Discovery of Novel Aminotetralines and Aminochromanes as Selective and Competitive Glycine Transporter 1 (GlyT1) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00300

The glycine transporter 1 (GlyT1) has emerged as a key novel target for the treatment of schizophrenia. Herein, we report the synthesis and biological evaluation of aminotetralines and aminochromanes as novel classes of competitive GlyT1 inhibitors. Starting from a high-throughput screening hit, structure-activity relationship studies led first to the discovery of aminotetralines displaying high GlyT1 potency and selectivity, with favorable pharmacokinetic properties. Systematic investigations of various parameters (e.g., topological polar surface area, number of hydrogen bond donors) guided by ex vivo target occupancy evaluation resulted in lead compounds possessing favorable brain penetration properties as for (7S,8R)-27a. Further optimization revealed compounds with reduced efflux liabilities as for aminochromane 51b. In an in vivo efficacy model (7S,8R)-27a, dose-dependently reversed L-687,414 induced hyperlocomotion in mice with an ED₅₀ of 0.8 mg/kg. All these results suggest (7S,8R)-27a and 51b as new GlyT1 inhibitors worthy of further profiling.

Full text of DOI:10.1021/acs.jmedchem.8b00300

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Article
Discovery of Novel Aminotetralines and Aminochromanes as
Selective and Competitive Glycine Transporter 1 (GlyT1) Inhibitors
Willi Amberg, Udo E.W. Lange, Michael Ochse, Frauke Pohlki, Berthold Behl, Ana Lucia Relo, Wilfried
Hornberger, Carolin Hoft, Mario Mezler, Jens Sydor, Ying Wang, Hongyu Zhao, Jason T. Brewer, Justin
Dietrich, Huanqiu Li, Irini Akritopoulou-Zanze, Yanbin Lao, Steven M Hannick, Yi-Yin Ku, and Anil Vasudevan
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Aug 2018
Downloaded from http://pubs.acs.org on August 6, 2018
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Discovery of Novel Aminotetralines and
Aminochromanes as Selective and Competitive
Glycine Transporter 1 (GlyT1) Inhibitors
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Willi Amberg,* Udo E. W. Lange, Michael Ochse, Frauke Pohlki, Berthold Behl, Ana
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†
†
†
‡
‡
Lucia Relo, Wilfried Hornberger, Carolin Hoft, Mario Mezler, Jens Sydor, Ying Wang,
‡
‡
‡
‡
‡
Hongyu Zhao, Jason T. Brewer, Justin Dietrich, Huanqiu Li, Irini Akritopoulou-Zanze,
‡
,§,
⊥
‡
‡
‡
Yanbin Lao,
Steven M. Hannick, Yi-Yin Ku, Anil Vasudevan
†
AbbVie Deutschland GmbH & Co. KG, Neuroscience Research, Knollstrasse, 67061
Ludwigshafen, Germany
‡
AbbVie Inc., 1 North Waukegan Rd., North Chicago, IL 60064, United States.
ABSTRACT: The glycine transporter 1 (GlyT1) has emerged as a key novel target for the
treatment of schizophrenia. Herein, we report the synthesis and biological evaluation of
aminotetralines and aminochromanes as novel classes of competitive GlyT1 inhibitors. Starting
from a highꢀthroughput screening hit, structureꢀactivity relationship studies led first to the
discovery of aminotetralines displaying high GlyT1 potency and selectivity, with favorable
pharmacokinetic properties. Systematic investigations of various parameters (e.g. topological
polar surface area, number of hydrogen bond donors) guided by ex vivo target occupancy
evaluation, resulted in lead compounds possessing favorable brain penetration properties as
(
7S,8R)-27a. Further optimization revealed compounds with reduced efflux liabilities as
aminochromane 51b. In an in vivo efficacy model (7S,8R)-27a doseꢀdependently reversed
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Lꢀ687,414 induced hyperlocomotion in mice with an ED of 0.8 mg/kg. All these results suggest
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(7S,8R)-27a and 51b as a new GlyT1 inhibitors worthy of further profiling.
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INTRODUCTION
Impaired NꢀmethylꢀDꢀaspartate receptor (NMDA receptor) mediated neurotransmission has been
implicated in schizophrenia. A role of hypofunctional NMDA receptor signaling supported by
clinical observations is that NMDA receptor antagonists can induce the whole spectrum of
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schizophrenia symptoms and by the discovery of schizophrenia susceptibility genes which are
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involved in or interfere with NMDA receptor signaling.
Neurophysiological findings on an imbalance between excitation and inhibition, on dysfunctional
microꢀcircuits and largeꢀscale disconnectivities further point to an involvement of impaired
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NMDA receptor signaling in the disease.
NMDA receptor function can be modulated by altering the concentrations of the coꢀagonists Dꢀ
serine or glycine. The concentration of glycine in the synaptic cleft is regulated by glycine
transporters (GlyT). One of the two glycine transporters, GlyT1, is localized at glutamatergic
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synapses and is physically attached to PSDꢀ95, a protein associated with NMDA receptors.
This indicates the role of GlyT1 in regulation of NMDA receptor activity. Preclinical studies
have shown beneficial effects of GlyT1 inhibitors in a variety of animal models for schizophrenia
1
2
addressing the whole spectrum of symptom domains.
Recently, HoffmannꢀLa Roche announced that two phase III studies of GlyT1 inhibitor RG1678
bitopertin) did not meet their clinical end points. GlyT1 inhibitors can be differentiated
(
1
3
according to their competitive or nonꢀcompetitive interaction with the glycine binding site on
GlyT1 (Figure 1). RG1678 is a nonꢀcompetitive inhibitor and the lack of clinical effects may be
related to its nonꢀcompetitive binding mode.
Competitive inhibitors may offer potential advantages with respect to their efficacy profile and
liability for peripheral side effects. The basis for the potential superiority of substrateꢀcompetitive
inhibitors is the dependence of the degree of inhibition on the concentration of glycine in the
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vicinity of the transporter. While low glycine concentrations would not interfere with the
inhibition, high glycine concentrations could displace a competitive inhibitor, and therefore
alleviate this block of glycine uptake through GlyT1.
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Figure 1. Examples of Nonꢀcompetitive and Competitive GlyT1 Inhibitors:
Nonꢀcompetitive Inhibitors:
Competitive Inhibitors:
In this paper we report the discovery of aminotetralines and aminochromanes as novel
chemotypes of competitive GlyT1 inhibitors. Starting from highꢀthroughput screening (HTS) hit
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(GlyT1 K = 124 nM), SAR studies accompanied by the early assessment of molecular
i
properties and metabolic stability of the synthesized derivatives quickly led to the identification
of a sulfonamide side chain attached in the 4ꢀposition that significantly increased potency (Table
1
). In addition, 7 displayed only moderate selectivity vs. several receptors and transporters (µꢀ
opioid, α , dopamine transporter) whereas compound 8 did not show significant receptor
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binding in the CEREP 75 receptor profile (data not shown).
