Scaffold Morphing Identifies 3-Pyridyl Azetidine Ureas as Inhibitors of Nicotinamide Phosphoribosyltransferase (NAMPT)

Article abstract of DOI:10.1021/acsmedchemlett.9b00325

Small molecules that inhibit the metabolic enzyme NAMPT have emerged as potential therapeutics in oncology. As part of our effort in this area, we took a scaffold morphing approach and identified 3-pyridyl azetidine ureas as a potent NAMPT inhibiting motif. We explored the SAR of this series, including 5 and 6 amino pyridines, using a convergent synthetic strategy. This lead optimization campaign yielded multiple compounds with excellent in vitro potency and good ADME properties that culminated in compound 27.

Full text of DOI:10.1021/acsmedchemlett.9b00325

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Letter
Scaffold morphing identifies 3-pyridyl azetidine ureas as
inhibitors of nicotinamide phosphoribosyltransferase (NAMPT)
Daniel S. Palacios, Erik L Meredith, Toshio Kawanami, Christopher M. Adams, Xin Chen,
Veronique Darsigny, Mark Palermo, Daniel Baird, Elizabeth L. George, Chantale Guy,
Jeffrey Hewett, Laryssa Tierney, Sachin Thigale, Louis Wang, and Wilhelm A. Weihofen
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00325 • Publication Date (Web): 10 Oct 2019
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ACS Medicinal Chemistry Letters
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Scaffold morphing identifies 3-pyridyl azetidine ureas as inhibitors
of nicotinamide phosphoribosyltransferase (NAMPT)
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Daniel S. Palacios,*† Erik L. Meredith,† Toshio Kawanami,† Christopher M. Adams,† Xin Chen,†
Veronique Darsigny,† Mark Palermo,† Daniel Baird,‡# Elizabeth L. George,‡ Chantale Guy, ‡ Jeffrey
Hewett, ‡ Laryssa Tierney, ‡ Sachin Thigale, ‡ Louis Wang,‡ Wilhelm A. Weihofen‡
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† Global Discovery Chemistry, Novartis Institute for Biomedical Research, 22 Windsor St, Cambridge MA, 02139
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‡ Chemical Biology and Therapeutics, Novartis Institute for Biomedical Research, 181 Massachusetts Ave, Cambridge MA, 02139
KEYWORDS: NAMPT, azetidine, structure based drug design, scaffold morphing, amino pyridine
ABSTRACT: Small molecules that inhibit the metabolic enzyme NAMPT have emerged as potential therapeutics in oncology. As
part of our effort in this area we took a scaffold morphing approach and identified 3-pyridyl azetidine ureas as a potent NAMPT
inhibiting motif. We explored the SAR of this series, including 5 and 6 amino pyridines, using a convergent synthetic strategy. This
lead optimization campaign yielded multiple compounds with excellent in vitro potency and good ADME properties that culminated
in compound 27.
The production of nicotinamide adenine dinucleotide (NAD+)
is a critical biochemical process in mammalian cells. NAD+ is
a substrate for enzymes such as poly (ADP-ribose) polymerases
(PARPs) and sirtuins and is a critical component of cellular
metabolism.1 Given the importance of this biochemical,
mammalian cells have evolved multiple methods to produce
NAD+,2 but the predominant pathway in mammals involves the
conversion of nicotinamide (NAM) and phosphoribosyl
pyrophosphate (PRPP) into nicotinamide mononucleotide
(NMN) by the enzyme nicotinamide phosphoribosyltransferase
(NAMPT) followed by a second reaction catalyzed by NMN
adenylyltransferase (NMNAT, Figure 1A).3,4 The NAMPT
catalyzed reaction is the overall rate limiting step in this process
and it therefore controls the availability of NAD+. Cancer cells
are highly active metabolically and as a consequence they
consume NAD+ at a higher rate than non-cancerous cells.5 In
this vein, small molecule inhibition of NAMPT in cancerous
cells has been shown to deplete cellular NAD+ and lead to cell
death and together these lines of evidence point to NAMPT as
a promising target to develop novel therapies for oncology.6-8
As shown in Figure 1B, we previously reported the elaboration
of urea 1 to the 3-pyridyl (S,S) cyclopropyl carboxamide 2.9 The
3-pyridyl (S,S) carboxamide was a promising motif that
potently inhibits NAMPT and led to a compound that
Figure 1. (A) The rate limiting step in the synthesis of NAD+ is the NAMPT catalyzed synthesis of NMN from NAM and PRPP; (B) Our
initial identification of the azetidine ureas came from a scaffold morph of the previously established cyclopropyl carboxamide NAMPT
inhibitors; (C) Synthesis of the five and six membered ring ureas demonstrates the superiority of the azetidine urea for NAMPT inhibition.
