Structure-based identification of ureas as novel nicotinamide phosphoribosyltransferase (Nampt) inhibitors

Article abstract of DOI:10.1021/jm400186h

Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target. Virtual screening identified a thiourea analogue, compound 5, as a novel highly potent Nampt inhibitor. Guided by the cocrystal structure of 5, SAR exploration revealed that the corresponding urea compound 7 exhibited similar potency with an improved solubility profile. These studies also indicated that a 3-pyridyl group was the preferred substituent at one inhibitor terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound 50 as a urea-containing Nampt inhibitor which exhibited excellent biochemical and cellular potency (enzyme IC₅₀ = 0.007 μM; A2780 IC₅₀ = 0.032 μM). Compound 50 also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model (TGI of 97% was observed on day 17).

Full text of DOI:10.1021/jm400186h

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Article
Structure-based Identification of Novel Nicotinamide
Phosphoribosyltransferase (Nampt) Inhibitors
xiaozhang zheng, paul bauer, Timm Baumeister, Alexandre J Buckmelter, Maureen Caligiuri,
Karl H Clodfelter, Bingsong Han, Yen-Ching Ho, Nikolai Kley, Jian Lin, Dominic J Reynolds,
Geeta Sharma, Chase C Smith, Zhongguo Wang, Peter S Dragovich, Angela Oh, Weiru
Wang, Mark Zak, Janet Gunzner-Toste, Guiling Zhao, Po-wai Yuen, and Kenneth W Bair
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400186h • Publication Date (Web): 25 Apr 2013
Downloaded from http://pubs.acs.org on May 17, 2013
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Structure-based Identification of Ureas as Novel
Nicotinamide Phosphoribosyltransferase (Nampt)
Inhibitors
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a
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Xiaozhang Zheng *, Paul Bauer , Timm Baumeister , Alexandre J. Buckmelter , Maureen
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Caligiuri , Karl H. Clodfelter , Bingsong Han , YenꢀChing Ho , Nikolai Kley , Jian Lin ,
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Dominic J. Reynolds , Geeta Sharma , Chase C. Smith , Zhongguo Wang , Peter S. Dragovich ,
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Angela Oh , Weiru Wang , Mark Zak , Janet GunznerꢀToste , Guiling Zhao , Poꢀwai Yuen ,
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Kenneth W. Bair
a
Forma Therapeutics, Inc., 500 Arsenal Street, Watertown, Massachusetts 02472, USA
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Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.
c
Pharmaron Beijing, Co. Ltd., 6 Tai He Road, BDA Beijing, 100176, P. R. China
RECEIVED DATE (to be automatically inserted after your manuscript is accepted)
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ABSTRACT: Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target.
Virtual screening identified a thiourea analog, compound 5, as a novel highly potent Nampt
inhibitor. Guided by the coꢀcrystal structure of 5, SAR exploration revealed that the
corresponding urea compound 7 exhibited similar potency with an improved solubility profile.
These studies also indicated that a 3ꢀpyridyl group was the preferred substituent at one inhibitor
terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor
structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound
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50 as a ureaꢀcontaining Nampt inhibitor which exhibited excellent biochemical and cellular
potency (enzyme IC50 = 0.007 ꢀM; A2780 IC50 = 0.032 ꢀM). Compound 50 also showed
excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft
model (TGI of 97% was observed on day 17).
INTRODUCTION
Nicotinamide phosphoribosyltransferase (Nampt, NAMPTase, PBEF, Visfatin, EC 2.4.2.12)
catalyzes the condensation of nicotinamide (NAM) with 5ꢀphosphoribosylꢀ1ꢀpyrophosphate
(PRPP) to form nicotinamide mononucleotide (NMN), an intermediate in the biosynthesis of
nicotinamide adenine dinucleotide (NAD). In mammalian cells, there are multiple pathways for
NAD biosynthesis: a de novo pathway using Tryptophan (Trp) as the precursor and three
pathways using exogenous vitamin sources as the precursors: NAM, nicotinic acid (NA), and
nicotinamide riboside (NR). Nampt is the rate limiting enzyme in the NAD biosynthetic
pathway using NAM. This NAMꢀdependent pathway is used in cells to recycle the NAM
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produced by NADꢀconsuming enzymes such as polyꢀADPꢀribose polymerases (PARPs) and
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sirtuins (Figures 1 and 2). Tumors have elevated PARP activity and thereby consume NAD at a
higher rate than normal tissues and are thus more dependent upon Nampt to maintain required
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NAD levels. NAD is an essential coꢀfactor in multiple energy producing processes such as
mitochondrial oxidative phosphorylation, beta oxidation, glycolysis, and the citric acid cycle.
