Identification of N -[(5-{[(4-Methylphenyl)sulfonyl]amino}-3-(trifluoroacetyl)-1 H -indol-1-yl)acetyl]- l -leucine (NTRC-824), a neurotensin-like nonpeptide compound selective for the neurotensin receptor type 2

Article abstract of DOI:10.1021/jm500857r

Compounds acting via the neurotensin receptor type 2 (NTS2) are known to be active in animal models of acute and chronic pain. To identify novel NTS2 selective analgesics, we searched for NTS2 selective nonpeptide compounds using a FLIPR assay and identif

Full text of DOI:10.1021/jm500857r

Brief Article
pubs.acs.org/jmc
Open Access on 08/26/2015
Identification of N‑[(5-{[(4-Methylphenyl)sulfonyl]amino}-3-
(trifluoroacetyl)‑1H‑indol-1-yl)acetyl]‑L‑leucine (NTRC-824), a
Neurotensin-like Nonpeptide Compound Selective for the
Neurotensin Receptor Type 2
James B. Thomas,*,† Angela M. Giddings,† Robert W. Wiethe,† Srinivas Olepu,† Keith R. Warner,†
Philippe Sarret,‡ Louis Gendron,‡ Jean-Michel Longpre,‡ Yanan Zhang,† Scott P. Runyon,†
and Brian P. Gilmour†
†Center for Organic and Medicinal Chemistry, Research Triangle Institute, P.O. Box 12194, 3040 Cornwallis Road, Research Triangle
Park, North Carolina 27709, United States
‡Department of Physiology and Biophysics, Faculty of Medicine and Health Sciences, Universite
North, Sherbrooke, Quebec J1H 5N4, Canada
́
de Sherbrooke, 3001, 12th Avenue
S
* Supporting Information
ABSTRACT: Compounds acting via the neurotensin receptor type 2
(NTS2) are known to be active in animal models of acute and chronic pain.
To identify novel NTS2 selective analgesics, we searched for NTS2 selective
nonpeptide compounds using a FLIPR assay and identified the title
compound (NTRC-824, 5) that, to our knowledge, is the first nonpeptide
that is selective for NTS2 versus NTS1 and behaves like the endogenous
ligand neurotensin in the functional assay.
from the study of the NTS2 selective peptide NT79 (1b)
support this notion as it possessed activity against visceral pain
but lacked the side effects described above.12,19
INTRODUCTION
■
The discovery of novel analgesics has been a major pursuit of
medicinal chemists for more than a hundred years. Although
opiates and opioids remain the treatment of choice for severe
acute pain, they produce serious side effects including
addiction, respiratory depression, and tolerance. Furthermore,
opioids often fail to provide relief from chronic pain, a
persistent form of pain resulting from nerve injury. Thus,
identification of nonopioid analgesics remains a key challenge
of discovery science.
The tridecapeptide neurotensin (NT, Glu-Leu-Tyr-Glu-Asn-
Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu), acting via two G-protein
coupled receptors NTS1 and NTS2 (NTR1, NTR2, etc.) and
the multiligand type-1 receptor sortilin, mediates many
biological functions including nonopioid analgesia.1−5 Com-
pounds acting via neurotensin receptor types 1 and 2 have been
reported to be active in animal models of both acute and
chronic pain.6−11 NT mediated analgesia is also synergistic,
with opioid analgesia suggesting that NT-based compounds
could function alone or as adjuncts to opioids in the
management of pain.12 Together, these findings underpin the
rational for identification of NT-based analgesics.
Surprisingly, only one nonpeptide compound has so far been
described to possess NT-based analgesic properties, the NTS2
versus NTS1 selective compound levocabastine (2), which
shows activity in both visceral and chronic pain models.10,20,21
Coupled with our desire to identify nonpeptide compounds,
the lower side effect profile demonstrated by the NTS2
selective peptide 1b prompted us to develop methods of
identifying NTS2 selective compounds.22
Literature reports with a CHO cell line stably expressing
rNTS2 indicated that the pyrazole compound 3a was an agonist
in the FLIPR assay and that NT was an antagonist of the
calcium release mediated by 3a.23 This suggested that we could
identify NT-like (antagonist) compounds by first activating
NTS2 with 3a and then screening for compounds that would
block this activity. The binding assay versus [125I]NT could
then be used as a secondary screen for active compounds to
verify interaction with NTS2.