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demonstrated in vivo efficacy in a mouse xenograft model.
approximately ten-fold in A2780 cells. We then determined if
the four membered nitrogen ring was the ideal ring size for
cellular activity. To this end, we prepared both enantiomers of
the
1
2
3
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5
6
7
8
Furthermore, the cellular potency of these molecules enabled
their translation into payloads for antibody-drug conjugates
(ADC).10 We were curious, though, if we could identify an
alternative warhead that could provide even greater potency for
use as a cytotoxic payload in antibody-drug conjugates because
of the known risk for on-target toxicity with this
mechanism.11,12 With this goal in mind, we reconsidered urea 1
in the context of the cellular potency exhibited by the
cyclopropane analogue 2. We reasoned that if small rings are
tolerated in this portion of the molecule then we may be able to
enclose the left hand nitrogen of the urea into a ring. As shown
in Figure 1B and the crystal structure in Figure 2, this concept
led to azetidine urea 3 and this compound was found to be very
potent in CellTiterGlo® (CTG) assays in A2780 and COR-L23
cells. With our scaffold morphing hypothesis validated, we
wanted to further explore this SAR of these ring containing
ureas.13
Before commencing an optimization campaign for this scaffold
we designed experiments to confirm that urea 3 is functioning
directly through NAMPT inhibition. First, we added the
biochemical product of NAMPT, NMN, to our cell based assays
and found that this completely rescues A2780 and COR-L23
cells from cell death induced by urea 3. Next, we demonstrated
that 3 directly inhibits NAMPT activity in a biochemical
inhibition assay (Figure 1B, IC50: 2.7 nM). Furthermore, we
obtained a co-crystal structure of urea 3 bound to NAMPT, as
shown in Figure 2, and found that the pyridine of 3 resides in
the NAM binding pocket, consistent with 3 acting as a NAMPT
substrate-based inhibitor.14 Interestingly, when urea 1,
cyclopropane 2, and urea 3 are overlaid in the NAMPT active
site (Figure 2) all three pyridine nitrogens are very closely
aligned,
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Scheme 1. Reagents and conditions: a) Zn, I2, Pd2(dba)3, N-Boc-3-
iodoazetidine, tri(o-tolyl)phosphine, DMF, 50 °C, 90 min
(Compounds 8 and 9); b) Zn, I2, Pd2(dba)3, N-Boc-3-iodoazetidine,
tri-2-furylphosphine; c) benzophenonimine, sodium tert-butoxide,
Pd2(dba)3, toluene, 100 °C, 3 hours, then 50% hydroxylamine in
water, MeOH, 25 °C, 18 hours; d) TFA, DCM, 25 °C, 3 hours.
pyrrolidine ring, compounds 5 and 6, as well as the achiral
piperidine urea 7. In our primary assays we found that all three
compounds were several orders of magnitude less potent
against both A2780 and COR-L23 cells. With this clarifying
result in hand, we decided to focus on the azetidine urea and
further elaborate the SAR of this series.
To further enable additional investigations into the structure
activity relationship (SAR) of this series we first prepared the
key 3-pyridyl azetidine building blocks 16,15 17 and 18 shown
in Scheme 1. The synthesis begins with either 3-bromo pyridine
8 or the bis-halogenated pyridines 9 and 10. A Negishi coupling
with 3-iodo N-Boc azetidine provides the essential carbon-
carbon bond for these compounds and yields the Boc protected
intermediates 11, 12 and 13. To then install an amine at either
the 5 or 6 position of the ring (see below for further details) we
employed benzophenonimine as a nitrogen source using
Buchwald-Hartwig conditions followed by deprotection to
generate anilines 14 and 15. Finally, the Boc group was
removed with trifluoroacetic acid to provide the azetidine
building blocks 16, 17, and 18.
Figure 2. Overlay of urea 1 (PDB: 6ATB), cyclopropane 2 (PDB:
6AZJ) and urea 3 (PDB: 6PEB) in the NAM binding pocket of
NAMPT. As shown above, there is a large degree of spatial overlap
for the three pyridine rings.
further evidence that the trajectory of the nitrogen is critical for
the substrate-based mechanism of these compounds14 and,
collectively, these data strongly suggest that NAMPT is the
cellular efficacy target of urea 3. Confident that analogs of 3
would also directly inhibit NAMPT, we chose to drive our
compound optimization program through cell based assays
given that our ultimate goal was a small molecule with in vivo
efficacy.
