Discovery of MK-4409, a novel oxazole FAAH inhibitor for the treatment of inflammatory and neuropathic pain

Article abstract of DOI:10.1021/ml5001239

We report herein the identification of MK-4409, a potent and selective fatty acid amide hydrolase (FAAH) inhibitor. Starting from a high throughput screening (HTS) hit, medicinal chemistry efforts focused on optimizing of FAAH inhibition in vitro potency,

Full text of DOI:10.1021/ml5001239

Letter
pubs.acs.org/acsmedchemlett
Discovery of MK-4409, a Novel Oxazole FAAH Inhibitor for the
Treatment of Inflammatory and Neuropathic Pain
Harry R. Chobanian,†,* Yan Guo,† Ping Liu,† Marc D. Chioda,† Selena Fung,† Thomas J. Lanza,†
Linda Chang,† Raman K. Bakshi,† James P. Dellureficio,† Qingmei Hong,† Mark McLaughlin,‡
Kevin M. Belyk,‡ Shane W. Krska,‡ Amanda K. Makarewicz,‡ Elliot J. Martel,‡ Joseph F. Leone,‡
Lisa Frey,‡ Bindhu Karanam,§ Maria Madeira,§ Raul Alvaro,§ Joyce Shuman,§ Gino Salituro,§
Jenna L. Terebetski,∥ Nina Jochnowitz,⊥ Shruti Mistry,⊥ Erin McGowan,⊥ Richard Hajdu,⊥
Mark Rosenbach,⊥ Catherine Abbadie,⊥ Jessica P. Alexander,○ Lin-Lin Shiao,○ Kathleen M. Sullivan,○
Ravi P. Nargund,† Matthew J. Wyvratt,† Linus S. Lin,† and Robert J. DeVita†
‡
§
∥
†Departments of Medicinal Chemistry, Process Chemistry, Drug Metabolism and Pharmacokinetics, Preclinical Development,
⊥Pharmacology, and ○Immunology, Merck Research Laboratories, Kenilworth, New Jersey 07033, United States
S
* Supporting Information
ABSTRACT: We report herein the identification of MK-4409, a potent and
selective fatty acid amide hydrolase (FAAH) inhibitor. Starting from a high
throughput screening (HTS) hit, medicinal chemistry efforts focused on optimizing
of FAAH inhibition in vitro potency, improving the pharmacokinetic (PK) profile,
and increasing in vivo efficacy in rodent inflammatory and neuropathic pain assays.
KEYWORDS: Fatty acid amide hydrolase, FAAH, oxazole, pyrazole, neuropathic pain, inflammatory pain, MK-4409, enzyme,
inhibitor, CNS
atty acid amide hydrolase (FAAH) is an integral,
Fmembrane-bound enzyme responsible for the breakdown
of fatty acid ethanolamide (FAE) signaling molecules, such as
the endocanabinoid arachidonyl ethanolamide (anandamide,
AEA), N-palmitoyl ethanolamide (PEA), and N-oleoyl
ethanolamide (OEA). FAAH is a member of the serine
hydrolase amidase signature family, which utilizes an unusual
serine−serine−lysine catalytic triad.1,2 Inhibition of FAAH
leads to elevated levels of these endogenous FAEs, which act on
cannabinoid receptors implicated in the suppression of pain
transmission.3 Levels of these FAEs were shown to be
Figure 1. Known covalent and noncovalent modifying FAAH
inhibitors.
significantly elevated in FAAH knockout (KO) mice as
compared to wild-type controls.4 Both genetic knockout of
inhibitors are the keto-oxazole class of FAAH inhibitors as
exemplified by OL-135,13 which reversibly forms an enzyme-
stabilized hemiketal through a particularly reactive electrophilic
carbonyl. More recently, however, several scaffolds have been
disclosed as reversible noncovalent modifying inhibitors of
FAAH and pharmacological modulation of FAAH activity
demonstrated reduced sensitivity to pain.5 Thus, FAAH
inhibitors are expected to provide therapeutic benefits in the
management of inflammatory and neuropathic pain.6−9
Several classes of covalent and noncovalent FAAH inhibitors
have been reported to date (Figure 1). Several covalent FAAH
inhibitors that irreversibly inhibit FAAH by carbamylation of
Ser241 have been reported and are exemplified by URB-
597,10−12 PF-3845, and PF-750.8 A second subclass of FAAH
Received: March 27, 2014
Accepted: April 10, 2014
Published: April 10, 2014
© 2014 American Chemical Society
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Letter
Figure 2. Key FAAH inhibitor pyrazole/oxazole compounds.