We tested this idea using the CHO cell line above and a
FLIPR Tetra and studied the peptide NT, the nonpeptide
levocabastine (2), and the two well-known nonpeptide
pyrazole-based ligands SR48692 (3a) and SR142948a (3b),
all of which are known to bind NTS2.24,25 In line with literature
precedent, we found that both pyrazole compounds (3a,b)
The search for such compounds is decades old, yet to date,
nearly all of the NT compounds reported to be active in animal
models of pain are peptides, variants of the terminal
hexapeptide fragment of NT (NT(8−13), 1a, Chart 1).12−16
The vast majority of these compounds produce analgesia that is
accompanied by hypothermia and hypotension, a feature
ascribed to interaction with the NTS1 receptor.17,18 Reports
Received: June 9, 2014
Published: August 26, 2014
© 2014 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
Chart 1
Scheme 1. Synthetic Scheme for Compounds 5 and 13
a
Reagents and conditions: (i) acetone, methyl bromoacetate, K2CO3,
RT, 4 days; (ii) 2N LiOH, 1,4-dioxanes, H2O, RT, 16 h; (iii) L-leucine
tert-butyl ester hydrochloride, HBTU, Et3N, CH2Cl2, RT, 4 h; (iv) H2,
Pd/C, EtOH, RT, 2 h; (v) tosyl chloride, 4-dimethylaminopyridine
(DMAP), CH2Cl2, RT, 16 h; (vi) TFA/CH2Cl2 (1:1), RT, 30 min to
12 h; (vii) trifluoroacetic anhydride/CH2Cl2, 30 min, RT.
to give intermediate 11 and then coupled to tosyl chloride to
give intermediate 12. Compound 13 was obtained from 12 by
treatment with trifluoroacetic acid (TFA) in CH2Cl2.
Compound 5 was obtained from 12 by treatment with
trifluoroacetic anhydride (TFAA) to give the acylated indole
derivative 14, which was subsequently deprotected with TFA to
give 5.
behaved as full agonists with 3b being more potent than 3a but
equally efficacious. Levocabastine showed potent partial agonist
activity (14−16% percent of 3b). NT, on the other hand, did
not induce calcium release but was an antagonist of 3b in the
FLIPR assay.
BIOLOGY
■
The binding affinities of the test compounds for the rNTS1 and
rNTS2 receptors, listed in Table 1, were determined using
While it seemed counterintuitive, our data showed clearly
that NT was an antagonist and levocabastine (2) a very low
efficacy potent partial agonist in the FLIPR assay, although
both are known to be antinociceptive in animal models of pain.
The two pyrazole compounds (3a,b), on the other hand, were
found to be agonists even though it is well documented that
both antagonize the analgesic action of NT-based compounds
in a variety of animal models. Overall, this pilot study suggested
that our search for novel NTS2-based analgesics should begin
with identification of compounds with in vitro profiles
mimicking either NT or 2 versus 3a or 3b.
We recently reported using this assay to drive an SAR study
that led to the identification of the NTS2 selective, low efficacy,
potent partial agonist 4 (NTRC-739).22 In this article, we
report a parallel study that identified the NTS2 selective
nonpeptide compound 5 that, like NT, is an antagonist of 3b in
the FLIPR assay. The details of this work are presented herein.
Table 1. Data for Reference Compounds NT and 3b and
Test Compounds 5 and 13 in the FLIPR Assay (NTS2) and
Binding Assays (NTS1 and NTS2 Receptors)
a
FLIPR assay
NTS2
binding assays
NTS1
NTS2
b
c
EC50
Emax
IC50
Ki
Ki
d
NT
3b
5
NA
114 24
1.1 0.3
1.4 0.4
>30 μM
>30 μM
28
6
22
5
100
6
5.8 1.1
202 44
1504 244
NA
38 11
13
NA
3322 556
a
b
c
[
125I]NT. EC50, IC50 and Ki values are nM SEM. Emax value is %
d
3b. Not active.
[
125I]NT in previously reported competitive binding assays.23
CHEMISTRY
The cells used were CHO-k1 cells (American Type Culture
Collection) engineered to overexpress either the rNTS1 or
rNTS2 receptor. Measures of functional agonist activity were
obtained by measuring changes in intensity of a calcium-
sensitive fluorescent dye as an indirect measure of changes in
internal calcium concentrations. Antagonism was measured as
inhibition of calcium mobilization induced by 3b. These
measurements were performed using a FLIPR Tetra (Molecular
Devices).