Scheme 2. Reagents and conditions: a) methyl 2-formylbenzoate,
DIPEA, sodium triacetoxyborohydride, DCM, 25 °C, 14 hours (for
20a); b) HATU, or COMU, DIPEA, DMF, 25 °C, 2 hours; c) Zn,
NH4Cl, EtOH/H2O, 50 °C, 1 hour; d) amine, DIPEA, phenyl
chloroformate, DCE 0 °C, 30 min then azetidine, 40 °C, 16 hours.
For our optimization campaign, we were interested in the (S)-
phenylethyl amide motif that we had previously identified9 to
determine if it was also optimal for the azetidine ureas. In this
vein, we prepared the collection of (S)-phenylethyl amide
As summarized in Figure 1C, we next generated the isoindol-1-
one azetidine urea 4 and found that removing one of the
pthalimide oxygens improves the activity of the compound
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building blocks 21a-f shown in Scheme 2. The synthesis of
these compounds began with commercially available (S)-1-(4-
nitrophenyl)ethan-1-amine 19 that was functionalized either
through a reductive amination/condensation sequence for
intermediate
1
2
3
Table 1: Cellular activity and in vitro ADME assays for compounds 22a-22f
4
5
6
7
MDCK MDR1
LEc A to B
(x10-6 cm/s)
CYP3A4
(midazolim,
µM)
A2780 IC50
(nM)
COR-L23
IC50 (nM)
MLMa (µL
min-1 mg-1)
HTb solubility
pH 6.8 (mM)
Compound
8
9
22a
22b
22c
22d
22e
22f
23a
23b
23c
23d
23e
23f
24a
24b
24c
24d
24e
24f
1.5
2.3
36
8
10
123.3
60.9
28.9
58
3.04
3.11
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
4.69
>25
>25
>25
>25
>25
0.130
0.011
0.930
0.380
>1.0
16.26
12.5
0.66
0.89
0.65
1.99
5.89
4.58
0.55
0.51
0.83
0.68
3.04
1.05
0.58
0.70
0.66
0.71
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130
58
17
5.7
25
38
40.8
31.4
<25
<25
<25
<25
<25
22
120
19
0.700
0.220
0.032
0.30
5.9
7.6
68
27
160
110
41
46
0.310
0.760
0.760
0.035
0.017
0.300
0.440
0.820
0.230
11
58
120
130
630
1380
970
200
1580
45
157.8
27.4
83.4
60.2
47.4
56.9
240
340
500
34
770
aMLM: Mouse liver microsomes; bHigh throughput; cMadin-Darby canine kidney multidrug resistance low expressing
22c eliminated CYP3A4 inhibition but this substitution also led
20a or an amide bond formation for intermediates 20b-f. Next,
the aryl nitro group was reduced with zinc to set the stage for
the final urea bond formation. Anilines 21a-f and pyridyl
azetidines 16-18 were then combined with phenyl
chloroformate in this convergent route to produce pyridines
22a-f, 6-amino pyridines 23a-f and 5-amino pyridines 24a-f.
Upon completion, the compounds were tested in our primary
assays and the results are shown in Table 1. In addition to
NAMPT activity, we obtained physiochemical (solubility,
permeability) and ADME data (mouse microsomal clearance,
CYP3A4 inhibition). We found that cellular activity was
broadly tolerant of structural modifications to the (S)-
phenylethylamide moiety. Initially, we were interested if the
(S)-phenylethylamine led to enhanced activity relative to the
unsubstituted benzyl amine. Comparing the matched
benzylamine isoindol-1one 4 (Figure 1B) and the (S)-
phenylethyl isoindol-1-one (22a, Table 1) we found that the
methyl group in this series afforded similar cellular potency
unlike the clear improvement observed in our previous
cyclopropyl carboxamide series.6 Even though a potency
enhancement was not observed, phenyl containing amides were
still sufficiently active in the A2780 and COR-L23 assays as
demonstrated by 22a and 3-fluoro phenyl amide 22b, but they
were both moderately potent in the CYP3A4 assay (3.04 and
3.11 µM, respectively). A heteroaryl amide such as pyrimidine
to an approximately ten fold decrease in potency and a
significant reduction in passive permeability as measure by the
MDCK assay.
