Imidazole-derived agonists for the neurotensin 1 receptor

Article abstract of DOI:10.1016/j.bmcl.2013.11.026

A scaffold-hop program seeking full agonists of the neurotensin-1 (NTR1) receptor identified the probe molecule ML301 (1) and associated analogs, including its naphthyl analog (14) which exhibited similar properties. Compound 1 showed full agonist behavior (79-93%) with an EC₅₀ of 2.0-4.1 μM against NTR1. Compound 1 also showed good activity in a Ca mobilization FLIPR assay (93% efficacy at 298 nM), consistent with it functioning via the G _q coupled pathway, and good selectivity relative to NTR2 and GPR35. In further profiling, 1 showed low potential for promiscuity and good overall pharmacological data. This report describes the discovery, synthesis, and SAR of 1 and associated analogs. Initial in vitro pharmacologic characterization is also presented.

Full text of DOI:10.1016/j.bmcl.2013.11.026

Accepted Manuscript
Imidazole - Derived Agonists for the Neurotensin 1 Receptor
Paul M. Hershberger, Michael P. Hedrick, Satyamaheshwar Peddibhotla,
Arianna Mangravita-Novo, Palak Gosalia, Yujie Li, Wilson Gray, Michael
Vicchiarelli, Layton H. Smith, Thomas D.Y. Chung, James B. Thomas, Marc
G. Caron, Anthony B. Pinkerton, Lawrence S. Barak, Gregory P. Roth
PII:
S0960-894X(13)01305-X
http://dx.doi.org/10.1016/j.bmcl.2013.11.026
BMCL 21053
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 October 2013
6 November 2013
11 November 2013
Please cite this article as: Hershberger, P.M., Hedrick, M.P., Peddibhotla, S., Mangravita-Novo, A., Gosalia, P., Li,
Y., Gray, W., Vicchiarelli, M., Smith, L.H., Chung, T.D.Y., Thomas, J.B., Caron, M.G., Pinkerton, A.B., Barak,
L.S., Roth, G.P., Imidazole - Derived Agonists for the Neurotensin 1 Receptor, Bioorganic & Medicinal Chemistry
Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.11.026
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Imidazole - Derived Agonists for the Neurotensin 1 Receptor
Paul M. Hershberger*,a, Michael P. Hedrickb, Satyamaheshwar Peddibhotlaa, Arianna
Mangravita-Novoa, Palak Gosalia, Yujie Li, Wilson Gray, Michael Vicchiarellia, Layton H.
Smitha, Thomas D.Y. Chungb, James B. Thomasd, Marc G. Caronc, Anthony B. Pinkertonb,
Lawrence S. Barakc, Gregory P. Rotha
aConrad Prebys Center for Chemical Genomics at Sanford-Burnham Medical Research Institute,
Orlando, Florida 32827, USA, bConrad Prebys Center for Chemical Genomics at Sanford-
Burnham Medical Research Institute, La Jolla, California 92037, USA and cDuke University
Medical Center, Durham, NC 27710, USA, RTI International, 3040 E Cornwallis Road,
Durham, North Carolina 27709, United States.
This is where the receipt/accepted dates will go; Received Month XX, 2012; Accepted Month XX, 2013 [BMCL RECEIPT]
Abstract— A scaffold-hop program seeking full agonists of the neurotensin-1 (NTR1) receptor
identified the probe molecule ML301 (1) and associated analogs, including its naphthyl analog
(14) which exhibited similar properties. Compound 1 showed full agonist behavior (79 - 93%)
with an EC50 of 2.0 – 4.1 µM against NTR1. Compound 1 also showed good activity in a Ca
mobilization FLIPR assay (93% efficacy at 298 nM), consistent with it functioning via the Gq
coupled pathway, and good selectivity relative to NTR2 and GPR35. In further profiling, 1
showed low potential for promiscuity and good overall pharmacological data. This report
describes the discovery, synthesis, and SAR of 1 and associated analogs. Initial in vitro
pharmacologic characterization is also presented.
*To whom correspondence should be addressed. Tel. +1-407-745-2000. E-mail: phershberger@sanfordburnham.org