Cancer cells have an increased demand for ATP and thus are more sensitive to perturbations of
these processes. Rapidly proliferating cancers also have additional biosynthetic requirements
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that require NAD as a coꢀfactor. In addition, NAD is essential in maintaining the reductive
environment that protects cells from reactive oxygen species (ROS), which are elevated in
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cancers. The combination of increased NAD consumption and dependence upon NADꢀdriven
biosynthetic and cellular redox homeostatic processes may make many cancer cells highly
dependent on Nampt activity for proliferation and/or survival. Nampt thus represents an
attractive target for the development of new cancer therapies.
Figure 1: Nampt catalyzed biochemical reaction.
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Figure 2: Schematic representation of NAD biosynthetic pathways found in mammalian cells.
At the outset of our Nampt program, only three classes of Nampt inhibitors were known in the
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literature.
Fujisawa/TopoTarget’s 1 (APO866) and Leo/GeminꢀX/Cephalon/Teva’s 2
(GMX1778) were the first two published Nampt inhibitors. 1 is currently in Phase 2 clinical
trials against cutaneous Tꢀcell lymphoma (CTCL), while 2 is in Phase 1/2 trials for metastatic
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melanoma. Triazoleꢀcontaining compounds were also reported to be potent Nampt inhibitors,
and a representative example, compound 3, exhibited an SHꢀSY5Y cytotoxicity IC value of 93
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nM. More recently, Myrexis, SanofiꢀAventis, and Abbott disclosed additional classes of
potent Nampt inhibitors. For example, compound 4 from Myrexis showed a Nampt enzyme IC50
of 0.5 nM and HCT116 cytotoxicity of 3.0 nM (Figure 3). In this paper, we report the
identification of highly potent ureaꢀcontaining smallꢀmolecule inhibitors of Nampt, which were
designed using the known crystal structure of the enzyme with an inhibitor present. During the
course of this work, Myrexis and Sanofi independently reported their own related ureaꢀ
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containing Nampt inhibitors.
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Figure 3: Reported Nampt Inhibitors.
RESULTS AND DISCUSSION
Structure-Based Identification of a New Chemical Scaffold to Target Nampt
Nampt acts both as a phosphoribosyltransferase and as an ATPase, with phosphorylation of
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His247 increasing the enzyme’s catalytic activity in a stoichiometric manner.
Structurally,
Nampt functions as a homodimer with two identical catalytic sites located at opposite ends of the
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dimer interface. The binding site of ATP overlaps with the PRPP and NAM binding sites. All
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three sites combine to form a protein surface of > 1000 Å , which represents a large area that a
potential competitive inhibitor could target. The NAM binding site lies at the entrance to a
tunnel through the protein that further extends along the dimer interface to a funnel shaped
opening. 1 occupies this tunnel with the piperidyl moiety overlapping with the NAM binding
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site (Figure 4).
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Figure 4: Slice through the coꢀcrystal structure of 1 (yellow) in complex with Nampt (important
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residues and solventꢀexcludedꢀsurface in grey) (PDBꢀID 2GVJ) . The crystallographically
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determined locations of the Nampt substrates NAM (2E5C) and PRPP (2E5D) are also shown
(green).
To pinpoint locations likely to be druggable within these regions, we utilized computational
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solvent mapping techniques employing CSꢀMap software. The consensus sites in the
Nampt binding region were used to define six pharmacophore points (AꢀF, Figure 5) with A and
BꢀC ranking significantly higher than the other three (see Supporting Information for details).
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Figure 5: Nampt crystal structure depicting virtual fragments which comprise CSꢀMap
consensus sites (carbons in green) and derived pharmacophore points (grey spheres). A =
pyridine, B = hydrogen bond acceptor, C = hydrogen bond donor, D = any ring, E = any polar
atom, F = any polar atom. Sphere sizes indicate radius of each point.
Three pharmacophore models were then created by separately combining the ABC points with
one of lower rank (DꢀF). The resulting models (ABCD, EABC, and FABC) were used to
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virtually screen the eMolecules database, resulting in a set of 228 compounds which were
subsequently purchased and tested in our biochemical and cellꢀbased assays. Of the three
pharmacophore models, only ABCD yielded compounds with subꢀmicromolar antiꢀNampt
activity. In particular, compound 5 (Table 3) showed a biochemical IC of 0.007 ꢀM and a
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cellular IC value of 0.19 ꢀM. The cellular activity of compound 5 was completely reversed by
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based. Thus, compound 5 represented an ideal starting point for additional SAR studies with
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respect to ligand efficiency (LE = 0.4) and lipophilicity efficiency (LipE:= 5.7).
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SAR discussion
To quickly expand SAR around compound 5, we identified analogues by chemical fingerprint
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similarity searches and purchased an additional 121 compounds from various commercial
sources. Table 1 summarizes the biological data generated for selected analogues. Moving the
pyridyl nitrogen atom from the 3ꢀ to the 4ꢀposition (6) was highly detrimental to the antiꢀNampt
activity. At the other end of the inhibitor structure, removal of the piperidine group led to a 100ꢀ
fold loss in biochemical potency (7: IC = 0.63 ꢀM), while removal of the entire sulfonamide
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group (8) led to complete loss of activity.