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Table 1. HTS Hit and Early Leads for Optimization
Cl
Cl
Cl
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NHSO nPr
2
HO
O
NH2
NH
7
8
9
GlyT1 Ki
124 nM
3.0 nM
35 nM
Mode of action
competitive
competitive
450/620
7%
competitive
17/95
mClint (r/h)*
µL/min/mg
3
43/130
< 1%
n.d.
F%** (rat)
31%
Brain/Plasma
0.54
0.05
(free)***
CYP (3A4)
inhibition (IC ,
> 10
0.62
> 10
5
0
µM)****
for details of all parameters see experimental part: determination of ADME parameters; * total
clearance of compound in rat and human liver microsomes; ** oral bioavailbility in rat; ***
brain/plasma ratio of free drug; **** inhibition of cytochrome P450 3A4 enzyme;
However, 8 was a moderate inhibitor of CYP 3A4. It also suffered from poor oral bioavailability
(F%) in rat. We assumed that this is caused by an oxidation/aromatization of the tetrahydroisoꢀ
quinoline core, which led to suboptimal liver microsomal stability. Among the numerous
attempts to prevent this aromatization, moving the nitrogen from an endoꢀ to an exoꢀcyclic
position turned out to be the crucial transformation. This change led to the first aminotetraline
compound 9, which possessed markedly increased liver microsomal stability and ultimately
better oral bioavailability while maintaining the GlyT1 potency.
Further studies led to highly potent, selective, drugꢀlike GlyT1 inhibitors, displaying excellent
metabolic stability without inhibition of major metabolizing CYP450 enzymes. Throughout SAR
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studies we were guided by target occupancy (TO) studies revealing good correlation between
unbound brain levels of our GlyT1 inhibitors, corrected by in vitro K , and the estimated TO.
i
Forebrain homogenates from mice intraperitoneally treated with different doses of a GlyT1
inhibitor, were tested for binding of a GlyT1 radioligand ex vivo. Furthermore, our compounds
doseꢀdependently reversed the hyperlocomotion induced in mice by (3R,4R)ꢀ3ꢀaminoꢀ1ꢀhydroxyꢀ
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ꢀmethylpyrrolidinꢀ2ꢀone (Lꢀ687,414), which is a known selective and brainꢀpenetrating glycine
site NMDA receptor antagonist.
RESULTS AND DISCUSSION
Synthesis. The aminotetraline and aminochromane derivatives examined in this study were
prepared as illustrated in Schemes 1 ꢀ 6. Scheme 1 shows the initial general synthesis method in
which the Nꢀprotected aminotetralines were utilized as intermediates for the synthesis of the
target sulfonamide containing aminotetralines.
Methoxytetralone 10 was first reacted with benzyl bromides 11 via an enamine to afford
20
benzylated tetralones 12, which were converted to aminotetralines 13 via reductive amination.
In all cases the cisꢀisomer was the major product, which could either be precipitated out directly
from the reaction mixture or isolated by flash chromatography. These compounds were first Nꢀ
protected with ethyl chloroformate in pyridine to aminotetralines 14, then demethylated with
BBr to compounds 15 and subsequently Oꢀalkylated with tertꢀbutyl (2ꢀbromoethyl)carbamate in
3
dimethylacetamide in the presence of a base to obtain compounds 16. Removal of the Boc group
produced the intermediates 17, which were converted to the sulfonamides 19 in the presence of
sulfonyl chlorides 18 followed by the removal of the ethyl carbamate with potassium hydroxide
in ethanol to afford compounds 20. The isolation and testing of the minor transꢀisomers was
ultimately abandoned in favor of the cisꢀisomers due to their superior overall profile.
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a
Scheme 1 . General Synthesis of Aminotetralines
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The synthesis of enantiomerically pure aminotetralines is shown in Scheme 2. Tetralone 12 was
first converted to the propionenamide 21 which was further transformed to compound (1R,2S)-22
by enantioselective hydrogenation mediated by chlorocyclooctadiene rhodium(I) dimer in the
presence of (S)ꢀ1ꢀ[(R )ꢀ2ꢀ(Diꢀ1ꢀnaphtylphosphino)ferrocenyl]ethyldiꢀtert.ꢀ
P
2
1ꢀ23
butylphosphine (Josiphos SLꢀJ216ꢀ2).
In all tested cases the observed enantiomeric excess
(ee) was higher than 90% which could be further increased to ee > 99% by recrystallization as
determined by HPLC using an Agilent 1100 HPLC (samples run on Chiralpak ADꢀH, for further
details see experimental section). Such a process was also exploited successfully in process scale
synthesis of these chiral compounds. Finally, the propionamide was hydrolyzed in the presence of
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sulfuric acid to obtain (1R,2S)-13 (R=H). The absolute configuration was assigned by single
crystal xꢀray analysis of the (S)ꢀ(+)ꢀmandelic acid salt of amine (1R,2S)-13 (R=H).
Demethylation afforded hydroxy compound (7S, 8R)-23, which was subsequently alkylated with
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,3ꢀdibromopropane or 1,4ꢀdibromobutane to give compounds (7S, 8R)-24. Aminotetraline
derivatives (7S,8R)-25 were then obtained by Oꢀalkylation with tertꢀbutyl (2ꢀ
bromoethyl)carbamate in dimethylacetamide in the presence of a base. Removal of the Boc group
produced (7S,8R)-26, which were further converted to final compounds (7S, 8R)-27 carrying an
azetidine or pyrrolidine residue.
a
Scheme 2 . Synthesis of Enantiopure Aminotetralines
H
H
N
a
b
c
O
NH2
O
O
O
N
O
O
O
(
1R,2S)-13
R=H
12 (R=H)
21
(1R,2S)-22
d
(CH2)n
f
(CH )
2 n
e
O
N
HO
N
HO
NH2
BocHN
g
(7S,8R)-25
(7S,8R)-24
(7S,8R)-23
O O
2S
(CH2)n
(CH )
2 n
h
O
N
O
N
H N
N
2
R
H
2
(7S,8R)-27a: n = 1; R = 1-Me-imidazole-4-yl
(7S,8R)-26
(7S,8R)-27
2
(7S,8R)-27b: n = 2; R = 1-Me-imidazole-4-yl
2
(7S,8R)-27c: n = 1: R = c-propyl-CH
2
a
Reagents and conditions. (a) Propionamide, pTsOH, toluene, reflux, 5 d; (b) Josiphos SL-J216-2, chlorocyclooctadiene
Rh (I) dimer, H2, methanol, 50-55°C, 4 bar, 40 h; (c) Sulfuric acid, acetic acid / water, reflux, 68 h (d) HBr, acetic acid,
1
10°C, 1.5 h (e) Alkyl dibromide, N,N diisopropylethylamine, MeCN, overnight; (f) tert-Butyl (2-bromoethyl)carbamate,
2
NaH, DMA, RT, 3 d; (g) HCl, iPrOH, RT, overnight (h) R SO Cl, DMAP, RT, overnight.
