The SAR of brain penetration for a series of heteroaryl urea FAAH inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.05.001

The SAR of brain penetration for a series of heteroaryl piperazinyl- and piperadinyl-urea fatty acid amide hydrolase (FAAH) inhibitors is described. Brain/plasma (B/P) ratios ranging from >4:1 to as low as 0.02:1 were obtained through relatively simple st

Full text of DOI:10.1016/j.bmcl.2016.05.001

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The SAR of brain penetration for a series of heteroaryl urea FAAH
inhibitors
⇑
John M. Keith , Mark S. Tichenor, Richard L. Apodaca, Wei Xiao, William M. Jones, Mark Seierstad,
Joan M. Pierce, James A. Palmer, Michael Webb, Mark J. Karbarz, Brian P. Scott, Sandy J. Wilson,
Michelle L. Wennerholm, Michele Rizzolio, Raymond Rynberg, Sandra R. Chaplan, J. Guy Breitenbucher
Janssen Pharmaceutical Companies of Johnson & Johnson, L.L.C. 3210 Merryfield Row, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The SAR of brain penetration for a series of heteroaryl piperazinyl- and piperadinyl-urea fatty acid amide
hydrolase (FAAH) inhibitors is described. Brain/plasma (B/P) ratios ranging from >4:1 to as low as 0.02:1
were obtained through relatively simple structural changes to various regions of the heteroaryl urea scaf-
fold. It was not possible to predict the degree of central nervous system (CNS) penetration from the vol-
umes of distribution (Vd) obtained from pharmacokinetic (PK) experiments as very high Vds did not
correlate with high B/P ratios. Similarly, calculated topological polar surface areas (TPSAs) did not consis-
tently correlate with the degree of brain penetration. The lowest B/P ratios were observed for those com-
pounds that were significantly ionized at physiological pH. However, as this class of compounds inhibits
the FAAH enzyme through covalent modification, low B/P ratios did not preclude effective central target
engagement.
Received 3 January 2016
Revised 29 April 2016
Accepted 2 May 2016
Available online xxxx
Keywords:
FAAH
Covalent inhibition
Blood brain barrier
BBB
Ó 2016 Elsevier Ltd. All rights reserved.
Heteroaryl urea
Rat PK
Target engagement
The brain is the primary moderator of numerous biological pro-
cesses for which pharmacological intervention may be of clinical
benefit. However, it is effectively separated from circulating blood
by the blood–brain barrier (BBB) and the blood-cerebrospinal fluid
barrier. Of the two, the BBB is the most important as its surface
area is several thousand fold greater than that of the blood-cere-
brospinal fluid barrier.1–3 The BBB lines the vasculature of the
CNS and consists of endothelial cells with continuous tight inter-
cellular junctions overlaying the basement membrane which in
turn overlays astrocytes.4 The BBB is also armed with an assort-
ment of efflux transporters and metabolic enzymes5 that collec-
tively tend to keep proteins and other large molecules
(mw > 500) as well as small polar or charged species from entering
the brain.6–11 While a certain amount of lipophilic character is
required for passive diffusion into the CNS,12 the relationship
between lipophilicity and brain uptake tends to be parabolic in
nature, with neither the exceptionally polar nor excessively lipo-
philic compounds penetrating readily.13–15 Published reports sug-
gest that a molecule’s ability to pass the BBB tends to be poor
+ O > 10.16–19 For many classes of CNS drugs, it has been found that
the ideal LogP lies between 2 and 3.5.20–26 Correlations between
the topological polar surface areas (TPSAs) of molecules and their
ability to penetrate the CNS have also been found.27,28 CNS pene-
trant compounds typically have TPSAs below 90 Å2, with many
below 60 Å2.29
In the course of an internal FAAH30,31 program,32–45 many very
similar compounds were profiled in vivo with particular interest
paid to their ability to penetrate the BBB. The compounds profiled
were heteroaryl piperazinyl and piperadinyl ureas; a class of com-
pounds, reported on previously by us and others (Fig. 1),46–50 that
inhibit the FAAH enzyme through forming a covalent adduct
between the active site serine (Ser241) and the urea carbonyl with
the concomitant loss of the aryl amine. The compounds are syn-
thetically modular and most are easily prepared in a few steps
(see Supporting information).