Table 1: Structureꢀactivity Relationships and Optimization of Hit Compound 5
Nampt
A2780
Entry
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IC (ꢀM)
IC (ꢀM)
5
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50
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> 10
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N
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a
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Nampt biochemical inhibition. A2780 cell proliferation inhibition. This inhibition can be
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reversed by addition of 0.33 mM of NMN. Unless otherwise noted, all biochemical and cellꢀ
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based assay results are reported as the arithmetic mean of at least 2 separate runs (n = 2). See
Figure 3 for the structures of 1 and 2. See Supporting Information for experimental details.
Simultaneously, in an attempt to better understand how compound 5 bound to Nampt and to
use this information to guide our further SAR optimization, a coꢀcrystal structure of 5 in complex
with the protein was generated (Figures 6 and 7). In the NAMꢀbinding region (corresponding to
pharmacophore points A, B, and C), the binding mode of compound 5 was noted to be similar to
that observed for 1 with the pyridyl group stacked between the aromatic side chains of Nampt
residues Phe193 and Tyr18’. A crystallographic water was also observed mediating a hydrogen
bonding network between the thiourea nitrogens, the backbone carbonyl of Val242, and the
Asp219 and Ser241 side chains. The hydroxyl group of Ser275 was located nearly perpendicular
to the thiourea moiety of 5, and this conformation did not allow for the formation of a typical
hydrogen bond with the nearby sulfur atom. Instead, hydrophobic contacts between the sulfur
and the side chains of Ile351 and Phe193 were observed. The phenyl ring of 5 occupied the
Nampt tunnel region and participated in an edgeꢀtoꢀface interaction with the side chain of
His191. The ligand’s sulfone oxygen atoms filled a hydrophobic pocket formed by the side
chains of Ala379, Ile351, and Ile309, but also made hydrogen bonds with crystallographic waters
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which, in turn, formed other hydrogen bonds to Nampt backbone carbonyl groups. The
piperidine moiety of 5 occupied space close to the piperidine present in 1, but was situated in a
perpendicular orientation. Hydrophobic contacts were noted between the piperidine C2 carbon
atom and the side chain of Ile309, as well as between the piperidine C3 and C4 carbons and the
Val242 and Pro273 residues.
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Figure 6: Coꢀcrystal structure of compound 5 (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 possible hydrogen bonds are
indicated with dashed orange lines. Hydrogen bonds which do not interact with the ligand, either
directly of via a water molecule, are not shown. The resolution of the structure is 1.9 Å.
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Figure 7: Schematic representation of the binding mode of compound 5 to Nampt. Waters are
depicted as ‘W’ and hydrogen bonds as dashed orange lines. Waters and hydrogen bonds which
do not interact with the ligand are not shown.
To facilitate further optimization, we investigated three distinct areas of these Nampt
inhibitors: the leftꢀhand side (LHS), the rightꢀhand side (RHS), and the linker (Figure 8). Since
we were unsure how improvements in one portion would translate to the others, our initial
inhibitor design strategy involved concurrent optimization of the various sections to define a
preferred pharmacophore. Accordingly, we first chose to optimize the linker region in tandem
with the RHS.
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Figure 8: Nampt inhibitor general structure.
Based on the coꢀcrystal structure information obtained with compound 5, the thiourea sulfur
atom did not form a hydrogen bond with the Nampt Ser275 residue (the serine hydroxyl oxygen
was within a suitable distance of 3.2 Å, but was perpendicular to the thiourea plane). The
corresponding urea compound (9) was therefore made to possibly enable a more favorable
binding geometry and at the same time reduce compound 5’s lipophilicity (calculated ClogP was
reduced from 2.4 to 1.5). In fact, compound 9 exhibited biological potency and microsomal
stability similar to that displayed by compound 5 (Table 2) and a significantly improved
solubility profile (82 vs 3.1 ꢀM). Accordingly, further SAR exploration was focused on
molecules containing the urea linker.
In the coꢀcrystal structure of compound 5, the phenyl ring sits in a narrow portion of the tunnel
where meta and particularly ortho substitution are sterically restricted by the tunnel walls vs para
substitution which points towards the opening. This was confirmed by the metaꢀattachment of
the sulfonamide RHS in compound 10 (Table 2). In the absence of further significant structural
guidance for RHS optimization, we initiated general exploratory SAR studies of the RHS with a
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broad survey of functional group tolerability (Table 2). Aryl or heteroarylsulfones (11 – 13)
were found to be equipotent compared to the aliphatic sulfonamide 9 and also displayed
improved mouse microsomal stability. However, the corresponding sulfide was found to exhibit
inferior activity in both biochemical and cellꢀbased assays (compare 13 with 14). Similarly,
compounds containing ether, ketone, or amide groups in their respective RHS moieties also
displayed weaker antiꢀNampt properties relative to the sulfones and sulfonamides (15 ꢀ 17, Table
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58
59
60
2
). The importance of maintaining an SO moiety in the inhibitor RHS is likely due to the need
2
to maintain hydrogen bonds to the structural water molecules observed in the Namptꢀ5 coꢀcrystal
structure. The attractive biological properties exhibited by the sulfonamide and sulfoneꢀ
containing inhibitors prompted the inclusion of these functional groups into subsequent Nampt
inhibitors.
