Discovery of β-Arrestin Biased, Orally Bioavailable, and CNS Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators

Article abstract of DOI:10.1021/acs.jmedchem.9b00340

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.

Full text of DOI:10.1021/acs.jmedchem.9b00340

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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
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Discovery of -Arrestin Biased, Orally Bioavailable and CNS
Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators
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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
addiction,21 it displayed low oral bioavailability (<5%). We
therefore embarked on an optimization campaign to improve
both potency and oral bioavailability. Herein we disclose the
discovery of SBI-553, an optimized derivative with a 10 fold
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 sub-
M 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
potency
improvement
and
significantly
improved
bioavailability that maintains good CNS penetration.
Figure 1: Selected previously reported NTR1 ligands
RESULTS AND DISCUSSION
Chemistry. The compounds described in this paper consist
of a quinazoline core structure with an N-linked piperdine or
piperazine in the 4 position. The synthesis of most analogs
followed our previously described routes20,22 (for full synthetic
details see Supporting information). A representative synthesis
is shown in Scheme 1. Starting from an appropriate substituted
carboxylic acid (9), conversion to the acid chloride followed by
reaction with a 2-cyanoaniline (11) gave intermediate 12.
Cyclization under basic conditions gave quinazoline 13. The
amino side chain 14 was introduced by a copper catalyzed
coupling reaction to give intermediate 15, which was
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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); Emax
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.
methylated using reductive alkylation to give 16. 16 was
coupled with the substituted piperdine 17 to provide the final
product 18.
b
1
2
3
4
5
6
7
8
9
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
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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48
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50
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55
56
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60
Table 2. Structures and NTR1 activities of analogs 20
a
Scheme 1. Representative synthesis of quinazoline compounds Reagents
o
and conditions: (a) (COCl)2, 35 C, 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%
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 (<5%). We therefore embarked on
an SAR campaign to improve both of these parameters. For our
primary SAR driving assay we used a high content assay
measuring NT receptor mediated -arrestin redistribution as
previously reported.23 The primary counterscreen was an assay
for NTR2 in the same format. Our initial SAR around the
ML314 scaffold investigated a wide range of substituents
around the quinazoline ring as well as around the pendant
phenyl ring on the piperazine. Of the >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.
Cpd
#
Emax
(%)b
5
6
7
EC50, µMa
89
89
19b
-H
-H
-OCH3
-N(CH3)2
-OCH3
-H
1.04  0.14
0.888 
0.11 (3)
20a
80
20b
20c
20d
-H
-H
-H
-H
0.56  0.13
60
68
6.71  1.54
-H
-H
(3)
0.732 
0.12
-H
-H
-H
86
86
10.02 
3.95
20e
20f
0.99  0.44
-CH3
(8)
68
3.01  0.18
20g
20h
20i
-H
-H
-H
-F
-H
-H
(6)
79
92
92
1.12  0.13
0.478 
0.08
Table 1. Structures and NTR1 activities of analogs 19
20j
20k
20l
-H
-H
-H
-H
-H
-H
0.81  0.10
6.72  1.59
2.78  0.57
82
79
82
Cpd #
7 (ML314)
19a
X
N
CH
N
Y
N
N
EC50, µMa
2.87  1.43 (171)
>20
Emax (%)b
0.81  0.25
20m
-H
-H
100
--
89
(8)
19b
CH
1.04  0.14
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of the compounds from this series showed activity in the NTR2 (>80 M)
93
78
79
0.64  0.29
counterscreen.
1
2
3
4
5
6
7
8
20n
20o
20p
-H
-H
-H
-H
-H
-H
(12)
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.
1.01  0.23
(8)
0.44  0.16
(8)
a-Arrestin2-GFP NTR1 potency measured relative to the EC100 (100 nM) of the
9
b
NT(8-13) peptide control average  SEM (n=4 unless otherwise noted); Emax
10
11
12
13
14
15
16
17
18
19
20
21
22
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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.
Table 4. Structures and NTR1 activities of analogs 22
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.
Cpd #
22a
R1
EC50, µMa
Emax (%)b
80
1.05  0.32
Interestingly the corresponding analogs with either
a
methoxyethyl (21c, 21d) or morpholinoethyl (21g-i) did not
show a similar potency boost.
78
3.88  1.12
22b
22c
22d
(6)
Ph-
70
96
4.23  0.88
0.27  0.05
Table 3. Structures and NTR1 activities of analogs 21
75
22e
1.48  0.38
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); Emax
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.
b
Emax
Cpd #
21a
R1
R2
-F
EC50, µMa
(%)b
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).