2
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Alternatively, chiral resolution of the racemates of final compounds or intermediates by
preparative HPLC or preparative SFC can be achieved (see experimental part for further details).
The general synthesis method for the aminochromanes on the other hand started with 6ꢀ
methoxychromanꢀ4ꢀone 28 as shown in Scheme 2. Transformation to 29 and a subsequent Neberꢀ
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rearrangement afforded amino ketone 30, which was Nꢀprotected to give 31 and further reacted
with benzylmagnesium chloride, to produce compound 32 after acidic workup.
a
Scheme 3 . Synthesis of Aminochromane Derivatives 36
Intermediate 33 was then prepared by hydrogenation of the benzylidene compound, which was
demethylated to 34 with BBr and deprotected under basic conditions affording 35. Further
3
conversion to the target compounds 36 was achieved following the protocol described in Scheme
2
for compounds 27 which involves the subsequent steps: formation of azetidine with 1,3ꢀ
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dibromopropane, Oꢀalkylation with tertꢀbutyl (2ꢀbromoethyl)carbamate in dimethylacetamide in
the presence of a base, removal of the Boc group, and conversion to the sulfonamides 36.
Scheme 4 shows the general synthesis of compounds with a truncated side chain and an azetidine
instead of a free amino group. Hydroxytetraline 37 was first converted to the triflate 38 which
was further transformed to nitrile 39 by Pdꢀmediated cross couplings. Deprotection of the amino
group produced compound 40 which were then converted to the azetidine 41 by alkylation with
1,3ꢀdibromopropane. Subsequent hydrogenation of the nitrile group afforded free amine 42,
which were converted to the sulfonamide 43 using the corresponding sulfonyl chlorides in the
presence of DMAP.
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8
9
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1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 4 . Synthesis of Aminotetralines with Short Sulfonamide Side Chain
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MonoꢀNꢀalkylated aminotetralines as shown in scheme 5 were prepared starting from the primary
amine 20k which was converted to amide 47 or from intermediates 19 or 45. Subsequent
reduction with LiAlH or BH SMe affords the desired compounds 44, 46 and 48.
4
3
2
a
1
1
1
1
1
1
1
1
1
1
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2
2
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0
1
2
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9
0
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2
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8
9
0
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 5 : Synthesis of Nꢀalkylated Aminotetralines
Scheme 6 depicts the synthesis of aminotetralines and aminochromanes carrying a rigidified side
chain. Triflate 49, was converted to the Bocꢀprotected intermediates 50 and 52 by Pdꢀmediated
crossꢀcouplings. Deprotection of the amino group and subsequent conversion to the sulfonamide
afforded final compounds 51 and 53.
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5
5
5
5
5
5
5
6
a
Scheme 6 . Synthesis of Aminotetralines and Chromanes with Rigidified Side Chains
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
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9
0
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0
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2
3
4
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6
7
8
9
0
Structure activity relationships: Early SAR studies aimed at maximizing GlyT1 potency and
further improvement of liver microsomal stability (rat/human). Liver microsomal metabolism
studies indicated that the nꢀpropyl residue of sulfonamide 9 was prone to oxidation and therefore
a metabolic hotspot. A targeted library of heterocyclic and alkyl sulfonamides was synthesized by
sulfonamide couplings with the corresponding sulfonyl chlorides (Scheme 1). In comparison to
related derivatives 9, 20a, 20b and 20d bearing sulfonamide moieties of similar size, the
cyclopropyl methyl compound 20c displayed improved potency. However, liver microsomal
stability was not improved, resulting in a dose of 14 mg/kg to achieve 50% TO in mice (i.p.
dosing). Methyl pyrazole 20e showed good potency but with similar turnover in rat liver
microsomes compared to compound 9. Methyl imidazole 20f emerged as a lead for further
optimization, showing improved potency and lower microsomal turnover relative to compound 9.
Replacing the imidazole or pyrrazole moiety by thienyl 20g or phenyl 20h led in turn to
compounds with much higher K s. Despite high potency, both GlyT1 inhibitors, 20e and 20f
i
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required a high dose to achieve half maximal TO, probably due to low brain availability (20e:
unbound, Cmax,brain = 0.2 nM at 2 mg/kg i.p.). Surprisingly the same trend with respect to potency
was also published for a series of GlyT1 inhibitors by Merck despite a very different core
2
5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
structure.
Table 2. In Vitro Inhibitory Activity of 9, 20aꢀh at hGlyT1 and their Metabolic Stability
mClint*
µL/min/mg]
rat / human
ED est. TO
50
(mice)
[mg/kg i.p.]
2
K [nM]
hGlyT1
i
Compound
R
[
clogP / TPSA
9
nꢀPropyl
Me
35
141
136
5.7
32
17 / 95
39 / 25
35 /173
25 / 92
49 / 55
49 / 44
10 / 36
95 / 99
40 / 78
n.d.
n.d.
n.d.
14
5.4 / 81
4.4 / 81
6.0 / 81
5.4 / 81
5.3 / 81
4.7 / 99
5.0 / 99
5.9 / 81
6.0 / 81
2
0a
20b
20c
nꢀButyl
2
0d
0e
0f
0g
0h
n.d.
> 20
> 20
n.d.
n.d.
2
1ꢀMeꢀpyrrazolꢀ4ꢀyl
1ꢀMeꢀimidazoleꢀ4ꢀyl
Thienꢀ2ꢀyl
1.2
2.6
163
660
2
2
2
Phenyl
K values and estimated ED for in vivo target occupancy (ED est. TO) was assessed as described in the
i
50
50
experimental part; * total clearance of compound in rat and human liver microsomes (see
experimental section)
GlyT1 inhibitors depicted in Table 2 have a clogP in the range of 4.4 to 6 and a TPSA in the
range of 81 to 99. Based on improved potency of 20f, we focused next on the optimization of the
benzyl moiety by reducing the lipophilicity, while keeping the 1ꢀMeꢀimidazoleꢀ4ꢀsulfonyl
functionality in place. Table 3 highlights selected compounds from this effort (clogP 3.6 to 5.1).
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5
5
5
5
5
6
High affinities were obtained with analogues carrying substituents in the 3ꢀ, 4ꢀ, or 5ꢀposition of
the benzyl residue (entries 20i, 20j and 20m, 20n), whereas substitution in the 2ꢀposition (20l)
afforded compounds with somewhat lower potency. However compound 20k carrying an
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
unsubstituted phenyl ring displayed one of the best affinities (K hGlyT1 = 3.6 nM) combined
i
with a good liver microsomal stability (mClint [µL/min/mg]: h < 23; r < 23). The unsubstituted
benzyl side at the aminotetraline core was therefore retained for further optimization.
