Computationally Guided Identification of Allosteric Agonists of the Metabotropic Glutamate 7 Receptor

Article abstract of DOI:10.1021/acschemneuro.8b00331

The metabotropic glutamate 7 (mGlu₇) receptor belongs to the group III of mGlu receptors. Since the mGlu₇ receptor can control excitatory neurotransmission in the hippocampus and cortex, modulation of the receptor may have therapeutic benefit in several CNS diseases. However, mGlu₇ remains relatively unexplored among the eight known mGlu receptors partly because of the limited availability of tool compounds to interrogate its potential therapeutic utility. Here we report the discovery of a new class of mGlu₇ allosteric agonists. Hits originating from virtual screening were followed up with further analogue searching and screening, leading to a novel series of mGlu₇ allosteric agonists. Guided by docking into a structural model of the mGlu₇ receptor the initial hit 5 was successfully optimized to analogues with comparable potencies and more attractive drug-like attributes than AMN082.

Full text of DOI:10.1021/acschemneuro.8b00331

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Article
Computationally Guided Identification of Allosteric
Agonists of the Metabotropic Glutamate 7 Receptor
José María Cid, Hilde Lavreysen, Gary Tresadern, Laura Pérez-
Benito, Fulgencio Tovar, Alberto Fontana, and Andrés A. Trabanco
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00331 • Publication Date (Web): 14 Sep 2018
Downloaded from http://pubs.acs.org on September 17, 2018
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Computationally Guided Identification of Allosteric
Agonists of the Metabotropic Glutamate 7 Receptor
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Jose María Cid,1,* Hilde Lavreysen,2 Gary Tresadern,2 Laura PérezꢀBenito,3 Fulgencio Tovar,4 Alberto
Fontana1 and Andrés A. Trabanco1,*
1 Janssen Research and Development, Calle Jarama 75A, Toledo, 45007, Spain. 2Janssen Research and
Development, Turnhoutseweg 30, 2440, Beerse, Belgium. 3Laboratori de Medicina Computacional
Unitat de Bioestadistica, Facultat de Medicina, Universitat Autonoma de Barcelona, Spain.
4Villapharma Research S.L., Parque Tecnológico de Fuente Álamo. Ctra. El EstrechoꢀLobosillo, Km.
2.5ꢀ Av. Azul 30320 Fuente Álamo de Murcia, Murcia, Spain.
*Corresponding Authors. (J.M.C.) Tel.: +34 925 245750. Eꢀmail: jcid@its.jnj.com; (A.A.T.) Tel.: +34
925 245750. Eꢀmail: atrabanc@its.jnj.com
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ABSTRACT
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The metabotropic glutamate 7 (mGlu7) receptor belongs to the group III of mGlu receptors. Since the
mGlu7 receptor can control excitatory neurotransmission in the hippocampus and cortex, modulation of
the receptor may has therapeutic benefit in several CNS diseases. However, mGlu7 remains relatively
unexplored amongst the eight known mGlu receptors partly because of the limited availability of tool
compounds to interrogate its potential therapeutic utility. Here we report the discovery of a new class of
mGlu7 allosteric agonists. Hits originating from virtual screening were followed up with further
analogue searching and screening leading to a novel series of mGlu7 allosteric agonists. Guided by
docking into a structural model of the mGlu7 receptor the initial hit 5 was successfully optimized to
analogues with comparable potencies and more attractive drugꢀlike attributes than AMN082.
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KEYWORDS: mGlu7, metabotropic, glutamate, allosteric, PAM, virtual screening, homology model.
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INTRODUCTION
The metabotropic glutamate 7 (mGlu7) receptor is a class C GPCR and belongs to the subfamily
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group III of metabotropic glutamate (mGlu) receptors along with mGlu4, mGlu6 and mGlu8. The mGlu7
receptor is widely distributed in the central nervous system (CNS)1,2,3,4,5,6.It is preferentially and
specifically enriched on excitatory terminals that contact inhibitory interneurons in the hippocampus and
cortex. It is thought to play a critical role in modulating normal neuronal function and synaptic
transmission making it particularly relevant in neuropharmacology.7,8,9,10,11,12,13,14,15,16 Its biology is
predicted to be distinct from the other group III mGlu members due to its specific localization and its
remarkably low affinity for glutamate compared to mGlu4 and mGlu8,17,18,19 being only activated by
millimolar (i.e. nonꢀphysiological) glutamate concentrations.
