Design and Synthesis of a Novel and Selective Kappa Opioid Receptor (KOR) Antagonist (BTRX-335140)

Article abstract of DOI:10.1021/acs.jmedchem.8b01679

κ opioid receptor (KOR) antagonists are potential pharmacotherapies for the treatment of migraine and stress-related mood disorders including depression, anxiety, and drug abuse, thus the development of novel KOR antagonists with an improved potency/selec

Full text of DOI:10.1021/acs.jmedchem.8b01679

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Drug Annotation
Design and Synthesis of a Novel and Selective Kappa
Opioid Receptor (KOR) Antagonist (BTRX-335140)
Miguel Guerrero, Mariangela Urbano, Eun-Kyong Kim, Ana Maria Gamo Albero,
Sean Riley, Lusine Abgaryan, Nora Leaf, Lori-Jean Van Orden, Steven J Brown,
Jennifer Xie, Frank Porreca, Michael D Cameron, Hugh Rosen, and Edward Roberts
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 01 Feb 2019
Downloaded from http://pubs.acs.org on February 1, 2019
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Design and Synthesis of a Novel and Selective Kappa Opioid Receptor
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(KOR) Antagonist (BTRX-335140)
9
Miguel Guerreroa, Mariangela Urbanoa, Eun-Kyong Kima, Ana M. Gamoa, Sean Rileya, Lusine Abgaryana,
Nora Leafa, Lori Jean Van Ordenb, Steven J. Browna, Jennifer Y. Xiec,d, Frank Porrecad, Michael D. Cameroneǡ
Hugh Rosena, Edward Robertsa*
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a
Department of Molecular Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA
92037, United States.
b BlackThorn Therapeutics, Inc. 780 Brannan St. San Francisco CA 94103, United States.
c
Current address: Department of Basic Sciences, New York Institute of Technology College of Osteopathic
Medicine @ Arkansas State University, Jonesboro, AR 72446, United States.
d Department of Pharmacology, University of Arizona, Tucson, AZ 85724, United States.
e Department of Molecular Medicine, The Scripps Research Institute, Jupiter, Florida 33458, United States
ABSTRACT: N opioid receptor (KOR) antagonists are potential pharmacotherapies for the treatment of
migraine and stress-related mood disorders including depression, anxiety and drug abuse, thus the development
of novel KOR antagonists with an improved potency/selectivity profile and medication-like duration of action
has attracted the interest of the medicinal chemistry community. In this paper, we describe the discovery of 1-
(6-ethyl-8-fluoro-4-methyl-3-(3-methyl-1,2,4-oxadiazol-5-yl)quinolin-2-yl)-N-(tetrahydro-2H-pyran-4-
yl)piperidin-4 amine (CYM-53093, BTRX-335140) as a potent and selective KOR antagonist, endowed with
favorable in vitro ADMET and in vivo pharmacokinetic profiles and medication-like duration of action in rat
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pharmacodynamic experiments. Orally administered CYM-53093 showed robust efficacy in antagonizing KOR
agonist-induced prolactin secretion and in tail-flick analgesia in mice. CYM-53093 exhibited a broad selectivity
over a panel of off-target proteins. This compound is in Phase 1 clinical trials for the treatment of
neuropsychiatric disorders wherein dynorphin is thought to contribute to the underlying pathophysiology.
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INTRODUCTION
The opioid receptors belong to the superfamily of G-protein coupled receptors and are generally classified into
four subtypes: P opioid receptor (MOR), G opioid receptor (DOR), N opioid receptor (.OR) and the
nociceptin/orphanin FQ (N/OFQ) receptor. The opioid receptors show a high degree of sequence homology,
however activation of these receptors by selective endogenous and exogenous ligands has been shown to
produce striking differences in pharmacological and physiological effects.1,2 The KOR is a Gαi/o-coupled
receptor primarily activated by endogenous dynorphin opioid peptides.3-4 The KOR is distributed throughout the
spinal cord, brain stem and human brain.5 In the brain, KORs are particularly expressed in the anterior cingulate
cortex, amygdala, insula, putamen, neocortical region, caudate, thalamus, globus pallidus, pons, substantia nigra
and hippocampus.5-9 Numerous lines of evidence from preclinical and clinical studies have suggested the KOR
as a central player in a variety of neuropsychiatric and neurological disorders such as depression, epilepsy,
Alzheimer’s disease, substance and alcohol abuse and schizophrenia.10-19 Studies suggest that the KOR may
play a role in post-traumatic stress disorder (consistent with the modulatory effects of dynorphin on reward,
mood, and stress) and in migraine prophylaxis.20-22 As a consequence of these findings, the development of
selective KOR antagonists has stimulated great interest in both academia and the pharmaceutical industry.
