Scheme 3. In the first method,
4-fluorobenzonitrile was condensed with commercially available

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4875–4885
4883
F
O
Br
Br
vi
F
O
O
O
R1
=
S
S
iii
O
N
O
O
v
O
O
69
70
Boc
NC
Br
Br
Br
i
ii
iv
iii
R1 OH
R
41
R
+
R
S
S
S
SH
65
O
O
O
O
66
67
68
vii
X
O
O
X
NC
NC
NC
N
NH
N
R2
iv
ix
viii
S
S
S
O
O
O
O
O
O
73
iv
71
72
R2= CH3, CH2CH3
O
x
O
Ketone =
O
O
Alkyl
NC
X = CH2, CH2CH2
N
iv
Alkyl = CH3, CH(CH3)2
41
S
O
O
74
Scheme 5. Synthesis of amines 41 required to prepare compounds 23–31. Reagents and conditions: (i) DIAD, PPh3, THF, 25 °C, 12 h, 41%; (ii) m-CPBA, CHCl3, 25 °C, 5 h, 81%;
(iii) Pd2(dba)3, dppf, ZnCN2, DMF, 120 °C, 12 h, 77%; (iv) Raney Ni, 2N NH3 in CH3OH, H2, 25 °C, 1 h, 59–69%; (v) HCl (2 M, aq), H2O, 60 °C, 3 h, 79%; (vi) DAST, CH2Cl2, 25 °C, 5 h,
36%; (vii) TFA, CH2Cl2, 25 °C, 5 h, 83%; (viii) RCOCl, (i-Pr)2NEt, CH2Cl2, 25 °C, 1 h, 92–94%; (ix) Ketone, Ti(OEt)4, NaBH3CN, AcOH, EtOH, 60 °C, 12 h, 62–78%; (x) CH2O or
acetone, NaBH3CN, AcOH, EtOH, 60 °C, 12 h, 42–67%.
As described in Scheme 3 above, reduction of the cyano groups
present in the tetrahydropyran-containing intermediates 68 with
hydrogen/Raney nickel27 afforded the desired amines (41). The
amine 41 required to synthesize difluoro-containing inhibitor 23
was prepared by acidic deprotection of the dioxolane-containing
intermediate 67 to give cyclohexanone 69. Reaction of 69 with
(diethylamino)sulfur trifluoride (DAST) afforded intermediate 70
which was transformed to the corresponding nitrile 68 (and subse-
quently amine 41) by the methods described above. The amines 41
required to synthesize inhibitors 26 and 27 were prepared by
removal of the Boc protecting group present in the piperidine-
containing intermediate 68 under acidic conditions followed by
derivitization of the free amine (71) with various acid chlorides. The
resulting acylated intermediates (72) were reduced as described
above to give the desired amines 41. Similarly, the alkylated
piperidines (41) required for the syntheses of inhibitors 28–31 were
prepared by alkylation or reductive amination of intermediate 71
followed by nitrile reduction as previously described.
The syntheses of the sulfonamide-containing benzylic amines
(41) required to prepare ureas 32–40 are depicted in Scheme 6.
Commercially available 4-cyanobenzene-1-sulfonyl chloride was
condensed with a variety of commercially available amines to give
the corresponding sulfonamides (75). Reduction of the nitrile moi-
eties present in 75 using the hydrogen/Raney nickel procedure de-
scribed in several other synthesis Schemes then afforded the
desired amines 41.
NC
R1
N
NC
i
ii
Cl
41
S
R2
S
O
O
O
O
75
Scheme 6. Synthesis of amines 41 required to prepare compounds 32–40. Reagents
and conditions: (i) Et3N, HNR1R2, CH2Cl2, 25 °C, 2 h, 67–85%; (ii) Raney Ni, 2 N NH3
in CH3OH, H2, 25 °C, 1 h, 82–98%.
sodium benzenesulfinate to afford the desired sulfone (59,
Ar = Ph).29 In the second method, 4-fluorobenzonitrile was reacted
with 3,5-difluorobenzenethiol to give the sulfide 60. This entity
was subsequently oxidized to the corresponding sulfone (59,
Ar = 3,5-di-F–Ph) by treatment with Oxone.
The syntheses of the benzylic amines (41) required to prepare
ureas 15, 16, 21, and 22 are depicted in Scheme 4. Sulfinate inter-
mediate 619 was condensed with 3-bromo-5-(trifluoromethyl)pyr-
idine to give sulfone 62 (Ar = 3-pyridyl-5-trifluoromethyl).26
Deprotection of the N-Boc moiety present in 62 under acidic con-
ditions then afforded the desired amine 41. Similarly, sulfinate
intermediate 6329 was coupled with a variety of boronic acids to
give the corresponding sulfones 64.26 Deprotection of the N-acetyl
moiety present in 64 under acidic conditions subsequently affor-
ded the desired amines 41.
The syntheses of the benzylic amines (41) required to prepare
ureas 23–31 are depicted in Scheme 5. 4-Bromobenzenethiol was
coupled under Mitsunobu conditions with a variety of secondary
alcohols to give sulfides 66.30 These entities were oxidized to the
corresponding sulfones, and the bromides present in these oxi-
dized molecules were replaced with nitrile groups using palla-
dium-catalyzed transformations (providing intermediates 68).31
Acknowledgements
We thank Drs. Krista Bowman and Jiansheng Wu and their
respective Genentech research groups for performing many
protein expression and purification activities, the DMPK group at
Genentech, especially Jonathan Cheong, Erlie Marie Delarosa,
Suzanne Tay, Jasleen Sodhi, and Ning Liu for their contributions

4884
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4875–4885
nicotinamide and PRPP; c.f.,. 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.
to the generation of the in vitro DMPK data, the Genentech in vivo
cell culture core and in vivo Pharmacology dosing technicians for
their support of the efficacy studies, Rashida Garcia-Dancey and
Shohini Ganguly for helping to generate the cellular data, and Agi-
lent Technologies (Woburn, MA) for generating the Rapidfire LCMS
data. We also thank Dr. Peter Jackson for many helpful discussions
regarding NAMPT.
