Potency enhancement of the κ-opioid receptor antagonist probe ML140 through sulfonamide constraint utilizing a tetrahydroisoquinoline motif

Article abstract of DOI:10.1016/j.bmc.2014.12.033

Optimization of the sulfonamide-based kappa opioid receptor (KOR) antagonist probe molecule ML140 through constraint of the sulfonamide nitrogen within a tetrahydroisoquinoline moiety afforded a marked increase in potency. This strategy, when combined wit

Full text of DOI:10.1016/j.bmc.2014.12.033

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Potency enhancement of the j-opioid receptor antagonist probe
ML140 through sulfonamide constraint utilizing a
tetrahydroisoquinoline motif
Kevin J. Frankowski a, Stephen R. Slauson a, Kimberly M. Lovell b, Angela M. Phillips b,
John M. Streicher b, Lei Zhou b, David A. Whipple a, Frank J. Schoenen a, Thomas E. Prisinzano a,
a,
Laura M. Bohn b, , Jeffrey Aubé
⇑
⇑
a University of Kansas Specialized Chemistry Center, 2034 Becker Drive, Lawrence, KS 66047-3761, USA
b Departments of Molecular Therapeutics and Neuroscience, The Scripps Research Institute, 130 Scripps Way, #2A2, Jupiter, FL 33458, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization of the sulfonamide-based kappa opioid receptor (KOR) antagonist probe molecule ML140
through constraint of the sulfonamide nitrogen within a tetrahydroisoquinoline moiety afforded a
marked increase in potency. This strategy, when combined with additional structure–activity relation-
ship exploration, has led to a compound only six-fold less potent than norBNI, a widely utilized KOR
antagonist tool compound, but significantly more synthetically accessible. The new optimized probe is
suitably potent for use as an in vivo tool to investigate the therapeutic potential of KOR antagonists.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 15 September 2014
Revised 12 December 2014
Accepted 16 December 2014
Available online xxxx
Keywords:
Kappa opioid receptor
Antagonist
Molecular constraint
Potency enhancement
Tetrahydroisoquinoline
1. Introduction
structurally-distinct KOR antagonists with novel pharmacological
properties, including a focus on KOR antagonists that are more
Activation of the kappa opioid receptor (KOR) by endogenous
neuropeptides, primarily dynorphin, initiates complex signaling
cascades. The downstream effects of KOR agonism vary greatly
and include antinociception, dysphoria and anxiety, though the
details of the pharmacological pathways are still being eluci-
dated.1,2 In contrast, KOR antagonists have been investigated as
potential therapeutic treatments for addiction, depression, post
traumatic stress disorder, eating disorders and other conditions
related to anxiety or aversion-reward responses.3–5 Many canonical
KOR antagonists (Fig. 1) are derived from, or bear a structural ele-
ment of morphinan opioids, such as the widely-utilized tool com-
pounds norBNI,6 50-GNTI7 and JDTic.8 Some KOR antagonists that
are widely used, such as norBNI, have a remarkable long duration
of action in animal models (weeks), which could introduce difficul-
ties in interpreting the effect of chronic antagonist exposure, or
some other effects of the drug that are not fully understood.9–12
Accordingly, one goal of our laboratories is to discover and evaluate
rapidly cleared in vivo. We recently disclosed a new sulfonamide
series of KOR antagonists, exemplified by the Molecular Libraries
probe molecule ML140 (Fig. 1).13
Although ML140 only exhibited modest potency in the Discov-
eRx barrestin2 PathHunter™ assay (403 nM), it was selective
against the
l and d receptors (IC50 values of >24 and >32 lM,
respectively). In contrast to the better-established chemotypes
noted, ML140 is highly modular, possesses no stereogenic centers
and bears little structural similarity to known opioid ligands.
Together, these properties inspired us to further investigate the
structure–activity relationship (SAR) of this chemotype. In our ori-
ginal report,13 we evaluated the effect on biological potency
through ca. forty structural changes focused primarily on the ‘east-
ern’ portion of the molecule (Fig. 2). We investigated both replac-
ing the substituents of the basic nitrogen as well as introducing
constraint into the eastern region of the molecule. Perhaps most
interestingly, we found that replacing the N-isopropyl with N-
tert-butyl can increase the potency by as much as 14-fold. Less suc-
cessful were our attempts to enhance antagonist potency through
constraint of the nitrogen within various ring systems as illus-
trated by the structures in Figure 2.
⇑
Corresponding authors. Tel.: +1 (561) 228 2227; fax: +1 (561) 228 3081
(L.M.B.); tel.: +1 (785) 864 4496; fax: +1 (785) 864 8179 (J.A.).
E-mail addresses: lbohn@scripps.edu (L.M. Bohn), jaube@ku.edu (J. Aubé).
http://dx.doi.org/10.1016/j.bmc.2014.12.033
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Frankowski, K. J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.033

2
K. J. Frankowski et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
two, we chose this assay for primary SAR determination. The ana-
NH
logues presented here were prepared in the manner of those previ-
ously reported,11 which is summarized in Scheme 1. Briefly, the
tetrahydroisoquinoline core was reacted with the appropriate sul-
fonyl chloride and subsequently saponified to afford the western
fragment bearing a carboxylic acid. Activation of the carboxylic
acid to the acid chloride and coupling with the diamine fragments
afforded the final analogues. All final compounds were purified by
mass-directed, reverse-phase HPLC and submitted to biological
assays at ꢀ 94% purity.
HN
N
NH2
N
HO
OH
O
N
OH
O
N
H
O
OH
N
H
OH
OH
norbinaltorphimine (norBNI)
5'-guanidinonaltrindole (GNTI)
We began this investigation by preparing the analogue where
both the sulfonamide and amide nitrogens of ML140 were methyl-
ated, which resulted in the loss of all KOR antagonist activity
(Table 1, entry 1a) and suggesting that at least one of or both N–
H groups were essential for activity. To determine if modification
at the sulfonamide nitrogen was allowable, we first investigated
the effect of tethering the sulfonamide nitrogen to the central ben-
zene ring. We chose the tetrahydroisoquinoline carboxylate scaf-
fold since it was both readily available and mapped directly onto
the template established by ML140. Thus, we synthesized twelve
tetrahydroisoquinoline analogues bearing modifications around
the basic nitrogen fragment and substitution on the aryl sulfon-
amide group. The results of screening these analogues in the three
KOR antagonist assays are presented in Table 1. To facilitate com-
parison to our previous SAR efforts and the broader KOR antagonist
literature, norBNI, ML140, and the bromo analogue of ML140, 1b,
were included in this study. Notably, the tetrahydroisoquinoline
analogues were all significantly more potent than either ML140
or 1b. Directly comparing 1b to the tetrahydroisoquinoline with
the same substitution, 1f, shows an eight-fold improvement in
potency. A further two-fold increase in potency was achieved
through a bromo to chloro replacement (i.e., 1f to 1e). The effect
of changing from isopropyl to tert-butyl was even more pro-
nounced than was observed in the context of ML140, providing
for the first time in this series compounds possessing single-digit
nanomolar potency. Halogen incorporation further improved the
potency, resulting in the nearly equipotent fluoro analogue 1j,
chloro analogue 1l and the corresponding bromide 1m. To date,
compound 1l is the most potent compound in the series, as mea-
sured by its activity in inhibiting G protein function. Throughout
this study, 2,4,6-trimethyl aryl sulfonamide substitution led to
notably less potent compounds when compared to either the ben-
zene sulfonamide, 4-methyl- or 4-ethyl-substituted aryl sulfon-
amide analogues. However they are still more potent than
ML140, nicely illustrating the positive influence of the tetrahydro-
isoquinoline moiety on the SAR of this chemotype.
w
e
s
t
e
r
Me
n
f
r
a
HO
Me
g
m
e
n
Me
O
t
S
ea
st
O
HO
N
H
e
N
O
Me
Me
r
n
f
ra
gm
H
N
e
nt
NH
N
H
O
N
JDTic
ML140
Figure 1. Representative widely utilized KOR antagonist ligands and ML140.
Each of these changes eradicated essentially all KOR activity,
suggesting that the constraints examined placed this portion of
the molecule into a conformation inconsistent with receptor activ-
ity, introduced unpalatable steric clashes, deprived the compound
of an important hydrogen bond, or some combination of these
effects. Herein we describe a more fruitful approach to conforma-
tional constraint by focusing on the modification of the sulfon-
amide portion of the molecule while retaining the promising
substitution patterns around the basic nitrogen. This process affor-
ded a series of antagonists with single-digit nanomolar potency in
G protein coupling and consistent SAR trends across the series.