80
1.45  0.31
(6)
84
60
0.92  0.28
21b
-N(CH3)2
-F
(6)
21c
-CH3
2.23  0.37
1.06  0.08
Table 5. Structures and NTR1 activities of analogs 23
86
21d
-F
-F
100
18
0.34  0.21
(SBI-553)
(99)
90
94
97
0.098 
21e
21f
-CH3
-CF3
0.01
0.34  0.21
(8)
Cpd
#
R1
EC50, µMa
Emax (%)b
21g
21h
21i
-F
1.72  0.13
3.36  0.28
95
100
93
88
87
91
98
95
23a
23b
23c
23d
23e
23f
23g
23h
5-F
8-F
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
71
78
-CH3
-CF3
5-Me
7-Me
8-Me
5-Cl
7-Cl
8-Cl
3.81  1.57
(8)
0.60  0.24 (23)
0.22  0.14
a-Arrestin2-GFP NTR1 potency measured relative to the EC100 (100 nM) of the
a-Arrestin2-GFP NTR1 potency measured relative to the EC100 (100 nM) of the
b
NT(8-13) peptide control average  SEM (n=4 unless otherwise noted); Emax
b
NT(8-13) peptide control average  SEM (n=4 unless otherwise noted); Emax
was calculated as the % of the response obtained with NT(8-13) peptide. None
was calculated as the % of the response obtained with NT(8-13) peptide. None
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of the compounds from this series showed activity in the NTR2 (>80 M)
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.
counterscreen.
1
2
3
4
5
6
7
8
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.
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.
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
Table 6. Selected mouse or rat PK parameters for compounds
18 and 21e
b
c
c
Compd Species
sa
Clp
Vdb
Cmax
AUCc
(ng.hr/mL)
t1/2 %F Brain:Plasma
@1 hrc
(hr)
(mL/min/kg) (L/kg) (ng/mL)
18
Mouse
44.8
6.16
1460
4824
5.28 45
0.54
(SBI-
553)
21e Mouse
79.4
81.0
7.68
7.02
627
3324
2693
2.90 52
2.23 48
0.35
0.98
18
Rat
3482
A: NTR1 -Arrestin2-GFP Assay
(SBI-
553)
[S B I-5 5 3 ],n M
0 .5
aCompounds dosed 5 mpk iv and 30 mpk po, biv, cpo
3 5 5 .6
0 .4
0 .3
0 .2
0 .1
0 .0
1 1 8 .5 3 3 3
3 9 .5 1 1 1 1
1 3 .1 7 0 3 7
4 .3 9 0 1 2 3
1 .4 6 3 3 7 4
0 .4 8 7 7 9 1
0
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
0 .0 0 1 0 .0 1
0 .1
1
1 0
[N T p e p tid e ], n M
B: NTR1 Ca2+ Flux Assay
2 .8
2 .4
2 .0
1 .6
1 .2
0 .8
[S B I-553], uM
2 .1 5
0 .7 1 6 6 6 7
0 .2 3 8 8 8 9
0 .0 7 9 6 3
0 .0 2 6 5 4 3
0 .0 0 8 8 4 8
0
0 .0 0 1
0 .0 1
0 .1
1
1 0
[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.
mechanism
of
action.
E n h a n c e m e n t o f N e u ro te n s in B in d in g
in th e P re s e n c e o f S B I-5 5 3
3 5 0
2 5 0
1 5 0
5 0
N e u ro te n s in
N e u ro te n s in
N
N
T 1 (h u m a n r e c e p to r )
T 2 (h u m a n r e c e p to r )
N
e u ro te n s in , n o n -s e le c tiv e (m o u s e re c e p to r)
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
-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
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the implications and benefits of this biased signaling in vitro
2a (55% at 5 M), dopamine D1 (36% at 5 M), histamine H1

1
2
3
4
5
6
7
8
and in vivo, including important differences in their toxicity
profiles relative to standard neurotensin ligands,24 and a
structural basis28 for their mechanism of action will be
published elsewhere.
(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
EXPERIMENTAL SECTION
All compounds were purified to >95% as determined by LC/MS
1
as well as H (and 13C in select cases) NMR and HRMS. The
9
analytical methods,
general chemistry, experimental
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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information, and syntheses of all other compounds are supplied
in the Supporting Information.
