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Brief Article
Discovery of b-Arrestin Biased, Orally Bioavailable and CNS Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators Anthony B Pinkerton, Satyamaheshwar Peddibhotla, Fusayo Yamamoto, Lauren Slosky, Yushi Bai, Patrick R. Maloney, Paul Hershberger, Michael P Hedrick, Bekhi Falter, Robert J Ardecky, Layton H. Smith, Thomas DY Chung, Michael Jackson, Marc G. Caron, and Lawrence S. Barak J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00340 • Publication Date (Web): 07 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019
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Journal of Medicinal Chemistry
Discovery of -Arrestin Biased, Orally Bioavailable and CNS Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators Anthony B. Pinkerton*,a Satyamaheshwar Peddibhotla,a Fusayo Yamamoto,a Lauren M. Slosky,b Yushi Bai,b Patrick Maloney,a Paul Hershberger,a Michael P. Hedrick,a Bekhi Falter,a Robert J. Ardecky,a Layton H. Smith,a Thomas D. Y. Chung,a Michael R. Jackson,a Marc G. Caron,b and Lawrence S. Barakb aConrad
Prebys Center for Chemical Genomics, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California 92037, United States bDuke
University Medical Center, Durham, North Carolina 27709, United States
ABSTRACT: Neurotensin receptor 1 (NTR1) is a G protein coupled receptor that is widely expressed throughout the central nervous system where it acts as a neuromodulator. Neurotensin receptors have been implicated in a wide variety of CNS disorders but despite extensive efforts to develop small molecule ligands there are few reports of such compounds. Herein we describe the optimization of a quinazoline based lead to give 18 (SBI-553), a potent and brain penetrant NTR1 allosteric modulator.
INTRODUCTION The tridecapeptide neurotransmitter neurotensin (NT)1 is widely distributed throughout the central and peripheral nervous systems2 where it acts as a neurotransmitter and neuromodulator, in particular for dopamine (DA) signaling.3 Neurotensin binds two G-protein-coupled receptors, NTR1 and NTR2,4,5 and neurotensin has been implicated in numerous CNS disorders such as schizophrenia,6 Parkinson’s disease7 and drug addiction.8 NTR1 is the most widely studied, mediates most of the known neurotensin effects, and holds the potential as an interesting therapeutic target. 9 In addition, two crystal structures of NTR1 have been recently reported.10,11 Despite the therapeutic promise of NTR1, it has proven to be difficult to develop ligands for the receptor. While there have been numerous reports of peptide agonists of NTR1,12,13 which in general suffer from poor oral bioavailability and CNS penetration, only a handful of small molecule antagonists and agonists (Figure 1) have been described. The most advanced compounds include the nM antagonists from Sanofi SR48692 (Meclinertant),14 which completed PhII clinical trials, and the related analog SR142948A.15 Positive modulators include subM compounds from RTI,16 which was derived from the SR compounds, and the related imidazole ML301.17 In addition, a weakly active indole based partial agonist from Wyeth18 and an optimized full agonist analog from Scripps (SR-12062) have been reported.19 We have reported on a series of -arrestin biased quinazoline positive modulators of NTR, exemplified by our probe compound ML314 (Figure 2).20 While ML314 was moderately potent, displayed good brain penetration after IP dosing and was active in vivo in a number of animal models of
addiction,21 it displayed low oral bioavailability (80 M) counterscreen.
With this result in hand, we then re-examined the SAR around the core (Table 2). Similar to the results with the piperazine linked compound 8, a dimethylamine group in the 6 position (20a) was reasonably potent. Increasing the size to an ethyl group improved activity (20b), although there were no further gains in potency going to the propyl analog (20d). Similar to previous SAR, the 6 position was preferred, with substitution in the 5 (compound 20c) or 7 (compound 20e) giving substantially less potent analogs. Adding an additional methyl (compound 20f) or fluoro (compound 20g) in the 7 position gave compounds of lower potency. A range of analogs that were substituted with a methoxy (20h), hydroxy (20i) or morpholino (20j) group showed that all modifications were tolerated, with hydroxy compound 20i being the most potent. Piperdine (20k,20l) and pyrrolidine (20m-p) substitution also gave compounds that showed good activity.