2. Results and discussion
In this investigation we measured the potency of all test com-
pounds by three complementary methods for assessing KOR antag-
onist potency:14
[ cS
35S]GTP binding,15 ERK activation,16 and
barrestin2 recruitment.17 Since our compounds, like norBNI, are
found to be most potent in the [35S]GTP
c
S assay over the other
Me
(all analogues IC50 > 18,000 nM)
Me
O
S
N
O
previous
efforts
H
O
H
N
N
S
N
H
O
ML140
O
H
N
N
([35S]GTPγS IC50 = 138 nM)
Me
Me
O
O
O
this
S
O
work
N
H
H
N
Me
N
O
S
O
O
N
H
S
O
N
H
N
N
H
N
N
O
Cl
O
1l
([35S]GTPγS IC50 = 1.6 nM)
Figure 2. Conformational constraints explored in ML140.
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K. J. Frankowski et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
i) SOCl2
ii)
H2N
N
R2
R3
R1
3
a
2 = iso-propyl
R
3 = H
i)
R
O
4 R2 = tert-butyl b R3 = fluoro
c R3 = chloro
R1
S
R1
O
Cl
O
O
HN
S
O
S
O
d R3 = bromo
ii) LiOH
(aq)
N
N
O
H
N
OH
N
R2
O
O
O
R3
R
1 = 4-methyl
2a
2b R1 = 4-ethyl
2c R1 = 2,4,6-trimethyl
Scheme 1. General scheme for the preparation of 2-(arylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxamide derivatives.
Table 1
KOR antagonist potency of ML140 analogues
IC50 (nM) S.E.M.a
Entry/compd
R1
R2
R3
[
35S]GTP
c
S
ERK
barr2
norBNI
ML140
—
—
—
—
—
—
0.28 0.03
138 54
4.6 0.7
591 87
2.5 0.3
403 86b
1a
Inactive
Inactive
Inactive
1b
168 48
750 170
937 190c
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
2,4,6-Me
4-Me
4-Me
4-Me
4-Et
4-Me
4-Et
4-Me
2,4,6-Me
4-Me
i-Pr
i-Pr
i-Pr
i-Pr
H
F
113 42
29.1 7.9
10.0 1.4
19.7 6.6
33 11
5.3 0.6
3.4 0.4
3.7 1.2
85 20
1120 110
406 80
400 87
258 93
314 56
49.1 9.7
143 23
76 24
930 150
478 110
392 91
388 85
350 68
Cl
Br
Br
H
H
F
i-Pr
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
tert-Bu
104
8
186 57
104 32
880 67
84 20
93 30
710 150
F
1200 300
107 17
87 17
Cl
Br
Br
1.6 0.5
2.9 1.2
106 16
1m
1n
4-Me
2,4,6-Me
1340 410
a
n P3. All compounds fully blocked U69,593 (>86%). Test compounds were run against the approximate EC80 of U69,593.
Previous testing of this compound at Sanford-Burnham Medical Research Institute measured a barrestin2 PathHunter™ assay potency of 860 nM; here we report the
b
contemporary and optimized value of 403 nM.13
C
Previous testing of this compound at Sanford-Burnham Medical Research Institute measured a DiscoveRx barrestin2 PathHunter™ assay potency of 660 nM; here we
report the contemporary and optimized value of 937 nM.13
Another interesting feature is the differential potencies noted
for many of the analogs against G protein function, as revealed in
downstream in both the G protein and barrestin2 pathways.
Although possibly intriguing in light of current interest in func-
tional selectivity in GPCR agonism, the functional relevance of
any such differences in the present series of antagonists is cur-
rently unknown.
the [35S]GTP
cS assay, relative to barrestin2 recruitment or ERK
phosphorylation. For example, simple comparison of the relevant
IC50 values obtained in each assay suggests that the compounds
may be more potent in inhibiting G protein function as opposed
to barrestin2 recruitment, although they are less potent than nor-
BNI in both assays. Whether this suggests a bias in the affinity
imparting differential active states for KOR remains to be deter-
mined as such mathematical modeling parameters have yet to be
defined for comparing compounds that do not reveal stimulatory
efficacy. Differences also appear in relative potencies for inhibiting
ERK phosphorylation, but may vary because the ERK is
We had previously profiled the lead compound for this study,
ML140, in radioligand binding assays and observed selectivity for
the KOR compared to the d opioid receptor (DOR) and modest
selectivity compared to the
l
opioid receptor (MOR).13 To deter-
mine if ML140 analogues had off target effects, 1f, 1h and 1l were
also profiled through the Psychoactive Drug Screening Program at
the University of North Carolina, Chapel Hill for KOR, DOR and
MOR binding affinity (Table 2). Gratifyingly, the more potent
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4
K. J. Frankowski et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
KOR, DOR and MOR binding affinity for ML140 and select analogues
Receptor binding affinity, Ki valuesa (nM)
Entry/compd
R1
R2
R3
KOR
DOR
1088
5190
2328
MOR
ML140
1f
1h
—
—
i-Pr
tert-Bu
tert-Bu
—
Br
H
50
73
37
53
306
8170
1749
4-Me
4-Me
4-Me
1l
Cl
>10,000
>10,000
a
Ki determinations calculated from a 12-point concentration response curve (n = 1) conducted by the Psychoactive Drug Screening Program using the rat KOR expressed in
HEK-293 cells. In those studies, the KD of U69,593 was determined to be 1 nM.
antagonists were found to be more selective in radioligand binding
assays than ML140. For example, 1l possessed a binding affinity
(Ki) at KOR of 53 nM and binding affinity (Ki) of >10,000 nM at both
the DOR and MOR.
sample) on an Agilent 1200 instrument with photodiode array
detector, an Agilent 6120 quadrupole mass spectrometer, and a
HTPAL LEAP autosampler. Fraction collection was triggered using
an MS and UV threshold determined by HPLC/MS analysis of the
crude sample. One of two column/mobile phase conditions was
chosen for both analysis and purification to promote the target
compound’s neutral state (0.02% formic acid with Waters Atlantis
3. Conclusion
T3 5
C18 5
l
l
M, 19 ꢂ 150 mm; or pH 9.8 NH4OH with Waters XBridge
We have improved the antagonist potency of the sulfonamide-
based KOR antagonist probe molecule ML140 from 138 nM to
M, 19 ꢂ 150 mm).
1.6 nM as measured by [35S]GTP
cS binding to afford a new molec-
ular tool appropriate to investigate the physiological role of the
KOR and the therapeutic potential of KOR antagonists. The con-
strained sulfonamide analogue 1l is almost 100-fold more potent
than ML140 and permits for the first time the potential use of this
chemotype in behavioral or in vivo efficacy models. These experi-
ments are currently planned as well as further pharmacological
characterization and the synthesis of additional analogues. The
results of these studies will be disclosed as appropriate.
4.2. Synthesis of carboxylic acid fragments 2a–2c
4.2.1. Methyl 2-tosyl-1,2,3,4-tetrahydroisoquinoline-6-
carboxylate
To a solution of methyl 1,2,3,4-tetrahydroisoquinoline-6-car-
boxylate (171 mg, 0.894 mmol) and triethylamine (271 mg,
2.68 mmol, 3.0 equiv) in CH2Cl2 (15 mL) was added p-toluenesulfo-
nyl chloride (256 mg, 1.34 mmol, 1.5 equiv). The reaction was stir-
red at rt for 15 h, diluted with 1 N HCl and extracted with CH2Cl2.
The combined organics were dried (Na2SO4), concentrated and
purified by silica gel chromatography to afford the sulfonamide
4. Experimental
4.1. General methods
product as
a white solid (231 mg, 0.669 mmol, 75% yield).
Mp = 143–145 °C; Rf = 0.34 (25% EtOAc/hexanes); 1H NMR
(400 MHz, CDCl3) d 2.41 (s, 3H), 2.96 (t, J = 6.0 Hz, 2H), 3.36 (t,
J = 6.0 Hz, 2H), 3.88 (s, 3H),4.28 (s, 2H), 7.10 (d, J = 8.0 Hz, 1H),
7.33 (d, J = 8.0 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H), 7.77 (s, 1H), 7.79
(d, J = 8.0 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence)
d d 21.5, 52.1, 126.6, 127.4, 127.7, 129.8, 130.2; u 28.8, 43.6, 47.7,
128.7, 133.2, 133.4, 136.9, 143.9, 166.7; IR 1718 cmꢁ1; HRMS (ESI)
m/z calcd for C18H20NO4S ([M+H]+) 346.1108, found 346.1116.