13. Aqueous solubilities were determined using a 24-hour high-throughput assay
employing DMSO compound solutions (see Supplementary data for additional
details). Accordingly, the reported aqueous solubilities are best utilized as
surrogates for more accurate (thermodynamic) solubility measurements
determined using crystalline materials. Our experience with both
assessments suggests that the high-throughput solubility values are unlikely
to improve with subsequent compound scale-up and that deprioritization of
compounds which perform poorly in the high-throughput assay is an effective
strategy to identify molecules with acceptable solubility properties.
Supplementary data
14. 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 (see Refs.
5a,6,9). It is also consistent with the inability to detect similar adduct
formation in related experiments with compound 10 (see Supplementary
data) and another cell-inactive compound that was nevertheless a potent
biochemical NAMPT inhibitor (Ref. 6). Once formed, the PRPP-adducts may
accumulate intracellularly and thereby enhance cell culture antiproliferation
effects (see Ref. 5a 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. In
addition, performing the PRPP-adduct assessments was a relatively resource-
intensive task and such determinations were therefore made for only a small
number of compounds.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.06.090.
References and notes
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2 for
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15. To better accommodate work schedules, the second dose of compound 18 was
given 10 h after the first dose on each day during the xenograft experiment.
Mouse PK parameters were determined using a similar dosing schedule.
16. NAMPT is believed to function as a symmetrical homodimer with two active
sites formed at opposite ends of the dimer interface. Accordingly, the protein
5. Two NAMPT inhibitors have been progressed to human clinical trials: (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.;
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7. (a) For other examples of urea-containing NAMPT inhibitors, see: Kumar, D. V.;
Slattum, P. M.; Yager, K. M.; Shenderovich, M. D.; Tangallapally, R.; Kim, S. -H.
WO 2012/177782.; (b) Willardsen, A. J.; Lockman, J. W.; Murphy, B. R.; Judd, W.
R.; Kim, I. C.; Kim, S. -H.; Zigar, D. F.; Yager, K. M.; Fleischer, T. C.; Terry-Lorenzo,
R. T.; Boniface, J. J.; Parker, D. P.; McAlexander, I. A.; Bursavich, M. G.; Dastrup,
D. M. WO 2011/109441.; (c) Arigon, J.; Bernhart, C.; Bouaboula, M.; Dimalta, A.;
Nardi, F.; Jegham, S.; Roux, R. WO 2012/038904.; (d) Bernhart, C.; Bouaboula,
M.; Casellas, P.; Jegham, S.; Arigon, J.; Combet, R.; Hilairet, S.; Fraisse, P. US
2010/0222319.
crystallized with such
a dimer present in the asymmetric unit. The two
monomer chains in the dimer were very similar, having an RMSD of only 0.12 Å
for 420 C-alpha atoms, and both active sites contained a molecule of compound
29 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. In
addition, the electron density clearly defined multiple inorganic phosphates
that likely originated from the crystallization buffer. See deposited PDB file for
additional structural details: code = 4KFP.
17. (a) Burgos, E. S.; Ho, M.-C.; Almo, S. C.; Schramm, V. L. Proc. Natl. Acad. Sci. USA
2009, 106, 13748; Wang, T.; Zhang, X.; Bheda, P.; Revollo, J. R.; Imai, S.-I.;
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Nat. Struct. Mol. Biol. 2006, 13, 582.
18. The chemical purities of all final compounds described in this work were >95%
at the time of their biological assessments as determined by LCMS analysis
with UV detection at 220 nm. We also noted, however, that some molecules
containing the 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived urea moiety
experienced oxidation upon prolonged storage (>1 month; particularly when
stored in solvated form; detected via LCMS analysis of stored materials). No
attempts were made to study or suppress this process and the oxidized
compounds were neither isolated nor biologically characterized. LCMS
fragmentation patterns suggested the oxidation products depicted below, but
no additional analyses were conducted to confirm these assignments.
O
O
O
O
N
N
N
N
and/or
N
N
R
HN R
HN
HN
R
O
8. For additional examples of NAMPT inhibitors, see: (a) You, H.; Youn, H.-S.; Im,
I.; Bae, M.-H.; Lee, S.-K.; Ko, H.; Eom, S. H.; Kim, Y.-C. Eur. J. Med. Chem. 2011, 46,
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cyclic, aliphatic amines, see Curtin, M. L.; Sorensen, B. K.; Heyman, H. R.; Clark,
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0122924.
12. The antiproliferative effects exhibited in cell culture by 8 were completely
eliminated (‘‘reversed’’) when the compound was tested in the presence of
0.33 mM NMN (the product of the NAMPT-catalyzed condensation of
27. (a) Depreux, P.; Lesieur, D.; Mansour, H. A.; Morgan, P.; Howell, H. E.; Renard,
P.; Caignard, D.-H.; Pfeiffer, B.; Delagrange, P.; Guardiola, B.; Yous, S.;
Demarque, A.; Adam, G.; Andrieux, J. J. Med. Chem. 1994, 37, 3231; (b) Amon,
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P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4875–4885
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