■
The chemistry used to prepare target compounds 5 and 13 is
illustrated in Scheme 1. Thus, 5-nitroindole (7) was alkylated
with methyl bromoacetate to give ester 8 that was subsequently
hydrolyzed to the corresponding acid intermediate 9 by
treatment with LiOH in dioxanes and water. Compound 9
was then coupled to leucine tert-butyl ester using O-
benzotriazol-1-yl-N,N,N,N′-tetramethyluronium hexafluoro-
phosphate (HBTU) and triethylamine to give intermediate
10. This was reduced using hydrogen and palladium on carbon
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LIBRARY SCREENING
■
Fan and co-workers reported that the nonpeptide compound
6a, Chart 1, possessed partial agonist activity at the NTS1
receptor.26 With the hope that some variant of this compound
might also be active at NTS2, we made a library of 66
compounds with general structure 6b using commercially
available sulfonyl chlorides and amino acid esters by following
the chemistry route described for compound 13 in Scheme 1.
We screened these for agonist and antagonist activity in parallel
at a single concentration (10 μM) using 3b as our NTS2
agonist standard and NT as our antagonist standard. Only one
compound (13) showed >50% inhibition of 3b induced
calcium mobilization in the NTS2 screen. Compound 13 was
also devoid of NTS2 agonist activity and showed no activity at
NTS1 (data not shown).
RESULTS AND DISCUSSION
Figure 1. Ke assays were run against dose−response curves of the
control agonist 3b. Compound 5 demonstrated the characteristics of
insurmountable antagonist activity versus 3b in the Ke experiment.
This was also seen for NT as reported earlier.22
■
In confirming the hit 13, an impurity was discovered that
represented 25% of the material in the well. Mass spectral and
19F NMR analysis suggested that the desired compound (13)
had incorporated a trifluoroacetyl group to give byproduct 5.
We postulated that this would have occurred at the indole 3-
position during deprotection with TFA and tested this by
synthesizing 5 from a different direction as described in Scheme
1
1. Comparison of the H NMR and mass spectral data of 5 to
that of the impurity confirmed that they were identical. Both
compounds 5 and 13 were carried forward into dose response
testing.
In Table 1, we provide the FLIPR assay (NTS2) and binding
assay (NTS1 and NTS2) data collected for the reference
compounds NT and 3b as well as the test compounds 5 and 13.
In the FLIPR assay, the reference compounds performed as
expected. NT showed no agonist activity up to 100 mM, but as
previously reported, the Ke experiment with NT at NTS2
versus 3b demonstrated insurmountable antagonist activ-
ity.22,27,28 For this reason, we determined and report here its
IC50 value of 114 nM. In the binding assay at NTS2, NT
provided a Ki value of 28 nM. Reference compound 3b gave an
EC50 of 22 nM (100% Emax) in the FLIPR assay and a Ki of 5.8
nM in the binding assay.
Figure 2. IC50 assays were run against the EC80 of 3b (73 nM final
concentration). The IC50 curves for 5 and NT in the NTS2 FLIPR
assay demonstrate that 5 is almost 3-fold more active than the
endogenous ligand NT.
Neither of the test compounds (5 or 13) showed activity in
the NTS1 FLIPR assay (data not shown), but in the NTS2
FLIPR assay, they both behaved like NT. Each compound
antagonized the action of 3b in the Ke experiment in an
insurmountable manner. The graph of the Ke experiment for 5
is illustrated in Figure 1. Both 5 and 13 were tested in the IC50
experiment and gave IC50 values of 38 and 3322 nM,
respectively. The results from the IC50 experiment of NT and
5 versus 3b are illustrated in Figure 2.
In the binding assays at NTS2, 5 and 13 showed Ki values of
202 and 1504 nM, respectively, versus 28 nM for NT. Testing
of compounds 5 and 13 at the NTS1 receptor showed no
binding activity in the NTS1 assay up to 30 μM, making 5
selective for NTS2 in both the binding and functional assays.