All reagents and materials were purchased from commercial
vendors (Sigma, Alfa Aesar, Oakwood or ASW Medchem) and used
as received. Ethyl ether, toluene, THF, MeCN and CH2Cl2 were
degassed with nitrogen and passed through two columns of basic
alumina on an Innovative Technology solvent purification system.
1H and 13C NMR spectra were recorded on a Bruker AM 400 spec-
trometer (operating at 400 and 100 MHz, respectively) in CDCl3
with 0.03% TMS as an internal standard, unless otherwise specified.
Chemical shifts are reported in parts per million (ppm) downfield
from TMS. 13C multiplicities were determined with the aid of an
APT pulse sequence, differentiating the signals for methyl and
methane carbons as ‘d’ from methylene and quaternary carbons
as ‘u’. The infrared (IR) spectra were acquired as thin films using
a universal ATR sampling accessory on a PerkinElmer Spectrum
100 FT-IR spectrometer and the absorption frequencies are
reported in cmꢁ1. Melting points were determined on a Stanford
Research Systems Optimelt automated melting point system inter-
faced through a PC and are uncorrected. ML140 and 1b were syn-
thesized as previously described.13
4.2.2. 2-Tosyl-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid
(2a)
To a solution of the above methyl ester (298 mg, 0.765 mmol) in
THF/MeOH/water (3:1:1, 10 mL) was added lithium hydroxide
monohydrate (160 mg, 3.83 mmol, 5 equiv) and the reaction stir-
red at rt for 15 h. The THF and MeOH were removed in vacuo
and the reaction concentrate was acidified with 2 N HCl, precipitat-
ing the carboxylic acid product as a sparingly soluble white solid
(213 mg, 0.643 mmol, 84% yield), which was filtered, washed with
water, dried under vacuum and used without further purification.
Mp = 234–240 °C; Rf = 0.58 (10% MeOH and 2% AcOH in CH2Cl2); 1H
NMR (400 MHz, DMSO-d6) d 2.39 (s, 3H), 2.91 (t, J = 6.0 Hz, 2H),
3.30 (t, J = 6.0 Hz, 2H), 4.25 (s, 2H), 7.28 (d, J = 8.0 Hz, 1H), 7.44
(d, J = 8.0 Hz, 2H), 7.70–7.74 (complex, 4H), 12.89 (br s, 1H); 13C
NMR (101 MHz, DMSO-d6, APT pulse sequence) d d 21.0, 126.7,
126.9, 127.4, 129.7, 129.9; u 27.9, 43.3, 47.3, 129.1, 133.0, 133.4,
136.7, 143.7, 167.0; IR 1678 cmꢁ1; HRMS (ESI) m/z calcd for C17H16
NO4S ([MꢁH]ꢁ) 330.0806, found 330.0807.
HPLC/MS analysis was carried out with gradient elution (5%
CH3CN to 100% CH3CN) on an Agilent 1200 RRLC with a photodiode
array UV detector and an Agilent 6224 TOF mass spectrometer
(also used to produce high resolution mass spectra). HPLC purifica-
tion was carried out by mass-directed fractionation (MDF) with
gradient elution (a narrow CH3CN gradient was chosen based on
the retention time of the target from LCMS analysis of the crude
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K. J. Frankowski et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
4.2.3. Methyl 2-((4-ethylphenyl)sulfonyl)-1,2,3,4-tetrahydro-
4.3. Conversion of carboxylic acids 2a–2c to the acid chlorides
isoquinoline-6-carboxylate
Methyl 1,2,3,4-tetrahydroisoquinoline-6-carboxylate (182 mg,
0.952 mmol) and 4-ethyl-benzenesulfonyl chloride (292 mg,
1.43 mmol, 1.5 equiv) were reacted according to the protocol in
Section 4.2.1. to afford the sulfonamide product as a white solid
(285 mg, 0.793 mmol, 83% yield). Mp = 128–130 °C; Rf = 0.40 (25%
EtOAc/hexanes); 1H NMR (400 MHz, CDCl3) d 1.23 (t, J = 7.6 Hz,
3H), 2.69 (q, J = 7.6 Hz, 2H), 2.95 (t, J = 6.0 Hz, 2H), 3.36
(t, J = 6.0 Hz, 2H), 3.87 (s, 3H), 4.28 (s, 2H), 7.09 (d, J = 8.4 Hz,
1H), 7.35 (d, J = 8.4 Hz, 2H), 7.74–7.79 (complex, 4H); 13C
NMR (101 MHz, CDCl3, APT pulse sequence) d d 14.9, 51.9,
126.4, 127.2, 127.7, 128.5, 130.0; u 28.62, 28.65, 43.5, 47.6,
4.3.1. 2-Tosyl-1,2,3,4-tetrahydroisoquinoline-6-carbonyl
chloride
The carboxylic acid 2a (145 mg, 0.438 mmol) was dissolved in
thionyl chloride (0.95 mL, 13.13 mmol, 30 equiv) and heated at
65 °C for 4 h. Excess thionyl chloride was removed in vacuo and
the residue azeotropically dried with toluene (3 ꢂ 5 mL) to afford
the acid chloride as a white solid (147 mg, 0.420 mmol, 96% yield),
which was used without further purification. 1H NMR (400 MHz,
CDCl3) d 2.36 (s, 3H), 2.94 (t, J = 5.9 Hz, 2H), 3.32 (t, J = 5.9 Hz,
2H), 4.25 (s, 2H), 7.11 (d, J = 8.2 Hz, 1H), 7.25–7.30 (m, 2H), 7.63–
7.69 (m, 2H), 7.77–7.85 (m, 2H).
128.6, 133.2, 133.3, 136.8, 149.8, 166.5; IR 1717 cmꢁ1
;
HRMS (ESI) m/z calcd for C19H22NO4S ([M+H]+) 360.1264, found
4.3.2. 2-((4-Ethylphenyl)sulfonyl)-1,2,3,4-tetrahydroisoquino-
line-6-carbonyl chloride
360.1274.
The carboxylic acid 2b (303 mg, 0.878 mmol) was reacted
according to the protocol in Section 4.3.1. to afford the acid chlo-
ride as a white solid (322 mg, 0.860 mmol, 98% yield), which was
used without further purification. 1H NMR (400 MHz, CDCl3) d
1.19 (t, J = 7.6 Hz, 3H), 2.65 (q, J = 7.6 Hz, 2H), 2.95 (t, J = 5.9 Hz,
2H), 3.33 (t, J = 5.9 Hz, 2H), 4.25 (s, 2H), 7.11 (d, J = 8.2 Hz, 1H),
7.27–7.32 (m, 2H), 7.65–7.71 (m, 2H), 7.77–7.85 (m, 2H).
4.2.4. 2-((4-Ethylphenyl)sulfonyl)-1,2,3,4-tetrahydroisoquino-
line-6-carboxylic acid (2b)
The above methyl ester (252 mg, 0.701 mmol) was reacted
according the protocol in Section 4.2.2. to afford the carboxylic acid
product as a sparingly soluble white solid (216 mg, 0.625 mmol,
89% yield), which was filtered, washed with water, dried
under vacuum and used without further purification. Mp = 208–
213 °C; Rf = 0.58 (10% MeOH and 2% AcOH in CH2Cl2); 1H NMR
(400 MHz, DMSO-d6) d 1.18 (t, J = 7.6 Hz, 3H), 2.68 (q, J = 7.6 Hz,
2H), 2.91 (t, J = 6.0 Hz, 2H), 3.31 (t, J = 6.0 Hz, 2H), 4.26 (s, 2H),
7.28 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.69–7.76
(complex, 4H), 12.91 (br s, 1H); 13C NMR (101 MHz, DMSO-d6,
APT pulse sequence) d d 15.0, 126.7, 126.8, 127.5, 128.7, 129.7; u
28.0, 43.3, 47.3, 129.1, 133.2, 133.4, 136.6, 149.6, 167.0; IR
4.3.3. 2-(Mesitylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-6-
carbonyl chloride
The carboxylic acid 2c (38 mg, 0.106 mmol) was reacted accord-
ing to the protocol in Section 4.3.1. to afford the acid chloride as a
white solid (39 mg, 0.103 mmol, 98% yield), which was used with-
out further purification. 1H NMR (400 MHz, CDCl3) d 2.31 (s,, 3H),
2.63 (s, 6H), 2.95 (t, J = 6.0 Hz, 2H), 3.493 (t, J = 6.0 Hz, 2H), 4.43
(s, 2H), 6.97 (s, 2H), 7.21 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.90 (d,
J = 8.4 Hz, 1H).