Table 2: Initial SAR exploration
Nampt
A2780
MLM
% left
HLM Solubility
Entry
X
S
R
d
e
a,c
b,c
f
IC (ꢀM)
IC (ꢀM)
% left
(ꢀM)
50
50
5
0.007
0.007
> 10
0.19
0.13
> 10
0.16
32
60
3.1
9
O
O
O
16
3.5
27
85
40
81
82
72
99
g
10
11
0.004
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
h
1
1
1
2
3
4
O
O
O
0.007
0.005
0.024
0.25
0.24
7.4
64
54
62
79
79
64
ALQ
17
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
i
BLQ
15
O
O
O
0.080
0.017
0.049
> 10
0.82
6.4
32
9.6
82
> 99
75
22
72
99
1
6
7
1
> 99
a
b
Nampt biochemical inhibition. A2780 cell proliferation inhibition. This inhibition can be
c
reversed by addition of 0.33 mM of NMN. Unless otherwise noted, all biochemical and cellꢀ
d
based assay results are reported as the arithmetic mean of at least 2 separate runs (n = 2).
e
Mouse liver microsomal stability ꢀ % remaining at 30 min. Human liver microsomal stability ꢀ
f
g
%
remaining at 30 min. Aqueous solubility. The Rꢀgroup was linked to the metaꢀposition of
h
i
the phenyl ring. Above Limit of Quantification (> 100 ꢀM). Below Limit of Quantification
(
< 0.01 ꢀM). See Supporting Information for experimental details.
With preliminary RHS SAR information in hand, our focus next turned to exploration of the
LHS (Table 3). We first examined the importance of the pyridyl nitrogen atom present in
compounds 9 and 11. The phenyl analogue corresponding to 9 (inhibitor 18) showed a dramatic
loss of potency in both biochemical and cellꢀbased assays. However, moving the nitrogen out of
the aromatic ring afforded a compound (19) with similar biochemical potency compared to the
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2
3
4
5
6
7
8
9
related pyridineꢀcontaining inhibitor (11). Next, we introduced a fluorine atom at different
positions on the phenyl ring in an attempt to mimic the electronꢀdeficient pyridyl moiety present
in 9. In doing so, we found that 2ꢀ or 3ꢀfluoroꢀsubstituted compounds (20 and 21) displayed
relatively potent biochemical activities that were much better than that exhibited by the 4ꢀfluoroꢀ
substituted compound 22. However, all three compounds lacked meaningful cellular activity.
Interestingly, introducing a nitrogen atom into the 3ꢀfluoroꢀbenzyl LHS substituent partially
restored the lost cell potency (23). These observations emphasized the importance of including
an appropriatelyꢀpositioned and sufficiently nucleophilic nitrogen atom in the LHS design with
respect to obtaining potent cellular activity. Additional details regarding the biological rationale
for this requirement are presented later in this work.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Continuing our efforts to search for novel LHS heterocycles, bicyclic LHS compounds were
synthesized and tested. As shown in Table 3, the imidazopyridine LHS compound 24 improved
antiꢀNampt cellular potency compared to compound 11, but poor MLM stability still remained
an issue. When the imidazopyridine system was partially saturated (25) much of the biological
activity was lost as compared with compound 24. Docking studies of 25 (not shown) suggested
that beneficial πꢀπ interactions were likely impeded by the loss in LHS planarity relative to
inhibitor 24. The unsaturated ring present in 23 also required a shift of the linker towards Ser275
which further reduced the efficiency of the hydrogen bond networks in the area. As a followꢀon
to compound 24, we converted the fused bicyclic system into a linked biaryl moiety (compound
26). This molecule, however, showed a 10ꢀfold biochemical activity loss likely due to steric
clashes with Nampt residues Tyr195 and Arg196. Replacing the pyridine of 11 with an alternate
heterocycle (e.g., a pyrazole ring; 27) resulted in a complete loss of activity. The described
exploration identified the 3ꢀpyridyl moiety as a preferred LHS substituent for obtaining both
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
potent biochemical and cellular antiꢀNampt activity. Accordingly, this functional group was
retained in the inhibitor design throughout the remainder of the SAR studies.