We next investigated the activity of compounds with
aliphatic amides, as exemplified by pyran 22d, N-methyl
proline 22e and ethyl 22f. The aliphatic groups were less active
in the cell based assays than their aromatic counterparts, but this
was compensated by improvement in other dimensions. For
example, all three of these compounds showed no measureable
activity in the CYP3A4 inhibition assay. In addition, these
compounds had a high aqueous solubility, especially when
compared to 3-fluorophenyl analogue 22b. In contrast,
however, the passive permeability of the aliphatic amides
significantly decreased relative to the aromatic amides (Table
1). Combined, the reduced permeability and cellular activity of
the aliphatic amides prevented their further progression.
Intrigued by the promising cellular activity of the aromatic
amides, we explored other avenues to improve NAMPT activity
while reducing CYP3A4 inhibition. Since 6-aminopyridine had
been shown to reduce CYP inhibition in the NAMPT inhibitor
literature16 we wanted to investigate the role of this motif in the
cellular activity of our scaffold. We also wanted to investigate
5-amino pyridines because, to our knowledge, this substructure
had not been utilized in a NAMPT inhibitor. The results of both
of these SAR investigations are summarized in Table 1.
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For the 6-amino pyridines, 23a-f, these compounds
Page 4 of 6
phenylethylamine since this modification did not offer any
advantage in terms of cellular potency when comparing
matched pairs 4 (A2780 IC50: 0.6 nM) and 22a (A2780 IC50: 1.5
nM). To this end, we prepared the compounds shown in Table
2 using a similar route as depicted in Scheme 2 (see supporting
information for further details of the syntheses).
1
2
3
4
5
6
7
8
generally suffer a moderate potency loss compared to the des-
amino analogues. Examining the most potent matched pair as
an example, the des-amino 22a has cellular IC50’s of 1.5 and 8
nM against A2780 and COR-L23 respectively, while the
analogous values for 6-amino 23a are 5.9 and 19 nM. However,
this loss in potency is compensated by a significant decrease in
mouse microsomal clearance (22a: 123 µL/min mg, 23a: <25
µL/min mg) and CYP3A4 inhibition (22a: 3.04 µM, 23a:>25
µM). In contrast to the improvements in these ADME
properties, adding the amino group to the scaffold led to a
significant reduction in passive permeability (compare the
permeability of 22a and 23a for example). Therefore, despite
the attractive improvement in clearance and CYP inhibition, the
reduction in cellular activity and permeability relative to the
des-amino analogues led to their deprioritization.
Altering the orientation of the fused aryl ring had the desired
effect and compounds 25-28 are among the more potent analogs
that we synthesized in this series of NAMPT inhibitors but do
not significantly exceed the potency of our previously identified
cyclopropylcarboxamides.9 Furthermore, we found that
replacing the amide with a heterocycle as in compounds 26-28
led to high levels of passive permeability, validating our
hypothesis. Given the single digit nanomolar potency of
benzoimidazole 27 we next wanted to determine the suitability
of this compound for in vivo studies. Time course oral
pharmacokinetics in nude mice was obtained for 27 up to 30
mg/kg and this data is plotted in Figure 3A as the fraction
unbound (27 is 96.2% bound to mouse plasma protein).
Comparing this exposure data to the COR-L23 IC50 (3.9 nM)
we found that we would need to dose at least 5 mg/kg twice
daily in order to maintain exposure above this level in mice. In
advance of an in vivo efficacy experiment, we first tested the
tolerability of nude mice to twice daily repeat oral dosing of 27.
As shown in Figure 3B, at all doses tested, there was a
significant amount of body weight loss in the mice relative to
vehicle. This is in contrast to our previous experience,9 and one
potential explanation lies in the differences in PK of the two
compounds. At a 10 mg/kg oral dose 27 has both a higher Cmax
(29,112 nmol/L) and AUC0-7hr (67,413 nmol*hr/L) compared to
cyclopropane SI-9 (Cmax=12,691 nmol/L; AUC0-7hr=19,187
nmol*hr/L, see supporting information for more details).9 It is
therefore possible that mutiple dosing of 27 led to toxic levels
of NAMPT inhibition yielding the observed intolerability in
mice. These data are consistent with the known risk of on-target
toxicity with this mechanism11,12 and are likely due to direct
target inhibition rather than an event specific to the azetidine
urea motif. Based on the data in Figure 3A, a twice daily dose
of 0.3 or 1 mg/kg could potentially provide the required target
coverage, but we did not have an opportunity to test the
tolerability of these doses. Given the weight loss data for 27 we
decided to stop further exploration of this compound in vivo.