;
1683 cmꢁ1 HRMS (ESI) m/z calcd for C18H20NO4S ([M+H]+)
346.1113, found 346.1114.
4.2.5. Methyl 2-((2,4,6-trimethylphenyl)sulfonyl)-1,2,3,4-
tetrahydroisoquinoline-6-carboxylate
4.4. Synthesis of diamine fragments 4a–4d
Methyl 1,2,3,4-tetrahydroisoquinoline-6-carboxylate (191 mg,
0.999 mmol) and 2,4,6-trimethyl-benzenesulfonyl chloride
(328 mg, 1.50 mmol, 1.5 equiv) were reacted according to the pro-
tocol in Section 4.2.1. to afford the sulfonamide product as a white
solid (188 mg, 0.503 mmol, 50% yield). Mp = 142–143 °C; Rf = 0.50
(25% EtOAc/hexanes); 1H NMR (400 MHz, CDCl3) d 2.29 (s, 3H),
2.63 (s, 6H), 2.91 (t, J = 6.0 Hz, 2H), 3.47 (t, J = 6.0 Hz, 2H), 3.89
(s, 3H), 4.40 (s, 2H), 6.96 (s, 2H), 7.12 (d, J = 8.0 Hz, 1H), 7.81
(m, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) d d
21.0, 22.8, 52.1, 126.6, 127.4, 130.3, 132.0; u 28.6, 41.9, 45.9,
4.4.1. 2-(Benzyl(tert-butyl)amino)acetonitrile
To a solution of N-benzyl-tert-butylamine (930 mg, 5.70 mmol)
in MeCN (15 mL) was added K2CO3 (1575 mg, 11.40 mmol,
2 equiv), potassium iodide (946 mg, 5.70 mmol, 1 equiv) and chlo-
roacetonitrile (1720 mg, 22.79 mmol, 4 equiv). The reaction was
heated at 75 °C for 16 h, cooled to rt and partitioned between
water (150 mL) and ethyl ether (3 ꢂ 75 mL). The combined organic
layers were washed with brine (50 mL), dried (Na2SO4), adsorbed
onto celite and purified by silica gel chromatography to afford
the nitrile product as a colorless oil (846 mg, 4.18 mmol, 73%
yield). Rf = 0.49 (10% EtOAc/hexanes); 1H NMR (400 MHz, CDCl3)
d 1.29 (s, 9H), 3.43 (s, 2H), 3.82 (s, 2H), 7.23–7.28 (m, 1H), 7.29–
7.36 (complex, 4H); 13C NMR (101 MHz, CDCl3, APT pulse
sequence) d d 27.4, 127.5, 128.5, 128.6; u 35.7, 51.4, 55.2, 118.0,
138.9; IR 2975, 1454, 1366, 1202 cmꢁ1; HRMS (ESI) m/z calcd for
C13H19N2 ([M+H]+) 203.1543, found 203.1536.
128.7, 131.7, 133.8, 137.3, 140.5, 142.9, 166.7; IR 1718 cmꢁ1
;
HRMS (ESI) m/z calcd for C20H24NO4S ([M+H]+) 374.1426, found
374.1424.
4.2.6. 2-((2,4,6-Trimethylphenyl)sulfonyl)-1,2,3,4-tetrahydro-
isoquinoline-6-carboxylic acid (2c)
The above methyl ester (146 mg, 0.391 mmol) was reacted
according to the protocol in Section 4.2.2. to afford the carboxylic
4.4.2. N1-benzyl-N1-(tert-butyl)ethane-1,2-diamine (4a)
To
a solution of the nitrile from Section 4.4.1. (255 mg,
acid product as
a sparingly soluble white solid (137 mg,
1.26 mmol) in THF (25 mL) was added LiAlH4 solution (1 M in
THF, 5.0 mL, 5.04 mmol, 4 equiv) at rt. The reaction was stirred at
rt for 4 h and quenched by the careful addition of Na2SO4ꢃ10H2O
(20 g). The reaction was filtered and the solids washed with ethyl
ether (2 ꢂ 10 mL). The combined organic layers were washed with
brine (50 mL), dried (Na2SO4) and purified by silica gel chromatog-
raphy to afford the diamine fragment 4a as a colorless oil (64 mg,
0.31 mmol, 25% yield). Rf = 0.49 (10% MeOH + 2% NH4OH in CH2
Cl2); 1H NMR (400 MHz, CDCl3) d 1.12 (s, 9H), 2.43 (t, J = 6.4 Hz,
2H), 2.62 (t, J = 6.4 Hz, 2H), 3.67 (s, 2H), 7.18 (t, J = 7.2 Hz, 1H),
0.381 mmol, 97% yield), which was filtered, washed with water,
dried under vacuum and used without further purification.
Mp = 222–233 °C; Rf = 0.55 (10% MeOH and 2% AcOH in CH2Cl2);
1H NMR (400 MHz, DMSO-d6) d 2.28 (s, 3H), 2.55 (s, 6H), 2.87 (t,
J = 5.6 Hz, 2H), 3.43 (t, J = 5.6 Hz, 2H), 4.36 (s, 2H), 7.08 (s, 2H),
7.31 (d, J = 7.6 Hz, 1H), 7.71–7.73 (m, 2H); 13C NMR (101 MHz,
DMSO-d6, APT pulse sequence) d d 20.5, 22.3, 126.8, 126.9, 129.9,
131.9; u 27.8, 41.6, 45.5, 129.2, 133.8, 137.2, 139.6, 142.6, 167.1;
IR 1683 cmꢁ1; HRMS (ESI) m/z calcd for C19H22NO4S ([M+H]+)
360.1264, found 360.1270.
Please cite this article in press as: Frankowski, K. J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.033

6
K. J. Frankowski et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7.27 (t, J = 7.2 Hz, 2H), 7.35 (d, J = 6.8 Hz, 2H); 13C NMR (101 MHz,
CDCl3, APT pulse sequence) d d 27.5, 126.4, 127.7, 128.1; u 42.2,
54.4, 55.1, 55.3, 143.6; IR 2967, 2869, 1576, 1469, 1452,
4.4.8. N1-(tert-Butyl)-N1-(4-bromobenzyl)ethane-1,2-diamine
(4d)
To a solution of the nitrile from 4.4.7. (195 mg, 0.69 mmol) in
THF (20 mL) was added LiAlH4 solution (1 M in THF, 0.7 mL,
0.69 mmol, 1 equiv) at 0 °C. The reaction was stirred at 0 °C for
4 h and quenched by the careful addition of Na2SO4ꢃ10H2O (20 g).
The reaction was filtered and the solids washed with ethyl ether
(2 ꢂ 10 mL). The combined organic layers were washed with brine
(50 mL), dried (Na2SO4) and purified by silica gel chromatography
to afford the diamine fragment 4d as a colorless oil (106 mg,
0.37 mmol, 54% yield). Rf = 0.46 (10% MeOH + 2% NH4OH in CH2
Cl2); 1H NMR (400 MHz, CDCl3) d 1.10 (s, 9H), 2.44 (t, J = 6.4 Hz,
2H), 2.61 (t, J = 6.0 Hz, 2H), 3.61 (s, 2H), 7.23 (d, J = 8.4 Hz, 2H),
7.39 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz, CDCl3, APT pulse
sequence) d d 27.4, 129.4, 131.4; u 42.2, 54.5, 54.7, 55.3, 120.1,
142.6; IR 2968, 2868, 1596, 1473, 1451, 1360 cmꢁ1; HRMS (ESI)
m/z calcd for C13H22BrN2 ([M+H]+) 285.0966, found 285.0960.
1361 cmꢁ1
;
HRMS (ESI) m/z calcd for C13H23N2 ([M+H]+)
207.1861, found 207.1848.
4.4.3. 2-((4-Fluorobenzyl)(tert-butyl)amino)acetonitrile
N-(4-Fluoromobenzyl)-tert-butylamine (900 mg, 4.97 mmol)
was reacted according to the protocol in Section 4.4.1. to afford
the nitrile product as a colorless oil (1038 mg, 4.71 mmol, 95%
yield). Rf = 0.49 (10% EtOAc/hexanes); 1H NMR (400 MHz, CDCl3)
d 1.29 (s, 9H), 3.42 (s, 2H), 3.80 (s, 2H), 7.00(t, J = 8.8 Hz, 2H),
7.31 (dd, J = 5.6, 8.8 Hz, 2H); 13C NMR (101 MHz, CDCl3, APT pulse
sequence) d d 27.4, 115.5 (d, J = 21.5 Hz), 130.1 (d, J = 8.0 Hz); u
35.6, 50.7, 55.3, 117.9, 134.5, 162.3 (d, J = 246.3 Hz); IR 2975,
1604, 1508, 1367, 1219 cmꢁ1
; HRMS (ESI) m/z calcd for
C13H18FN2 ([M+H]+) 221.1449, found 221.1438.