Table 1 contains a selection of data generated from compounds
on which only the heteroaryl amine was varied. Included in the
table are the in vitro potency of the molecules reported as apparent
IC50s (due to the time dependent nature of the inhibition), PK data
from Sprague–Dawley rats when available, the calculated topolog-
ical polar surface areas (TPSAs) and ALogP values, and the brain to
plasma ratio of compound concentrations (Cb/Cp) obtained after
dosing of the compound by oral gavage. Interestingly, the range
when it possesses any of the following characteristics:
P
mw > 450 g/mol; LogP > 4; hydrogen bond donors > 5;
N
⇑
Corresponding author.
E-mail address: jkeith@its.jnj.com (J.M. Keith).
http://dx.doi.org/10.1016/j.bmcl.2016.05.001
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Keith, J. M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.001

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
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J. M. Keith et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 1
The effect of heteroaryl substituents on CNS penetration
O
HetAr
Cl
N
H
N
N
O
a
a
h
Compd
HetAr
hFAAH apparent IC50
(nM)
rFAAH apparent IC50
(nM)
PK properties
TPSA
(Å2)
ALogP pKa
Cb/Cp ratio (dose)
Cl (L/h/
Vss (L/
F (%)
–
kg)
kg)
(1)
6
4
298 86
33 8.7
–
–
44.8
57.7
4.7
3.5
–
3.64 0.33 (10 mg/kg)c
N
N
(2) JNJ-
40355003
53–
69
2.62 0.24 (0.6, 6 &
20 mg/kg)g
1.4 0.41
1.3 0.55
0.69b
2.79b
3.2
5.25
3.85
(3)
20
9
3.33
18
57.7
4.2
1.18 0.30 (20 mg/kg)
Cl
N
(4)
(5)
17 5.5
8.7 6.3
17 4.3
84 34
–
–
–
81.5
70.6
3.4
2.9
1.9
0.6
3.28 0.26 (20 mg/kg)d
4.24 0.21 (20 mg/kg)
CN
N
0.93b
4.11
35
N
O N
(6)
(7)
(8)
30 8.5
32 5.4
81 12
615 140
154 81
46 11
0.6b
0.5b
0.2c
2.6
2
50
70.8
73.5
99.3
3.1
3.3
2.8
À2.97 3.34 0.32 (20 mg/kg)
HN
N
1.28 0.11 (3, 10 &
75
2.5
30 mg/kg)f
HN
N
N
1.1
100
4.9
0.023 0.004 (6 mg/kg)
0.86 0.05 (3 mg/kg)
N
O N
(9)
10 2.3
46 16
8
2.5
0.56b
3.8d
2.48
10.7
44
60
70.8
62.1
4.9
3.6
À4.7
N
N
(10)
118 53
5.7 1.1
6.8
6.8
0.33 0.06 (20 mg/kg)
0.33 0.11 (20 mg/kg)e
N
N
(11)
2.7 0.4
–
–
–
62.1
3.6
a
b
c
1 h incubation with the enzyme, errors are the standard error of the mean (SEM).
Vehicle was 75% pharmasolve, 20% cremaphore and 5% aqueous dextrose.
30% SBCD with 1 M HCl.
50% PEG 400/water.
Purified water.
At 2 h time point.
d
e
f
g
h
5% aqueous dextrose.
pKa values are for the heterocycle and substituent (if any) absent the urea.
target and thus the time to onset of pharmacological effect. In those
instances where target inhibition is slowly or non-reversible, then
it’s possible for the pharmacology induced by the molecule to per-
sist beyond the time it takes for it to be cleared from the organism.
For those indications where onset of centrally mediated pharma-
cology needs to be rapid (such as analgesia), a quickly absorbed
compound with a high Cb/Cp ratio may be preferred. For chronic
conditions, a compound with a lower Cb/Cp ratio may be preferable
in order to minimize the potential for off-target side effects result-
ing from accumulation of the compound in the brain.