A comparison of FLIPR data for compounds 5 and 13
revealed that the impurity 5 was not only 90-fold more active
than the target compound 13 but also 3-fold more active than
the endogenous ligand NT. This result was unexpected, as the
impurity was found to be far more interesting than the intended
compound. Indeed, it is unlikely that this discovery would have
been made if not by accident as substitution with the
trifluoroacetyl group is not a common practice in medicinal
chemistry. Overall, however, we have identified a molecular
region of 5 that is very important to antagonism of calcium
mobilization at NTS2.
The activity of both 5 and 13 in the NTS2 FLIPR and
binding assays provides additional evidence that the calcium
release mediated by 3b is an NTS2 mediated event. Also, a
comparison of their binding data at NTS2 revealed that the
trifluoroacetyl substitution of 5 did not increase the binding
affinity to the same degree observed for the FLIPR assay. We
found a 90-fold boost to antagonist activity for 5 versus 13 in
the FLIPR assay and a 7.5-fold boost to binding affinity.
Although these need not necessarily be equivalent, we suspect
that this could be a consequence of measuring the two end
points using two different reference compounds. In the FLIPR
assay, 5 is believed to compete directly with 3b, while in the
binding assay 5 is competing against NT. In most assay
systems, the endogenous ligand (or another more active
compound) serves as both the reference compound in the
functional assay and the radioligand in the binding assay. In this
case, however, the endogenous ligand (NT) does not mobilize
calcium. Thus, we would require radioactive 3b in order to
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and the reaction stirred at RT for 2 h. This mixture was then
concentrated to dryness and the residue dissolved in a minimal
amount of CH2Cl2, and then 2 mL each of ethyl ether and hexanes was
added to precipitate the acid. This was filtered, washed with hexanes,
and then dried under vacuum to provide 5 (67.3 mg, 86%) as an off-
white powder. 1H NMR (CD3OD) δ 8.71 (d, J = 8.0 Hz, 1H), 8.29 (d,
J = 1.6 Hz, 1H), 7.99 (d, J = 2.0 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H),
7.37 (d, J = 8.9 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.20 (dd, J = 2.0, 8.9
Hz, 1H), 4.97−5.15 (m, 2H), 4.38−4.53 (m, 1H), 2.35 (s, 3H), 1.58−
1.79 (m, 3H), 0.94 (dd, J = 5.9, 19.5 Hz, 6H). 13C NMR (CD3OD) δ
21.39, 21.69, 23.35, 26.11, 41.51, 50.53, 52.35, 103.00, 104.59, 110.56,
112.39, 116.26, 120.93, 128.39, 128.54, 130.51, 135.49, 136.26, 137.95,
achieve parity. While this is possible, radioactive NT is
commercially available and far more convenient to obtain.29
Empirically, however, we observe that every compound so far
identified that inhibits 3b in the FLIPR assay also competes
with NT in the binding assay, suggesting that the difference in
magnitude of effect does not prevent the detection of NTS2
active compounds.
As noted above, we do not know that 5 is competing directly
with 3b for its binding site, this is only a working assumption.
There is, however, empirical evidence that supports the
assumption that the binding sites for 3b and NT are, at least
partially, overlapping. The most important observation is that
NT, 3a, 3b, and 5 all rely heavily upon a free carboxylic acid
group for binding to NTS2 (and NTS1). In studies of the
NTS1 receptor, the ionic interaction established by the binding
of this group to an arginine residue in the receptor, formed the
principle ligand to receptor stabilizing interaction.30 Along with
this, a partially overlapping scenario has been postulated for NT
and 3a binding sites based on evidence obtained from
molecular modeling and point mutation studies at the NTS1
receptor.31 Although not conclusive, these observations do offer
support to the notion that these compounds all share partially
overlapping binding sites.
142.01, 142.09, 144.94, 168.73, 175.58. 19F NMR (CD3OD) δ −74.02.
25
D
MS (ESI): 552.7 (M − H)−; [α]
(C25H26F3N3O6S): C, H, N.
−17.1 (c 0.62, CH3OH). Anal.