FAAH. Aminopyrimidine 514 and sulfonamide 615 are chief
among these novel classes of FAAH inhibitors. This approach,
in our view, would decrease potential safety concerns over the
creation of a long-lived covalent adduct between a compound
and the FAAH enzyme.
a
Scheme 1. Synthesis of Analogues 11−13
In this letter, we report the identification of a fully reversible,
noncovalently modifying FAAH inhibitor derived from a high
throughput screening campaign culminating in pyrazole 7
(Figure 2). Pyrazole 7 suffers from moderate potency as well as
poor preclinical PK.16 However, when dosed iv, the compound
did show moderate efficacy (at 30 mg/kg) in the complete
Freund’s adjuvant (CFA) inflammatory pain model, which
generated considerable interest within the team.17 In addition,
because of a lack of the presence of any obvious covalent
modifying site, we directed our studies toward optimization of
this pyrazole lead series to identify a selective, potent analogue
with desirable pharmacokinetic properties in three preclinical
species (rat, dog, and rhesus). Coupled with excellent in vitro
potency (<10 nM), we sought to identify a compound that was
fully CNS penetrant, maintained enzyme inhibition over a 24 h
time frame, displayed selectivity over related enzymes, and
possessed desirable efficacy in the CFA pain model at 10 mg/kg
or less.
a
Reagents and conditions: (a) Hunig’s base, THF, rt; then DMF-
DMA, 100 °C, 59% (18a), 86% (18b), 78% (18c); (b) hydrazine,
EtOH, 120 °C, 89% (19a), 86% (19b), 83% (19c); (c) for 11,
iodobenzene, K2CO3, K3PO4, CuI, trans-N,N′-dimethyl-1,2-cyclo-
hexane diamine, CH3CN, 77%, for 12, 4-F iodobenzene, K2CO3,
K3PO4, CuI, trans-N,N′-dimethyl-1,2-cyclohexane diamine, CH3CN,
59%; (d) 3-iodopyridine, K2CO3, CuI, D,L-proline, DMSO, 80 °C,
96%; (e) HO-NH2 (aq), EtOH, 80 °C, quant; (f) triethylorthofor-
mate, TsOH, 80 °C, 94%.
The synthesis of our key analogues was achieved through
several different routes. To synthesize pyrazole analogues 11
and 12, 4-chlorothiophenol was condensed with a bromoace-
tophenone of interest (Scheme 1). The resulting adduct was
then treated with DMF-DMA and hydrazine to afford the
desired pyrazoles 19a−c. Arylation at the N-1 position was
accomplished through a copper mediated coupling to yield
analogues 11 and 12. Alternatively for analogue 13, copper
mediated C−N bond formation was achieved on the
corresponding 4-CN phenyl derivative 18c followed by 1,2,4-
oxadiazole formation under standard conditions to afford 13.18
For the oxazole derivatives 16 and 17, the corresponding
oxazole triflates as previously described by Panek and co-
workers19 were used as the key starting material for each
analogue (Scheme 2). One-pot boronate formation and
subsequent Suzuki coupling was accomplished for compounds
23a and 23b.20 Halogenation and cross coupling with the
desired aryl thiol gave rise to 16 and 17.21
Our initial structure−activity relationship (SAR) efforts
focused primarily upon improvements in potency and
pharmaceutical properties in the pyrazole-containing system
7. Given the low molecular weight and moderate in vitro
potency, we believed that numerous modifications could be
made to compound 7 to optimize for drug-like properties. We
initially sought to eliminate the sulfur-linked aryl ring.
Heteroatom modification of the S atom led to a deleterious
effect on in vitro potency for FAAH inhibition (S = 119 nM
and O = 1007 nM) for compound 8 (Figure 2). Oxidation of
the S atom to its various higher oxidation states also led to a
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ACS Medicinal Chemistry Letters
Letter
channel blocker.23 During this process, we had noticed that
upon introduction of an oxazole central ring as a replacement
for the pyrazole, a noticeable improvement in MK-499 binding
potency was observed. The closely related analogues 12 and 15,
which both maintained a phenyl methyl sulfone moiety as the
active pharmacophore, had MK-499 binding values of 637 nM
and 28 μM, respectively, implying that the oxazole core had
quite an advantageous effect on MK-499 over the pyrazole
(Table 1). In addition, with the data collected for compounds
16 and 17, it was readily apparent that VMAT inhibition had
serendipitously improved upon modification of the central core
to the oxazole. Lastly, compound 17 took advantage of the fact
that throughout our earlier SAR, we found that optimization of
the core thioether ring system to a 2-pyridyl substitutent not
only helped with PK, but also improved in vivo efficacy in our
current pain models. Several of these compounds were further
studied in vivo for rat PK and efficacy based on these
improvements in the in vitro profiles.