Compounds 20f and 20i of table 3 still displayed low brain availability which led to an
unfavorable ED in our ex vivo binding assay. The favorable physicochemical space (topological
5
0
polar surface area (TPSA) = 45, clogP = 2.8, number of hydrogen bond donors (#HBD) = 1, pKa
2
6ꢀ28
= 8.4, MW 305), for CNS drugs has been described in numerous publications.
Table 3. In Vitro Inhibitory Activity of Different Benzyl Derivatives 20f, 20iꢀ20n at hGlyT1
ED est. TO
5
0
K [nM]
hGlyT1
i
Compound
R
(mice)
clogP / TPSA
[
mg/kg]
20f
20i
3,4ꢀCl
3ꢀCl
4ꢀCl
H
2
2.6
1.5
1.1
3.6
75
> 20
5.0 / 99
4.3 / 99
4.3 / 99
3.6 / 99
4.3 / 99
4.5 / 99
4.5 / 99
15
2
0j
0k
0l
0m
0n
n.d.
n.d.
n.d.
n.d.
n.d.
2
2
2ꢀCl
2
5ꢀCl, 3ꢀF
3ꢀCF
1.2
0.8
2
3
K values and estimated ED for in vivo target occupancy (ED est. TO) was assessed as described in the
i
50
50
experimental part
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Compounds within this space have a higher likelihood of being brain penetrant and displaying a
benign safety profile. Reducing the TPSA (TPSA = 99 for all compounds in table 3) and the
number of hydrogen bond donors (#HBD = 3 for all compounds in table 3) was therefore the next
step.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
1
2
3
4
5
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7
8
9
0
A reduction of HBDs can be achieved by elimination of protic hydrogen atoms, whereas TPSA
can be reduced most easily by replacing nitrogen, oxygen or protic hydrogen atoms by carbon
atoms, and the number of rotatable bonds by rigidification of the sulfonamide bearing side chain.
An iterative approach was employed to rapidly develop SAR. Table 4 displays selected
compounds resulting from these extensive studies, in which TPSA and number of HBDs were
reduced stepwise.
Table 4. In Vitro Inhibitory Activity of Different Amine Derivatives at hGlyT1
ED est.
HBD TO (mice)
5
0
2
1
K [nM] TPSA
i
Compound
R
R
2
hGlyT1
[Å ]
[
mg/kg]
2
0k
7S,8R)-27a
7S,8R)-27b
7S,8R)-27c
NH
2
3.6
0.8
1.2
2.6
1.2
99
76
76
59
67
3
1
1
1
2
n.d.
(
1.4
1.6
0.5
1.4
(
(
(
7S,8R)-44
NHMe
NHMe
43a
3.0
9.5
63
58
1
2
1.8
2.0
4
6
nꢀPropyl
K values and estimated ED for in vivo target occupancy (ED est. TO) was assessed as described in the
i
50
50
experimental part
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5
5
5
5
6
As assumed these variations led to compounds with improved brain availability, demonstrated by
low single digit doses to achieve 50% TO in mice. Compound (7S,8R)ꢀ27c combines low TPSA
with the least number of HBDs and also exhibited an good brain to plasma drug concentration
ratio of 0.8 (unbound, Cmax,brain = 9.9 nM at 2 mg/kg i.p.) in rat and achieved an ED50 of 0.5
mg/kg (i.p.) in our ex vivo binding assay.
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
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1
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4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(7S,8R)ꢀ27c showed relatively high liver microsomal clearance (mClint [µL/min/mg]: h = 113; r
= 137), which could be a reason for its low oral bioavailability in rat (Fp.o. = 8%). Additionally, it
proved to be a very potent hERG inhibitor with an IC50 of 80 nM. Compared to (7S,8R)ꢀ27c,
2
compound (7S,8R)ꢀ44 has a higher TPSA (67Å ) and 2 HBDs but still achieved a good unbound
drug concentration in the brain (Cmax,brain = 2.3 nM at 2 mg/kg i.p.) resulting in an ED50 of 1.4
mg/kg in the ex vivo binding assay. Liver microsomal clearance was still an issue but oral
bioavailability in rat was 22%. Compound 46 represents an analogue with a shorter sulfonamide
side chain. It carries a methylated amino group leading ultimately to an ED of 2.0 mg/kg in the
50
ex vivo binding assay. Further reduction of the number of HBDs by introduction of an azetidine
afforded 47a, which achieved again a good unbound drug concentration in the brain (Cmax,brain
=
1
3 nM at 2 mg/kg i.p) and an ED of 1.8 mg/kg in the ex vivo assay despite the replacement of
5
0
the nꢀpropyl group by a more polar imidazole moiety. In addition compound 43a displayed good
oral bioavailability in preclinical species (Fp.o. = 51% in rat and 75% in dog).
Compounds (7S,8R)ꢀ27a and (7S,8R)ꢀ27b showed the best overall profile. Both proved to be
very selective with no significant receptor binding in the CEREP profile (75 receptors tested at 10
µM) with an ED50 of 1.4 mg/kg and 1.6 mg/kg respectively in the ex vivo binding assay. The
unbound drug concentration in brain was determined to be 2 nM for both, (7S,8R)ꢀ27a and
(7S,8R)ꢀ27b at a dose of 1 mg/kg i.p. Liver microsomal stability was moderate without inhibition
of major metabolizing CYP450 enzymes and oral bioavailability in preclinical species was 17%
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(
rat) / 75% (dog) for (7S,8R)ꢀ27a and 29% (rat) / 49% (dog) for (7S,8R)ꢀ27b. In addition both
GlyT1 inhibitors displayed an excellent solubility in water of more than 1 mg/mL, and high
ꢀ6
ꢀ6
permeability in the MDCK assay of 22 ꢁ 10 cm/s (7S,8R)ꢀ27a and 17 ꢁ 10 cm/s (7S,8R)ꢀ27b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
indicating BCS I like properties.
In order to study the mode of inhibition of the advanced GlyT1 inhibitors, the functional
1
3
inhibition was studied with two different glycine concentrations, 10 µM and 3000 µM. The K
i
of RG1678 for GlyT1 was 47 nM and for (7S,8R)-27a 0.8 nM as determined in our ligand
binding assay. 300 nM RG1678 inhibited the 10 µM glycine current by 67 ± 8% (n=4), and
blocked the current of 3000 µM glycine by 95 ± 1% (n=6). In contrast, (7S,8R)-27a demonstrated
inhibition of 97 ± 1% (n=4) at 10 µM and only 39 ± 3% (n=5) at 3000 µM glycine (Fig. 2). These
results indicate that while RG1678 appears to be a nonꢀcompetitive inhibitor of GlyT1c, (7S,8R)-
2
7a competitively inhibits glycine transport.