4.4.4. N1-(tert-Butyl)-N1-(4-fluorobenzyl)ethane-1,2-diamine
(4b)
4.5. Synthesis of compounds 1a, 1c–1m
The nitrile from Section 4.4.3. (582 mg, 2.64 mmol) was reacted
according to the protocol in Section 4.4.2. to afford the diamine
fragment 4b as a colorless oil (422 mg, 1.88 mmol, 71% yield).
Rf = 0.46 (10% MeOH + 2% NH4OH in CH2Cl2); 1H NMR (400 MHz,
CDCl3) d 1.11 (s, 9H), 2.42 (t, J = 6.4 Hz, 2H), 2.61 (t, J = 6.4 Hz,
2H), 3.63 (s, 2H), 6.96 (t, J = 8.8 Hz, 2H), 7.30 (dd, J = 6.4, 8.8 Hz,
2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) d d 27.4,
114.8 (d, J = 21.2 Hz), 129.0 (d, J = 7.8 Hz); u 42.1, 54.3, 54.5, 55.1,
139.1, 161.6 (d, J = 244.6 Hz); IR 2970, 2870, 1603, 1506,
4.5.1. N-(2-(Benzyl(isopropyl)amino)ethyl)-N-methyl-4-((N,4-
dimethylphenylsulfonamido)methyl)benzamide (1a)
To a solution of ML140 (41 mg, 0.085 mmol) in DMF (1 mL) was
added sodium hydride, 60% dispersion in mineral oil (10 mg,
.256 mmol, 3 equiv). The reaction was stirred for 10 min at rt and
methyl iodide (30 mg, 0.215 mmol, 2.5 equiv) was added. The reac-
tion was stirred at rt for 17 h and partitioned between aqueous
NaHCO3 and CH2Cl2. The organics were separated and the aqueous
layer extracted with CH2Cl2 (3 ꢂ 5 mL) and the combined organic
layers were dried (Na2SO4), concentrated and purified by silica
gel chromatography to afford 1a as a light yellow oil (36 mg,
0.071 mmol, 83% yield). Rf = 0.49 (75% EtOAc/hexanes); 1H NMR
1362 cmꢁ1
;
HRMS (ESI) m/z calcd for C13H22FN2 ([M+H]+)
225.1767, found 225.1756.
4.4.5. 2-((4-Chlorobenzyl)(tert-butyl)amino)acetonitrile
N-(4-Chlorobenzyl)-tert-butylamine (700 mg, 3.54 mmol) was
reacted according to the protocol in Section 4.4.1. to afford the
nitrile product as a white solid (685 mg, 2.89 mmol, 82% yield).
Mp = 65–67 °C; Rf = 0.49 (10% EtOAc/hexanes); 1H NMR
(400 MHz, CDCl3) d 1.27 (s, 9H), 3.41 (s, 2H), 3.78 (s, 2H), 7.27(s,,
4H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) d d 27.2,
128.6, 129.8; u 35.6, 50.7, 55.2, 117.7, 133.0, 137.4; IR 2975,
1597, 1490, 1368, 1201 cmꢁ1; HRMS (ESI) m/z calcd for C13H18ClN2
([M+H]+) 237.1153, found 237.1141.
(400 MHz, CDCl3) (ca. 1:1 mixture of rotomers) d 0.92 (d,
J = 6.4 Hz, 3H), 1.06 (d, J = 6.4 Hz, 3H), 2.46 (s, 3H), 2.50 (t,
J = 7.2 Hz, 1 H), 2.54 (s, 1.5H), 2.59 (s, 1.5H), 2.73 (m, 1.5H), 2.85
(s, 1.5H), 2.93 (s, 1.5H), 3.04 (m, 0.5H), 4.14 (m, 1 H), 3.41 (s, 1
H), 3.49 (t, J = 6.0 Hz, 1 H), 3.63 (s, 1 H), 4.13 (d, J = 3.6 Hz, 2 H),
7.19–7.37 (complex, 11H), 7.73 (d, J = 8.0 Hz, 2 H); 13C NMR
(101 MHz, CDCl3, APT pulse sequence) (mixture of rotomers) d d
17.9, 21.5, 33.6, 34.3, 34.5, 38.6, 49.8, 50.5, 126.7, 126.9, 127.0,
127.3, 127.5, 128.2, 128.4, 128.7, 129.8; u 46.9, 47.1, 47.9, 51.0,
53.9, 54.6, 134.4, 136.4, 136.5, 136.9, 137.0, 140.4, 140.9, 143.6,
170.7, 171.5; IR 2964, 2928, 1629 cmꢁ1; HRMS (ESI) m/z calcd for
4.4.6. N1-(tert-Butyl)-N1-(4-chlorobenzyl)ethane-1,2-diamine
(4c)
C
29H38N3O3S ([M+H]+) 508.2628, found 508.2629; HPLC
purity = 97.4%.
The nitrile from Section 4.4.5. (384 mg, 1.62 mmol) was reacted
according to the protocol in Section 4.4.2. to afford the diamine
fragment 4c as a colorless oil (248 mg, 1.03 mmol, 64% yield).
Rf = 0.51 (10% MeOH + 2% NH4OH in CH2Cl2); 1H NMR (400 MHz,
CDCl3) d 1.10 (s, 9H), 2.44 (t, J = 6.4 Hz, 2H), 2.61 (t, J = 6.4 Hz,
2H), 3.63 (s, 2H), 7.22–7.30 (complex, 4H); 13C NMR (101 MHz,
CDCl3, APT pulse sequence) d d 27.4, 128.2, 128.9; u 42.2,
4.5.2. N-(2-(Benzyl(isopropyl)amino)ethyl)-2-(mesitylsulfonyl)-
1,2,3,4-tetrahydroisoquinoline-6-carboxamide (1c)
To a solution of 2-(mesitylsulfonyl)-1,2,3,4-tetrahydroisoquino-
line-6-carbonyl chloride (20 mg, 0.054 mmol) in CH2Cl2 (3 mL) was
added diamine fragment 3a (10 mg, 0.054 mmol) and triethyl-
amine (0.019 mL, 0.14 mmol, 2.6 equiv). The reaction was stirred
for 48 h at rt, aqueous, saturated sodium bicarbonate solution
(2 mL) was added and all solvents removed in vacuo. The residue
was extracted with a solution of CH2Cl2/MeOH (5:1, 6 mL). The fil-
trate was dried (Na2SO4), evaporated and purified by mass-direc-
ted, reverse phase preparative HPLC to afford 1c (9 mg,
0.017 mmol, 31% yield). 1H NMR (400 MHz, DMSO-d6) d 1.00 (d,
J = 6.6 Hz, 6H), 2.24 (s, 3H), 2.57 (s, 6H), 2.61 (t, J = 5.8 Hz, 2H),
2.83 (t, J = 5.9 Hz, 2H), 2.94 (sep, J = 6.6 Hz, 1H), 3.29 (q,
J = 5.0 Hz, 2H), 3.41 (t, J = 5.9 Hz, 2H), 3.49 (s, 2H), 4.33 (s, 2H),
6.47 (br s, 1H), 6.90 (s, 2H), 7.03 (d, J = 8.0 Hz, 1H), 7.15–7.30 (m,
6H), 7.39 (s, 1H); 13C NMR (101 MHz, DMSO-d6) d 18.0, 21.0,
22.9, 28.7, 37.5, 41.9, 45.7, 47.8, 49.7, 53.7, 124.4, 126.6, 127.0,
127.9, 128.5, 128.6, 131.6, 132.0, 133.3, 133.8, 135.4, 140.6,
140.8, 142.9, 166.7; IR 2966, 1648, 1544, 1494 cmꢁ1; HRMS (ESI)
54.5, 54.6, 55.2, 131.9, 142.2; IR 2969, 2869, 1488, 1363 cmꢁ1
;
HRMS (ESI) m/z calcd for C13H22ClN2 ([M+H]+) 241.1472, found
241.1462.