Methamphetamine addiction remains a substantial public health issue1, and currently no small molecule therapies
are available for its treatment. The tridecapeptide neurotensin2 (NT) has neurological function known to influence reward
behavior.3-6 Neurotensin receptor 1 (NTR1) peptide agonists produce behaviors that are exactly opposite of the
psychostimulant effects observed with methamphetamine abuse, such as hyperactivity, neurotoxicity, psychotic episodes,
and cognitive deficits, and repeated administrations of NTR1 agonists do not lead to the development of tolerance7,8.
Recent data from the Hanson laboratory9 suggesting that NT receptor agonists may have a role in addiction therapy are:
(a) in a methamphetamine self-administration rat model the substitution of the peptide NT agonist (Lys(CH2NH)lys-
Pro,Trp-tert-Leu-Leu-Oet) for methamphetamine did not significantly affect motor activity but dramatically reduced lever
pressing (b) the peptide agonist was not self-administered, and (c) the effects were associated with nucleus accumbens
dopamine D1 receptors. These findings strengthen the hypothesis that neurotensin receptors are valid targets for
antagonizing drug seeking behaviors and preventing relapses. Although NTR1 has been known for many years, there have
been few reports of relevant non-peptide ligands. A review of the isolation, cloning, localization, and binding properties
of the accepted receptor subtypes (NTR1, NTR2, and NTR3) and the molecules known to bind at these receptors was
published in 200910. Also in 2009, the partial agonists II-a, II-b, and II-c (Table 1), structural analogs of the potent
NTR1 antagonists SR48692 (I-a) and SR142948 (I-b), were identified using a Ca mobilization FLIPR assay11,12
.
Replacement of the adamantyl group in I-a with a panel of amino acid derived substituents revealed that the isobutyl
group derived from L-leucine gave the best results. Eliminating one of the methoxy groups in II-a reduced the potency
but improved efficacy (II-c). Researchers at Wyeth reported two different chemotypes, III and IV, each of which showed
partial agonist activity for NTR1 in the FLIPR assay13.
Table 1. Chemical Structures of Previously Reported Small Molecule NTR1 Agonists and Antagonists.
Cl
N
H
CO2H
N
N
O
O
N
O
II-a, from L-leucine
II-b, from D-leucine
II-c
I-a (SR48692)
I-b (SR142948A)
IV
III, Fmoc-leucine
ML314
This project sought novel and improved small molecule neurotensin receptor agonists using a primary assay based
upon the ability of a β-arrestin fluorescent reporter to directly recognize the activated state of NTR1. We recently
reported14 the discovery of the quinazoline ML314 (Table 1), a nonpeptidic β-arrestin biased agonist, which resulted from
a screening program using the NIH Molecular Libraries Small Molecules Repository (MLSMR) of >300,000 compounds
followed by medicinal chemistry driven SAR studies.
In parallel, a scaffold-hop program was conceived from our evaluation of existing literature (Table 1). As assays
were already being developed for the screening program, the opportunity to examine scaffold-hop molecules was an

attractive way to extend our work to chemotypes not present in the MLSMR. This led to the identification of the
imidazole-based agonist ML301 and associated analogs described herein.
The new compounds were synthesized according to the two routes outlined in Scheme 1. Although the alternate
method was fewer steps, its low yields and the high cost of 4-aminoquinoline building blocks made the longer method
more preferred and practical for most cases of interest. In addition to standard Pinner conditions, which generally do not
work well for cases involving sterically hindered benzonitriles, many amidine-forming conditions were attempted15-20 for
the generation of G. Conditions (h), although inefficient, were the best we found for this sterically and electronically
disfavorable case. For some analogs without the 2,6-dimethoxy moiety, the shorter route was more preferred.
Importantly, both routes produced exclusively the key intermediate regioisomer D shown in the box, and there is ample
literature precedent15,21-23 for this assignment via the amidine (alternate) route. While it may appear that the preferred
route could be shortened by performing the N-arylation step on the trifluoromethyl intermediate followed by CF3
hydrolysis and amino acid coupling, this was not possible because the CF3 hydrolysis failed if the imidazole was N-
arylated, and the N-arylation was not efficient when performed on the acid intermediate. Thus, it was necessary to first
hydrolyze the CF3 group (b), and then esterify (c) prior to N-arylation (d).
Overall, variation at the R1 and R2 positions was emphasized, as a survey of R3 groups for the pyrazole scaffold
was already disclosed11. Table 2 shows that R1 is preferably both electron donating and sterically demanding; hence, the
preference for 2,6-dimethoxy (1, 2, 5, 14). A large loss of activity was observed when electronics were maintained but the
steric factor was reduced via the 2,4-dimethoxy case (11). When the steric factor was maintained but the electron donating
effect was either changed to electron withdrawing via the 2,6-dichloro case (12) or lowered via the 2,6-dimethyl case (13),
agonism was maintained but the potency fell by 2.6x (12) and 4.6x (13). Other examples with lesser steric and / or
electronic factors at the R1 position were also much less active; namely, the 2-methoxy case (3) and the unsubstituted case
of R1 = H (4). The R2 position accommodated 7-chloroquinoline (1), 7-H-quinoline (5), and 1-naphthyl (14) motifs.
Generally, compounds bearing the 7-Cl group were better than the corresponding 7-H cases (1, 3, and 4 versus 5-7). For
example, the 7-Cl compounds were more efficacious and, both 4 and 5 retained low potency whereas 6 and 7 were
completely inactive. When the bicyclic aryl system at R2 was replaced by either a 4-pyridyl (8-10) or a 4-methylphenyl
(15) group, activity was lost. Although the amino acid derived region (R3) has not yet been explored in detail for the
imidazole scaffold, inverting the stereochemistry from the natural S- to the unnatural R-configuration led to a 4.7-fold loss
of potency and substantially lower efficacy (2). Replacement of the leucine – derived isobutyl group with the adamantyl
moiety (16) led to much lower potency but retention of agonist behavior.
Table 2. SAR of Imidazole-Scaffold Neurotensin-1 Agonists.
NTR1
EC50 µM
NTR1
Emax, %NT
Entry
1
R1
R2
R3
2,6-di-OMe
2 - 4
9.4
56
79 - 93
50
(R)
2,6-di-OMe
2-OMe
H
2
3
4
100
72
100