Table 3: LHS SAR.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Nampt
A2780
MLM
HLM Solubility
Entry
R1
R2
d
e
a,c
b,c
f
IC (ꢀM)
IC (ꢀM)
% left % left
(ꢀM)
5
0
50
9
A
B
0.007
0.004
0.13
0.16
16
27
85
81
82
99
11
1
8
A
B
0.47
> 10
2.5
0.1
57
75
79
2.0
4.2
1
9
0
0.011
F
g
2
A
0.026
> 10
BLQ
57
0.1
2
1
2
A
A
0.039
2.0
> 10
> 10
26
62
62
0.1
4.3
2
0.7
g
2
3
4
A
0.024
0.75
BLQ
65
4.3
2
B
B
0.003
0.13
0.048
2.6
21
54
49
83
19
75
h
2
5
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3
4
5
6
7
8
9
2
6
7
B
B
0.041
> 10
> 10
> 10
45
84
77
0.1
88
2
> 99
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
Nampt biochemical inhibition. A2780 cell proliferation inhibition. This inhibition can be
c
reversed by addition of 0.33 mM of NMN. Unless otherwise noted, all biochemical and cellꢀ
d
based assay results are reported as the arithmetic mean of at least 2 separate runs (n = 2).
e
Mouse liver microsomal stability ꢀ % remaining at 30 min. Human liver microsomal stability ꢀ
f
g
h
% remaining at 30 min. Aqueous solubility. Below Limit of Quantification (< 0.01 ꢀM).
Racemic compound. See Supporting Information for experimental details.
We next turned our attention to the linker region (Table 4). Given the expected participation of
both urea NH groups in an intricate, waterꢀmediated hydrogenꢀbond network (c.f., Figure), we
anticipated that perturbation of either moiety might lead to reduced Nampt inhibition activity.
β
Accordingly, compound 28 confirmed that NH was critical for biological potency. However,
α
replacing NH with an oxygen (29) or carbon atom (30 and 32) retained potent biochemical
activity while causing a complete loss in cellular potency. The lack of cellular activity exhibited
by these molecules may be due to incorrect positioning of the pyridyl nitrogen atom with respect
to the PRPP coꢀfactor in the NAMPT active site (see additional discussion later in this work). In
α
contrast, methylation of the NH (31) caused more than a 100ꢀfold loss in biochemical potency
compared with compound 11. Although the Namptꢀ5 coꢀcrystal structure suggested that space
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
may exist for methylation at the LHS benzylic position, compound 33 showed that branching at
this location was not tolerated.
Following this discovery, we examined the effect of varying the linker lengths. As shown by
compound 34, shortening the linker abolished all activity (compare to compound 11; Table 4). A
oneꢀcarbon extension on either side of the urea moiety present in 9 was somewhat tolerated and
afforded similar biochemical activity but also a 5ꢀfold loss of cellular potency (35 and 36).
Transfer of the methylene group of compound 9 to the right side of the linker (37) led to a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
roughly 5ꢀfold loss of biochemical activity and almost a complete loss of cellular potency (IC =
50
2.1 ꢀM) compared to the parent molecule (9). When we replaced the urea moiety present in
compound 11 with the corresponding sulfonylurea, the resulting compound (38) lost almost all
biological activity. Additionally, many of the described linker modifications had significant
negative impacts on the microsomal stability and solubility of the resulting compounds. For
example, inhibitors 30 - 33, 35, and 38 all exhibited inferior metabolic stability toward mouse
and/or human liver microsomes relative to the parent compounds 9 and 11. Similarly, inhibitors
29 and 34 exhibited drastically reduced solubility as compared with 9 and 11. The origins of
these changes are not known with certainty, but may involve unfavorable alterations in the
lipophilicity and/or hydrogenꢀbonding potential of the described molecules. The preceding SAR
studies confirmed the urea linker moiety present in compounds such as 9 and 11 as optimal for
obtaining potent antiꢀNampt biochemical and cellular activity. This functional group was
therefore incorporated into all subsequent inhibitors described in this work.
Table 4: Linker SAR.
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2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Nampt
A2780
MLM
% left
HLM
% left
Solubility
(ꢀM)
Entry
Linker
R
d
e
a,c
b,c
f
IC (ꢀM)
IC (ꢀM)
5
0
50
9
A
B
0.007
0.004
0.13
0.16
16
27
85
81
82
99
1
1
g
g
g
2
8
A
B
B
B
B
B
B
A
A
4.8
> 10
> 10
> 10
> 10
> 10
> 10
> 10
0.73
0.61
nd
nd
nd
29
0.016
0.027
1.8
12
1.0
1.0
1.0
2.0
81
75
1.0
79
7.0
81
92
36
63
1.0
7.9
16
20
72
0.5
81
86
3
0
31
3
2
0.028
0.93
h
3
3
3
3
3
4
5
6
> 10
0.010
0.019
0.2
15
3
3
7
8
A
B
0.025
1.8
2.1
7.8
15
71
23
83
63
> 10
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3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
b
Nampt biochemical inhibition. A2780 cell proliferation inhibition. This inhibition can be
c
reversed by addition of 0.33 mM of NMN. Unless otherwise noted, all biochemical and cellꢀ
d
based assay results are reported as the arithmetic mean of at least 2 separate runs (n = 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
e
Mouse liver microsomal stability ꢀ % remaining at 30 min. Human liver microsomal stability ꢀ
f
g
h
% remaining at 30 min. Aqueous solubility. Not determined. Racemic compound. See
Supporting Information for experimental details.