4.4.7. 2-((4-Bromobenzyl)(tert-butyl)amino)acetonitrile
N-(4-Bromobenzyl)-tert-butylamine (920 mg, 3.80 mmol) was
reacted according to the protocol in Section 4.4.1. to afford the
nitrile product as a white solid (906 mg, 3.22 mmol, 85% yield).
Mp = 64–66 °C; Rf = 0.49 (10% EtOAc/hexanes); 1H NMR
(400 MHz, CDCl3) d 1.26 (s, 9H), 3.40 (s, 2H), 3.76 (s, 2H), 7.21(d,
J = 8.4 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H); 13C NMR (101 MHz, CDCl3,
APT pulse sequence) d d 27.2, 130.1, 131.5; u 35.6, 50.7, 55.1,
117.6, 121.1, 137.9; IR 2976, 1592, 1485, 1366, 1203 cmꢁ1; HRMS
(ESI) m/z calcd for C13H18BrN2 ([M+H]+) 281.0648, found 281.0644.
Please cite this article in press as: Frankowski, K. J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.033

K. J. Frankowski et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
m/z calcd for C31H39N3O3S ([M+H]+), 534.2790, found 534.2804;
HPLC purity = 98.7%.
DMSO-d6) d 15.0, 17.8, 27.98, 28.03, 40.4, 43.4, 47.2, 48.6, 49.5,
53.1, 119.3, 124.7, 126.3, 127.47, 127.55, 128.7, 130.3, 130.8,
132.92, 132.93, 133.1, 134.6, 140.5 149.5, 165.6; IR 2965, 1646,
4.5.3. N-(2-((4-Fluorobenzyl)(isopropyl)amino)ethyl)-2-tosyl-
1,2,3,4-tetrahydroisoquinoline-6-carboxamide (1d)
1543, 1486 cmꢁ1
; HRMS (ESI) m/z calcd for C30H36BrN3O3S
([M+H]+), 600.1719, found 600.1740; HPLC purity = 98.1%.
2-Tosyl-1,2,3,4-tetrahydroisoquinoline-6-carbonyl
chloride
(20 mg, 0.054 mmol) and diamine fragment 3b (11 mg,
0.054 mmol) were reacted according the protocol in Section 4.5.2.
to afford 1d (15 mg, 0.028 mmol, 52% yield). 1H NMR (400 MHz,
DMSO-d6) d 0.96 (d, J = 6.4 Hz, 6H), 2.39 (s, 3H), 2.51 (m, 2H),
2.82–2.90 (m, 3H), 3.22 (m, 2H), 3.29 (t, J = 6.4 Hz, 2H), 3.54 (s,
2H), 4.21 (s, 2H), 7.06 (t, J = 8.8 Hz, 2H), 7.23 (d, J = 8.4 Hz, 1H),
7.36 (dd, J = 6.4, 8.8 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.56 (m, 2H),
7.72 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) d 17.9,
21.0, 28.0, 40.4, 43.4, 47.2, 48.4, 49.3, 53.0, 114.7 (d, J = 21.1 Hz),
124.7, 126.4, 127.45, 127.49, 129.9 130.0, 132.90, 132.96, 132.99,
134.6, 137.0 (d, J = 2.8 Hz), 143.7, 161.0 (d, J = 242.3 Hz), 165.6;
IR 2966, 1649, 1508 cmꢁ1; HRMS (ESI) m/z calcd for C29H35FN3O3S
([M+H]+), 524.2383, found 524.2401; HPLC purity = 97.0%.
4.5.7. N-(2-(Benzyl(tert-butyl)amino)ethyl)-2-tosyl-1,2,3,4-
tetrahydroisoquinoline-6-carboxamide (1h)
2-Tosyl-1,2,3,4-tetrahydroisoquinoline-6-carbonyl
chloride
(20 mg, 0.054 mmol) and diamine fragment 4a (11 mg,
0.054 mmol) were reacted according the protocol in Section 4.5.2.
to afford 1h (26 mg, 0.049 mmol, 91% yield). 1H NMR (400 MHz,
CDCl3) d 1.10 (s, 9H), 2.36 (s, 3H), 2.76 (t, J = 6.0 Hz, 2H), 2.89 (t,
J = 6.0 Hz, 2H), 3.12 (q, J = 5.6 Hz, 2H), 3.30 (t, J = 6.0 Hz, 2H), 3.64
(s, 2H), 4.21 (s, 2H), 6.22 (br s, 1H), 6.98 (d, J = 8.0 Hz, 1H), 7.09
(m, 1H), 7.16–7.22 (m, 3H), 7.27–7.29 (complex, 5H), 7.66 (d,
J = 8.4 Hz, 2H); 13C NMR (101 MHz, CDCl3) d 21.6, 27.5, 28.9, 39.9,
43.7, 47.6, 50.1, 55.2, 55.6, 124.5, 126.5, 126.8, 127.7, 127.8,
127.9, 128.5, 129.9, 133.2, 133.36, 133.42, 134.9, 142.9, 143.9,
166.7; IR 2989, 1628, 1538, 1497, 1460 cmꢁ1; HRMS (ESI) m/z calcd
4.5.4. N-(2-((4-Chlorobenzyl)(isopropyl)amino)ethyl)-2-tosyl-
for C
30H37N3O3S ([M+H]+), 520.2634, found 520.2650; HPLC
1,2,3,4-tetrahydroisoquinoline-6-carboxamide (1e)
purity = 97.5%.
2-Tosyl-1,2,3,4-tetrahydroisoquinoline-6-carbonyl
chloride
(20 mg, 0.054 mmol) and diamine fragment 3c (12 mg,
0.054 mmol) were reacted according the protocol in Section 4.5.2.
to afford 1e (13 mg, 0.024 mmol, 45% yield). 1H NMR (400 MHz,
DMSO-d6) d 0.95 (d, J = 6.4 Hz, 6H), 2.31 (s, 3H), 2.55 (m, 2H),
2.85 (m, 2H), 3.24–3.28 (m, 4H), 3.41 (s, 2H), 4.17 (s, 2H), 6.97
(d, J = 8.0 Hz, 1H), 7.07–7.12 (complex, 4H), 7.15 (s, 1H), 7.19 (m,
1H), 7.22 (d, J = 8.0 Hz, 2H), 7.31 (br s, 1H), 7.62 (d, J = 8.4 Hz,
2H); 13C NMR (101 MHz, DMSO-d6) d 17.9, 21.0, 28.0, 40.4, 43.4,
47.2, 48.6, 49.5, 53.0, 124.7, 126.4, 127.4, 127.5, 127.9, 129.86,
129.95, 130.2, 130.9, 132.9, 133.0, 134.6, 140.1 143.6, 165.6; IR
4.5.8. N-(2-(Benzyl(tert-butyl)amino)ethyl) -((4-
ethylphenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline-6-
carboxamide (1i)
2-((4-Ethylphenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline-6-
carbonyl chloride (160 mg, 0.44 mmol) and diamine fragment 4a
(91 mg, 0.44 mmol) were reacted according the protocol in Sec-
tion 4.5.2. to afford 1i (66 mg, 0.12 mmol, 28% yield). 1H NMR
(400 MHz, CDCl3) d 1.11 (s, 9H), 1.20 (t, J = 7.6 Hz, 3H), 2.67 (q,
J = 7.6 Hz, 2H), 2.77 (t, J = 6.1 Hz, 2H), 2.90 (t, J = 5.9 Hz, 2H), 3.12
(q, J = 5.9 Hz, 2H), 3.31 (t, J = 5.9 Hz, 2H), 3.65 (s, 2H), 4.21 (s,
2H), 6.37 (br s, 1H), 7.00 (d, J = 8.0 Hz, 1H), 7.07–7.11 (m, 1H),
7.16–7.21 (m, 2H), 7.24–7.27 (m, 1H), 7.29–7.33 (m, 5H), 7.67–
7.71 (m, 2H); 13C NMR (101 MHz, CDCl3) d 15.0, 27.3, 28.7, 28.8,
39.9, 40.9, 43.6, 47.5, 50.0, 55.1, 124.5, 126.4, 126.6, 127.6,
127.76, 127.81, 128.3, 128.6, 133.1, 133.2, 134.7, 142.8, 149.9,
166.6; IR 2968, 1645, 1540, 1494 cmꢁ1; HRMS (ESI) m/z calcd for
2966, 1647, 1543, 1491 cmꢁ1
29H35ClN3O3S ([M+H]+), 540.2088, found 540.2104; HPLC
purity = 100.0%.