c
Scheme 2. Synthesis of 16 and 17
c
Reagents and conditions: (a) bis-pinacolatodiboron, Pd(dppf)Cl2·
CH2Cl2, dppf, KOAc, dioxane, 100 °C; (b) R2X, Pd(PPh3)Cl2, 2.0 M
Na2CO3, dioxane, 90 °C, 67% over 2 steps; (c) NBS, TFA, DCM, rt,
60−66%; (d) R3SH, K2CO3, NMP, 120 °C, 69−85%.
reduction in potency (S(O) = 2192 nM, SO2 = >3000 nM) as
seen in compounds 9 and 10. This data led us to abandon this
strategy and to explore capping of the free pyrazole with various
groups. A simple Ph ring directly attached to the N-1 of the
pyrazole led to a greater than 4-fold improvement to the
intrinsic FAAH potency as exemplified by compound 11.
We next turned our attention to scission of the dioxane ring,
which was deemed imperative in order to improve upon the PK
properties of this series of compounds. Initial data pointed out
clearly that the para position has the greatest potential and
flexibility toward structural diversification. Methyl sulfone and
1,2,4-oxadiazole replacements for the dioxane ring were
potency-enhancing. The optimized N-subtituent is shown in
compound 12 as a para-F phenyl or as a 3-pyridyl group as
exemplified in compound 13. Subsequent SAR studies showed
importantly that a 4-fold improvement was realized upon
introduction of various heterocyclic tertiary alcohol moieties at
the 4-position such as that shown in compound 14 while
maintaining the para-F phenyl ring, which capped the pyrazole.
Compound 7, 11, 12, and 13 were further evaluated for off-
target activities as well as pharmacokinetic (PK) studies (Table
1). An off-target activity detected in a Pan Laboratories screen
Compounds 12 and 13 were dosed for rat PK studies at 1
mg/kg iv and 2 mg/kg po (Table 2), and both compounds
Table 2. Rat PK data on FAAH inhibitors 12, 15, 16 and 17
d
AUCNpo
half-life
(h)
Vd
e
compd ((μM h kg)/mg)
Cl (mL/min·kg) (L/kg) F (%)
ab
,
12
13
14
17
1.06
0.39
1.07
2.2
1.79
2.57
4.9
34
8.4
100
35
ac
,
34
7.8
ab
,
33.8
20
15.8
6.2
96
ac
,
4.3
120
a
Dosing vehicle DMSO/PEG400/H2O 20/60/20, 1 mg/kg iv.
b
c
Dosing vehicle Imwitor/Tween 1/1, 2 mg/kg po. Dosing vehicle
d
PEG400/Tween80/H2O 40/10/50, 2 mg/kg po. Dose normalized
AUCN. Bioavailability.
e
showed similar half-life, clearance, and volume of distribution. A
3-fold lower bioavailability for compound 13 to compound 12
may be attributed to poor oral absorption despite the improved
solubility of compound 13 as compared to compound 12
(FaSSIF solubility for 12 was measured to be 0.003 mg/mL
versus 0.027 mg/mL for compound 13). Furthermore, changes
of the pharmacophore from the p-methanesulfonyl benzene
ring to a pyrazine ring substituted with a 4-tertiary alcohol (14)
provided a rather substantial boost in terminal half-life while
maintaining the oral exposure observed in compound 12.
Lastly, compound 17, which combined optimized substituents
by replacing the 4-pyrazinyl tertiary alcohol moiety with a 4-
pyridyl tertiary alcohol, along with the thiopyridine substitutent
drastically improved the rat PK to levels, which could translate
to QD dosing for in vivo efficacy models. To further
characterize these compounds, they were dosed in our in vivo
efficacy pain models.