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Studies using mGlu7 receptor knockꢀout animals have provided important insights into the potential
role of this receptor in numerous neurological and psychiatric disorders including schizophrenia,10
depression,20 anxiety,21 and epilepsy.22 Likewise numerous polymorphisms in the metabotropic
glutamate receptor 7 gene (GRM7) have been linked to autism, depression, ADHD, and
schizophrenia.23,24,25,26 Academic groups and pharmaceutical companies have mostly concentrated on
the development of ligands acting at group I and II receptors. Both orthosteric and allosteric ligands
have progressed into clinical trials with encouraging results in various indications.27 However, much
less has been discovered regarding the activation or blockade of group III mGlu receptors,16 amongst
which mGlu7 is arguably the least explored. This is partly due to the difficulties to develop tool
compounds with appropriate selectivity profiles to aid in performing proofꢀofꢀconcept studies regarding
the therapeutic potential of each receptor subtype.28 After the discovery of early selective group III
receptor orthosteric agonists, such as Lꢀ2ꢀaminoꢀ4ꢀphosphonobutyric acid (LꢀAP4),29,30 research efforts
shifted towards the search of allosteric modulators. Allosteric ligands could offer numerous advantages
over orthosteric ligands such as improved subtype selectivity amongst other members of the mGlu
receptor family, more drugꢀlike structures, and the ability to enhance receptor activity only when and
where biologically needed.31,32 Thus, N,Nꢀdibenzhydrylethaneꢀ 1,2ꢀdiamine dihydrochloride (AMN082,
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1) was identified as the first mGlu7 receptorꢀselective allosteric agonist, that is, a ligand that is capable
of directly activating the receptor from an allosteric site in the absence of an orthosteric agonist,33 and
has been widely used to interrogate the role and therapeutic potential of the mGlu7 receptor (Figure
1).34,35 AMN082 has been shown to directly activate mGlu7 via an allosteric binding site acting as the
first full allosteric agonist at an mGlu receptor.9 Unlike positive allosteric modulators (PAMs) that
usually increase the potency of orthosteric agonists, AMN082 has little or no effects on the potency of
glutamate or the orthosteric group III selective agonist LꢀAP4.28. However, AMN082 is rapidly
metabolized in vivo. Nꢀbenzhydrylethaneꢀ1,2ꢀdiamine, a major metabolite of this compound, has been
shown to exhibit significant offꢀtarget effects via modulation of multiple monoamine transporters.
Concerns have been raised that interaction with these offꢀtargets could confound the outcomes from in
vivo behavioral models by producing a nonꢀmGlu7 receptorꢀmediated response.36 This limits its value as
a tool compound and there is a significant need for improving mGlu7 allosteric ligands.
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Recently, Niswender et al. have described the first group III selective PAMs: VU0422288 (2) and
VU0155094 (3).37 Unlike allosteric agonist, a PAM potentiates an agonistꢀmediated receptor response.33
Despite the lack of selectivity for mGlu7 vs mGlu4 and mGlu8 both compounds represented valid tools
to selectively probe a role for mGlu7 due to its restricted expression in SCꢀCA1 synapses in adult
animals. More recently, Lindsley et al. identified a series of pyrazolo[1,5ꢀa]pyrimidines from a high
throughput screen (HTS) that display a range of group III mGlu receptors selectivity38. Among these,
the selective mGlu7/8 PAM VU6005649 (4) demonstrated proꢀcognitive effects on associative learning
in a mouse contextual fear conditioning model. Here we report a new chemical series of mGlu7
allosteric modulators. The series was optimized with the help of modelling the binding mode in the 7ꢀ
transmembrane (7TM) domain of the mGlu7 receptor. With pIC50 values of around 7 and relatively good
physicochemical properties they offer potential for future lead optimization.