; HRMS (ESI) m/z calcd for
C
4.5.5. N-(2-((4-Bromobenzyl)(isopropyl)amino)ethyl)-2-tosyl-
1,2,3,4-tetrahydroisoquinoline-6-carboxamide (1f)
2-Tosyl-1,2,3,4-tetrahydroisoquinoline-6-carbonyl
chloride
C
31H39N3O3S ([M+H]+), 534.2790, found 534.2801; HPLC
(20 mg, 0.054 mmol) and diamine fragment 3d (15 mg,
0.054 mmol) were reacted according the protocol in Section 4.5.2.
to afford 1f (18 mg, 0.30 mmol, 56% yield). 1H NMR (400 MHz,
CDCl3) d 0.98 (d, J = 6.4 Hz, 6H), 2.35 (s, 3H), 2.58 (t, J = 5.6 Hz,
2H), 2.88–2.94 (m, 3H), 3.26–3.31 (m, 4H), 3.42 (s, 2H), 4.21 (s,
2H), 6.39 (br s, 1 H), 7.01 (d, J = 8.0 Hz, 1H), 7.08 (d, J = 8.4 Hz,
1H), 7.22–7.28 (complex, 5H), 7.34 (s, 1H), 7.66 (d, J = 8.4 Hz,
2H); 13C NMR (101 MHz, CDCl3) d 18.1, 21.6, 29.0, 37.8, 43.7,
47.6, 48.2, 49.9, 53.2, 120.7, 124.4, 126.6, 127.75, 127.82, 129.9,
130.4, 131.6, 133.2, 133.3, 133.7, 135.1, 139.9 144.0, 166.9; IR
purity = 94.7%.
4.5.9. N-(2-((4-Fluorobenzyl)(tert-butyl)amino)ethyl)-2-tosyl-
1,2,3,4-tetrahydroisoquinoline-6-carboxamide (1j)
2-Tosyl-1,2,3,4-tetrahydroisoquinoline-6-carbonyl
chloride
(20 mg, 0.054 mmol) and diamine fragment 4b (12 mg,
0.054 mmol) were reacted according the protocol in Section 4.5.2.
to afford 1j (28 mg, 0.052 mmol, 96% yield). 1H NMR (400 MHz,
CDCl3) d 1.08 (s, 9H), 2.35 (s, 3H), 2.73 (t, J = 6.1 Hz, 2H), 2.87 (t,
J = 5.9 Hz, 2H), 3.10 (q, J = 5.6 Hz, 2H), 3.29 (t, J = 5.9 Hz, 2H), 3.58
(s, 2H), 4.20 (s, 2H), 6.10 (br s, 1H), 6.77–6.84 (m, 2H), 6.97 (d,
J = 8.0 Hz, 1H), 7.18–7.28 (m, 6H), 7.64–7.67 (m, 2H); 13C NMR
(101 MHz, CDCl3) d 21.5, 27.4, 28.8, 40.1, 43.6, 47.5, 50.0, 54.5,
55.5, 115.2 (d, J = 21.2 Hz), 124.3, 126.4, 127.6, 127.7, 129.3 (d,
J = 8.0 Hz), 129.8, 133.2, 133.3, 133.5, 135.0, 138.3 (d, J = 4.0 Hz),
143.9, 161.6 (d, J = 245.4 Hz), 166.7; IR 2970, 1643, 1541,
1506 cmꢁ1; HRMS (ESI) m/z calcd for C30H36FN3O3S ([M+H]+),
538.2540, found 538.2554; HPLC purity = 96.3%.
2965, 1646, 1541, 1486 cmꢁ1
29H34BrN3O3S ([M+H]+), 586.1562, found 586.1585; HPLC
purity = 94.6%.
; HRMS (ESI) m/z calcd for
C
4.5.6. N-(2-((4-Bromobenzyl)(isopropyl)amino)ethyl)-2-((4-
ethylphenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline-6-
carboxamide (1g)
2-((4-Ethylphenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline-6-
carbonyl chloride (160 mg, 0.44 mmol) and diamine fragment 3d
(119 mg, 0.44 mmol) were reacted according the protocol in Sec-
tion 4.5.2. to afford 1g (122 mg, 0.21 mmol, 47% yield). 1H NMR
(400 MHz, DMSO-d6) d 0.95 (d, J = 6.8 Hz, 6H), 1.15 (t, J = 7.6 Hz,
3H), 2.55 (t, J = 6.0 Hz, 2H), 2.61 (q, J = 7.6 Hz, 2H), 2.83–2.90 (m,
3H), 3.22–3.28 (m, 4H) 3.40 (s, 2H), 4.18 (s, 2H), 6.52 (br s, 1H),
6.99 (d, J = 8.0 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H) 7.21–7.27 (complex,
5H), 7.33 (s, 1H), 7.65 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz,
4.5.10. N-(2-((4-Fluorobenzyl)(tert-butyl)amino)ethyl)-2-
(mesitylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-6-
carboxamide (1k)
2-(Mesitylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonyl
chloride (20 mg, 0.054 mmol) and diamine fragment 4b (12 mg,
0.054 mmol) were reacted according the protocol in Section 4.5.2.
to afford 1k (14 mg, 0.024 mmol, 45% yield). 1H NMR (400 MHz,
Please cite this article in press as: Frankowski, K. J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.033

8
K. J. Frankowski et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
CDCl3) d 1.08 (s, 9H), 2.35 (s, 3H), 2.54 (s, 6H), 2.73 (t, J = 6.1 Hz, 2H),
2.87 (t, J = 5.9 Hz, 2H), 3.10 (q, J = 5.6 Hz, 2H), 3.29 (t, J = 5.9 Hz, 2H),
3.58 (s, 2H), 4.20 (s, 2H), 6.10 (br s, 1H), 6.77–6.84 (m, 2H), 6.97 (d,
J = 8.0 Hz, 1H), 7.18–7.28 (m, 6H), 7.64–7.67 (m, 2H); 13C NMR
(101 MHz, CDCl3) d 21.5, 27.4, 28.8, 40.1, 43.6, 47.5, 50.0, 54.5,
55.5, 115.2 (d, J = 21.2 Hz), 124.3, 126.4, 127.6, 127.7, 129.3 (d,
J = 8.0 Hz), 129.8, 133.2, 133.3, 133.5, 135.0, 138.3 (d, J = 4.0 Hz),
143.9, 161.6 (d, J = 245.4 Hz), 166.7; IR 2971, 1644, 1604, 1543,
1507 cmꢁ1; HRMS (ESI) m/z calcd for C32H41FN3O3S ([M+H]+),
566.2853, found 566.2871; HPLC purity = 99.1%.
U69,593 was prepared in ethanol as a 10 mM stock, norBNI was
prepared in water as a 10 mM stock, and test compounds were
prepared as 10 mM stocks in DMSO (Fisher). All compounds were
then diluted to working concentrations in vehicle for each assay
without exceeding 1% DMSO or ethanol concentrations. [35S]GTP
cS
was purchased from PerkinElmer Life Sciences. Phospho-ERK1/2
and total ERK1/2 antibodies were purchased from Cell Signaling
(Beverly, MA) and Li-Cor secondary antibodies (anti-rabbit
IRDye800CW and anti-mouse IRDye680LT) were purchased from
Li-Cor Biosciences.
4.5.11. N-(2-((4-Chlorobenzyl)(tert-butyl)amino)ethyl)-2-tosyl-
4.6.2. Cell lines and cell culture
1,2,3,4-tetrahydroisoquinoline-6-carboxamide (1l)
Chinese hamster ovary (CHO) cells were virally transfected to
express HA-tagged recombinant human kappa opioid receptors
(CHO-hKOR cell line) and maintained in DMEM/F-12 media (Invit-
rogen) supplemented with 10% fetal bovine serum, 1% penicillin/
2-Tosyl-1,2,3,4-tetrahydroisoquinoline-6-carbonyl
chloride
(20 mg, 0.054 mmol) and diamine fragment 4c (13 mg,
0.054 mmol) were reacted according the protocol in Section 4.5.2.
to afford 1l (25 mg, 0.046 mmol, 85% yield). 1H NMR (400 MHz,
CDCl3) d 1.09 (s, 9H), 2.39 (s, 3H), 2.64 (m, 2H), 2.87 (t, J = 6.0 Hz,
2H), 3.04 (m, 2H), 3.28 (t, J = 6.0 Hz, 2H), 3.69 (s, 2H), 4.20 (s,
2H), 7.20 (d, J = 8.0 Hz, 1H), 7.31 (m, 2H), 7.39–7.45 (m, 4H), 7.53
(m, 2H), 7.71 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz, CDCl3) d
21.0, 27.1, 28.0, 40.4, 43.4, 47.2, 49.9, 53.4, 54.8, 124.7, 126.3,
127.42, 127.45, 127.8, 129.3, 129.9, 130.5, 132.77, 132.84, 132.9,
134.6, 142.1, 143.6, 165.5; IR 2969, 1644, 1541, 1489 cmꢁ1; HRMS
(ESI) m/z calcd for C30H37ClN3O3S ([M+H]+), 554.2244, found
554.2261; HPLC purity = 99.2%.
streptomycin, and 500 l
g/ml geneticin as previously described.14
A DiscoveRx PathHunter™ U2OS cell line expressing barrestin2
and hKOR (U2OS-hKOR-barrestin2-DX) was purchased from Dis-
coveRx Corporation (Fremont, CA) and maintained MEM with
10% fetal bovine serum, 1% penicillin/streptomycin, 500
lg/ml
geneticin and 250 g/ml hygromycin B. All cells were grown at
l
37 °C (5% CO2 and 95% relative humidity).