Archetypical KOR antagonists nor-BNI (1), GNTI (2) and non-morphinan JDTic (3) (Figure 1) exhibit a delay
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in the onset of action of hours or days, and their antagonism effects are measurable even for several weeks at
minimally-effective doses; in contrast, these compounds show a rapid decline in plasma levels.23 Concerns
about the feasibility of developing medications with archetypical KOR-antagonists have centered on their
abnormal long duration of action. These concerns have led to the development of KOR antagonists with
medication-like duration of action from which JNJ-67953964 (4) (formerly known as LY-2456302 and CERC-
501) and PF-04455242 (5) have been evaluated in clinical trials (Figure 1).24 5 showed single digit nanomolar
activity at the KOR and good selectivity against the DOR, but poor selectivity against the MOR.25 Phase 1
clinical trials of 5 were terminated due to toxicology findings in animals exposed to the compound for three
months.26 4 displayed in vitro sub-nanomolar KOR antagonism with a selectivity of approximately 21-fold over
the MOR and 135-fold over the DOR, and efficacy in animal models of substance abuse and depression.24, 27, 28,
29 4 was until recently the only KOR antagonist undergoing clinical development as monotherapy and has been
shown to be safe in humans with mild to moderate side effects at daily doses of 10 mg (p.o.).30,31 Remarkably, 4
has shown clinically meaningful results in a phase II proof of concept study for treating patients with treatment-
resistant depression32 but dose escalation may be limited as effects of MOR antagonism begin to emerge.31,33
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The compound ALKS-5461 is a combination of buprenorphine (6) (partial MOR-agonist and KOR-antagonist)
and samidorphan (7) (MOR-antagonist) that has been postulated to act primarily as a KOR-antagonist.34 ALKS-
5461 is well tolerated in humans with limited concerns for abuse potential. The compound has been studied as
adjunctive therapy for patients with treatment-resistant depression and showed clinically meaningful
antidepressant effects in a few randomized double-blind placebo-controlled trials.35 As a result of this work a
new drug application (NDA) has recently been filed for ALKS-5461 for the adjunctive treatment of major
depressive disorder (MDD) in patients with inadequate response to standard antidepressant therapy.36
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RESULTS AND DISCUSSION
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In an effort to identify novel selective KOR antagonists with desirable pharmacokinetic and pharmacodynamic
properties, a high-throughput screening (HTS) campaign of the Molecular Libraries-Small Molecule Repository
(MLSMR) was carried out by our laboratories and identified the hit 3-ethyl-5-methyl-2-(4-((2-
hydroxycyclohexyl)amino)piperidin-1-yl)-6-methylpyridine-3,5-dicarboxylate 8 (Figure 2) as a weak KOR
antagonist (IC50 = 410 nM) with poor selectivity against the MOR (11-fold).37 The initial structure–activity
relationship (SAR) investigations of the hit were focused on increasing the potency while trying to improve the
selectivity against the MOR. Iterative SAR led to the discovery of the pyridine CYM-50202 (9) as a potent
KOR antagonist (IC50 = 12.6 nM) with good and moderate selectivity against the DOR and MOR (278- and 27-
fold), respectively (Figure 2). 9 displayed high solubility, an encouraging pharmacokinetic (PK) profile in rat
and excellent CNS-penetration. Perhaps more importantly, 9 did not display long lasting pharmacodynamic
effects observed with prototypical KOR antagonists.37 From a panel of 52 pharmacologically relevant off-target
proteins, 9 exhibited >50% inhibition at only the sodium channel site 2 (IC50 = 1.8 μM) and ORL-1 (IC50 = 3
μM).37 Unfortunately, we identified a number of issues with this molecule. First, 9 showed significant inhibition
of CYP2D6 at 10μM raising concerns of potential drug-drug interaction liabilities. In addition, the activity at
sodium channel site 2 raised concerns of potential cardiovascular liabilities. Moreover, the methyl ester and
bromide group were identified as undesirable structural motifs of this chemotype due to the likely ester cleavage
in vivo and a structural alert in the case of the bromide. Consequently, additional efforts to develop KOR
antagonists with improved potency (single-digit nanomolar), selectivity (>100 fold against MOR) and safety
profile were undertaken and the results of the SAR studies are reported in this manuscript. For the purpose of
exploring the SAR of 9, the molecule was divided into three fragments: the pyridine head group A, the
piperidine linker B and the amine tail C.
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Figure 1. Prototypical and Clinical Stage KOR Antagonists
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Figure 2. Early KOR Antagonist HTS Hit 8 and 9
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We started our iterative SAR studies by replacing the ester group with an oxadiazole isostere and by changing
the bromine for small alkyl groups while exploring single diastereomers in the piperidine-region B. These
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efforts culminated in the discovery of single digit nanomolar KOR antagonist 16 (IC50 = 1.3 nM) with modest
and high selectivity against the MOR and DOR (24 and >100-fold), respectively (Table 1). All synthesized
compounds were evaluated for antagonist activity at the MOR, DOR, and KOR using our established assays.37
16 is endowed with physicochemical properties that are consistent with CNS-penetrant compounds38 (cLogP =
2.2, tPSA = 81.8, MW = 371) and devoid of the structural liabilities of 9. Unfortunately selectivity over the
MOR remained suboptimal (optimal >100 fold). The synthesis of 16 is depicted in Scheme 1. Amine 12 was
synthesized through reductive-amination of diastereomerically-pure amine 10 with cyclohexanone followed by
Cbz-hydrogenolysis. The oxadiazole intermediate 15 was synthesized from the carboxylic acid 13 via coupling-
cyclization with acetamidoxime 14 in the presence of EDCI and HOBt. The final compound 16 was obtained
via microwave-assisted nucleophilic aromatic substitution (SNAr) of chloride 15 with amine 12 (Scheme 1).