Figure 2. Oocyte Experiments with RG1678 (1) and (7S,8R)-27a
****
****
100
75
50
25
0
Surmountability of GlyT1 inhibitors. Oocytes expressing GlyT1c were stimulated with 10 µM
open bars) or 3000 µM (dark bars) glycine to induce a current through the transporter. 300 nM
(
RG1678 (left) or 300 nM (7S,8R)-27a (right) were added to the glycine solution, and the
inhibition of the current calculated. Data are represented as mean +/ꢀ SD, N>4, **** p<0.0001.
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
4
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5
5
5
6
Furthermore, compound (7S,8R)ꢀ27a doseꢀdependently reversed Lꢀ687,414ꢀinduced
29
hyperlocomotion in mice (Figure 3).
Lꢀ687,414 ((3R,4R)ꢀ3ꢀaminoꢀ1ꢀhydroxyꢀ4ꢀ
3
0
methylpyrrolidinꢀ2ꢀone) is a known selective and brainꢀpenetrant glycine site NMDA receptor
antagonist. This behavioral effect was also described with the nonꢀcompetitive GlyT1 inhibitors
RG1678 and the sarcosineꢀbased compound Org25935 (2), and likely results from the increased
extracellular level of glycine in the brain through GylT1 transport inhibition. This increased
glycine level subsequently displaces the NMDA glycineꢀsite antagonist. (7S,8R)ꢀ27a reached
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0% inhibition of Lꢀ687,414ꢀinduced hyperlocomotion at a dose of 0.8 mg/kg after i.p.
administration, which is comparable to the dose necessary to achieve 50% TO.
Figure 3. Reversal of Lꢀ687,414ꢀinduced Hyperlocomotion by Compound (7S,8R)ꢀ27a
3500
+
50 mg/kg L-687,414 (i.p.)
ED =0.8 mg/kg
3
2
000
500
5
0
2000
500
1000
*
1
5
00
0
0
0
0.3
1.0
3.0
(7S,8R)-27a (mg/kg, ip)
Nonꢀhabituated mice were injected i.p. with (7S,8R)ꢀ27a or its vehicle 120 min prior to test. 15
min prior to the test, animals were then treated with 50 mg/kg (i.p.) of Lꢀ687,414 or its vehicle
and the locomotor activity was measured for 60 min. WilcoxonꢀMannꢀWhitney test revealed a
significant stimulatory effect of Lꢀ687,414 (Mann Whitney test, p=0.0006). (7S,8R)ꢀ27a dose
dependently reduced the Lꢀ687,414ꢀinduced hyperlocomotion reaching statistical significance at
3
mg/kg (Dunn's multiple comparisons test, *p<0.05). Data are shown as mean ± SEM for n=8
mice per group.
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However, a high efflux ratio (ER) in the MDR1 transwell assay was observed ((7S,8R)ꢀ27a: ER
=
+
12; (7S,8R)ꢀ27b: ER = 7.2). Consistently, in a subsequent study with (7S,8R)ꢀ27b in MDRIa/b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
/+ and ꢀ/ꢀ mice, the brain to blood ratio of MDR1a/b ꢀ/ꢀ mice was 26ꢀfold higher than in the
MDR1a/b +/+ mice. This strengthens our finding in the in vitro studies and suggest that
compound (7S,8R)ꢀ27b is likely an in vivo substrate of the efflux transporter Pꢀglycoprotein.
These results sparked the initialization of a second round of optimization.
We first examined the effect of reducing TPSA and the number of hydrogen bond donors on
3
1
lowering ER. Following this strategy, we identified compound 27c, with a superior ER of 1.6.
However, the compound proved to be a very potent inhibitor of hERG (IC : 80 nM). As a second
5
0
3
2ꢀ34
approach, we speculated that a reduction of basicity could positively influence the ER,
since
compound 48, a close analogue of (7S,8R)ꢀ27a, with a calculated pKa of 5.6, displayed an ER of
only 1.4. As shown in table 5, inhibition of GlyT1 was still moderate K = 69 nM, but liver
i
microsomal clearance was too high to allow further development. Since the same effect on
microsomal stability was also seen for additional compounds with fluorine containing alkyl side
chains, we suspended these variations and instead pursued an exchange of our central core to the
amino chromanes. The amino group of this scaffold has a calculated pKa value of 8.0 and offers
the possibility to prepare compounds, which have a lower degree of protonation at neutral pH.
Indeed, compound 36a displayed a reduced ER of 3.5, compared to (7S,8R)ꢀ27a (ER = 12). In
general, amino chromanes were well tolerated in rodents, but the resulting compounds often
displayed a higher liver microsomal clearance compared to the aminotetralines. In summary, up
to this point both series, aminotetralines and ꢀchromanes had suboptimal properties, but
rigidification of the sulfonamide bearing side chain could potentially reduce liabilities. A number
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of derivatives with different linkers were synthesized and studied. The summary of these
approaches is shown in Table 6 for some selected compounds.
Table 5. Derivatives of Compound 20k: Calculated pKa and Influence on ER
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
K
i
[nM]
mClint (h / r)
[µL/min/mg]
pKa
(calc.)
Compound
R
X
ER
hGlyT1
(7S,8R)-27a
(3R,4R)-36a
48
CH
2
2
0.8
10
37
55
9.4
8.0
5.6
12
3.5
1.4
O
3.3
277
103
NHCH
CF
CH
69
125
2
3
K values and estimated ED for in vivo target occupancy (ED est. TO) was assessed as described in the
i
50
50
experimental part
Compared to (7S,8R)ꢀ27c, the potency of compound 36b is much reduced and liver microsomal
clearance is higher. Shortening of the ethanol amino side chain to a methylene amino linker
afforded compound 43b, however, also this derivative displayed lower potency. Compounds
(7S,8R)ꢀ51a – (3R,4R)ꢀ51d carry an azetidine linker, which had the highest impact on all desired
parameters in both series. This side chain retains excellent K , shows improved microsomal
i
stability and reduced Pꢀgp substrate liability, combined with low hERG inhibition, validating our
hypothesis that lowering pKa of the central core and/or rigidification of the linker would lead to
improved in vitro parameters. In addition, it could be shown that a more polar pyrrolidinone
linker was tolerated giving equally potent, metabolically stable compound N-(R)-(7S,8R)-53
(clogP 3.5, TPSA 70) with low ER. Compound (7S,8R)ꢀ51a is a selective and potent competitive
GlyT1 inhibitor with no significant receptor binding in the CEREP profile (75 receptors tested at
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1
0 µM). It has an oral bioavailability in rat comparable to that of compound (7S,8R)ꢀ27a (F =
po
2
5%) but achieved unbound drug concentrations of 18.7 nM in the brain at (2 mg/kg i.p.).