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11
12
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19
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25
26
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Table 2: SAR of amide replacements
O
R
N
N
H
N
MDCK
MDR1
LE A to
B (x10-6
cm/s)
A2780
IC50
(nM)
COR-
L23 IC50
(nM)
Compou
nd
R
O
O
25
26
27
0.4
0.2
0.7
3.2
4.3
3.9
12.30
16.25
18.61
N
N
MeO
N
N
EtO
N
n-Bu
N
28
0.9
5
18.44
N
O
Next, the cellular activity of the 5-amino compounds 24a-f were
interrogated. This substitution led to a significant reduction in
cellular activity relative to the des-amino or 6-amino analogues.
For example the A2780 IC50 of 3-fluoro phenyl 24b is 240 nM
which is a 100-fold drop in potency relative to des-amino 22b.
Similar declines in potency were observed across the 5-amino
analogues and they were deprioritized from further
investigations.
Since we were unable to improve the potency of this scaffold
by modifying the pyridine ring, we returned to the potential of
the amide to improve cellular activity. The potency of the fused
isoindol-1-one stood out to us and we were curious if changing
the position of the fused aryl ring would impact activity.
Furthermore, we were inspired by the use of heterocycles as
amide replacements in the angiotensin II receptor blocker
literature,17 and we hypothesized that replacing the amide could
improve the passive permeability of the series. Finally, we
wanted to revisit compounds that lacked the (S)-
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Corresponding Author
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3
*Daniel Palacios, email: daniel.palacios@novartis.com
Current addresses
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9
# Casma Therapeutics, 400 Technology Square, Suite 201,
Cambridge, MA 02139
Notes
The authors were all employees of the Novartis Institutes for
Biomedical Research during the time when this work was
completed.
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Funding Sources
This research was funded by Novartis Inc.
ACKNOWLEDGMENT
We thank the in vitro ADME group for generating the data in
Tables 1 and 2, Scott Busby for the biochemical inhibition data in
Figure 1, Giuliano Berellini for contributing to Figure 3A and
Dallas Bednarczyk for data analysis.
ABBREVIATIONS
NAD, nicotinamide adenine dinucleotide; PARP, poly (ADP-
ribose) polymerase; NAM, nicotinamide; PRPP, phosphoribosyl
pyrophosphate; NMN, nicotinamide mononucleotide; NAMPT,
nicotinamide phosphoribosyltransferase; NMNAT, nicotinamide
mononucleotide adenylyltransferase; ADC, antibody-drug
conjugate, ADME, absorption, distribution, metabolism and
excretion; CTG, Cell Titer Glo®; SAR, structure activity
relationship; MLM. mouse liver microsome; CYP, cytochrome
P450; HT, high throughput; MDCK, Madin-Darby canine kidney;
MDR1, multidrug resistance; LE, low expressing; PK,
pharmacokinetics; MTD maximum tolerated dose.
Figure 3. (A) Time course of calculated unbound plasma
concentrations of 27 at oral doses of 30, 5, 1 and 0.3 mg/kg. 27 is
96.2% bound to mouse plasma protein. (B) Percent weight loss
induced by oral dosing of 27 to nude mice. 27 was dosed twice per
day.
In summary, a scaffold morph from a cyclopropyl
carboxamide led to the identification of 3-azetidine urea
NAMPT inhibitors. Through biochemical experiments we were
able to show that these compounds were acting directly on
NAMPT function and initiated a lead optimization campaign
based on this substructure. A convergent synthetic route was
developed, including the highly versatile bifunctional pyridine
building blocks 17 and 18, to explore this SAR. We found that
a broad range of substituents was tolerated on the eastern
portion of the molecule and that the unsubstituted pyridine
provided the best blend of cellular potency and ADME
properties. These efforts culminated in the highly potent
NAMPT inhibitor 27, though we were unable to achieve our
stated aim of improving upon the potency of the cyclopropane
series. This compound was tested in a repeat dose tolerability
study in nude mice in preparation for an in vivo efficacy
experiment but we found that it was not tolerated at 5 mg/kg
twice daily. Based on these data that highlight the potential for
on-target NAMPT mediated intolerability, we decided not to
pursue further evaluation of this compound.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Detailed synthesis procedures and analytical data for all compound,
descriptions of the in vitro assay and in vivo experiments (PDF)
(9) Palacios, D.S.; Meredith, E.; Kawanami, T.; Adams, C.; Chen, X.;
Darsigny, V.; Geno, E.; Palermo, M.; Baird, D.; Boynton, G.; Busby,
S.A.; George, E.L.; Guy, C.; Hewett, J.; Tierny, L.; Thigale, S.;
Weihofen, W.; Wang, L.; White, N.; Yin, M.; Argikar, U.A. Structure
AUTHOR INFORMATION
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