2,6-di-OMe
2-OMe
H
3.8
>80
>80
66
59
5
6
-
-
7
2,6-di-OMe
2-OMe
H
100
-
8
>80
>80
>80
5.2
9
-
10
11
12
2,4-di-OMe
2,6-di-Cl
-
80
2,6-di-Me
9.2
2.3
78
93
13
14
2,6-di-OMe
2,6-di-OMe
>80
-
15
2,6-di-OMe
46
100
16
A high-content imaging assay was used to measure the modulation of the NTR1 receptor in U2OS cells expressing the receptor and a β-arrestin
fluorescent reporter. The location of the receptor-arrestin complex was monitored upon receptor activation. The EC50 and Emax values were calculated
relative to the efficacy of the NT peptide at a saturating concentration of 100 nM.
Table 3 shows data for key prior art pyrazole compounds, which were synthesized independently via a solution
phase adaptation of the published route11. As was observed for the imidazole scaffold, potency declined when the 2,6-
dimethoxy case was switched to 2-methoxy (18). Two other prior art pyrazoles7,8 I-a (19) and I-b (20) containing the
adamantyl moiety and different R2 groups were purchased and found to be less active as well.
Table 3. SAR of Pyrazole-Scaffold Neurotensin-1 Agonists.
NTR1
EC50 µM
NTR1
Emax, %NT
Entry
17
R1
R2
R3
2,6-di-OMe
0.75
0.97
100
98
(R)
2-OMe
18