Methyl (5-Nitro-1H-indol-1-yl)acetate (8). Into a 250 mL RBF
with a magnetic stir bar are placed 5-nitroindole (7, 4.864 g, 0.030
mol) and K2CO3 (4.561 g, 0.033 mol) and 90 mL of acetone. The
methyl bromoacetate (5.048 g, 0.033 mol, 3.10 mL) was added over a
minute via syringe and then stirred 4 days at RT. The reaction was
then concentrated, poured into water, and extracted with EtOAc (2×),
and the combined organics were washed (H2O, brine) and dried
(Na2SO4) and concentrated to give a yellow oil. This was
chromatographed on an ISCO silica gel column (80 g) using a 0−
60% EtOAc/hexanes gradient and then crystallized from ethyl acetate
1
to give the desired product as a yellow solid (4.38 g, 65% yield). H
NMR (CDCl3) δ 8.60 (d, J = 2.1 Hz, 1 H), 8.14 (dd, J = 2.2, 9.1 Hz, 1
H), 7.26 (d, J = 9.1 Hz, 1 H), 7.25 (d, J = 3.3 Hz, 1 H), 6.75 (d, J = 3.2
Hz, 1 H), 4.92 (s, 2 H), 3.78 (s, 3 H).
SUMMARY
■
The work presented herein provides additional evidence that
3b can be used as an agonist in the NTS2 FLIPR assay to
identify novel NTS2 active compounds. Because NT is an
antagonist of 3b, novel NT-like antagonist compounds can be
identified. To our knowledge, 5 is the first nonpeptide
compound that behaves like, and is more active than, the
endogenous ligand in this functional assay. This work
compliments our discovery of compound 4, providing an
additional nonpeptide compound whose in vitro properties
align with those of compounds that are known to be active in
models of pain. Together, 4 and 5 should be useful in
additional studies related to the biology of the NT receptors.
We are evaluating these compounds in animal models of pain
to establish the relevance of this assay with respect to
identification of potential NTS2-based analgesics. Additional
SAR studies with 5 are in progress and will be reported in the
near future.
(5-Nitro-1H-indol-1-yl)acetic Acid (9). To a solution of 8 (10.10
g, 0.045 mol) in 50 mL of 1,4-dioxane was added 50 mL of 1 N LiOH,
and this was stirred overnight at RT. The reaction was concentrated to
remove dioxane and then diluted with 100 mL of H2O and acidified to
pH 2. This was then extracted with 100 mL of ethyl acetate (3×), and
the combined organic layers were dried over Na2SO4. Concentration
1
gave 9 (8.72 g, 88%) as a white solid. H NMR (DMSO-d6) δ 13.13
(br s, 1H), 8.58 (d, J = 2.3 Hz, 1 H), 8.03 (dd, J = 2.3, 9.0 Hz, 1 H),
7.66−7.58 (m, 2 H), 6.76 (d, J = 3.2 Hz, 1 H), 5.16 (s, 2 H). MS
(ESI): 219.3 (M − H)−.
tert-Butyl N-[(5-Nitro-1H-indol-1-yl)acetyl]-L-leucinate (10).
Into a 100 mL RBF with stir bar containing 9 (2.202 g, 0.010 mol),
(S)-(L)-leucine tert-butyl ester HCl salt (2.461 g, 0.011 mol), and
HBTU (4.172g, 0.011 mol) was added CH2Cl2 (50 mL) and then
Et3N (3.036 g, 0.030 mol, 4.18 mL), and this was stirred at RT
overnight. The reaction mixture was then concentrated and chromato-
graphed on an 80 g ISCO silica gel column using a 10−60% gradient
of EtOAc in hexanes to give 10 (4.07 g) as a yellow oil which
crystallized when dried under vacuum to give a yellow−orange solid
1
EXPERIMENTAL SECTION
(3.859 g, 99%). H NMR (CDCl3) δ 8.59 (d, J = 2.2 Hz, 1 H), 8.13
■
(dd, J = 2.2, 9.0 Hz, 1 H), 7.35 (d, J = 9.0 Hz, 1 H), 7.31 (d, J = 3.3
Hz, 1 H), 6.80 (dd, J = 0.5, 3.2 Hz, 1 H), 5.96 (d, J = 8.3 Hz, 1 H),
4.88 (s, 2 H), 4.50 (dt, J = 5.2, 8.5 Hz, 1 H), 1.59−1.42 (m, 2 H), 1.40
(s, 9 H), 0.85 (dd, J = 3.3, 6.2 Hz, 6 H). MS (ESI): 360.5 (M + H)+.
tert-Butyl N-[(5-Amino-1H-indol-1-yl)acetyl]-L-leucinate (11).