Scheme 1. Synthesis of 16
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Encouraged by these data we sought to improve the selectivity of 16 by undertaking a multiparameter-SAR
optimization of the three molecular regions of 16 (A, B and C). For the optimization process we outlined
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specific tactics for each molecular region: 1) in region A to replace the pyridine for a quinoline ring while
keeping the oxadiazole fixed in position 3 (Scheme 2); 2) in region B to simplify the piperidine ring by
removing the 3-hydroxy group to rapidly prepare a variety of analogs in the quinoline ring and tail-amine in a
parallel-chemistry format; 3) in region C to explore a variety of structurally-diverse amines with the goal of
optimizing the physicochemical properties (pKa, tPSA, MW, cLogP and hydrogen bond donors) for good oral
bioavailability and brain penetration.38
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The synthesis of these compounds is depicted in Scheme 2 and their biological activities are listed in Table 1.
Oxadiazole intermediates 19 and 20 were synthesized from the corresponding commercially available
carboxylic acids 17 and 18 via acyl chloride formation followed by coupling-cyclization with acetamidoxime
14. Ketones 24 and 25 were synthesized via microwave-assisted SNAr of 19 and 20 with amine 21 followed by
ketal deprotection. Finally, reductive amination of ketones 24 and 25 with a variety of amines 26 furnished the
final compounds 27-39.
Based on iterative SAR optimization cycles, the 6-position of the quinoline ring was found to be an essential
structural feature to enhance selectivity against the MOR and DOR. For example, the 6-ethyl analogs 30 and 31
showed similar potency at the KOR and enhanced selectivity against the DOR and MOR (>1700-fold) as
compared to the 6-methyl counterparts 27 and 28. This SAR analysis also revealed that the location of a
hydrogen bond acceptor in the amine tail was important for selectivity. For instance, both 6-methylquinoline 4-
THP derivative 29 (CYM-51317) and 6-ethylquinoline 4-THP analog 32 showed single-digit nanomolar
activity at the KOR but modest selectivity against the MOR (26-fold). Further modifications at the tail region
were carried out while keeping the ethyl group at the 6-position. The enantiomeric pair of 3-THP-analogs 36
and 37 showed similar activity at KOR but (R)-3-THP 36 was more selective against the MOR. The 5-
membered ring analogs (R)-3-THF 38 and (S)-3-THP 39 were ~12 and ~17-fold less potent than their
corresponding 6-membered ring enantiomers 36 and 37, and showed good selectivity against the MOR.
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Interestingly, the bulkier and lipophilic analog 33 as well as the polar aminoalcohol 34 showed sub-nanomolar
potency at the KOR and exquisite selectivity against the MOR (4382- and 988-fold, respectively). Furthermore,
the oxetane 35 showed nanomolar potency at the KOR and remarkable selectivity against the MOR. This
diverse set of molecules highlighted the potential of the amine tail to tune-in the desired pharmacokinetic
properties while maintaining potency and selectivity.
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Scheme 2. Synthesis of 27-39
Table 1. KOR, MOR, DOR Antagonist Activity of Compounds 16, 27-39
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IC50 (nM)a
Selectivity
Calculated
Physicochemical
Properties50, 51
Compound
R
R1
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KORb MOR
DOR
2410
MOR/KOR
24
cLogP
2.2
tPSA cpKa
16
27
1.3
1.3
31.1
176
82
62
10.2
10.7
Me
Me
>10000
>10000
135
377
3.4
2.9
28
2.2
3.0
829
79
62
71
10.6
10.5
29 (CYM-
51317)
Me
4410
26
1.6
30
31
Et
Et
1.3
1.7
4390 >10000
2930 >10000
3377
1723
3.9
3.4
62
62
10.7
10.6
32
33
34
Et
Et
Et
7
183
>10000
26
2.1
4.2
3.4
71
62
82
10.5
10.8
10.2
0.76
0.59
3330 >10000
4382
988
583
>10000
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35
36
Et
Et
4.5
>10000 >10000
1460 >10000
>2222
1896
2.1
3.0
71
71
10.3
9.9
0.77
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37
Et
1.5
9.5
1500 >10000
>10000 >10000
1000
3.0
71
9.9
38
39
Et
Et
>1053
>971
2.6
2.6
71
71
9.8
9.8
10.3 >10000 >10000
a Values are reported as mean of n = 3 determinations.
bThis assay uses Tango ꢀꢁꢂꢃͳ-bla U2OS cells which express KOR linked to a GAL4-VP16 transcription
factor via a TEV protease site. Stimulation of the KOR by U-50488 causes migration of the β-arrestin fusion
protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. Assay protocols are
described in ref 37.