In order to ascertain in vivo efficacy, inflammatory pain was
induced by intraplantar injection of complete Freund’s adjuvant
(CFA) into the hind paws of rats.17 Mechanical allodynia was
assessed at 1 and 3 h post a single oral dose. This study was
performed on compounds 13, 14, 16, and 17 (Table 3). A
major objective was to try to achieve reasonable efficacy in the
CFA at a moderate dose (10 mg/kg) as a criterion for a
potential development compound. Compound 13 when dosed
at 30 mg/kg showed efficacy at the 1 and 3 h time points (49
and 62% inhibition, respectively). However, the VMAT and
MK-499 binding activity led us to focus on other more
structurally diverse analogues. The dose responses of
a
Table 1. In Vitro Data of Key FAAH Inhibitors
human FAAH
lysate IC50
(nM)
RatFAAH
lysate IC50
(nM)
human
MK-499 binding VMAT EC50
assay, Ki, (nM)
b
compd
(nM)
7
119
27
16
13
32
13
28
11
125
8
1538
>30000
637
ND
11
12
13
14
15
16
17
ND
6
363
4.8
10
6
5214
740
137
1157
ND
28 540
2500
1254
21
11
14000
>20000
a
Assay protocols are provided in the Supporting Information. In vitro
assay results are the average of at least three duplicates. Binding
b
displacement of [35S] MK-499.
for compounds 12−14 was human vesicular monoamine
transporter (VMAT) inhibition. This is of particular concern
since this particular transporter is centrally expressed.22
Additionally, the pyrazole lead series was also plagued by
hERG activity. Potent compounds were screened against the
hERG K+ channel using a binding assay that measures the
displacement of [35S]MK-499, a well characterized hERG K+
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ACS Medicinal Chemistry Letters
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Table 3. CFA Data Key FAAH Compounds
a
b
dose
compd (mg/kg)
% reversal of allodynia 1 h
% reversal of allodynia
3 h time point
time point
13
14
14
16
17
17
30
30
10
10
30
10
49
58
42
46
70
62
40
38
44
71
51
49
a
b
Oral dose in mg/kg. Average percent reversal is calculated as
(postdose − predose)/(preinjury − predose) for each rat (n = 6).
Figure 4. SNL efficacy of compound 17 (MK-4409) at 50% reversal
plotted against rota-rod impairment at 20% (adverse effect) compared
to current standards of care or positive controls.
compound 14 at both 10 and 30 mg/kg indicated comparable
efficacy to compound 13 at both doses, which suggested to us
that the heterocyclic tertiary alcohol modification not only
improved PK but also provided comparable in vivo efficacy to
compound 13 but at a lower dose. More importantly, with both
an improved off-target profile and PK profile, compound 17
also showed reduction of inflammatory pain on par with the
standard of care, naproxen, at both 10 and 30 mg/kg in a model
for inflammatory pain. Taking compound 17 one step further,
we decided to evaluate its efficacy in the rat spinal nerve
ligation model (SNL) for neuropathic pain.24
Additionally, free fraction was measured across species (rat,
2.9%; dog, 1.2%; rhesus, 0.8%; human, 1.6%) indicating a high
level of plasma protein binding for compound 17. An additional
concern we had for this mechanism was tachyphylaxis. To
alleviate this concern, we initiated a chronic paradigm in the
CFA pain model versus a CB1 and CB2 agonist, WIN 55,212-2
known to produce CB1 desensitization and loss of anti-
nociception under chronic dosing.26
The pan CB agonist WIN 55,212-2 loses its analgesic
properties in the CFA assay at roughly day 5. Compound 17,
however, continues to show excellent efficacy out to 10 days
(Figure 5). Plasma exposures were also obtained at both the 3
Using the SNL model as a measure of efficacy for
neuropathic pain, compound 17 was evaluated in vivo at
doses of 3, 30, and 100 mg/kg with excellent response observed
at all three doses (Figure 3). The efficacy of compound 17 at a
Figure 3. SNL efficacy of compound 17 (MK-4409).
Figure 5. Chronic dosing of compound 17 versus WIN 55,212-2 in
CFA pain model along with plasma exposure.
level of roughly 50% analgesia when compared to standards of
care Pregabalin, Gabapentin, and Duloxetine was shown to be
quite favorable. A secondary effect of dosing these standards of
care into rodents is loss in cognition due to drowsiness and
motor skill impairment. In order to ensure that a FAAH
inhibitor would not cause the same type of motor skill
impairment seen with other analgesics, we also implemented
the rota-rod assay as a secondary assay along with the SNL as a
standard counterscreen for these undesirable cognitive effects.