With the completion of LHS and linker region optimizations, we turned our SAR exploration
efforts back to the RHS with a focus not only on further potency improvements but also on
improving microsomal stability and solubility (Table 5). We first investigated the impact of
cyclic sulfonamide variations with respect to these parameters and found that the ring size
correlated strongly with enzyme and cellular potency (compounds 9, 39ꢀ41). Although the 7ꢀ
membered sulfonamide 39 was comparable to the 6ꢀmembered analogue 9, it did not offer any
advantages with respect to microsomal stability and solubility (39 was less stable in MLM and
was also less soluble relative to 9). In general, aliphatic primary sulfonamides such as
compound 42 were less potent than the cyclic secondary sulfonamides, especially in cellꢀbased
assays. Phenyl sulfonamides, such as compound 43, didn’t have good activity and were
therefore not extensively explored. Nonꢀcyclic secondary sulfonamides such as compound 44
were also much weaker in the cellular assay relative to the cyclic analogues and were similarly
deprioritized.
With the above structureꢀactivity relationships in hand, we focused further optimization efforts
on piperidineꢀbased sulfonamides (the optimization of biaryl sulfoneꢀcontaining Nampt
inhibitors will be described in a separate disclosure). In general, methyl substitution at the 2ꢀ, 3ꢀ
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4
5
6
7
8
9
or 4ꢀposition of the piperidine ring resulted in equivalent cellular potency (compare 45-47 with
9
; Table 7). However, the methylated compounds exhibited inferior microsomal stability
properties relative to 9 and additional alkylated piperidines were therefore not pursued. In
contrast, introduction of a primary amide at the piperidine 4ꢀposition (48) dramatically improved
MLM stability compared to 9. However, this benefit was realized at the expense of cell potency,
possibly due to the highly polar nature of the amide substituent. We therefore prepared the
morpholineꢀcontaining compound 49 to test whether incorporation of a less polar electronꢀ
withdrawing 4ꢀsubstituent would also afford good metabolic stability properties. Encouragingly,
inhibitor 49 exhibited improved microsomal stability relative to compound 9 albeit with a
concomitant 7ꢀfold loss in cellular activity. Satisfyingly, a compound containing a bridged
morpholine moiety (50) showed an excellent overall profile with respect to biochemical and
cellular potency, microsomal stability, and solubility. Compared to the initial lead compound
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(5), compound 50 not only maintained excellent ligand efficiency but also greatly improved
lipophilicity efficiency (5 vs 50; LE: 0.4 vs 0.4; LipE: 5.7 vs 7.2, respectively).
Table 5: Sulfonamide RHS SAR.
Nampt
A2780
MLM
% left
HLM
% left
Solubility
(ꢀM)
Entry
R
d
e
a,c
b,c
f
IC (ꢀM)
IC (ꢀM)
50
50
9
0.007
0.004
0.13
0.13
16
85
82
39
9.4
41
62
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
4
4
0
1
2
0.030
0.060
0.009
0.059
0.014
0.011
0.009
0.005
1.4
1.6
19
27
42
19
20
3.4
18
19
> 99
98
99
93
85
89
94
82
58
61
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.46
2.2
84
43
80
64% inh
4
4
88
g
@
1ꢀM
N
i
4
5
6
0.32
0.067
0.20
29
Me
i
4
43
4
4
7
49
8
0.012
1.4
89
83
95
h
4
5
9
0
0.031
0.007
1.0
68
57
> 99
92
ALQ
94
0.032
a
b
Nampt biochemical inhibition. A2780 cell proliferation inhibition. This inhibition can be
c
reversed by addition of 0.33 mM of NMN. Unless otherwise noted, all biochemical and cellꢀ
d
based assay results are reported as the arithmetic mean of at least 2 separate runs (n = 2).
e
Mouse liver microsomal stability ꢀ % remaining at 30 min. Human liver microsomal stability ꢀ
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2
3
4
5
6
7
8
9
f
g
po% remaining at 30 min.
Aqueous solubility.
% Inhibition was determined at 1 ꢀM
h
i
concentration.
Above Limit of Quantification (> 100 ꢀM).
Racemic compound. See
Supporting Information for experimental details.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Given the encouraging biological profile of compound 50, the molecule was progressed to in
vivo mouse PK and xenograft efficacy studies to evaluate its oral exposure and antitumor effects.
As summarized in Table 6, compound 50 demonstrated reasonable PK properties in mice with
oral bioavailability of 26%. It showed a moderate clearance (2.7 ± 0.36 L/h/kg) and a high
volume of distribution (1.1 ± 0.22 L/kg). The compound also exhibited maximum plasma
exposures that were about 30ꢀfold greater than the corresponding cell IC value, and its plasma
5
0
levels remained at least 5ꢀfold above this IC for 2 hours (all values uncorrected for protein
5
0
binding effects).