A set of selected compounds 27, 29, 33, 34, 35, 36 were assayed for stability in rat, mouse and human liver
microsomes (Table 2). The tested compounds showed relatively high intrinsic clearance in human hepatic
microsomes and, in general, compounds with lower cLogP values (e.g. 29, 35) displayed lower intrinsic
clearance in hepatic microsomes than compounds with higher cLogP values (e.g. 27, 33). Representative
compounds 29, 34, 35, 36 were further profiled in additional pharmacokinetic and off-target assays (Table 3).
The compounds were moderately bound by plasma proteins (64.0-98.1% bound) across species and a
correlation was observed between cLogP values and plasma protein binding. Remarkably, many compounds (at
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10μM concentration) showed both low cytochrome P450 inhibition and hERG inhibition profile. Specifically,
29 was tested in a panel of four relevant cardiac ion channels in a patch clamp assay and found devoid of
activity at 10 μM (<50% inhibition). Additionally, 29 showed high membrane permeability (A-B) in a Caco-2
cell line with low efflux (Table 3). Based on these results we decided to examine the in vivo pharmacokinetic
profile of 29 in rats (Table 4). 29 distributed well into tissues (Vss = 13 L/kg) and had a high clearance (CL)
after 1 mg kg-1 intravenous dosing (i.v.) and exhibited plasma exposures out to 6 hours (110.16 ng mL-1 at Cmax
and 1705 min·ng mL-1 AUC0-∞), after a dose of 2 mg kg-1 p.o. The absolute oral bioavailability (F) was 20%
(Table 4). Additionally, 29 displayed a brain to plasma ratio of 2.9 in mice at 30 minutes and of 3.8 at 120
minutes post intraperitoneal (i.p.) administration of a 10 mg kg-1 dose (Table 4). Although 29 was only
moderately selective against the MOR, it showed promising overall pharmacokinetic properties and off-target
profile thus it was selected as a tool compound for further in vivo studies.
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Table 2. Intrinsic Clearance (Clint) in Hepatic Microsomes of Compounds 27, 29, 33, 34, 35 and 36
Compound
Calculated Physicochemical
properties
Microsomal Clint (mL/min/kg)a
MW tPSA
cLogP
3.4
human
49.5
15.1
46.5
36.1
23.1
40.3
rat
mouse
ND
27
29
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34
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36
379
407
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435
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421
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82
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ND
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3.4
2.1
3.0
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aTest compounds (1 μM) were incubated with 0.2 mg mL-1 pooled human, rat and
mouse hepatic microsomes at 37°C with continuous shaking. Further details for the
assays can be found in the experimental section and reference 37.
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Table 3. ADMET Properties of 29, 34, 35, 36
Compound
29
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64
78
4
34
98.1
95.5
97
35
91.7
89
36
human
mouse
96.5
91.4
96
Protein plasma binding (% bound) at 1 PMa
rat
92.4
-13
-40
-11
10
CYP3A4
CYP2D6
CYP2C9
CYP1A2
-28
9
-22
1
20
-6
5
Human cytochrome P450 inhibition at 10PM (%)a
-13
13
-18
20
Voltage-gated sodium (Nav1.5)
hERG
32
35
72
77
19
6
Ion channel %
Inhibition at
10 PMa,b
Voltage-gated calcium (Cav1.2) <25
Inward-rectifying voltage-gated potassium (Kir2.1) <25
Hyperpolarization-activated cyclic nucleotide-gated <25
potassium (HCN4)
28
ND
ND
ND
ND
ND
ND
<25
<25
Caco-2 cells
A-B (10-6 cm/s) 26.2
ND
ND
ND
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Efflux ratio
1.0
ND
ND
ND
ND = No data. A-B = movement of the compound from the apical to the basal side of the membrane; a
surrogate for permeability
8
9
aFurther details for the assays can be found in the experimental section and reference 37.
bCompounds were profiled in the Eurofin cardiac profiler panel. Further details can be found in the
supporting information.
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Plasma Pharmacokinetic Profile of Compound 29 in Rodentsa
Rat (n = 3, mean values)
i.v. (1 mg·kg-1)
AUC∞
p.o. (2 mg kg-1)
T
Cmax
AUC0-∞
min.ng/mL
1,705
F
½
CL (mL·min-1·kg-1)
83
Vss (L·kg-1)
12.95
T (h)
½
(h)
min.ng/mL
12,740
(ng/mL)
110.16
(%)
20
1.98
1.56
30 min
120 min
2.9
3.8
Brain:plasma ratio in mice (i.p. administration, 10 mg kg-1, mean values)
a 29 was dosed as the monotartrate salt.