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Cl
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H
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H
Cl
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, AMN082
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O
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O
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O
, VU0155094
, VU6005649
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Figure 1. Structures of group III mGlu allosteric agonists and PAMs
RESULTS AND DISCUSSION
We have recently reported on our efforts to identify novel small molecule ligands for mGlu7 receptors
by means of a virtual screen using proteochemometric modelling.39 The approach used a combined inꢀ
house and public dataset of around 2800 compounds tested for activation or inhibition of signaling
versus mGlu receptors. The data was used to build computational models that could predict mGlu7
receptor activity of molecules from the Janssen corporate compound collection. This method was well
suited to take advantage of the many bioactive molecules for targets such as mGlu1, mGlu2 and mGlu5
receptors, and use this information to make predictions for a similar but less explored receptor such as
mGlu7. Compounds were selected and screened and one particularly interesting hit molecule A, referred
to as molecule 3 in the original report, was identified (Figure 2).3938 The hit showed structural similarity
to AMN082 due to the presence of a similar distal benzhydryl group. This molecule A was followed up
with immediate closeꢀanalogue searching. In brief, the analogue searches used 2D ECFP6 circular
topological fingerprints and Tanimoto similarity well suited for similarity comparison. The closest
compounds from the Janssen compound library, as well as analogues from substructure searches were
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combined giving over 400 unique compounds that were selected and screened. This led to compound 5
that was an acceptable micromolar starting point to begin building structure activity relationships
(SAR).
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N
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A
mGlu7 pEC50 5.8; Emax 76%
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Z
H
N
W
R
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Y
R
R
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mGlu7 pEC50 5.8; Emax 58%
This work
Figure 2. Structure of hits A and 5 derived from an mGlu7 receptor virtual screen, and the general
scheme of mGlu7 allosteric agonists presented here.
Chemistry
The majority of the analogues 5-16, 19 and 23-25 were prepared in one step via alkylation of the
corresponding commercially available haloacetamides (26a-f) with an array of benzylamines and benzyl
alcohols (27a-j) as outlined in Scheme 1. Additionally, compound 17 was synthesized as shown in
Scheme 2. Thus, Nꢀalkylation of tertꢀbutyl Nꢀ(2ꢀaminoꢀ2ꢀphenylethyl)carbamate (27k) with the
chloroacetamide (26e) afforded compound 28. Subsequent Boc protection of the benzylic nitrogen led to
the corresponding bisꢀBoc derivative (29) that was treated with MeI to afford the intermediate
compound 30. Finally, removal of the Boc protecting groups in acidic media led to the target compound
17.
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Compounds 18, 20-22 were following a reaction sequence similar to that described for the synthesis
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of 17 (Scheme 3). Alkylation of the benzylalcohols 27l, m with chloroacetamides (26e, f) to obtain 31a,
b, followed by coupling with 2ꢀnitrobenzenesulfonyl chloride yielded intermediates 32a, b. Subsequent
alkylation of the sulfonamide nitrogen yielded intermediates 34a-d. Removal of the nosyl group from
intermediates 34a-c led to the final compounds 18, 20 and 21 in low to moderate yields. Finally
compound 22 was obtained from intermediate 34d by Suzuki cross coupling with pyridylꢀ4ꢀboronic acid
and subsequent cleavage of the Nꢀnosyl protecting group.
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The chemical structures of compounds 5−25 are shown in Tables 1 and 2.
O
O
Z
Hal
W
R
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R
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N
R
N
R
(i)
X
X
+
W
Z
Y
Y
R
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3
27a: W = NH2; R1 = H; R2 = H
27b: W = NH2; R1 = Ph; R2 = H
27c: W = NH2; R1 = CyPr; R2 = H
27d: W = NH2; R1 = CH3; R2 = H
27e: W = NH2; R1 = CF3; R2 = H
27f: W = NH2; R1 = CH2OH; R2 = H
27g: W = NH2; R1 = CH2OCH3; R2 = H
26a: Hal = Br; X,Y,Z = CH; R3 = H
5-16; 19; 23-25
26b: Hal = Cl; X,Z = CH; Y= N; R3 = H
26c: Hal = Cl; X,Y = CH; Z = N; R3 = H
26d: Hal = Cl; X = O; Y,Z = CH; R3 = H
26e: Hal = Cl; X,Y,Z = CH; R3 = H
26f: Hal = Cl; X,Y,Z = CH; R3 = F
27h: W = OH; R1 = CH2N(CH3)2; R2 = H
27i: W = OH; R1 = CH2NHCH3; R2 = F
27j: W = OH; R1 = CH2NHCH3; R2 = H
Scheme 1. Reagents and conditions: (i) a) Et3N, CH3CN, rt, 3 h, 10ꢀ49% for 6ꢀ9 and 12ꢀ16 or b) NaH,
DIPEA, DMPU, rt, 3 days, 4ꢀ17% for 540, 10 and 11 or c) NaH, THF, rt, 16 h, 25ꢀ38% for 19 and 23ꢀ25.