10 (3.894 g, 0.010 mol) was dissolved in ethanol (200 mL) and
reduced using a Parr apparatus with 10% Pd/C (400 mg) under 40
psig of H2. The reaction mixture was filtered through Celite, the cake
was washed with EtOH, and the filtrate was concentrated under
vacuum. Drying under HVAC gave 11 (3.620 g, 100%) as a red oil.
[NOTE: This material darkens with exposure to air.] 1H NMR
(CDCl3) δ 7.09 (d, J = 8.6 Hz, 1 H), 7.01 (d, J = 3.1 Hz, 1 H), 6.96 (d,
J = 2.1 Hz, 1 H), 6.72 (dd, J = 2.2, 8.6 Hz, 1 H), 6.44 (d, J = 2.7 Hz, 1
H), 5.62 (d, J = 8.5 Hz, 1 H), 4.74 (s, 2 H), 4.45 (dt, J = 5.1, 8.7 Hz, 1
H), 1.50−1.41 (m, 2 H), 1.36 (s, 9 H), 1.31−1.20 (m, 1 H), 0.81 (dd,
J = 6.4, 7.8 Hz, 6 H). MS (ESI): 358.5 (M − H)−, 360.3 (M + H)+.
tert-Butyl N-({5-[(4-Methylphenylsulfonyl)amino]-1H-indol-
1-yl}acetyl)-L-leucinate (12). 11 (3.595 g, 0.010 mol) was dissolved
in CH2Cl2 (50 mL) in a 250 mL RBF equipped with a stir bar. DMAP
(1.344 g, 0.011 mol) was added, followed by a solution of tosyl
chloride (2.002 g, 0.011 mol) in CH2Cl2 (20 mL). The reaction was
Reactions were conducted under a nitrogen atmosphere using oven-
dried glassware as required. All solvents and chemicals used were
reagent grade. 5-Nitroindole (7) was purchased from Combi-blocks,
Inc. Flash column chromatography was carried out using a Teledyne
ISCO Combiflash Rf system and Redisep Rf gold prepacked HP silica
columns. Flow rates are automatically determined by column size.
Fractions were collected as “peaks only” over 30 min. The purity of
target compounds was determined to be ≥95% by combustion
analysis. NMR spectra were recorded on a Bruker Avance DPX-300
(300 MHz). Low-resolution mass spectra were obtained using a
Waters Alliance HT/Micromass ZQ system (ESI) in both positive and
negative mode. Optical rotations were measured on an Auto Pol III
polarimeter at the sodium D line. Thin layer chromatography (TLC)
was performed on EMD precoated silica gel 60 F254 plates, and spots
were visualized with UV light and I2 or phosphomolybdic acid stain.
CHO-k1 cells were from the American Type Culture Collection, and
[
125I]NT was from PerkinElmer. Calcium 5 dye was from Molecular
Devices. Cell culture reagents were from Life Technologies.
N-{[5-{[(4-Methylphenyl)sulfonyl]amino}-3-(trifluoroacetyl)-
1H-indol-1-yl]acetyl}-L-leucine (5). 14 (78.0 mg, 0.14 mmol) was
dissolved in dry CH2Cl2 (2 mL), and to this was added TFA (2 mL)
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stirred overnight and then concentrated and chromatographed on an
80 g silica gel cartridge with a 0−80% EtOAc/hexanes gradient. The
product crystallized from the fractions upon standing to give 12
(4.65g, 91%) as colorless crystals. 1H NMR (CDCl3) δ 7.61 (d, J = 8.2
Hz, 2 H), 7.39 (d, J = 1.9 Hz, 1 H), 7.18 (s, 2 H), 7.16 (s, 1 H), 7.12−
7.09 (m, 1 H), 6.95 (dd, J = 1.9, 8.8 Hz, 1 H), 6.52 (d, J = 3.2 Hz, 1
H), 5.75 (d, J = 8.5 Hz, 1 H), 4.77 (s, 2 H), 4.46 (dt, J = 5.1, 8.5 Hz, 1
H), 2.35 (s, 3 H), 1.53−1.41 (m, 3 H), 1.37 (s, 9 H), 0.79 (t, J = 6.2
Hz, 6 H). MS (ESI): 514.3 (M + H)+.
Data Analysis. To determine EC50 and IC50 values, data were fit to
a three-parameter logistic equation using GraphPad Prism software. Ki
values for radioligand binding assays were determined from IC50 values
using the equation of Cheng and Prusoff. All data are from at least
three independent experiments run in duplicate wells.
