2
J. M. Keith et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
O
O
N
N
H
N
N
H
N
N
N
S
N
JNJ-1661010/Takeda
PF 750
N
N
N
O
N
O
CF3
Cl
N
H
N
N
H
N
N
O
O
PF-04457845
JNJ-40355003
Figure 1. Aryl piperazinyl and piperadinyl ureas.
of Cb/Cp ratios obtained was quite broad and they did not correlate
with TPSAs or pKas. Likewise, the ALogP values for these com-
pounds (ranging from 2.8 to 4.9) did not offer a predictive trend
with regard to brain penetration. The simple phenyl derivative
(1) serves as a reference point and its Cb/Cp ratio was an impressive
3.64.
ether tail (Table 2). Replacement of the chlorine atom of JNJ-
40355003 with a bromine (12), fluorine (13) or CF3 (14) group
leads to a modest reduction in Cb/Cp. Introduction of a chloropy-
ridine group onto the fluoro derivative (15) leads to a substantial
further decrease of Cb/Cp, and as was observed with (3), gave a
compound that was rapidly cleared.
It has been reported that the presence of pyridine nitrogens in a
molecule tends to lead to decreased brain penetration relative to
phenyl.51–53 In the present series of molecules, replacing the phe-
nyl with a 3-pyridyl group to give JNJ-40355003 (2) modestly
decreased CNS penetration to give a Cb/Cp ratio of 2.62. While
JNJ-40355003 retained excellent brain penetration properties, sim-
ply adding a chlorine atom to the 4-position of the pyridine ring (3)
led to a significantly lower Cb/Cp ratio. Addition of the chlorine
atom also appears to have introduced greater metabolic instability
as (3) had a 4-fold higher rate of clearance than JNJ-40355003.
Counterintuitively, the 4-cyano pyridine derivative (4) actually
had a higher B/P ratio than JNJ-40355003, although it too gets
cleared rather quickly. The non-basic pyrazine derivative (5) exhib-
ited the highest Cb/Cp ratio (4.2:1) of any of the compounds in this
data set despite having two azine nitrogen atoms.
The greatest variation in B/P ratios was obtained with 5-mem-
bered heteroarenes. The simple 3-isooxazole derivative (6) is
highly brain-penetrant (Cb/Cp ratio of 3.3), but replacement of the
oxygen with a hydrogen-bond donating N–H results in a com-
pound only slightly favoring partitioning into the brain over the
plasma. Perhaps not surprisingly, the tetrazole (8), which would
be completely deprotonated under physiological conditions (tetra-
zole pKa ꢀ 4.9,54 >99% deprotonated at pH 7.2),55 is the least brain
penetrant compound of this series with a Cb/Cp of 0.02:1 (Cp/Cb of
>45:1). However, the very poor CNS penetration ability of (8) does
not preclude effective central target engagement (vide infra).
Hydrogen-bond donating and acidic groups are not the only
chemical features that lead to decreased brain penetration. Fusion
of a benzo ring to the isoxazole to give (9) also leads to a large
decrease in the Cb/Cp ratio, greater in fact, than replacement of
the oxygen with an N–H.56 The two imidazopyridine derivatives
(10 and 11) are interesting cases as they are significantly proto-
nated57 at physiological pH (imidazopyridine pKa ꢀ 6.8,58 60% pro-
tonated at pH 7.2). These compounds favor the plasma over the
brain by about 3:1 and, at least with (10), appear to be cleared
quickly. This compound too, should serve as a reminder that high
volumes of distribution as obtained from simple PK experiments,
in this case Vss = 10.7, does not necessarily predict good brain
penetration.
Introduction of a chloropyridine group onto the fluoro deriva-
tive (13 ? 15) leads to a similarly potent compound that exhibits
a lower Cb/Cp, and as was observed with (3), was rapidly cleared.
However, the high clearance of (15) did not prevent the increase
in the concentrations of FAAs even as its plasma levels were rapidly
decreasing.
Introduction of a chlorine atom to the benzyl ring of the biaryl
ether had a significant deleterious impact on the PK properties of
(16) relative to (2). The PK profile of (16) suggested very slow
absorption from the gut was taking place, such that after 4 h, com-
pound levels in the plasma and brain were still rising and the Cb/Cp
gradually increasing (Fig. 2).
Surprisingly, a much smaller effect was observed when the
piperazine nitrogen was replaced with a C–H. Compound (17) gave
a Cb/Cp ratio very close to that of JNJ-40355003. Interestingly, re-
introduction of a nitrogen atom into the biaryl ether (18) had a
robust deleterious effect on CNS penetration (Cb/Cp ratio of 0.45).
While lacking a direct comparator for (19), it would appear that
moving the side-chain of the piperidine ring from the 4- to the 3-
position did not benefit CNS penetration.
As mentioned above, a strong preference for partitioning into
the brain over the plasma is not always necessary for effective tar-
get engagement. Using the most CNS penetrant compound (5) as a
reference (Fig. 3), it was determined that residual FAAH activity
was reduced to below 10% within a half-hour and remained sup-
pressed throughout the experiment. The profile for the highly
potent, but plasma preferring compound (10), is somewhat differ-
ent. Thirty minutes after dosing, (10) has only inhibited approxi-
mately 60% of FAAH present in the brain, but by 1 h there is less
than 10% active enzyme and by 4 h residual activity is below 5%.
Even the tetrazole (8; Cb/Cp ratio of 0.02:1) is able to inhibit FAAH
centrally to below 10% residual enzyme activity but it took approx-
imately 4 h to do so. PEA and OEA concentrations began increasing
at lesser degrees of FAAH inhibition because they are hydrolyzed
by the enzyme more slowly than AEA, which is the preferred sub-
strate of FAAH. It is possible that AEA levels increased 7–10 h post
dosing, but data for those time points were not collected. Interest-
ingly, a complete return of enzymatic activity took place by 24
hours post dose, whereas complete reversal of inhibition with
the more CNS penetrant JNJ-40355003 took approximately 48 h.
Mechanism-based enzyme inhibitors don’t necessarily need to
exhibit a high Cb/Cp to engage the target, but low concentrations
in the brain will affect the rate at which inhibitors encounter the
We have been able to affect large changes in the CNS penetrat-
ing ability of these ureas simply through the modification of the
pendant heteroarenes of the urea, but we can also modulate Cb/
Cp through small changes to the piperazine core and the biaryl