Due to recent events in the clinic with BIA 10-2474, it is impor-
tant to extensively characterize the selectivity and secondary phar-
macology of any FAAH inhibitor (particularly a mechanism-based
inhibitor) that has the potential to enter the clinic. In addition,
while there is the potential for the secondary pharmacology to
influence the PK and Cb/Cp ratio of a compound, it is hypothesized
that this effect would be minimal. The secondary pharmacology of
every compound described in this manuscript has not been
studied, but those that have and other compounds within this class
of compound were shown to be highly selective after having been
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J. M. Keith et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 2
The effect of small changes to the core and biaryl ether tail on CNS penetration
Compd
hFAAH apparent
IC50 (nM)
rFAAH apparent
IC50 (nM)
TPSA (Å2)
ALogP
Cb/Cp ratio (dose)
a
a
N
N
N
N
O
O
O
O
O
O
O
O
Br
F
2.4 1.3
20 6.0
290 12
18 3.5
57.7
3.6
1.58 0.33f (20 mg/kg)b
N
H
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
16
5
57.7
57.7
57.7
57.7
54.5
67.4
54.5
3.1
3.8
3.8
4.2
4.5
3.8
4.1
1.57 0.14 (20 mg/kg)c
1.99 0.13g (20 mg/kg)b
0.67 0.16 (20 mg/kg)d
1.26 0.09 (20 mg/kg)b
2.26 0.26 (20 mg/kg)c
0.45 0.07 (20 mg/kg)d
0.33 0.03 (20 mg/kg)e
N
H
CF3
1.0 0.5
3.3 0.8
8.0 2.1
4.3 2.2
N
H
F
48
4
N
H
Cl
N
Cl
Cl
Cl
Cl
76 16
215 15
22 2.9
41 14
N
H
N
N
N
N
H
1.0
28
0
8
N
H
N
O
O
N
H
F
a
b
c
d
e
f
1 h incubation with the enzyme, errors are the s.e.m.
Vehicle = water.
Vehicle = 20% hydroxypropyl cyclodextrin.
Vehicle = 50% PEG 400/water.
Dosed as a suspension in 75% pharmasolve, 20% cremaphore and 5% aqueous dextrose;
PK parameters Cl = 0.8 L/h/kg, Vss = 2.8 L/kg, F ꢀ 100%.
g
PK parameters Cl = 0.6 L/h/kg, Vss = 4.0 L/kg, F ꢀ 100%.
extensively profiled (radioligand binding, functional assays, ester-
ase/serine hydrolase enzymatic inhibition assays, and activity-
based protein profiling (proteomics)). These data will be included
in a subsequent manuscript describing the further characterization
of biology and pharmacology of these compounds.
In conclusion, we have shown that the brain penetrating ability
of heteroaryl piperazine and piperadine ureas can be modulated
through small structural changes to the heteroarenes, the piper-
azine or piperidine core, and the biaryl ether tail. Compounds
exhibiting Cb/Cp ratios ranging as high as 4.2:1 to as low as
0.02:1 have been preparable. The degree of brain penetration
was not predictable from PK experiments as large volumes of dis-
tribution did not correlate with a high Cb/Cp. As the described com-
pounds are mechanism-based inhibitors of the FAAH enzyme, low
Cb/Cp ratios did not preclude central target engagement, but did
slow the onset of inhibition.
Compound exposure of (16)
(20 mg/kg in H20, n=3)
10
Plasma
Brain
8
6
4
2
0
0
1
2
3
4
Time post-dose (hr)
Figure 2. Compound exposures for the slowly absorbed (16).
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J. M. Keith et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Ex vivo brain FAAH activity
Ex vivo brain FAAH activity
(5)
(10)
(20 mg/kg in 1:4:15 Pharmasolve:cremaphor:D5W; n=3)
(20 mg/kg in H20, n=3)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Vehicle
0.5
1
4
Veh
0.5
1
2
6
Time post-dose (hr)
Time post-dose (hr)
Ex vivo brain FAAH activity
(8)
(6 mg/kg in 30% SBCD, n=3)
120
100
80
60
40
20
0
Veh 0.5
1
2
4
6
24
Time post-dose (hr)
Figure 3. Time-course of brain FAAH inhibition by (5), (10) and (8).
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ates, as well as their respective characterization data (annotated 1H
NMR and MS data), is included in the Supporting information. Also
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and fatty acid amide elevation and residual enzyme activity
graphs.
Supplementary data associated with this article can be found, in
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Please cite this article in press as: Keith, J. M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.001