2,6-di-OMe
2,6-di-OMe
31
100
19
20
>80
A high-content imaging assay was used to measure the modulation of the NTR1 receptor in U2OS cells expressing the receptor and a β-arrestin
fluorescent reporter. The location of the receptor-arrestin complex was monitored upon receptor activation. The EC50 and Emax values were calculated
relative to the efficacy of the NT peptide at a saturating concentration of 100 nM.
The imidazole 1 and the prior art pyrazole 17 were further profiled in counterassays, and in β-arrestin and Ca2+
mobilization assays (Table 4). Both compounds were selective for NTR1 over NTR2 and GPR35, but only the imidazole
1 retained activity in the DiscoveRx β-arrestin assay. Consistent with their functioning via the Gq-coupled pathway, both
compounds exhibited good functional activity in a Ca mobilization assay, with EC50s at least 5 times better than in the
primary NTR1 assay. Additionally, preincubation with the known antagonist I-b (IC50 = 0.24 nM)25 inhibited 1 (EC100
10 µM) mediated NTR1 activation with an IC50 of 62 nM, which supports the hypothesis that 1 acts via NTR1 binding
(Figure 2).
=
Table 4. Additional Profiling Assays for Neurotensin-1 Agonists.
NTR2
GPR35
β-arrestin
Ca2+ Mobilization
NTR1
EC50
µM
2.0
Entry
Emax
% NT
79
EC50
µM
Emax
% NT
EC50
Emax
% NT
EC50
µM
6.1
Emax
% NT
100
EC50
µM
0.298
<0.156
Emax
% NT
93
µM
>40
>40
>80
>80
-
-
-
-
1
17
0.75
100
>33
-
63
A high-content imaging assay was used to measure the modulation of the NTR1, NTR2, and GPR35 receptors in U2OS cells expressing the receptor
of interest and a β-arrestin fluorescent reporter. The location of the receptor-arrestin complex was monitored upon receptor activation.
The β-arrestin assay utilizes the DiscoveRx PathHunter system in a CHO-K1 cell line in which the NTR1 receptor is fused in frame with the small
enzyme fragment ProLink™ and co-expressed in cells stably expressing a fusion protein of β-Arrestin and the larger, N-terminal deletion mutant of
β-gal (called enzyme acceptor or EA). Activation of the GPCR stimulates binding of β-Arrestin to the ProLink-tagged GPCR and forces
complementation of the two enzyme fragments, resulting in the formation of an active β-gal enzyme. This interaction leads to an increase in enzyme
activity that can be measured using chemiluminescent reagents.
The NTR1 Ca2+ Flux assay was performed by ChanTest (Rockville, MD0 and used a CHO cell line which stably expressed the NTR1 receptor.
Overall, it is notable that although the imidazole 1 and the pyrazole 17 possess closely related chemical structures
they exhibit substantially different SAR in some respects. First, 1 is less potent (IC50), but similarly efficacious in the
NTR1 assay (Tables 2 and 3). However, 1 maintained full efficacy in a Ca mobilization assay (Table 4), whereas the
efficacy of 17 dropped to 63%, consistent with the previously reported11 value of 54%. Secondly, 1 and 17 gave different
responses in the β-arrestin assay (Table 3). These differences were deemed important, as identifying a robust full agonist
was a key project goal. Thirdly, the two scaffolds apparently respond very differently to structural perturbations. For
example, replacing the 7-quinolinyl group with 1-naphthyl reportedly led to potent antagonist behavior11 for the pyrazole
17. In contrast, identical perturbation of the imidazole scaffold of 1 led to potent and full agonist behavior (14).
The imidazole 1, its naphthyl analog 14, and the pyrazole 17 were advanced to pharmacological screening (Table
5). Overall, despite its structural similarity to the pyrazole 17, 1 showed substantial advantages on plasma and
microsomal stability. The three compounds exhibited good solubility due to the presence of the carboxylic acid moiety.
The PAMPA (Parallel Artificial Membrane Permeability Assay) assay is used as an in vitro model of passive,
transcellular permeability. The compounds exhibited overall permeability inversely related to the pH of the donor
compartment. Because these NTR1 agonists are envisioned as predecessors of psychoactive drugs, a preliminary
assessment of their potential to cross the blood brain barrier (BBB) was performed. When incubated with an artificial
membrane that models the BBB, much lower permeability was observed in contrast to our recently reported ML314