Assuming dose linearity, a drug concentration of 0.9 nM (equivalent to K ) is achieved in brain at
i
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
doses in the range of 0.2 mg/kg. Indeed a subsequent ex vivo TO study, revealed an estimated
ED of 0.2 mg/kg for compound (7S,8R)ꢀ51a, which correlates well with the predicted value. A
5
0
complete evaluation of free brain levels vs. K in all TO studies confirms this further (Figure 4).
i
Table 6. In Vitro Inhibitory Activity of Aminotetralines and Aminochromanes with Different
4
Moieties R at hGlyT1
K [nM]
mClint (h / r)
[µL/min/mg]
hERG
i
4
compound
R
X
hGlyT1
0.8
ER
12
IC [µM]
50
(7S,8R)-27a
CH
2
10
38
55
15
(
3R,4R)-36a
O
CH
O
3.3
2.6
277
137
940
3.5
1.6
n.d.
0.08
n.d.
(
7S,8R)-27c
2
113
239
3
6b
3b
239
n.d.
4
CH
2
71
122
302
n.d.
n.d.
(
7S,8R)-51a
1b
7S,8R)-51c
1d
CH
O
2
0.9
18
< 23
< 23
< 23
< 23
85
130
42
1.7
1.0
1.4
0.9
4.4
11.4
6.8
5
(
CH
2
2.6
44
5
O
66
n.d.
N-(R),
7S,8R)-53
CH2
28
< 23
33
1.6
4.5
(
K values and estimated ED for in vivo target occupancy (ED est. TO) was assessed as described in the
i
50
50
experimental part
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The receptor occupancy from ex vivo binding was determined as a function of the free brain
concentration, over a dose range and the free brain EC50 values were calculated for each
compound by fitting to an Emax model. Figure 4 shows the correlation of the free brain EC and
50
2
0
1
2
3
4
5
6
7
8
9
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the in vitro K for the available dataset. The correlation is very good (Pearson r = 0.86; r = 0.73),
i
indicating that the in vitro K value is a relevant measure to predict the receptor occupancy in
i
vivo, when considering the free concentration of the compound in the brain.
Figure 4. Correlation between the Free Brain EC Target Occupancy and in vitro K
i
5
0
The apparent brain EC50 values were determined by curve fitting from the receptor occupancy
curve over the free concentration/K , using the brain concentrations corrected for the fraction
i
unbound in brain (fu,brain). The dashed line represents the linear curve fit of the data set (Pearson r
2
=
0.86; r = 0.73). Red symbols represent the compounds described in detail in the chemistry
section of this publication.
The relevance of the development of competitive over nonꢀcompetitive inhibitors for GlyT1 has
1
3
been discussed in the literature. The alleviation of GlyT1 inhibition through competitive
inhibitors by high glycine could make it possible to eliminate high glycine concentrations
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associated with efficacyꢀcompromising and potentially toxic effects, within the glutamatergic
synapse, but more importantly also in inhibitory glycinergic synapses, while still allowing a
moderate increase of glycine in order to enhance the activity of the NMDA receptor. It is known
that different glycine levels are required for the activation of NMDA receptors (efficacy
mediating target) and the induction of unwanted effects (efficacy compromising effects or
peripheral side effects), which are in part mediated by the strychnineꢀsensitive glycine receptor.
1
1
1
1
1
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1
2
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7
8
9
0
NMDA receptors are activated by low glycine concentrations, as indicated by the K of glycine
d
35,36
for the modulatory site of NMDA receptors in the low µM range.
Much higher concentrations
of glycine are required to activate strychnineꢀsensitive glycine receptors as suggested by the
3
7,38
lower affinity of glycine for these receptors with K values in the high µM range.
Activation
d
of strychnineꢀsensitive glycine receptors has been shown to suppress longꢀterm potentiation
39
(
LTP), a NMDA receptor mediated effect induced by lower concentrations of glycine. This
could explain the bellꢀshaped concentrationꢀresponse curve of LTP induction mediated by the
4
0
nonꢀcompetitive inhibitor RG1678 (1). Internalization of NMDA receptors is a further
mechanism which could compromise NMDA receptor activity at high glycine levels.
41
Internalization occurs at glycine concentrations in the range of 40 µM, again much higher than
the concentration required for NMDA receptor activation.
Since high concentrations of glycine displace competitive inhibitors from their binding side, these
efficacyꢀcompromising effects may be less pronounced in the case of a competitive inhibitor as
compared to a nonꢀcompetitive inhibitor. This displacement is particularly relevant in glycinergic
synapses, where the range of concentrations of glycine is large. In that case, functioning of GlyT1
transport in these synapses by competition of the inhibitor by high synaptic glycine
concentrations can lead to an alleviation of the inhibition and a normalization of synaptic
functioning of these inhibitory synapses.
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Compared to extracellular glycine concentrations in the brain, peripheral extracellular glycine
42
levels are much higher. Therefore the liability for peripheral side effects such as a reduced
4
3
heme synthesis, which are due to blockade of glycine uptake into peripheral cells, should be
0
1
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9
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9
0
significantly lower for competitive inhibitors.
The described function of competitive GlyT1 inhibitors may lead to less central and peripheral
sideꢀeffects and would make competitive inhibitors more promising drug candidates compared to
nonꢀcompetitive GlyT1 blockers. This hypothesis will need to be further substantiated by
elaborate pharmacological experiments.
CONCLUSION
In summary, chemical modifications of the original tetrahydroquinoline core led to the discovery
of aminotetralines as new series of GlyT1 inhibitors. We successfully improved potency and
4
4
brain availability and identified compound (7S,8R)ꢀ27a as a highly potent and selective GlyT1
inhibitor, exhibiting excellent pharmacokinetic properties. These resulted in low doses to achieve
half maximum TO and high efficacy in the Lꢀ687,414ꢀinduced hyperlocomotion assay. Further
4
5,46
SAR studies revealed compound (7S,8R)ꢀ51a
displaying further improvements over (7S,8R)ꢀ
27a regarding the ER and ex vitro binding efficacy. The biological characterization of these
compounds will be reported in further detail in forthcoming publications.
EXPERIMENTAL SECTION
Chemistry. All commercially available chemicals and solvents were used without further
purification. In general, reaction mixtures were magnetically stirred at the respective temperature
under nitrogen atmosphere. Organic solutions were concentrated under reduced pressure using a
Buechi rotovap. Reactions under microwave irradiation conditions were carried out in a Biotage
Initiator EXP instrument. Normal phase chromatography was performed on an ISCO CombiFlash
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Companion MPLC system using RediSep prepacked columns with silica, and reverse phase
chromatography was performed using Chromabond C18 cartridges. Reactions were monitored by
thin layer chromatography using HPTLC Silicagel 60 F254 plates from Merck KGaA and were
visualized using 254 nm ultraviolet light and/or exposure to silica gel impregnated with iodine.