Figure 2: Quinazoline NTR1 modulators
Table 2. Structures and NTR1 activities of analogs 20 Scheme 1. Representative synthesis of quinazoline compounds Reagents and conditions: (a) (COCl)2, 35 oC, 1.5h; (b) Pyridine, DCM, rt, 2h 86% over 2 steps; (c) NaOH, H2O2, EtOH, reflux, 12 h, 66%; (d) K3PO4, CuI, proline, DMSO, 100 oC, 12h; (e) NaBH(OAc)3, HCHO, MeOH, rt, 1h, 39% over 2 steps; (f) BOP, DBU, CH3CN, rt, 12h, 87% a
SAR. We have previously reported the preliminary SAR around the quinazoline scaffold to give the probe compound 7, ML314.20 However, ML314, while selective, displays only moderate potency (~2.8 M) and modest pharmacokinetics, with low oral bioavailability (100 analogs synthesized (data not shown) only one, the dimethylamine analog 8 displayed activity higher than the parent compound (NTR1 EC50= 0.71 M). During the course of our preliminary investigation we also examined the piperazine linker; only 6 membered rings gave active compounds, and there was strong preference for a nitrogen-linkage to the quinazoline core. As shown in Table 1, piperdine 19b was approximately three fold more potent than the piperazine, while 19a, with the reverse linkage, was not active. Table 1. Structures and NTR1 activities of analogs 19
Cpd # 7 (ML314) 19a 19b
X N CH N
Y N N CH
EC50, µMa 2.87 1.43 (171) >20 1.04 0.14
Emax (%)b 100 -89
Cpd # 19b
5
6
7
EC50, µMa
-H
-OCH3
-OCH3
20a
-H
-N(CH3)2
-H
1.04 0.14 0.888 0.11 (3)
20b
-H
20c
-H
Emax (%)b 89 89 80
-H
0.56 0.13
-H
6.71 1.54 (3)
60
-H
0.732 0.12
68
10.02 3.95
86
20d
-H
20e
-H
20f
-H
-CH3
0.99 0.44 (8)
86
20g
-H
-F
3.01 0.18 (6)
68
20h
-H
-H
1.12 0.13
20i
-H
-H
0.478 0.08
20j
-H
-H
0.81 0.10
20k
-H
-H
6.72 1.59
20l
-H
-H
2.78 0.57
20m
-H
-H
0.81 0.25 (8)
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-H
79 92 92 82 79 82
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Journal of Medicinal Chemistry
20n
-H
-H
0.64 0.29 (12)
20o
-H
-H
1.01 0.23 (8)
20p
-H
-H
0.44 0.16 (8)
93
78
79
a-Arrestin2-GFP
NTR1 potency measured relative to the EC100 (100 nM) of the NT(8-13) peptide control average SEM (n=4 unless otherwise noted); bEmax was calculated as the % of the response obtained with NT(8-13) peptide. None of the compounds from this series showed activity in the NTR2 (>80 M) counterscreen.
We then turned to examining the effect of substitution on the cyclopropyl ring off the quinazoline core (Table 3). Substitution at the 2 and 3 positions of the cyclopropyl ring were generally poorly tolerated (data not shown); however substitution at the 1 position was tolerated and also improved the PK properties (vide infra). While in some cases potency was not improved (for example 21a compared to 20d or 21e to 20a), the combination of the hydroxyethyl group with either a fluorine (18, SBI-553) or methyl group (21e) gave up to a five fold increase in potency. Interestingly the corresponding analogs with either a methoxyethyl (21c, 21d) or morpholinoethyl (21g-i) did not show a similar potency boost.
of the compounds from this series showed activity in the NTR2 (>80 M) counterscreen.
We also investigated a range of other groups in place of the cyclopropyl group (Table 4). Cyclobutyl (22a) was well tolerated, but potency dropped off when the ring size was increased to cylcopentyl (22b). A phenyl substituent (22c) was also less active. As with the cyclopropyl compounds 18 and 21e, substitution of the cyclobutyl group in the 1 position with either F (22d) or methyl (22e) gave potent compounds, although activity was not substantially improved in the case of methyl analog 22e, which is in contrast with the corresponding analog 21e. Table 4. Structures and NTR1 activities of analogs 22
Cpd #
EC50, µMa
Emax (%)b 80
22a
1.05 0.32
22b
3.88 1.12 (6)
78
4.23 0.88
70 96
22c
Table 3. Structures and NTR1 activities of analogs 21
R1
Ph-
22d
0.27 0.05
22e
1.48 0.38
75
a-Arrestin2-GFP
Cpd #
R1
21a 21b 21c
-N(CH3)2
R2
EC50, µMa
-F
1.45 0.31 (6)
-F
0.92 0.28 (6)
-CH3
2.23 0.37
Emax (%)b 80 84 60 86
21d
-F
1.06 0.08
18 (SBI-553)
-F
0.34 0.21 (99)
100
21e
-CH3
0.098 0.01
90
21f
-CF3
0.34 0.21 (8)
94
21g
-F
1.72 0.13
97
71 21h
-CH3
3.36 0.28
21i
-CF3
3.81 1.57 (8)
a-Arrestin2-GFP
78
NTR1 potency measured relative to the EC100 (100 nM) of the NT(8-13) peptide control average SEM (n=4 unless otherwise noted); bEmax was calculated as the % of the response obtained with NT(8-13) peptide. None
NTR1 potency measured relative to the EC100 (100 nM) of the NT(8-13) peptide control average SEM (n=4 unless otherwise noted); bEmax was calculated as the % of the response obtained with NT(8-13) peptide. None of the compounds from this series showed activity in the NTR2 (>80 M) counterscreen.