ASSOCIATED CONTENT
* Supporting Information
■
S
Elemental analysis results for test compounds 5, 13, and 14.
This material is available free of charge via the Internet at
http://pubs.acs.org.
N-[(5-{[(4-Methylphenyl)sulfonyl]amino}-1H-indol-1-yl)-
acetyl]-L-leucine (13). 12 (51.4 mg, 0.1 mmol) was dissolved in dry
CH2Cl2 (2 mL), and then TFA was added (2 mL) and the reaction
stirred at RT for 30 min. This was then concentrated to dryness and
the residue dissolved in a minimal amount of CH2Cl2/MeOH and
then triturated with 2 mL each of ethyl ether and hexanes to
precipitate the acid as an off-white powder. This was collected by
filtration and washed with hexanes, and then the solids were dried
under vacuum. This was then purified on a 4 g ISCO silica gel column
using a gradient of 0−10% MeOH/NH4OH (10:1) in CH2Cl2 to
provide the 13 (11.7 mg, 26%) as a off-white solid. 1H NMR
(CD3OD) δ 7.55 (d, J = 8.3 Hz, 1H), 7.23−7.31 (m, 1H), 7.16−7.23
(m, 1H), 7.02−7.15 (m, 2H), 6.98 (t, J = 8.5 Hz, 1H), 6.89 (dt, J = 2.0,
4.3 Hz, 1H), 6.82 (dd, J = 2.0, 8.8 Hz, 1H), 6.73 (dd, J = 2.0, 8.8 Hz,
1H), 6.40 (d, J = 3.0 Hz, 1H), 4.72−4.86 (m, 2H), 4.40−4.62 (m,
1H), 2.16−2.41 (m, 3H), 1.52−1.79 (m, 4H), 0.81−0.98 (m, 6H). 13C
NMR (CD3OD) δ 21.42, 21.75, 23.38, 26.05, 41.57, 50.04, 52.20,
102.88, 110.73, 116.78, 119.64, 128.37, 130.38, 130.42, 130.90, 131.35,
AUTHOR INFORMATION
Corresponding Author
*Phone: 919-541-6375. Fax: 919-541-6499. E-mail: jbthomas@
rti.org.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
136.16, 138.10, 144.60, 170.62, 175.76. MS (ESI): 456.3 (M − H)−;
We gratefully acknowledge the NIMH Chemical Synthesis and
Drug Supply Program for providing us with the samples of
SR142948a (3b), and SR48692 (3a). This research was
supported by the National Institute on Drug Abuse, grant
DA029961.
25
[α]
−8.84 (c 0.37, CH3OH). Anal. (C23H27N3O5S): C, H, N.
D
tert-Butyl N-{[5-{[(4-Methylphenyl)sulfonyl]amino}-3-(tri-
fluoroacetyl)-1H-indol-1-yl]acetyl}-L-leucinate (14). 12 (128.4
mg, 0.25 mmol) was dissolved in CH2Cl2 (5 mL) and to this was
added trifluoroacetic anhydride (57.8 mg, 0.04 mL, 0.28 mmol) and
the reaction was stirred at RT for 2 h. The reaction was then
concentrated to a solid film and purified on a 4 g ISCO silica gel
column using a 0−60% EtOAc/hexanes gradient. The desired fractions
were concentrated to provide 14 (85.9 mg, 56%) as a colorless film.
1H NMR (CDCl3) δ 7.92 (d, J = 1.5 Hz, 1H), 7.84 (s, 1H), 7.76 (s,
ABBREVIATIONS USED
■
NT, neurotensin; NTS1, neurotensin receptor 1; NTS2,
neurotensin receptor 2; CNS, central nervous system 5; H1,
histamine receptor 1; FLIPR, fluorometric imaging plate reader;
CHO, Chinese hamster ovary
1H), 7.61 (d, J = 8.3 Hz, 2H), 7.10−7.19 (m, 4H), 6.83 (d, J = 8.3 Hz,
1H), 4.88 (s, 2H), 4.49−4.62 (m, 1H), 2.31 (s, 3H), 1.51−1.68 (m,
3H), 1.46 (s, 9H), 0.90 (d, J = 3.9 Hz, 6H). MS (ESI): 609.2 (M −
H)−.
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