Optical rotations were obtained on a PerkinElmer polarimeter 341. All new compounds gave
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
satisfactory H NMR and LC/MS. On the basis of LCMS, all final compounds were >95% pure
unless otherwise noted. NMR spectra were obtained on a Bruker Avance I 400 MHz or Avance
III 500 or 600 MHz NMR spectrometer using residual signal of deuterated NMR solvent as
internal reference; chemical shifts for protons are reported in a parts per million scale downfield
from tetramethylsilane. Analytical LCMS data were obtained using an Agilent 1100 series HPLC
system with DAD and SQ mass spectrometer with ESI in positive mode and a scan range of
1
00−700 amu. Samples were run on a Chromasil 80 ODSꢀ7pH column 4ꢂm, 40 mm × 2 mm;
gradient elution 5−100% B over 10 min; solvent A, H O/0.1% TFA; solvent B, MeCN/0.1%
2
TFA; flow rate, 0.5 mL/min; temperature, 60°C. Chiral compounds were analyzed by chiral
HPLC using an Agilent 1100 HPLC with samples run on Chiralpak ADꢀH 4.6 mmID × 250 mm;
5
ꢂm; with nꢀhexane/EtOH/MeOH 20/40/40 as mobile phase; flow rate, 0.6 mL/min and
detection at 300 nm wavelength. Chemical names were generated using ChemDraw Professional
1
5.0 (PerkinElmer Informatics).
N-(2-((cis-7-Amino-8-(3,4-dichlorobenzyl)-5,6,7,8-tetrahydronaphthalen-2-
yl)oxy)ethyl)propane-1-sulfonamide hydrochloride (9). Step 1. To a solution of 7ꢀ
methoxyꢀ3,4ꢀdihydronaphthalenꢀ2(1H)ꢀone (15 g, 85 mmol) in 200 mL of dry MeOH under
nitrogen was added dropwise pyrrolidine (6.7 g, 94 mmol). The mixture was stirred for 1 h. Then
the solvent was reduced under vacuum and the residue was dissolved in MeCN. At 5°C 4ꢀ
(bromomethyl)ꢀ1,2ꢀdichlorobenzene (22.5 g, 94.0 mmol) dissolved in MeCN was added and the
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
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mixture was stirred overnight at RT. The solvent was reduced under vacuo and the residue was
mixed with MeOH/DCM/H O 1:1:1 (50 mL, 50 mL, 50 mL) after which 10 mL of glacial acid
2
was added. The mixture was stirred overnight. The reaction mixture was poured into a mixture of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
ice and H O and extracted 3x with DCM. The combined organic layers were washed 1x with
2
NaHCO solution and 1x with saturated NaCl solution. The organic phase was dried over MgSO4
3
and the solvent was evaporated. The residue (31.5 g) was purified by flashꢀchromatography on
silica gel with heptane/EtOAc 2:1, resulting in desired 1ꢀ(3,4ꢀdichlorobenzyl)ꢀ7ꢀmethoxyꢀ3,4ꢀ
+
dihydronaphthalenꢀ2(1H)ꢀone (24.1 g, 71.7 mmol, 84%). LC−MS (ESI, m/z) 335.0 [M + H] ,
3
37.0, 339.9
Step 2. To a solution of 1ꢀ(3,4ꢀdichlorobenzyl)ꢀ7ꢀmethoxyꢀ3,4ꢀdihydronaphthalenꢀ2(1H)ꢀone
(5.20 g, 15.5 mmol) in MeOH was added ammonium acetate (12.0 g, 155 mmol) and sodium
cyanoborohydride (1.46 g, 23.3 mmol) under nitrogen. The mixture was stirred for 4 d at RT. The
solvent was reduced under vacuo and extracted with EtOAc after addition of H O. The organic
2
layer was washed with NaCl, dried over MgSO and the solvent was removed. The residue was
4
dissolved in iPrOH and HCl in iPrOH (6N) was added. After crystallization overnight the HClꢀ
salt was separated from the mother liquor and converted to the free base with NaOH (1N). An oil
was obtained which afforded cisꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ7ꢀmethoxyꢀ1,2,3,4ꢀtetrahydroꢀ
naphthalenꢀ2ꢀamine hydrochloride (1.9 g, 5.8 mmol, 37%) after treatment with HCl and
+
crystallization. LC−MS (ESI, m/z) 336.2 [M + H] , 338.2, 340.2
Step 3. To a solution of cisꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ7ꢀmethoxyꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀ
amine hydrochloride (1.9 g, 5.8 mmol) in pyridine (10 mL) was added slowly ethyl
chloroformate (1.0 g, 9.3 mmol) under nitrogen. The mixture was stirred overnight at RT, then
the solvent was reduced and extracted with DCM. The organic layer was washed subsequently
with HCl (1N), saturated NaHCO solution, brine and dried over MgSO . The mixture was
3
4
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filtered and concentrated affording ethyl (cisꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ7ꢀmethoxyꢀ1,2,3,4ꢀ
tetrahydronaphthalenꢀ2ꢀyl)carbamate as an orange oil that crystallized after a few hours (2.1 g,
+
5
.1 mmol, 89%). LC−MS (ESI, m/z) 408.1 [M + H] , 410.1, 412.1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Step 4. To
a
solution of ethyl (cisꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ7ꢀmethoxyꢀ1,2,3,4ꢀ
tetrahydronaphthalenꢀ2ꢀyl)carbamate (2.1 g, 5.1 mmol) in DCM (50 mL) was added BBr (3.87
3
g, 15.4 mmol) at ꢀ10°C after which the reaction was allowed to warm up to RT and stirred for 2
h. A mixture of ice and H O was added to the mixture which was extracted with DCM. The
2
organic layer was washed subsequently with saturated NaHCO solution, brine, and dried over
3
MgSO . Then the mixture was filtered and concentrated affording ethyl (cisꢀ1ꢀ(3,4ꢀ
4
dichlorobenzyl)ꢀ7ꢀhydroxyꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀyl)carbamate as a brown oil. LC−MS
+
(
ESI, m/z) 394.1 [M + H] , 396.1, 398.1.
Step 5. To a suspension of NaH (55% in paraffin, 34.5 mmol) in dimethylacetatamide (80 mL)
was
added
ethyl
(cisꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ7ꢀhydroxyꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀ
yl)carbamate (6.80 g, 17.3 mmol) dissolved in dimethylacetatamide (40 mL) and the mixture
stirred for 1 h at RT. Then tertꢀbutyl (2ꢀbromoethyl)carbamate (11.6 g, 51.7 mmol) was added in
portions and the mixture was stirred for further 3 d at RT. Brine was added to the reaction
mixture which was extracted with EtOAc. The organic layer was washed subsequently with H O,
2
brine, and dried over MgSO . Then the mixture was filtered, concentrated and the residue was
4
purified on silica gel (flash chromatography DCM/MeOH 98:2) affording ethyl (cisꢀ7ꢀ(2ꢀ((tertꢀ
butoxycarbonyl)amino)ethoxy)ꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀ
yl)carbamate as an yellow oil (9.27 g, 17.2 mmol) which crystallized after a few hours. LC−MS
+
+
(
ESI, m/z) 481.1 [MꢀtꢀBu + H] , 483.1, 485.1, 559.2 [M + Na] , 561.2, 563.2.