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O
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H2N
N
H
O
O
O
O
H
N
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O
Cl
O
O
(ii)
27k
N
N
N
H
O
N
N
N
H
O
(i)
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26e
28
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O
H
N
O
O
O
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(iii)
(iv)
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H
N
N
N
O
30
17
Scheme 2. Reagents and conditions: (i) K2CO3, CH3CN, 80 ºC, 16 h, 51%; (ii) BOC2O, Et3N, DCM, rt,
24 h, 47 %; (iii) MeI, NaH, THF, rt. 48 h, 51% (iv) HCl, rt, 85%.
O
O
NO
2
O
O
HO
O
O
S
(ii)
NH2
N
NH2
N
R
N
H
or
26e 26f
(i)
R
R
R
R
2
2
1
2
1
: R = H; R = H
: R = H; R = H
1 2
31a
32a
(S)-
(S)-
: R = H
27l
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2
: R = F; R = Br
: R = F; R = Br
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32b
: R = Br
27m
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2
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2
O
NO
O
2
O
O
O
S
O
R3-X
(iii)
: R = CH ; X = I
(iv), (v)
33a-c
N
N
N
NH
R
R
3
3
33a
33b
33c
3
3
R
R
R
R
2
1
2
1
: R = CH CH ; X = I
3
2
3
: R = CH CF ; X = OSO (CF ) CF
3
3
2
3
2
2 3
: R = H; R = H; R = CH
3
34a
34b
34c
34d
18, 20-22
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2
3
: R = H; R = H; R = CH CH
3
1
2
3
2
: R = H; R = H; R = CH CF
3
1
2
3
2
: R = F; R = Br; R = CH CF
3
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2
3
2
Scheme 3. Reagents and conditions: (i) NaH, THF, 0 ºC to rt, 24 h, 61ꢀ100 %; (ii) oꢀ
nitrobenzenosulfonyl chloride, Et3N, DCM, rt, 16 h, 84 %; (iii) Cs2CO3, THF, 0 ºC to rt, 70ꢀ99 %; (iv)
34aꢀc, 2ꢀmercaptoethanol, LiOH, DMF, rt, 16 h, 14ꢀ77 %, for 18, 20 and 21; (v) a) 34d, 4ꢀpyridinyl
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boronic acid, PdCl2(dppf)2, Na2CO3, 1,4ꢀdioxane, 90 ºC, 6 h, 87 %; b) 2ꢀmercaptoethanol, LiOH, DMF,
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rt, 16 h, 57 % for 22.
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O
H
N
R
1
N
X
Z
Y
9
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Table 1. Functional activity and metabolic stability of representative mGlu7 receptor allosteric agonists
1, 5-17a.
mGlu7 mGlu7
pEC50
CLint microsomes
Entry
X
Y
ꢀ
Z
ꢀ
R1
ꢀ
cLogPb
E
max(%)a
(ꢀL/min)/mg)(h/r)c
a
ꢀ
6.8
5.8
62
58
69
90
44
97
73
98
65
40
3
5.9
3.7
3.7
3.7
3.3
4.7
4.1
4.1
2.5
1.0
1.0
1.9
3.3
2.9
nt
nt
1
5
6
7
8
9
10
11
12
13
14
15
16
17
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
CH CH (RS)ꢀCH3
CH CH (R)-CH3
CH CH (S)-CH3
CH CH
CH CH Ph
CH CH (RS)ꢀCyPr
CH CH (RS)-CF3
CH CH (S)-CH2OH
N
CH
5.59
6.06
<4.5
6.66
6.62
6.19
5.63
<4.3
<4.3
5.62
5.75
>346/>346
>346/>346
68/>346
>346/>346
>346/>346
>346/>346
147/>346
nt
H
CH (S)-CH2OH
(S)-CH2OH
N
nt
CH CH (S)-CH2OH
CH CH (S)-CH2OCH3
46
58
77
43/240
>346/>346
90/>346
CH2
CH2
CH CH (S)-CH2NHCH3 6.14
aMost values represent the mean for at least two experiments, bcLogP calculated with Biobyte software, c(h/r) human/rat.