Lastly, we examined substitution of the quinoline core as outlined in Table 5. Compared with the unsubstituted analog (20i) we observed only modest variations in potency with F, Cl or methyl substitution, with the 8 position being preferred for substitution (compare 23b, 23e and 23h). Table 5. Structures and NTR1 activities of analogs 23
Cpd # 23a 23b 23c 23d 23e 23f 23g 23h a-Arrestin2-GFP
R1
EC50, µMa
Emax (%)b
5-F 8-F 5-Me 7-Me 8-Me 5-Cl 7-Cl 8-Cl
0.59 0.08 (8) 0.16 0.04 (8) 1.26 0.36 (8) 0.56 0.11 0.16 0.04 (8) 0.50 0.16 0.60 0.24 (23) 0.22 0.14
95 100 93 88 87 91 98 95
NTR1 potency measured relative to the EC100 (100 nM) of the NT(8-13) peptide control average SEM (n=4 unless otherwise noted); bEmax was calculated as the % of the response obtained with NT(8-13) peptide. None
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Journal of Medicinal Chemistry
Table 6. Selected mouse or rat PK parameters for compounds 18 and 21e Compd Species sa
18 Mouse (SBI553)
Clpb
Vdb
Cmaxc
AUCc t1/2c %F Brain:Plasma (ng.hr/mL) @1 hrc (mL/min/kg) (L/kg) (ng/mL) (hr) 44.8
6.16
1460
4824
5.28 45
0.54
21e
Mouse
79.4
7.68
627
3324
2.90 52
0.35
18 (SBI553)
Rat
81.0
7.02
3482
2693
2.23 48
0.98
Compounds dosed 5 mpk iv and 30 mpk po, biv, cpo
a
In vitro profiling. This series of compounds display a unique profile in that they are allosteric modulators of NTR1 that selectively enhance the interaction with -arrestin while blocking the classical Ca2+ signaling. Extensive in vitro and in vivo characterization, including the consequences of this biased signaling, will be reported elsewhere.24 Similar to ML314, 18 (SBI-553) alone showed no activity in a Ca2+ flux assay.20 Also, as observed with ML314, 18 (SBI-553) appears to act as a positive allosteric modulator of neurotensin peptide binding. 18 (SBI-553) was evaluated by Eurofins Panlabs Discovery Services25 in a radioligand binding assay that examined the binding of radiolabeled neurotensin (NT) to membranes prepared from cells overexpressing human NTR1 or NTR2, or mouse NTR. Unlike orthosteric ligands, which displace neurotensin, significant enhancement of neurotensin peptide binding to the human NTR1 and mouse NTR1, but not human NTR2, was observed in the presence of increasing concentrations of 18 (SBI-553) (Figure 3), indicating both the selectivity of the compound as well as confirming its unique mechanism of action. E n h a n c e m e n t o f N e u r o t e n s in B in d in g in t h e P r e s e n c e o f S B I- 5 5 3 350
The presence of 18 (SBI-553) had no effect on the potency of NT in the NTR1 high-content (β-arrestin2-GFP) assay (Figure 4A). EC50 values for NT in this assay ranged from 0.37 to 0.40 nM across the range of 18 (SBI-553) doses tested (0.5 – 356 nM), while the EC50 for NT in the absence of 18 (SBI-553) was 0.40 nM. In contrast, the EC50 for NT in the Ca2+ flux assay (Figure 4B) was shifted to the right over the tested concentration range of 18 (SBI-553), 0.0088-2.15 µM, but did not quite reach saturation as indicated by the separation of the response curves at higher concentrations. In the absence of 18 (SBI-553), the EC50 for NT in this assay was 0.035 nM, while the EC50 for the NT was shifted >30-fold (1.18 nM) in the presence of 2.15 µM 18 (SBI-553). A Schild analysis of these results (Figure S1) supports the notion that 18 (SBI-553) acts functionally similar to a competitive antagonist over this range. Thus, from Figures 3,4, and S1, 18 (SBI-553) can be considered a β-arrestin-biased modulator and acts functionally like a “PAM antagonist” of the Ca2+ pathway with respect to its effects on NT/NTR1 signaling. A: NTR1 -Arrestin2-GFP Assay R a ti o T o t. S p o tI n t e n s i ty :C e l l In t e n s i ty
Pharmacokinetics. We examined a range of analogs for their rodent PK properties, with the results for the fluoro-cyclopropyl (18, SBI-553) and methyl-cyclopropyl (21e) analogs shown in Table 6. Both analogs showed high clearance but also a high volume of distribution in both mouse and rat with good oral bioavailability (~50%) and half life in both species. In addition, the brain:plasma ratio 1 hour post dose was good for SBI-553 in both mouse (0.54) and rat (0.98). Similar brain:plasma ratios in rat were observed 4 and 8 hours post dose (PO, data not shown). Due to the combination of potency, bioavailability and brain penetration, 18 (SBI-553) was selected for further in vitro and in vivo pharmacological profiling.