Synthesis of 2-({2-(1-Fluoro-cyclopropyl)-4-[4-(2-methoxy-
phenyl)-piperidin-1-yl]-quinazolin-6-yl}-methyl-amino)-
ethanol (18): Steps a,b: To
a solution of 1-fluoro-
cyclopropanecarboxylic acid (Oakwood Products, West
Columbia, SC, 9, 50.8 g, 0.49 mol) in methylene chloride (500
mL), was added oxalyl dichloride (74.7 g, 0.59 mol) and DMF
o
(0.5 mL). The mixture was stirred at 35 C for 1.5 h. The
resulting solution was added slowly to a solution of 2-amino-5-
bromo-benzonitrile (Sigma Aldrich, St. Louis, MO, 11, 96 g,
0.49 mol) in methylene chloride (1 L) and pyridine (300 mL).
The reaction was stirred at room temperature for 2 h, then was
washed with water (1 L), 1N HCl (1 L) and saturated NaHCO3
solution (1 L), dried over Na2SO4 and filtered. The filtrate was
concentrated and the residue was triturated with MeOH to give
1-fluoro-cyclopropanecarboxylic acid (4-bromo-2-cyano-
phenyl)-amide (12, 78 g).22 The mother liquid was concentrated
and purified by silica gel column (petroleum ether/ethyl acetate
= 10/1) to give additional 1-fluoro-cyclopropanecarboxylic acid
(4-bromo-2-cyano-phenyl)-amide (12, 41 g, total yield: 86%) as
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<0.05-p<0.001 Vehicle vs. SBI-553; #p<0.05-p<0.001
Vehicle vs. PD149163; ~p<0.05-p<0.001 SBI-553 vs. PD149163, using a two-
way, repeated measures ANOVA (FTime(23, 1679)=13.5, p<0.0001; FTreatment(2,
73)=45.1, p<0.0001; FInteraction(46, 1679)=10.4, p<0.0001) followed by the post
hoc Tukey test. (B) Time course data in (A) was summed to determine the total
distance traveled in the 2 hr post-treatment. ***,###,~~~p<0.001, as indicated,
using one-way ANOVA (F(2, 72)=46.6, p<0.0001) followed by the post hoc
Tukey test. Data are represented as mean ± SEM of 18-29 animals per group. (C)
Animals received SBI-553 10, 30 or 100 mg/kg or vehicle (distilled H2O) p.o.
Beam breaks in 5 min intervals were recorded to generate horizontal distance
traveled per time bin. *p<0.05-p<0.001 Vehicle vs. 100 mg/kg SBI-553; #p<0.05
Vehicle vs. 30 mg/kg SBI-553; ~p<0.05 10 mg/kg SBI-553 vs. 100 mg/kg SBI-
553, using a two-way, repeated measures ANOVA (FTime(23, 989)=8.6,
p<0.0001; FTreatment(3, 43)=6.8, p<0.001; FInteraction(69, 989)=1.1, p=0.2726)
followed by the post hoc Tukey test. (D) Time course data in (C) was summed
to determine the total distance traveled in the 2 hr post-treatment. **p<0.01,
#p<0.05,~p<0.05, as indicated, using one-way ANOVA (F(3, 43)=6.8, p<0.001)
followed by the post hoc Tukey test. Data are represented as mean ± SEM of 11-
12 animals per group.