In a mouse tail-flick assay 29 did not show a long duration of action in blocking the antinociceptive effect of the
KOR-agonist, U69,593.22 Perhaps most importantly, in an injury-free rat model of cephalic pain with features of
migraine and medication overuse headache, oral administration of 29 showed preventive benefits on blocking
stress induced allodynia thus suggesting that KOR antagonists could provide a new therapeutic strategy in
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preventing migraine.22 In parallel, the potential mutagenic activity of this compound class was tested on
selected compounds 33, 34, 35 and 36 in a bacterial reverse mutation assay (Ames assay). Four Salmonella
typhimurium strains TA100, TA1535, TA1537, TA98 as well as the E. coli strain WP2uvrA were used in the
presence and absence of an exogenous metabolic activation system (S9). Both 34 and 35 exhibited mutagenicity
in the TA1537 strain in the presence and absence of S9; 33 and 36 also showed a mutagenic response in the
TA1537 strain but only in the absence of S9. Positive mutagenic activity in the TA1537 strain often reflects
intercalation of the tested molecules with DNA, and this intercalation is commonly associated with the
planarity, π-stacking and hydrogen bonding interaction capabilities of molecules.39 Based on the hypothesis that
the quinoline A was the portion of the molecule involved in the intercalation with the DNA, two strategies were
evolved to address the genotoxicity liability: 1) reduce the basicity of the quinoline-nitrogen to disrupt
hydrogen-bond interactions between DNA and the quinoline ring and 2) disrupt the planarity of the quinoline-
oxadiazole axis to hinder π-stacking interactions. To implement these strategies, a fluorine atom was introduced
at the 8-position of the quinoline-ring to substantially reduce the pKa of the quinoline nitrogen (up to 2.5 pKa
units). In addition, a methyl group was installed in the 4-position of the quinoline ring in an attempt to disrupt
the planarity.
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58
59
60
For the construction of poly-substituted quinoline rings we decided to use the Friedländer methodology since it
is one of the most robust strategies for the modification and manipulation of quinoline rings.40 The synthesis of
these compounds is described in Scheme 3 (without a 4-methyl group) and Scheme 4 (with a 4-methyl group)
and their biological activity reported in Table 5. The aldehyde 41 was synthesized from carboxylic acid 40 via
reduction with BH3-THF complex followed by oxidation with manganese dioxide in dichloromethane. The
quinoline intermediate 43 was synthesized by condensation of acetic acid 42 with aldehyde 41 in phosphorus
oxychloride. A SNAr reaction of 43 with amine 21, followed by Suzuki-Miyaura coupling with Et3B and ketal
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deprotection afforded the key ketone-intermediate 45. Finally, reductive amination of ketone 45 with a series of
amines 26 furnished the final compounds 46-50 (Scheme 3). Preparation of ketone 54 was accomplished via
iodination of aniline 51 followed by Heck coupling with ethylene glycol vinyl ether 53.41 Condensation of
acetic acid 42 with ketone 54 in phosphorus oxychloride afforded the poly-substituted quinoline 55. A SNAr
reaction of amine 21 with 55 followed by ketal deprotection afforded the ketone intermediate 57. The final
compounds 58-62 were synthesized via reductive amination of 57 with amines 26 (Scheme 4).
Scheme 3. Synthesis of 46-50
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58
59
60
Scheme 4. Synthesis of 58-62
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For the selection of tail-amines we sought to modulate the physicochemical properties in a range compatible
with oral bioavailability and CNS penetration utilizing the SARs gleaned from the previous series. The THP
analogs 47 (CYM-53052), 48 and 46 were approximately equipotent to the non-fluorinated analogs 36, 37 and
32, with higher selectivity against the MOR in the case of 46 and 48 and lower in the case of 47. Furthermore,
the 3-amino-THF enantiomers 49 and 50 were ~4-fold more potent than the non-fluorinated analogs 38 and 39,
and both enantiomers (49 and 50) retained excellent selectivity against the MOR. The introduction of a methyl
group in the 4-position of the quinoline ring generally increased potency at the KOR while decreasing the
selectivity against the MOR. For example, the 4-amino-THP 58 (CYM-53093, BTRX-335140) and the 3-
amino-THP enantiomers 59 and 60 were ~4-fold more potent than the demethylated analogs 46-48 but less
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selective against the MOR. The 5-membered ring analogs 61 and 62 were ~3-fold more potent than 49 and 50 at
the KOR and moderately less selective against the MOR. From these analogs, compounds 58 and 47 were
selected as representative molecules of the 4-methyl- and non-methylated series for Ames assays. Neither
analog showed mutagenicity in the Ames assays, with or without the addition of S9, highlighting the success of
the aforementioned stereoelectronic design principles.