Step 6. To a solution of ethyl (cisꢀ7ꢀ(2ꢀ((tertꢀbutoxycarbonyl)amino)ethoxy)ꢀ1ꢀ(3,4ꢀ
dichlorobenzyl)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀyl)carbamate (9.27 g, 17.2 mmol) in DCM (200
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2
2
2
2
2
2
2
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mL) was added HCl (6M in iPrOH). The reaction was stirred at RT overnight. Diethyl ether was
added and ethyl (cisꢀ7ꢀ(2ꢀaminoethoxy)ꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀ
yl)carbamate hydrochloride precipitated out and was isolated by filtration (5.85 g, 12.3 mmol).
+
0
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2
3
4
5
6
7
8
9
0
LC−MS (ESI, m/z) 437.1 [M + H] , 439.1, 441.1
Step 7. To a solution of ethyl (cisꢀ7ꢀ(2ꢀaminoethoxy)ꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ1,2,3,4ꢀ
tetrahydronaphthalenꢀ2ꢀyl)carbamate hydrochloride (635 mg, 1.34 mmol) and DMAP (327 mg,
2.68 mmol) in DCM (15 mL) was added nꢀpropaneꢀ1ꢀsulfonyl chloride (191 mg, 1.34 mmol)
dissolved in DCM (15 mL). The reaction mixture was stirred overnight at RT. The mixture was
partitioned with H O, the organic fraction was collected, and the aqueous fraction was washed
2
with DCM. The organic fractions were combined, washed subsequently with HCl (1M), saturated
NaHCO and brine, dried over MgSO , concentrated and triturated with EtOAc/diisopropylether.
3
4
The
mixture
was
filtered
to
obtain
ethyl
(cisꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ7ꢀ(2ꢀ
(propylsulfonamido)ethoxy)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀyl)carbamate as a white solid (600
+
mg, 1.10 mmol). LC−MS (ESI, m/z) 543.2 [M + H] , 545.2, 547.2
Step 8. A solution of ethyl (cisꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ7ꢀ(2ꢀ(propylsulfonamido)ethoxy)ꢀ
1
,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀyl)carbamate (600 mg, 1.10 mmol) in 30 mL KOH (20% in
EtOH) was refluxed for 2 h. The mixture was partitioned with half concentrated brine/H O
2
solution, the organic fraction was collected, and the aqueous fraction was washed with EtOAc.
The organic fractions were combined, washed with brine, dried over MgSO , concentrated,
4
triturated with EtOAc and filtered affording Nꢀ(2ꢀ((cisꢀ7ꢀaminoꢀ8ꢀ(3,4ꢀdichlorobenzyl)ꢀ5,6,7,8ꢀ
tetrahydronaphthalenꢀ2ꢀyl)oxy)ethyl)propaneꢀ1ꢀsulfonamide (125 mg, 0.270 mmol). The solvent
was concentrated, the obtained residue dissolved in MeOH and treated with 1M HCl. Then the
MeOH was evaporated and the obtained solid was filtered and dried affording Nꢀ(2ꢀ((cisꢀ7ꢀ
aminoꢀ8ꢀ(3,4ꢀdichlorobenzyl)ꢀ5,6,7,8ꢀtetrahydronaphthalenꢀ2ꢀyl)oxy)ethyl)propaneꢀ1ꢀ
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Page 29 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
+
sulfonamide hydrochloride (325 mg, 0.639 mmol). LC−MS (ESI, m/z) 471.1 [M + H] , 473.1,
1
4
75.1, H NMR (400 MHz, DMSOꢀd ) δ (ppm) 8.32 (s, 3H), 7.55 (d, J = 8.3 Hz, 1H), 7.43 (d, J =
6
2
.0 Hz, 1H), 7.23 (t, J = 5.9 Hz, 1H), 7.15 – 6.99 (m, 2H), 6.74 (dd, J = 8.5, 2.6 Hz, 1H), 5.70 (d,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J = 2.6 Hz, 1H), 3.73 (dt, J = 10.9, 5.7 Hz, 1H), 3.55 (dt, J = 10.6, 5.3 Hz, 2H), 3.27 – 3.06 (m,
4
–
H), 3.00 – 2.88 (m, 3H), 2.82 (dt, J = 17.5, 8.9 Hz, 1H), 2.59 – 2.53 (m, 1H), 1.99 (s, 2H), 1.71
1.56 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H).
N-(2-((cis-7-Amino-8-(3,4-dichlorobenzyl)-5,6,7,8-tetrahydronaphthalen-2-
yl)oxy)ethyl)methanesulfonamide hydrochloride (20a). Compound 20a (80 mg, 80%
over last two steps) was prepared according to representative example 9 from ethyl (cisꢀ7ꢀ(2ꢀ
aminoethoxy)ꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀyl)carbamate (100 mg,
0.229 mmol) by using methyl sulfonyl chloride in place of nꢀpropaneꢀ1ꢀsulfonyl chloride. White
+
1
solid. LC−MS (ESI, m/z) 443.1 [M + H] , 445.1, 447.1, H NMR (400 MHz, DMSOꢀd ) δ (ppm)
6
δ 8.46 – 8.26 (m, 3H), 7.56 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.21 (t, J = 6.0 Hz, 1H),
7.09 (dd, J = 8.3, 2.0 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.75 (dd, J = 8.4, 2.6 Hz, 1H), 5.70 (d, J =
2
3
.7 Hz, 1H), 3.80 – 3.70 (m, 1H), 3.60 – 3.51 (m, 2H), 3.27 – 3.14 (m, 3H), 3.14 – 3.07 (m, 1H),
.00 – 2.89 (m, 1H), 2.88 (s, 3H), 2.85 – 2.76 (m, 1H), 2.60 – 2.53 (m, 1H), 2.08 – 1.92 (m, 2H).
N-(2-((cis-7-Amino-8-(3,4-dichlorobenzyl)-5,6,7,8-tetrahydronaphthalen-2-
yl)oxy)ethyl)butane-1-sulfonamide hydrochloride (20b). Compound 20b (78 mg,
6
8% over last two steps) was prepared according to representative example 9 from ethyl (cisꢀ7ꢀ
2ꢀaminoethoxy)ꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ1,2,3,4ꢀtetrahydronaphthalenꢀ2ꢀyl)carbamate (100 mg,
.229 mmol) by using nꢀbutylꢀ1ꢀsulfonyl chloride in place of nꢀpropaneꢀ1ꢀsulfonyl chloride.
(
0
+
1
White solid. LC−MS (ESI, m/z) 485.1 [M + H] , 487.1, 489.1, H NMR (400 MHz, DMSOꢀd ) δ
6
(
ppm) 8.27 (s, 3H), 7.55 (d, J = 8.3 Hz, 1H), 7.42 (d, J = 2.0 Hz, 1H), 7.22 (t, J = 5.9 Hz, 1H),
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