a
white solid. Step c: To a suspension of 1-fluoro-
cyclopropanecarboxylic acid (4-bromo-2-cyano-phenyl)-amide
(12, 78 g, 276 mmol) in EtOH (1.6 L) and H2O2 (30%, 300 mL),
was added NaOH (13.23 g, 0.33 mol). The mixture was stirred
o
at 85 C overnight. The reaction was worked up together with
another batch (using 1-fluoro-cyclopropanecarboxylic acid (4-
bromo-2-cyano-phenyl)-amide, (12, 41 g, 145 mmol), EtOH,
850 mL, H2O2 (30%, 150 mL) and NaOH (7 g, 0.17 mol)). The
combined reaction solution was concentrated to 800 mL and
poured into cold water (1.2 L). The precipitate was collected by
filtration to give 6-bromo-2-(1-fluoro-cyclopropyl)-quinazolin-
4-ol (13, 39 g).22 The filtrate was treated with excess Na2SO4
and extracted with ethyl acetate (0.6 L x2). The organic layer
was concentrated to 200 mL and filtered to remove Na2SO4. The
filtrate was taken up with ethyl acetate (100 mL) and stored at
room temperature for 2 days. The solid that was formed was
collected by filtration, the cake was washed with H2O, and dried
to give 6-bromo-2-(1-fluoro-cyclopropyl)-quinazolin-4-ol (13,
40 g of recrystallized product, total of 79 g, total yield: 66%)22
1
as a light yellow solid. H NMR (300 MHz, DMSO-d6): δ =
12.71 (brs, 1H), 8.17 (d, J = 2.4 Hz, 1H), 7.92 (dd, J = 8.7, 2.1
Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 1.60-1.48 (m, 4H). Steps d,e:
A mixture of 6-bromo-2-(1-fluoro-cyclopropyl)-quinazolin-4-
ol (13, 67 g, 237 mmol), 2-amino-ethanol (14, 21.6 g, 355
mmol), CuI (4.5 g, 23.7 mmol), L-proline (2.73 g, 23.7 mmol)
and K3PO4 (100 g, 474 mmol) in DMSO was stirred at 100 oC
under N2 overnight. The reaction was concentrated to give
CONCLUSION
In conclusion we have optimized a series of quinazoline based
NTR1 modulators to give a potent and orally available
compound. In the process we have maintained a unique
mechanism of action, wherein the compound enables biased -
arrestin interactions and acts as an antagonist on the Ca2+
pathway for Gq mediated activation. An extensive account of
crude
2-(1-fluoro-cyclopropyl)-6-(2-hydroxy-ethylamino)-
quinazolin-4-ol (15). The crude material was dissolved in
MeOH (800 mL) and water (400 mL). To the mixture was
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Journal of Medicinal Chemistry
added formaldehyde solution (37%, 45 g), NaBH(AcO)3 (59 g,
Page 6 of 8
habituated to oral dosing via 5 consecutive daily sessions in
which 10 ml/kg H2O was delivered p.o. After treatment on test
days, animals were immediately returned to activity monitors
and locomotor activity was recorded over the next 2 hr. Infrared
beam breaks in 5 min intervals were recorded to generate
horizontal distance traveled per time bin. Distance values were
averaged across mice, and the means ± SEM were plotted. Total
distance traveled in the 2 hr post-treatment was calculated by
summing the time bins between 30 and 150 min. Data were
plotted and analyzed using GraphPad Prism V.7.
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5
6
7
8
280 mmol) and NaBH3CN (27 g, 420 mmol). The reaction was
stirred at room temperature for 1 h. The solution was poured
into water (3 L) and extracted with ethyl acetate (1.5 L x3). The
organic layers were combined, dried over Na2SO4, concentrated
and purified by silica gel column (methylene chloride/MeOH =
20/1) to give 2-(1-fluoro-cyclopropyl)-6-[(2-hydroxy-ethyl)-
methyl-amino]-quinazolin-4-ol (16, 32 g, yield: 39% over 2
steps)22 as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ =
12.25 (brs, 1H), 7.43 (d, J = 9.2 Hz, 1H), 7.28 (dd, J = 9.2, 3.2
Hz, 1H), 7.17 (d, J = 2.8 Hz, 1H), 4.75 (brs, 1H), 3.57-3.47 (m,
4H), 3.01 (s, 3H), 1.52-1.36 (m, 4H). Step f: To a suspension of
2-(1-fluoro-cyclopropyl)-6-[(2-hydroxy-ethyl)-methyl-amino]-
quinazolin-4-ol (16, 14.2 g, 51.3 mmol) and BOP (34 g, 77
mmol) in MeCN (500 mL) was added DBU (31.2 g, 205 mmol)
and the mixture was stirred for 5 min. To the reaction, 4-(2-
methoxy-phenyl)-piperidine (17, HCl salt, 12.8 g, 56.4 mmol)
was added. The reaction was stirred at room temperature
overnight and filtered. The filter cake was washed with MeCN
(100 mL) and dried to give 2-({2-(1-fluoro-cyclopropyl)-4-[4-
(2-methoxy-phenyl)-piperidin-1-yl]-quinazolin-6-yl}-methyl-
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ASSOCIATED CONTENT
Supporting Information. This material is available free of
charge via the Internet at http://pubs.acs.org. Experimental
procedures and spectroscopic data for selected compounds,
details of the PK studies, and assay protocols for the binding
studies. (PDF).
AUTHOR INFORMATION
1
amino)-ethanol (18, 20 g, yield: 87%) as a yellow solid. H
Corresponding Author. *Phone: 01-858-795-5320. E-mail:
apinkerton@sbpdiscovery.org ORCID ID Anthony B.