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SAR and model discussion
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Structural analogy of the original virtual screening hit A as well as molecule 5 with AMN082
prompted us to start our SAR studies by exploring the alpha position of the benzylamine (see Figure 2).
Compounds 5−25 were evaluated for mGlu7 agonistic activity (see methods) and data is shown in
Table 1 and 2. As a point of reference, compound 1 (AMN082) had a pEC50 of 6.8 with 62%
potentiation in our agonist assay compared with a reported EC50 of 64 nM.8 Replacement of the alkyl
linker of compound 5 with a single enantiomer chain produced enantiomers 6 (R) and 7 (S) with pEC50
values of 5.59 and 6.06 respectively. Although relatively close in activity, the preference for the Sꢀ
isomer conformation in the linker region led us to maintain this for further derivative synthesis.
Potency was remarkably improved by installation of a phenyl group in 9 that showed a 0.86 log unit
increase in potency (pEC50 = 6.66) compared to initial hit 5, Table 1. Compound 9 had comparable
activity to AMN082 and also confirmed the similarity with the benzhydryl amino fragment present in
AMN082. The activity of 9 and similarity with AMN082 provided confidence to consider additional
computational methods to help design new compounds. Recently, we have shown how combined
experimental and computational approaches can be used to generate binding mode hypotheses for
different series of mGlu2 receptor PAMs and NAMs.41,42,43, A robust binding mode was proposed and
further study led to a hypothesis of the origin of functional activity for mGlu allosteric modulators. They
achieve their effect by modifying the movement of amino acids in crucial positions of the GPCR 7TM,
referred to as the Triger and Transmission switches.44 Amino acids at the same positions in TMs 3, 5,
and 6 are known to be important for class A activation.45,46
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Based on this knowledge of mGlu receptor modelling and allosteric ligand function we derived a
structureꢀbased hypothesis for the binding mode of mGlu7 receptor allosteric agonist 9. Firstly, a
structural model of the active state of the mGlu7 receptor (Uniprot code Q14831) 7TM was built. The
crystal structure of the mGlu5 receptor47 (PDB 4OO9, 50% sequence identity in the 7TM region) was
used as a template for most of the receptor. Extracellular loop 2, which is not resolved in the mGlu5
structure, was modelled using the mGlu1 receptor structure48 (PDB 4OR2). To model the active state,
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the conformation of the intracellular TM6 of mGu7 receptor (N785ꢀM794) was modified based on the
active conformation in the β2ARꢀGs complex49 (PDB 3SN6). Models were constructed with MOE
(Chemical Computing Group, https://www.chemcomp.com/). The final model was chosen as the best
quality result having a low RMSD (root mean square deviation) to the average models (i.e. not a
structural outlier), no outliers from a Ramachandran dihedral or bond length plots, correct
stereochemistry and no high energy contacts. These factors along with the sequence identity to the
mGlu5 template in the 7TM region provided confidence in the final model. Molecule 9 was docked
using the MOE induced fit docking approach. The binding site and flexible amino acids were defined as
all the atoms of the mGlu7 sidechains within a 6 Å radius around the position of the allosteric binding
site. The allosteric site was defined based on structural alignment to the mGlu1 crystal structure bound
with FITM47 (PDB 4OR2). A tether factor (10x) was used to constraint sidechain flexibility and results
were submitted to further minimization.
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Docking of 9 delivered multiple binding modes as expected, but one was common to the best scoring
solutions, Figure 3. In this orientation, the benzhydrylamine group penetrated deepest into the receptor.