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Table 5. KOR, MOR, DOR Antagonist Activity of Compounds 29, 46-50, 58-62
Compound
R
R1
IC50(nM)a
Selectivity
Calculated
Physicochemical
Properties50, 51
KORb MOR
DOR
4410
MOR/KOR
cLogP
tPSA
71
cpKa
29
46
3.0
3.6
79
26
1.6
2.2
10.5
10.5
H
H
1200 >10000
333
71
47
48
49
1.1
2.1
2.3
1400 >10000
1273
2952
2783
3.1
3.1
2.7
71
71
71
9.9
9.9
9.8
H
H
6200 >10000
6400 >10000
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50
58
59
H
2.4
0.8
6500 >10000
2708
138
2.7
2.4
3.3
71
71
71
9.8
10.5
9.9
Me
Me
110
100
6500
4500
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21
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33
34
35
36
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39
40
41
42
43
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.26
384
60
Me
0.53
400
>10000
754
3.3
71
9.9
61
62
Me
Me
0.78
0.88
1100 >10000
1400 >10000
1410
1591
2.9
2.9
71
71
9.8
9.8
a Values are reported as mean of n = 3 determinations.
bThis assay uses Tango ꢀꢁꢂꢃͳ-bla U2OS cells which express KOR linked to a GAL4-VP16 transcription
factor via a TEV protease site. Stimulation of the KOR by U-50488 causes migration of the β-arrestin fusion
protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. Assay protocols are
described in ref 37.
In a variety of in vitro pharmacokinetic assays, 47 and 58 exhibited low intrinsic clearance in human hepatic
microsomes and hepatocytes and moderate to high clearance in other species (Table 6). In vivo pharmacokinetic
studies in rats (Table 7) indicated that 58 distributed well into tissues (high apparent Vss of 13.8 L kg-1) and had
a high plasma clearance rate (105 mL min kg-1) after a 1 mg kg-1 i.v. dose. After a 5 mg kg-1 p.o. dose, 58
-1
exhibited plasma exposure levels out to 8 hours (39.1 ng mL-1 at Cmax and 232 h·ng mL-1 AUC0-t). The
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absolute oral bioavailability was 30%. The pharmacokinetic parameters observed in mice were similar. In rats,
58 distributed well into the CNS with a high brain to plasma ratio of 2.2:1 and adequate CSF exposure (~4 fold
its IC50) 1 hour after an oral administration of 5 mg kg-1 (Table 8).
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Table 6. Intrinsic Clearance (Clint) in Hepatic Microsomes and Hepatocytes for 47, 49, 58, 59, 61 and 62
Compd Physicochemical Properties
Microsomal Clint (mL/min/kg)
human rat mouse dog
Hepatocytes Clint (mL/min/kg)
human rat mouse dog
MW
439
tPSA
71
cLogP
3.1
47
12.0
22.7
ND
54.4
14.8
209
ND
ND
49
58
59
61
62
425
453
453
439
439
71
71
71
71
71
2.7
2.4
3.3
2.9
2.9
6.69
1.95
36.2
21.0
22.0
83.0 112.0 ND
17.5 49.1 55.2
ND
8.3
ND
118
ND
ND
ND
ND
ND
ND
ND
ND
ND
84.7
ND
ND
ND
57.5
54.6
89.5
ND
ND
ND
ND
ND
ND
ND
ND
39.5
ND = No data.
aDetails for the assays can be found in the supporting information. Compounds were profiled by Agilux.
Table 7. Pharmacokinetic Profile of Compound 58 in Rodentsa
i.v. (rat, 1 mg·kg-1; mice, 3 mg·kg-1)
p.o. (rat, 5 mg kg-1; mice, 10 mg·kg-1)
T ½ Cmax (ng AUC0-t (h·ng F
CL (mL·min-
1·kg-1)
66.5
T ½
(h)
AUC0-t (h·ng
mL-1)
Vss
(L·kg-1)
7.72
(h)
mL-1)
80.9
39.1
mL-1)
265
(%)
12
Mice
Rats
1.91
1.81
725
2.57
6.19
105
153
13.8
232
30.2
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(fed)
5
6
a 58 was dosed as the monotartrate salt.
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Table 8: CNS Exposure of 58 in Male Rats (n = 2, mean values)a
Plasma μM Brain μM CSF μM Brain:plasma
0.276 0.54 0.0031 2.2
58
p.o., 5 mg kg-1, 1h
(fasted)
a 58 was dosed as the monotartrate salt.
Having a few compounds in the series with promising in vitro and in vivo attributes in hand, we sought to assess
the potential for our KOR antagonists to elicit an on-target pharmacodynamic response in an in vivo model.
Prolactin (PRL) is a hormone involved in many biological processes that is mainly secreted by lactotroph cells
of the anterior pituitary gland. Prolactin is primarily regulated via tonic inhibition by dopamine released from
the tuberoinfundibular dopaminergic (TIDA) neurons.42,43 Activation of the opioid system, particularly of the
KOR believed to be localized on dopaminergic neurons within TIDA and in the mesolimbic-mesocortical
system modulates dopamine release.44,45,46 PRL overproduction by KOR agonists has been investigated in
clinical and preclinical models and its modulation has been postulated as a KOR-mediated translatable
biomarker,44,46-48 thus reduction of KOR agonist-induced plasma PRL in rats by KOR antagonists may be used
as an estimation of KOR antagonist target engagement. We selected this preclinical pharmacodynamic model as
a screen to guide the decision-making process for the further advancement of our KOR antagonists. For
practical reasons the rat PRL model was adapted to mice and leveraged to rapidly screen KOR antagonist
compounds. U69,593 was chosen as a reference KOR agonist and administered via intraperitoneal injection.