4.6.3. G Protein coupling assay
[
35S]GTP
cS binding assay was performed following a previously
published protocol.14,18 Briefly, cells were serum-starved for 1 h
and membranes were prepared. Each reaction was performed at
4.5.12. N-(2-((4-Bromobenzyl)(tert-butyl)amino)ethyl)-2-tosyl-
1,2,3,4-tetrahydroisoquinoline-6-carboxamide (1m)
room temperature and contained 15
l
g of membrane protein,
2-Tosyl-1,2,3,4-tetrahydroisoquinoline-6-carbonyl
chloride
40
l
M GDP, ꢄ0.1 nM [35S]GTP
cS and increasing concentrations of
(20 mg, 0.054 mmol) and diamine fragment 4d (15 mg,
0.054 mmol) were reacted according the protocol in Section 4.5.2.
to afford 1m (7.8 mg, 0.013 mmol, 24% yield). 1H NMR (400 MHz,
CDCl3) d 1.08 (s, 9H), 2.35 (s, 3H), 2.74 (t, J = 6.0 Hz, 2H), 2.89 (t,
J = 5.9 Hz, 2H), 3.13 (q, J = 5.8 Hz, 2H), 3.27–3.32 (m, 2H), 3.56 (s,
2H), 4.21 (s, 2H), 6.07 (br s, 1H), 7.00 (d, J = 8.0 Hz, 1H), 7.11–
7.20 (m, 3H), 7.23–7.30 (m, 5H), 7.64–7.68 (m, 2H); 13C NMR
(101 MHz, CDCl3) d 21.6, 27.3, 28.9, 40.1, 43.6, 47.5, 50.2, 54.6,
55.6, 120.3, 124.3, 126.5, 127.6, 127.7, 129.5, 129.8, 131.5,
133.21, 133.23, 133.6, 135.0, 141.9, 143.8, 166.7; IR 2980, 1652,
compounds in assay buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl,
5 mM MgCl2, 1 mM EDTA). Directly after the addition of test com-
pounds, 100 nM U69,593 was added to yield a total volume of
200 lL. After 1 h, reactions were quenched by rapid filtration
through GF/B filters and radioactivity was counted with a Top-
Count NXT high throughput screening microplate scintillation
and luminescence counter (PerkinElmer Life Sciences). In some
cases, the assays were also carried out as above except that 1
U69,593 was used; analogous results were obtained.
lM
1521, 1489 cmꢁ1
;
HRMS (ESI) m/z calcd for C30H36BrN3O3S
4.6.4. bArrestin2 recruitment (DiscoveRx PathHunter™) assay
The PathHunter™ assay was performed according to the manu-
facturer’s protocol with slight modification and following previ-
ously published protocols.14,18 Briefly, 5000 cells/well were
plated overnight in Opti-MEM containing 1% fetal bovine serum,
1% penicillin/streptomycin. Cells were pretreated with antagonist
([M+H]+), 600.1719, found 600.1740; HPLC purity = 94.1%.
4.5.13. N-(2-((4-Bromobenzyl)(tert-butyl)amino)ethyl)-2-
(mesitylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-6-
carboxamide (1n)
2-(Mesitylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonyl
chloride (20 mg, 0.054 mmol) and diamine fragment 4d (15 mg,
0.054 mmol) were reacted according the protocol in Section 4.5.2.
to afford 1n (10 mg, 0.016 mmol, 29% yield). 1H NMR (400 MHz,
CDCl3) d 1.17 (s, 9H), 2.33 (s, 3H), 2.66 (s, 6H), 2.83 (m, 2H), 2.92 (t,
J = 6.0 Hz, 2H), 3.33 (q, J = 6.0 Hz, 2H), 3.50 (t, J = 6.0 Hz, 2H), 3.66
(s, 2H), 4.42 (s, 2H), 6.18 (br s, 1H), 6.99 (s, 2H), 7.12 (d, J = 8.0 Hz,
1H), 7.12 (d, J = 8.4 Hz, 2H), 7.28 (m, 1H), 7.34 (d, J = 8.4 Hz, 2H),
7.42 (s, 1H); 13C NMR (101 MHz, CDCl3) d 21.1, 23.0, 27.5, 28.8,
40.2, 42.0, 45.8, 50.3, 54.8, 55.7, 120.4, 124.3, 126.8, 127.9, 129.6,
131.6, 131.7, 132.1, 133.4, 134.0, 135.6, 140.7, 142.0, 143.0, 166.9;
IR 2972, 1647, 1533, 1486 cmꢁ1; HRMS (ESI) m/z calcd for C32H40-
BrN3O3S ([M+H]+), 628.2032, found 628.2049; HPLC purity = 98.6%.
for 15 min at 37 °C followed by the addition of 1 lM U69,593
and a 90 min incubation at 37 °C. PathHunter™ detection reagent
was added and cells incubated at room temperature for 60 min.
Chemiluminescence was detected using a SpectraMaxÒ M5e Multi-
mode Plate Reader (Molecular Devices).
4.6.5. In-cell western ERK1/2 phosphorylation
Antagonist inhibition of U69,593-induced ERK phosphorylation
was determined by in-cell westerns as previously described.14,18
Briefly, hKOR-CHO cells were plated in 384-well plate at 15,000
cells per well and incubated at 37 °C overnight. After an hour
serum-starve, cells were treated with compound followed by the
addition of 100 nM U69,593 and a 10 min incubation at 37 °C. Cells
were fixed, permeabilized, blocked, and stained with primary anti-
bodies for phosphorylated ERK1/2 and total-ERK1/2 (1:300 and
1:400, respectively) at 4 °C overnight. Cells were then incubated
with Li-Cor secondary antibodies (anti-rabbit IRDye800CW,
1:500; anti-mouse IRDye680LT, 1:1500) and imaged with the
Odyssey Infrared Imager (Li-Cor Biosciences, Lincoln, NE) at 700
and 800 nM.
4.6. In vitro assay methods
4.6.1. Compounds and reagents
(+)-(5a,7a,8b)-N-Methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4.5)-
dec-8-yl)-benzeneacetamide (U69,593) and nor-binaltorphimine
dihydrochloride (norBNI) were purchased from Sigma Aldrich.
Please cite this article in press as: Frankowski, K. J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.033

K. J. Frankowski et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
4.6.6. Data analysis and statistics
References and notes
GraphPad Prism 6.01 software (GraphPad) was used to generate
sigmoidal concentration-response curves using a three-parameter,
non-linear regression analysis. All compounds were run in parallel
assays in 2–4 replicates per individual experiment. All studies were
performed n ꢀ 3 independent experiments in multiple replicates.
For determination of antagonist inhibition, each individual experi-
ment was normalized to the percentage of maximal U69,593 stim-
ulation. The efficacy and potency values were obtained from the
averages of the nonlinear regression analysis performed on each
individual curve and are reported as the mean S.E.M.
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Acknowledgments
We gratefully acknowledge financial support from the National
Institute on Drug Abuse (Grant R01 DA031927 to L.M.B. and J.A.).
We thank Ben Neuenswander for performing HPLC compound
purification and high resolution mass determinations. Ki determi-
nations were generously provided by the National Institute of
Mental Health’s Psychoactive Drug Screening Program, Contract
# HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is
directed by Bryan L. Roth MD, PhD at the University of North Car-
olina at Chapel Hill and project officer Jamie Driscoll at NIMH,
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.12.033.
Please cite this article in press as: Frankowski, K. J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.033