which gave Pe 399 cm/s in the same assay.14 The imidazoles 1 and 14 were 4 times less permeable than 17. These
observations are consistent with the carboxylic acid function in the compounds, and may present an opportunity for future
enhancements. Compounds 1 and 14 exhibited substantial plasma protein binding, but lower than that of 17. Importantly,
the unbound fraction for 1 and 14 was about five to ten times greater than for 17. The differences observed for 1 and 14
when compared to 17 in the BBB and protein binding models may be due in part to zwitterionic character associated with
the imidazoles. The imidazole 1 also showed excellent stability in plasma, significantly better than that of either 14 or 17.
Additionally, 1 showed excellent stability in human and modest stability in mouse liver homogenates, in contrast to the
pyrazole 17 which gave values of 76% and 46% in the same assays run in triplicate. None of the compounds showed
toxicity (>50 µM) toward human hepatocytes after 24 hours.
Table 5. Summary of in vitro ADME/T Properties of NTR1 Agonists.
ADME/T Assay Panel Component
1
14
17
Aqueous Solubility in pION’s buffer (µg/mL) [µM]a
pH 5.0/6.2/7.4
>52 / >52 / >52
[>99 / >99 / >99]
102.6 / >145 / >145
[247 / >297 / >297]
52.9 / >155 / >155
[113 / >296 / >296]
PAMPA Permeability, Pe (x10-6 cm/s)
363 / 17 / 6
1.2
953 / 145 / 12
1.1
1267 / 725 / 70
4.8
Donor pH: 5.0 / 6.2 / 7.4 Acceptor pH: 7.4
BBB-PAMPA Permeability, Pe (x10-6 cm/s)
Donor pH: 7.4 Acceptor pH: 7.4
Plasma Protein Binding
(% Bound)
98.7 / 98.7
92.2 / 91.4
97.9 / 98.0
96.4 / 95.4
99.6 / 99.7
96.6 / 97.1
Human 1 µM / 10 µM
Mouse 1 µM / 10 µM
Plasma Stability (%Remaining at 3 hrs) Human/Mouse
100 / 100
100 / 59
76.0 / 76.0
100 / 76
88.9 / 70.7
76 / 46
Hepatic Microsome Stability (% Remaining at 1hr)
Human/Mouse
Toxicity Towards Fa2N-4 Immortalized Human
>50
>50
>50
Hepatocytes LC50 (µM), after 24 hours.
a Solubility also expressed in molar units (µM) as indicated in italicized [bracketed values], in addition to µg/mL units.
To assess the potential for promiscuous activity across a range of GPCRs, 1 and 17 were submitted to the
Psychoactive Drug Screening Program (PDSP) at the University of North Carolina (Bryan Roth, PI)26. The results
(Figure 1) indicate 1 shows very little potential for promiscuity across a range of GPCRs at 10 µM concentration.
Contrarily, the pyrazole 17 showed a somewhat higher potential for promiscuity. Follow up dose response studies
revealed Ki values of >10 µM (DAT) and 10 µM (NTS1) for 1, and >10 µM (DAT), 5.2 µM (DOR), 3.4 µM (MOR), and
3.3 µM (NTS1) for 17. It is not known whether these activities in binding assays are translated into functional
modification of the activities of these receptors.
In summary, the imidazole 1 was designed via a scaffold hop approach based on the previously disclosed pyrazole
17, which was designed in part from knowledge of SR48692 (I-a, Table 1). Taken together, these advances underscore
the value of drug design via iterative / intuitive structural enhancement of an identified scaffold. Despite having
substantial structural similarity to the pyrazole 17, the imidazole 1 exhibits intriguing differences / advantages in chemical
and biological properties. Although 1 is less potent than 17 and comparably potent to 14, it is a more effective agonist
than 17 in the calcium mobilization assay. Identification of a full agonist was a primary objective for this program. The
imidazole 1 also showed a much better pharmacology profile, including lower protein binding along with improved
plasma and hepatic microsomal stability. These improvements may enable 1 or future analogs of it to achieve a
distribution profile more favorably disposed toward in vivo activity. Compound 1 also showed minimal promiscuity. As
it is tractable from a synthetic chemistry perspective and appears more tolerant of variation at the R2 position, it represents
a strong platform on which to launch a medicinal chemistry-based program for further enhancement.
Acknowledgements

This work was supported by NIH grants 1 R03 MH089653-01 to LSB and an NIH Molecular Libraries grant (U54
HG005033-03) to the Conrad Prebys Center for Chemical Genomics at the Sanford Burnham Medical Research Institute,
one of the comprehensive centers of the NIH Molecular Libraries Probe Production Centers Network (MLPCN).
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Scheme 1, Preferred route: Synthesis of 1, conditions: a.24 1. NaOAc, water, 1,1-dibromo-3,3,3-
trifluoroacetone, 100 °C, 30 min. 2. 2,6-dimethoxybenzaldehyde, ammonium hydroxide, water, methanol, 52%; b.24
NaOH, water, 100 °C, 94%; c. SOCl2, EtOH, 75 °C; d. 7-chloro-4-iodoquinoline, cesium carbonate, butyronitrile, 110 °C,
20 h, 43%; e. KOH, EtOH, water; f. L-leucine t-butyl ester, EDC, HOBt, triethylamine, DMF, 83%; g. trifluoroacetic
acid, DCM, reverse phase HPLC purification, 82%.
Scheme 1, Alternate route: Synthesis of 1, conditions: h. 7-chloro-4-aminoquinoline, EtMgBr, diethyl ether,
tetrahydrofuran, 75 °C, 9%; i. 1. Ethyl bromopyruvate, sodium bicarbonate, EtOH, reflux, 22 h, work up. 2. pTsOH,
toluene, reflux, 4 h, 17%.
Figure 1. Comparison of 1 and 17 in a GPCR panel of assays (% inhibition at 10 µM).
Figure 2. Inhibitory dose response of NTR1 antagonist I-b (SR142948A) in the presence of agonists 1 (10 µM)
and NT(8-13) (5 nM).

Scheme 1

Figure 1

Figure 2