Pinkerton: 0000-0003-4571-152X
NMR (500 MHz, CDCl3) δ 7.98 (d, J = 9.2 Hz, 1H), 7.41 (dd, J
= 9.3, 2.6 Hz, 1H), 7.24 (t, J = 7.4 Hz, 2H), 6.99 (t, J = 7.9 Hz,
2H), 6.91 (d, J = 8.2 Hz, 1H), 4.42 (d, J = 13.2 Hz, 2H), 3.90 (t,
J = 5.7 Hz, 2H), 3.87 (s, 3H), 3.60 (t, J = 5.6 Hz, 2H), 3.36 –
3.27 (m, 1H), 3.19 (t, J = 12.6 Hz, 2H), 3.08 (s, 3H), 2.30 (broad
s, 1H), 2.00 (d, J = 12.8 Hz, 2H), 1.95 – 1.85 (m, 2H), 1.59 –
Author Contributions. This work represents a collaborative
effort from Sanford Burnham Prebys Medical Discovery
Institute (SBP) and Duke University led by senior investigators
from both institutions (A.B.P., L.S.B., M.G.C.). Investigators at
SBP were primarily responsible for SAR studies and
investigators at Duke were primarily responsible for in vivo
studies. All authors participated in preparing the manuscript.
Notes. The authors declare the following competing financial
interest: Patent applications covering SBI-553 and related
derivatives have been filed by SBP and Duke University.
1.50 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 164.33 (d, J C-F
=
3.6 Hz), 159.17 (d, JC-F = 20.3 Hz), 156.80, 146.99, 144.97,
133.65, 129.07, 127.17, 126.48, 121.63, 120.65, 116.49,
110.41, 103.79, 79.01 (d, JC-F = 218.0 Hz), 60.08, 55.47, 55.30,
50.51, 38.95, 35.74, 31.81, 15.00 (d, JC-F = 10.8 Hz). HRMS:
calculated for C26H31FN4O2: m/z = 451.2509; (M+H+) and
observed m/z = 451.2528. HPLC Purity: 99%.
In Vivo Studies. PD149163 was purchased from
MilliporeSigma (St. Louis, MO). All compounds were freshly
prepared in indicated vehicles on the day of testing. Animals.
Experiments employed adult male and female DAT KO mice27
maintained on a C57BL/6J background. Animals within studies
were 12-24 week old, 18–30 g body weight and were age- and
sex-matched. Animals were maintained on a 12/12 hour
light/dark cycle and supplied tap water and standard laboratory
chow ad libitum, except during testing. Mice were randomly
assigned to treatment groups, and all experiments were carried
out at the beginning of the light cycle. All mouse studies were
conducted in accordance with the National Institutes of Health
Guidelines for Animal Care and Use and with approved animal
protocols from the Duke University Animal Care and Use
Committee. Locomotor activity. Locomotor activity was
evaluated by open field automated activity monitors (AccuScan
Instruments, Columbus, OH) featured with eight photo-beams
equally distributed along the border, as previously described.21
To assess the effect of NTR1 ligands on the basal
hyperlocomotion exhibited by DAT KO animals, adult male
and female DAT KO mice were acclimated to the open field for
30 min prior to treatment. For i.p. dosing studies, treatments
included 12 mg/kg SBI-553, 1 mg/kg PD149163 or vehicle (4%
DMSO in saline) delivered in a 10 ml/kg volume. For p.o.
dosing studies treatments included 10, 30, or 100 mg/kg SBI-
553 as the HCl salt or vehicle (distilled H2O) delivered in a 10
ml/kg volume. Prior to oral dosing studies animals were
ACKNOWLEDGEMENTS
Funding. This work was supported by National Institutes of
Health grants R21/33 DA038019 to A.B.P. and 5P30DA029925
to M.G.C. and L.S.B. We acknowledge Peter Wilson at Almac,
Craigavon, United Kingdom for scale up work on 18, WuXi,
Shanghai, China, for measurements of the PK parameters,
Eurofins, Taipei, Taiwan, for the binding studies in Figure 3,
and Alexander Braddock and Emmanuel Theodorakis at the
University of California, San Diego for assistance with
measuring optical rotations.
ABBREVIATIONS USED
NT, neurotensin; NTR1, neurotensin receptor 1; NTR2,
neurotensin receptor 2; DA, dopamine; DAT, dopamine
transporter; GFP, green fluorescent protein; PAM, positive
allosteric
modulator.
BOP,
benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate ;
DBU, 1,8-Diazabicyclo[5.4.0]undec-7-ene.
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biased allosteric modulator. Unpublished results.
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