Table 6. Compound 50 in vivo mean mouse PK parameters ± standard deviation (SD)
IV 1 mg/kg
po 5 mg/kg
PK Parameter
Mean (n = 3) ± SD
Mean (n = 3) ± SD
t
1/2(h)
max (h)
max (ꢀM)
AUCinf (ꢀM*h)
ss (L/kg)
0.27 ± 0.03
0.08 ± 0.00
2.7 ± 0.28
0.92 ± 0.11
1.1 ± 0.22
2.7 ± 0.36
1.3 ± 0.22
T
0.50 ± 0.00
C
1.1 ± 0.35
1.4 ± 0.32
V
ꢀ
ꢀ
Cl (L/h/kg)
F (%)
26
In an efficacy study, compound 50 exhibited significant antitumor effects against an A2780
human ovarian carcinoma tumor xenograft model without significant body weight loss. In mice
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
bearing A2780 xenografts, oral administration of the compound for 10 days (125 mg/kg b.i.d.)
produced rapid tumor regression (Figure 9). Tumor growth inhibition effects were also noted
after cessation of compound dosing during the duration of the xenograft experiment. For
example, a tumor growth inhibition value (TGI) of 97% was observed on day 17 of the study.
The described xenograft results confirmed that a ureaꢀcontaining Nampt inhibitor with
appropriate biological and DMPK properties can function as a potent in vivo antiꢀcancer agent.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
700
500
300
100
Vehicle
2 2
V e h ic le
Compound 48 (125 mpk PO BIDX10)
C o m p o u n d 4 8 ( 1 2 5
m p k P O B ID X 1 0 )
2
0
9
00
700
00
300
00
1 8
5
1
1
6
4
1
0
6 8
10
12
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16
18
20
22
24
26
28 30
Days
0
6
8
1 0
1 2
1 4
1 6
1 8
2 0
2 2
2 4
2 6
Dosing
D a y s
D o s in g
Figure 9: Left panel: compound 50 in vivo efficacy results in A2780 human ovarian carcinoma
tumor xenograft model. Right panel: body weight losl as a result of compound 50 administration
in xenograft experiment. Data reflect mean values (± standard deviation) of 8 animals in each
group.
Phosphoribosylated adduct detection and additional biochemical studies
During the LHS SAR optimization process described above, we identified several compounds
which exhibited potent inhibition in the Nampt biochemical assay but little activity in the A2780
cell culture assessment (e.g., 20-22, 29, 30, and 32). In the case of inhibitors 20ꢀ22, these
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outcomes seemed to be associated with the absence of a nucleophilic nitrogen atom in the LHS
portions of the molecules. It was previously shown that other Nampt inhibitors bearing similar
nucleophilic nitrogen atoms (e.g., compound 2) could serve as phosphoribosylation substrates
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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29
30
31
32
33
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
19
and form adducts with PRPP via a Namptꢀcatalyzed process. We suspected that the described
ureaꢀcontaining inhibitors might function in a related manner and therefore conducted detailed
mass spectrometry experiments to verify this hypothesis. Accordingly, compound 21 (a nonꢀ
nitrogen containing LHS inhibitor devoid of cell activity) and compound 50 (a nitrogen
containing LHS inhibitor with potent cell activity) were incubated with the Nampt enzyme under
the standard biochemical assay conditions, and the formation of the corresponding PRPPꢀderived
phosphoribosylated adducts was assessed by LCꢀMS/MS. The phosphoribosylated adduct was
only detected for compound 50 (Figure 10) and was not observed for compound 21 (data in
Supporting Information). Importantly, the 50ꢀPRPP adduct was not detected when the Nampt
enzyme was omitted from the biochemical experiment, strongly suggesting that the adduct was
formed through a Namptꢀcatalyzed process (Supporting Information). These results indicate that
the ability of a given ureaꢀcontaining Nampt inhibitor to form a phosphoribosylated adduct may
be a necessary requirement for obtaining potent antiꢀNampt cell activity.
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9
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0
Figure 10. LC/MS/MS Chromatograms of compound 50 enzyme reaction mixture: (a) Multiple
Reaction Monitoring (MRM) of transition from m/z 615.2 (adduct M+H ion) to 403.2 (parent
M+H ion) indicating presence of phosphoribosylated adduct (b) MRM of transition from m/z
403.2 (parent M+H ion) to 135.1 (representing fragmentation of the left hand side of the parent)
indicating presence of both parent (50) and phosphoribosylated adduct. See Supporting
Information for experimental details.
However, the PRPPꢀderived ribose adducts corresponding to cellꢀinactive compounds 29, 30,
and 32 were also detected by mass spectrometry in similar biochemical experiments (Supporting
Information). These data suggest that adduct formation itself is not sufficient to impart potent
cell activity to a given Nampt inhibitor and that other factors also influence these outcomes.