KOR antagonist compounds were initially assessed via intraperitoneal (i.p.) dosing at a single concentration,
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typically at 10 or 30 mg kg-1, and those demonstrating inhibition of PRL release were further evaluated at lower
concentrations. Compounds that showed robust i.p. PRL inhibition were tested orally (p.o.). Each KOR
antagonist was injected 1h prior to U69,593 in the i.p. experiments, while in the p.o. experiments the KOR
antagonists were administered by oral gavage 2h before U69,593. From these p.o. studies we found that at 30
and 10 mg kg-1 47 and 49 were effective in reducing plasma prolactin to basal levels but were ineffective at
lower doses; at 3 mg kg-1, 58 and 62 were effective in reducing U69,593-stimulated plasma prolactin secretion
to basal levels (Figure 3 and 4). Furthermore, 58 showed effective PRL reduction at 0.1 mg kg-1 after oral
administration (Figure 4).
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Figure 3. Inhibition of KOR Agonist Stimulated Prolactin Via p.o. Administration of 47, 49 and 62a,b
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a KOR antagonists were dosed as the monotartrate salts.
4
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6
b 3 mice per dose group where 2 samples were collected per mouse.
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Figure 4. Inhibition of KOR Agonist Stimulated Prolactin Via p.o. Administration of 58a, b
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a 58 was dosed as the monotartrate salt.
b 4 mice per dose group where 2 samples were collected per mouse.
From this set, 47, 49, 58 and 62 were tested in further pharmacokinetic and off-target assays (Table 9). 58 was
93.4% bound to human plasma proteins and 98.4% bound to rat brain homogenate. The other tested compounds
were protein bound to a similar extent in human plasma and in plasma across other species. Moderate solubility
was observed for this series of compounds (70-85 μM) and permeability was moderate to high for 58 with low
efflux ratio (7.1 and 20.9 10-6 cm/s in Caco-2 cells and MDCK-MDR1 cells, respectively; ER <2.3).
Furthermore, 58 showed minimal inhibition of CYP450 enzymes 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4 with
IC50 values of >30 μM and there was no change in the potency of 58 upon pre-incubation for any of the CYP
enzymes. 58 also was profiled in a very comprehensive panel of over 500 off-targets including ion channels,
kinases, GPCRs and other receptors and transporters and there were no hits identified with IC50 values <1μM.
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Few off-target hits were identified with IC50 values between 10 and 1 μM; in a patch clamp assay conducted a
physiological temperature, the compound inhibited the hERG channel with an IC50 of 1 μM.
7
8
9
Table 9. Additional ADMET Properties of 47, 49, 58, and 62a
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48
49
50
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58
59
60
47
92.4
ND
ND
49
58
62
97.3
ND
ND
Plasma protein binding
(% bound)
human
rat
94.8
94.3
96.3
93.4
91.5
91.5
dog
Binding in rat brain
ND
ND
98.4
ND
homogenate (2μM) (% bound)
MDCK-MDR1 cell line (P-gp)
A-B (10-6 cm/s)
Efflux ratio
8.76
1.5
13.5
0.896
ND
7.1
2.3
4.6
5.2
ND
ND
70
Caco-2 cell line
A-B (10-6 cm/s)
Efflux ratio
19.2
0.493
75
20.9
0.38
74
ND
Solubility (μM) at pH 7.4
84
a KOR-antagonists were used as monotartrate salts. ND = No data. A-B = movement of the compound from the
apical to the basal side of the membrane; a surrogate for permeability.
In a radioligand binding assay using [3H]diprenorphine, 58 exhibited a Ki of 1.5 nM at the human KOR and
300-fold selectivity over the human MOR (Ki = 450 nM).
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Based on these results, we decided to evaluate in vivo the duration of action of 58 in a hot water (50°C) tail-flick
test in male mice by assessing its ability to antagonize the antinociceptive effect of U-50488. 58 or vehicle were
administered to mice at 1 and 24 h prior to the administration of U-50488 (15 mg kg-1, i.p.) and latencies for
tail-flick were measured at baseline, 30 and 60 minutes after agonist injection (Figure 5). Baseline tail-flick
latency of naïve mice was 5.76 ± 0.16 s. Injection of U-50488 produced antinociception indicated by a
significant increase in tail-flick latency, which peaked at 30 min post-dose. Unlike nor-BNI22, 58 (1 mg kg-1,
i.p.) blocked the U-50488-induced antinociception at 1 h, but not at 24 h pretreatment time, indicating a
medication-like duration of action in blocking the KOR in vivo (Figure 5A-D). There was no observable change
in the rat tail-flick latency response induced by i.p. 58 (1 mg kg-1) alone at 1 h post-dose (Figure 5E, F).
Remarkably, oral administration of 58 (3, 10, and 30 mg kg-1) also blocked U-50488-induced antinociception in
a dose-dependent manner (Figure 5G, H).