Compound 17 not only provided 50% reversal of allodynia at a
low dose (3 mg/kg) but also did not show any effect in the
concomitant roto-rod assay at doses as high as 100 mg/kg
(Figure 4). This was in line with URB-597 (1), a previously
described covalent modifying FAAH inhibitor that required a
21 mg/kg dose to achieve comparable efficacy to 17 in our
hands.
and 24 h time points following dosing at 30 mg/kg. One
interesting observation is that compound 17 reduced edema
observed in the animals over several days, which may contribute
to its anti-inflammatory properties.
In conclusion, we discovered compound 17 (MK-4409) as a
potent and selective reversible noncovalent modifying FAAH
inhibitor. MK-4409 showed excellent efficacy in numerous
preclinical models including the CFA and SNL pain models. In
addition, no cognitive effects were observed for this brain
penetrant FAAH inhibitor (measured rat brain/plasma ratio
was 2). A chronic dosing paradigm in the CFA assay showed
overall anti-inflammatory activity and no tachyphylaxis apparent
after 10 days of dosing. MK-4409 (17) was accepted as a
development candidate based on the promising preclinical
profile. Additional human clinical data will be reported in
subsequent publications.
On the basis of the promising in vivo and in vitro data
generated for compound 17, a full PK profile was obtained
across preclinical species.25 The resulting PK parameters
supported the development of a QD compound in human.
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hexylcarbamic acid 3′- or 4′-substituted biphenyl-3-yl esters as fatty
acid amide hydrolase inhibitors: synthesis, quantitative structure−
activity relationships, and molecular modeling studies. J. Med. Chem.
2004, 47, 4998.
(12) Liu, P.; Hamill, T. G.; Chioda, M.; Chobanian, H. R.; Fung, S.;
Guo, Y.; Chang, L.; Bakshi, R.; Hong, Q.; Dellureficio, J.; Lin, L. S.;
Abbadie, C.; Alexander, J.; Jin, H.; Mandala, S.; Shiao, L.-L.; Li, W.;
Sanabria, S.; Williams, D.; Zeng, Z.; Hajdu, R.; Jochnowitz, N.;
Rosenbach, M.; Karanam, B.; Madeira, M.; Salituro, G.; Powell, J.; Xu,
L.; Terebetski, J. L.; Leone, J. F.; Miller, P.; Cook, J.; Holahan, M.;
Joshi, A.; O’Malley, S.; Purcell, M.; Posavec, M.; Chen, T.; Riffel, K.;
Williams, M.; Hargreaves, R.; Sullivan, K. A.; Nargund, R. P.; DeVita,
R. J. Discovery of MK-3168: A PET tracer for imaging brain fatty acid
amide hydrolase. ACS Med. Chem. Lett. 2013, 4, 509.
ASSOCIATED CONTENT
* Supporting Information
Synthetic procedures and analytical data of selected FAAH
inhibitors and conditions for all the biological assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*(H.R.C.) Phone: (908) 740-5131. E-mail:harry_chobanian@
merck.com.
■
Notes
(13) Boger, D. L.; Miyauchi, H.; Du, W.; Hardouin, C.; Fecik, R. A.;
Cheng, H.; Hwang, I.; Hedrick, M. P.; Leung, D.; Acevado, O.;
Guimaraes, C. R. W.; Jorgensen, W. L.; Cravatt, B. F. Discovery of a
potent, selective, and efficacious class of reversible alpha-ketohetero-
cycle inhibitors of fatty acid amide hydrolase effective as analgesics. J.
Med. Chem. 2005, 48, 1849.
(14) Gustin, D. J.; Ma, Z.; Min, X.; Li, Y.; Hedberg, C.; Guimaraes,
C.; Porter, A. C.; Lindstrom, M.; Lester-Zeiner, D.; Xu, G.; Carlson, T.
J.; Xiao, S.; Meleza, C.; Connors, R.; Wang, Z.; Kayser, F.
Identification of potent, noncovalent fatty acid amide hydrolase
(FAAH) inhibitors. Bioorg. Med. Chem. Lett. 2011, 21, 2492.
(15) Wang, X.; Sarris, K.; Kage, K.; Zhang, D.; Brown, S. P.; Kolosa,
T.; Surowy, C.; Kouhen, O. F.; Muchmore, S. W.; Brioni, J. D.;
Stewart, A. O. Synthesis and evaluation of benzothiazole-based
analogues as novel, potent, and selective fatty acid amide hydrolase
inhibitors. J. Med. Chem. 2009, 52, 170.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The author wishes to thank the department of Laboratory
Animal Resources for their assistance in animal dosing and
sampling.
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