doses of 18 (SBI-553). The EC50 for SBI-553 at the human receptor for this experiment was 0.14 μM, with a maximum efficacy of 140% (Data were plotted and analyzed using GraphPad Prism V.7, n=2). Efficacy is relative to the maximal displacement of orthosteric ligands (i.e. -100%). Data point excluded from curve fitting is shown in red. NT peptide in these assays give Kds of 0.082 nM (hNTR1), 0.59 nM (hNTR2) and 0.17 nM (mNTR) respectively.
[S B I- 5 5 3 ] , n M 0 .5
3 5 5 .6 1 1 8 .5 3 3 3
0 .4
3 9 .5 1 1 1 1
0 .3
1 3 .1 7 0 3 7 0 .2
4 .3 9 0 1 2 3 1 .4 6 3 3 7 4
0 .1
0 .4 8 7 7 9 1 0 .0
0 0 .0 0 1
0 .0 1
0 .1
1
10
[N T p e p tid e ], n M
B: NTR1 Ca2+ Flux Assay 2 .8
[S B I-5 5 3 ], u M
2 .1 5
2 .4
M a x R a tio
of the compounds from this series showed activity in the NTR2 (>80 M) counterscreen.
% E n h a n c e m e n t o f lig a n d b i n d in g
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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0 .7 1 6 6 6 7 0 .2 3 8 8 8 9
2 .0
0 .0 7 9 6 3 1 .6
0 .0 2 6 5 4 3 0 .0 0 8 8 4 8
1 .2
0
0 .8 0 .0 0 1
0 .0 1
0 .1
1
10
[ N T p e p tid e ], n M
Figure 4. Dose response of NT peptide in the presence of 18 (SBI-553) in the -Arrestin (A) and Ca2+ flux (B) assays. Data were plotted and analyzed using GraphPad Prism V.7 and are presented as an average SEM (n=4). The compound shows divergent behavior, with no effect on the EC50 in the -Arrestin assay but a strong rightward shift in the Ca2+ flux assay.
N e u r o t e n s in N T 1 ( h u m a n r e c e p t o r )
250
N e u r o t e n s in N T 2 ( h u m a n r e c e p t o r ) N e u r o t e n s in , n o n - s e le c t iv e ( m o u s e r e c e p t o r )
150
50
-5 0 0 .0 0 1
0 .0 1
0 .1
1
[ ], u M
Figure 3. Augmentation of [125I]-NT binding (0.02 nM) to cell membranes in the presence of increasing
As with ML314, 18 (SBI-553) showed no cross reactivity against a range of related and unrelated GPCRs using the same high-content assay format (NTR2, GPR35, GPR55, -opioid). 18 (SBI-553) was also screened in a Eurofins/Panlabs panel of 80 targets and showed moderate promiscuity with binding (>30%) against 10 receptors at 10 M concentration. Subsequent dose titrations indicated binding at the adrenergic
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Journal of Medicinal Chemistry 2a (55% at 5 M), dopamine D1 (36% at 5 M), histamine H1 (75% at 5 M) and 5-HT2B (42% at 5 M) receptors. In vivo efficacy. We then examined the effect of 18 (SBI-553) in vivo, after both IP (Figure 5, Panels A and B) and PO (Panels C and D, Figure 5) in the dopamine transporter knockout mouse model model of hyperdopaminergic activity.26 Unlike the peptide NTR1 agonist PD149163, which causes significant hypotension and hypothermia, animals dosed with 18 did not show any adverse effects. In addition, 18 attenuated hyperlocomotion after both IP and PO dosing. Full in vivo profiling of 18 will be reported elsewhere. 24
Figure 5: 18 (SBI-553) attenuates basal hyperlocomotion in dopamine transporter knockout (DAT-/-) mice. Locomotor activity was evaluated using open field automated activity monitors (AccuScan Instruments, Columbus, OH). Male and female DAT-/- mice were acclimated to the open field chamber for 30 min prior to administration of NTR1 ligands (indicated by arrow). After treatment, animals were immediately returned to chambers and horizontal locomotion was monitored over the next 2 hrs. (A) Animals received SBI-553 (12 mg/kg), PD149163 (1 mg/kg) or vehicle (4% DMSO in saline; 10 ml/kg) i.p. Beam breaks in 5 min intervals were recorded to generate horizontal distance traveled per time bin. *p