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59
60
CONCLUSIONS
In summary, we have developed a novel chemical class of potent and selective KOR antagonists via
multiparametric optimization approach originating from a sub-micromolar HTS-hit 8. 29 showed favorable
pharmacokinetic properties including good CNS penetration. Remarkably, 29 was tested in a novel injury-free
rat model of migraine and was found to ameliorate stress-induced allodynia, thus providing support for the
hypothesis that KOR antagonists could have a therapeutic role in the preventive treatment of migraine. 29 and
58 displayed reversibility of analgesic effects induced by a KOR-agonist within 24h in a tail-flick assay in mice
indicating medication-like duration of action of their KOR antagonist activity in vivo. The pharmacokinetic
attributes of 58, including its CNS exposure provide confidence in the drug-like properties and developability of
the compound. A low oral dose of 58 to mice effectively reduced the elevation of U69,593-induced plasma
PRL. 58 did not exhibit significant inhibition of the major cytochrome P450 isoforms and was highly selective
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for KOR against a broad off-target panel. 58 was advanced into Phase 1 clinical trials49 based on its overall in
vitro and in vivo properties across nonclinical species as well as its safety profile.
6
7
8
9
Figure 5. Blockade of Antinociceptive Response by 58 in the Mouse Tail-Flick Assay
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Figure 5. KOR blockade by 58 in the mouse tail-flick assay. A, B. Systemic injection of U50488 (15 mg kg-
1, i.p.) induced robust antinociception within 30 min in the mouse tail-flick test (50°C water bath), which was
blocked by 58 (1 mg kg-1, i.p.) given at 1 h prior to U50488. C, D. 58 (1 mg kg-1, i.p.) given at 24 h prior to
U50488 (15 mg kg-1, i.p.) failed to block U50488-induced antinociception assessed at 30 min post-U50,488. E,
F. 58 (1 mg kg-1, i.p.) alone did not alter tail flick latency at 60 min post-dose. G, H. Oral 58 (3, 10, 30 mg kg-1,
p.o.) given at 1 h prior to U50488 (15 mg kg-1, i.p.) dose-dependently blocked U50488-induced antinociception
assessed at 30 min post-U50488. Data represent mean ± SEM. %Maximum Possible Effect (MPE) = (Post dose
latency – Pre dose latency)/(Cutoff – Pre dose latency) x 100. Cutoff = 15 sec to prevent tissue damage.
Statistics performed using two-way ANOVA followed by Sidak's multiple comparisons post-hoc test (A-F) or
linear regression (G, H). *P<0.05 vs. baseline (BL) of the corresponding treatment group. Group sizes are
shown in the figure. 58 was dosed as the monotartrate salt.
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Acknowledgments: We thank Ronald B. Franklin and Paul C. Anderson for insightful scientific consultation
during the course of the project and for their help in preparing the manuscript. This work was supported by the
National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) and
BlackThorn Therapeutics. The grant is funded as part of the Blueprint Neurotherapeutics Network (BPN) of the
NIH Blueprint for Neuroscience Research grant 1UH2 NS093030-01 (Edward Roberts, Hugh Rosen, Frank
Porreca).
EXPERIMENTAL SECTION
Methods and Materials
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Page 29 of 67
Journal of Medicinal Chemistry
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Plasma protein binding
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Plasma protein binding of compounds 29, 34, 35 and 36 to human, mouse, and rat plasma was evaluated using
equilibrium dialysis (Pierce RED system). Compound (1.0 μM) was added to the plasma compartment and after
eight hours the concentration of drug was measured in the plasma and buffer compartments by LC-MS/MS. The
concentration in the buffer compartment is considered to be the free fraction of compound.
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Plasma protein binding of compounds 47, 49, 58 and 62 were performed by Agilux Laboratories (now Charles
River Laboratories) (Worcester, MA) (Assay protocols are described in supporting information).
Hepatic microsome stability
Test compounds (27, 29, 33, 34, 35 and 36) (1 μM) were incubated (separately) with 0.2 mg mL-1 pooled
human, mouse and rat hepatic microsomes at 37 °C with continuous shaking to determine the rate of
metabolism. Samples were collected at 0, 5, 10, 20, 40, and 60 min and acetonitrile was added to quench the
reactions and precipitate the proteins. Samples were then centrifuged and the concentration of test compound
was determined using HPLC coupled to a triple quadrupole mass spectrometer (LC/MS-MS).
Microsomal hepatic stability of compounds 47, 49, 58, 59, 61 and 62 and hepatic stability of 47 and 58 were
performed by Agilux Laboratories (now Charles River Laboratories) (Worcester, MA) (Assay protocols are
described in supporting information).
Cytochrome P450 inhibition
The metabolism by CYP1A2 (phenaceten demethylated to acetaminophen), CYP2C9 (tolbutamide
hydroxylated to hydroxytolbutamide), CYP2D6 (bufuralol hydroxylated to 4'-hydroxybufuralol), and CYP3A4
(midazolam hydroxylated to 1'-hydroxymidazolam) in the presence or absence of 10 μM test compound (29,
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