4
D.S. Palacios et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
(compound 10) significantly increases the activity of the scaffold in
the CORL23 CTG assay. This interaction is also highly stereoselec-
tive as the corresponding (R)-N-1-phenylethylamide (compound
11) is approximately 100 fold less active than the (S) enantiomer.
Next, we were interested to discover if the combination of the (S,
S)-cyclopropane and the (S)-N-1-phenylethylamide would further
enhance cellular activity but we found, contrary to our expectation,
that urea 10 and cyclopropane 12 are approximately equipotent.
While cyclopropane 12 displays excellent in vitro potency, we
found 12 to have poor solubility, permeability and in vitro clear-
ance. To address these poor properties, we set out to reduce the
overall size and hydrophobicity of the molecule. However, as
shown in Fig. 3B, both the (S)-N-1-phenylethylamide 13 and
cyclopropane 14 were very weakly active in our cell based assays.
Next, despite the equipotency of urea 10 and cyclopropane 12,
when we merged the analogous compounds 13 and 14 we were
surprised to find that cyclopropane compound 15 gained greater
than 50-fold potency in both the A2780 and CORL23 CTG assays
versus the parent compounds. In addition, as we had hypothesized,
the smaller ethylamide 15 is both more soluble and metabolically
stable than benzyl morpholine 12. Thus, ethylamide 15 revealed
the utility of combining the cyclopropane and (S)-N-1-phenylethy-
lamide to enable the installation of relatively small groups on the
eastern portion of the molecule to improve compound properties.
Furthermore, this result was achieved by challenging our assump-
tion based on cyclopropane 12 that the (S,S) cyclopropane and (S)-
N-1-phenylethylamide would not be additive in terms of com-
pound activity. For our chemistry strategy, this was a lesson
learned that SAR between closely related series may not always
track and one has to always question preconceived wisdom regard-
ing scaffold optimization.
Based on the SAR shown in Table 3, we decided to keep the
methyl group fixed and commenced with an interrogation of the
amide SAR. As shown in Scheme 1, the synthesis of these analogues
began with the Heck reaction of 3-bromo pyridine 20 and t-butyl
acrylate to give the alpha-beta unsaturated ester 21. The t-butyl
ester is critical because we found that the corresponding methyl
ester was hydrolyzed during the Corey-Chaykovsky cyclopropana-
tion and the resulting acid proved difficult to isolate. Deprotection
of the ester with TFA followed by chiral chromatography (see Sup-
porting information for details) then afforded enantiomerically
pure 23. Through this sequence, we were able to generate multi-
gram quantities of the key non-racemic intermediate 23 in signif-
icantly fewer steps than was previously reported.14 Diversification
of the amide began with the commercially available secondary
amine 24 which was coupled to a range of acids using standard
amide bond forming conditions. Reduction of the aryl nitro group
then afforded the corresponding anilines 26a–i and the ultimate
step was a second amide bond formation to give compounds
27a–i.
As shown in Table 4, the eastern amide was broadly tolerant of
diverse substructures, enabling the identification of a number of
potent compounds with diverse physiochemical properties. Ali-
phatic heterocycles such as tetrahydropyran 27b and N-methyl
proline 27d have single digit nanomolar potency and good solubil-
ity. In addition, N-methyl proline 27d shows an improved perme-
ability profile, likely due to the formation of an intramolecular
hydrogen bond between the amide proton and the proline nitro-
gen.18 An unsubstituted phenyl group was active and though the
in vitro clearance was increased, this could be attenuated through
the addition of electron withdrawing groups to the four position of
the ring, as demonstrated by fluorine 27f and nitrile 27g.
Given the utility of the (S)-N-1-phenylethylamide, we next
probed the SAR of this position to determine if the methyl group
was ideal for activity and, as shown in Table 3, there is limited tol-
erance at this position with small non-polar groups being pre-
ferred. Even adding an additional carbon with ethyl 17 reduces
the activity of the compound about tenfold relative to the methyl
matched pair (compound 27e Table 4). In addition, adding a polar
atom, as demonstrated by primary alcohol 18, deleteriously
impacts the cell based activity of the scaffold (200 nM A2780 CTG).
In addition, heterocycles such pyridine 27h and pyrimidine 27i
can also replace the phenyl group with a small loss in activity but a
significantly decreased in vitro clearance. Significantly, the CYP3A4
inhibition of these compounds is attenuated relative to previously
reported cyclopropylcarboxamide NAMPT inhibitors14 (Table 4).
This may in part be due to the potency combination of the cyclo-
propane and (S)-N-1-phenylethylamide which enables the installa-
tion of relatively small amide appendages.
Given the promising in vitro properties of phenyl amide 27e, we
decided to investigate the in vivo metabolic stability of this com-
pound. As shown in Fig. S2 (supporting information), 27e has good
in vivo properties with a half-life of 90 min and a markedly reduced
intrinsic clearance than was predicted by the mouse liver
microsomes.
Table 3
SAR of the (S) benzyl position.
R
O
The good in vivo exposure of 27e combined with its concise syn-
thesis suggests that this small molecule would be an excellent tool
to explore NAMPT biology both in vivo and in vitro. With this in
mind, we employed a mouse CORL23 xenograft model and found
that phenylamide 27e, when dosed orally at 10 mg/kg bid, signifi-
cantly reduced the growth of the implanted tumor (Fig. 4A). The
antitumor efficacy that we observe for 27e is comparable to the
result shown in Fig. 4B for the known NAMPT inhibitor 28 (see
Supporting information). Furthermore, we also found that the
observed efficacy of 27e correlates to the blockade of glycolytic
flux, which requires NAD for turnover, as measured by the accu-
mulation of the glycolysis intermediates fructose-6-phosphate
and glucose-6-phosphate (Supporting information).19
To conclude, our pursuit of compounds that inhibit the function
of the NAD+ producing enzyme NAMPT began with phenotypic
screening hit 1. The poor physiochemical properties and complex
synthesis of pyrrolo-pyrimidine 1 catalyzed the search for alterna-
tive chemical matter and a consequent in silico screen identified 2-
amino pyridine 2. The promising cellular activity and much
improved access to analogues of 2 enabled a structure based lead
optimization effort to improve overall physiochemical properties.
N
H
N
Cmpd
R
A2780 CTG (nM)
832
CORL23 CTG (nM)
2310
16
17
18
19
43
200
1
137
630
4