While compound permeability and protein binding characteristics are two likely determinants of
cell potency, we suspected that, due to high structural similarity, these parameters would not
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vary significantly among the cell potent (50) and cellꢀinactive (21, 29, 30, and 32) molecules
described above. We therefore conducted more detailed biochemical experiments with these
compounds in an attempt to better understand their cell culture behavior.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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29
30
31
32
33
34
35
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42
43
44
45
46
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48
49
50
51
52
53
54
55
56
57
58
59
60
As shown in Figure 11 and Table 7, competition experiments indicated that all these molecules
inhibit Nampt through a NAMꢀcompetitive manner with linear increases in IC values observed
50
2
0
with increasing NAM concentrations.
Importantly, varying NAM concentrations had a
relatively minor impact on the IC value of the cell active compound (50) as compared with
5
0
effects on IC outcomes for the other (cellꢀinactive) molecules studied. This behavior resulted in
50
21
compound 50 having the most potent calculated K value among these inhibitors (Table 7).
i
Thus, the ability of the compounds and/or their PRPPꢀribose adducts to effectively inhibit Nampt
in the presence of NAM may also influence their ability to function as potent cellꢀactive
2
2,23
agents.
However, given the similarities between the cellꢀactive molecule 50 and the cellꢀ
inactive compound 29 in these experiments, it is possible that additional factors also contribute
2
4
to the cellular outcomes.
NAM effects on IC 's
5
0
2
.0
.5
1.0
.5
.0
Cpd-21
Cpd-29
Cpd-30
Cpd-32
Cpd-50
1
0
0
100
200
300
ꢀM NAM
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 11. Effects of varying NAM concentrations on the ability of selected compounds to
inhibit Nampt biochemical activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 7. Nampt biochemical inhibition values determined in the presence of various NAM
concentrations.
a
Nampt IC (ꢀM)
5
0
b
Entry
K (ꢀM)
i
1
ꢀM NAM 5 ꢀM NAM 25 ꢀM
125 ꢀM
0.756
0.205
0.476
1.247
0.083
250 ꢀM
1.504
0.525
1.213
>3.0
21
0.030
0.015
0.012
0.021
0.010
0.034
0.024
0.033
0.064
0.013
0.121
0.051
0.082
0.195
0.019
0.027
0.009
0.021
0.044
0.003
2
9
30
3
5
2
0
0.171
a
Nampt biochemical inhibition. As a result of independent experiments, the IC50 values
determined using 1 ꢀM NAM for the indicated compounds differ slightly from those listed in
b
Tables 3ꢀ5. Calculated K value for each inhibitor (see reference 21). Results are reported as
i
the arithmetic mean of at least 2 separate runs (n = 2).
Chemistry
The urea derivatives were prepared by utilizing the general procedures shown in Schemes 1 ꢀ
5
.
Scheme 1 summarizes the isocyanate approach. Pyridinꢀ3ꢀylmethanamine 51a was
condensed with several commercially available isocyanates (52) to give the ureas (15, 53, and
6). Treatment of the sulfonyl chloride (53) with different amines/anilines (54) then afforded
5
compounds 9, 39 – 40, and 48 – 49. To speed up our SAR exploration, a oneꢀstep parallel
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synthesis protocol (reaction of 53 with 54) was also developed to provide the final compounds
42 – 46. Reduction of the sulfonyl chloride group present in intermediate 53 gave the
corresponding sulfinate (55) which was coupled with 1ꢀmethylꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
dioxaborolanꢀ2ꢀyl)ꢀ1Hꢀpyrazole to provide compound 12. Basic hydrolysis of the methyl ester
contained in 56 gave acid 57 which was coupled with piperidine to afford final compound 17.
Alternatively, treatment of various anilines or phenols (58) with triphosgene followed by
coupling with benzylic amines or pyridinylmethanamines provided compounds 10 ꢀ 11, 14, 16,
18, 20 – 24, 26 – 28, 34 – 37, 41, 47, 50, and 59 (Scheme 2). Oxidation of the thioether (14)
with mCPBA led to compound 13. Removal of the Boc protecting group present in intermediate
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
59 afforded the final compound 19. As shown in Scheme 3, condensation of the commercially
available aniline 58b with various chloroformates (60) formed the corresponding carbamate
intermediates (61) which were then coupled with the appropriate alcohols or amines (62) to
provide compounds 25, 29, 31, and 33. The synthesis of the amideꢀcontaining compounds 30
and 32 is shown in Scheme 4 and was accomplished by simply treating aniline 58b with the
appropriate acid chlorides (63). Coupling of aniline 58b with chlorosulfonyl isocyanate and 2ꢀ
chloroethanol provided the intermediate sulfamoylcarbamate (64) which was then reacted with
pyridinꢀ3ꢀylmethanamine 51a to afford the sulfonyl urea (38) as the final compound (Scheme 5).
Scheme 1: Urea derivatives synthesis ꢀ isocyanate approach.
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