The 7TM binding cavity was hydrophobic and interestingly contains as many as three methionine
residues making direct contact with the ligand, M6663.40c, M7675.51c, and M8297.36c. The upper part of
the binding pocket was lined with lipophilic amino acids interacting with the tetrahydroquinoline,
L6623.36c, I7565.40c, L7605.44c, W8016.50c, F8046.53c and L8257.32c. The amide carbonyl of 9 was predicted
to act as a hydrogen bond acceptor from the side chain of S7635.47c. Meanwhile the benzhydryl group
entered the lower lipophilic pocket, surrounded by the three methionine residues and sitting on top of
the mGlu receptor conserved and functionally important Y6703.44c. The amino acids M6663.40c
,
S7635.47c, W8016.50c are at the same structural position as the sideꢀchains associated with the trigger
switch of mGlu2 PAM activity,42 hence it was consistent to see similar interactions with the mGlu7
allosteric agonists here. With only a limited amount of SAR it was difficult at this stage to confirm the
binding mode, thus we designed molecules to test it further.
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Figure 3. The docked binding mode of compound 9 at the 7TM domain of the mGlu7 receptor. A: The
binding mode with the benzhydryl group penetrating deepest, TM helices are numbered, and amino
acids labelled. B: Cutꢀthrough surface side view of the ligand binding cavity, coloring is according to
nearby atom types. C: The 2D interaction map showing more details of the amino acids surrounding the
ligand.
With this binding mode in mind we designed smaller and less lipophilic groups to replace the
benzhydryl substituent. Thus, compounds 10-12 were synthesized to investigate if more drugꢀlike motifs
were tolerated. Removing the R1 methyl group, as shown in 8, resulted in a loss of potency. The
replacement of the phenyl group by cyclopropyl produced 10, that exhibited potency similar potency to
9 (10: pEC50 = 6.62 vs 9: pEC50 = 6.66). Slight reduction in potency was found with the CF3 substitution
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(11: pEC50 = 6.19). The surface view in Figure 3B suggested that more polar groups may be permitted
in the deeper part of the pocket and interact with acceptor groups represented by the redꢀsurface
coloring. Interestingly, introduction of a group such as CH2OH (12) did indeed result in active
compounds (pEC50 = 5.63). The activity seen for compound 12 stimulated us to exploit the CH2OH
substitution in combination with other variations. Thus, starting from 12, we reduced the lipophilicity of
the bicyclic tetrahydroquinoline warhead. Unfortunately, analogues 13 and 14 in which a Nꢀatom is
introduced into the benzopyran nucleus had a deleterious effect on activity. These results were likely
due to unfavorable interactions with the many lipophilic amino acids around this upper pocket.
However, the 1,4ꢀbenzoxazine 15 that had the oxygen atom at the benzylic position of the bicyclic core
showed comparable activity to that of its corresponding carbon analogue (12: pEC50 = 5.63 vs 15: pEC50
= 5.62). The overall favored tetrahydroquinoline group was kept intact for subsequent exploration.
Further exploration of the deep pocket by introducing alternative heteroatom containing substituents, led
to compounds 16 and 17. The ether derivative displayed comparable potency to the alcohol 12 (12:
pEC50 = 5.63 vs 16: pEC50 = 5.75). Introduction of an aliphatic amine (17) was somewhat more
favorable than methoxy (pEC50 = 6.14) and also attractive from the druglike perspective for potentially
improving solubility and reducing lipophilicity.
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Table 2. Functional activity and metabolic stability of representative mGlu7 receptor agonists 18-25a
1
2
3
4
O
O
R
1
5
6
N
7
8
9
10
11
12
13
14
15
16
17
18
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60
R
R
2
3
mGlu7
pEC50
mGlu7
CLint microsomes
Entry
R1
R2
R3
cLogPb
Emax(%)a
(ꢀL/min)/mg) (h/r)c
a
(S)-CH2NHCH3
H
H
H
H
H
H
H
H
F
H
F
F
6.96
6.7
71
60
68
81
5
47
57
32
3.0
3.6
3.6
3.8
4.6
3.2
3.2
3.3
80/>346
nt
96/>346
>346/>346
nt
54/>346
68/>346
42/>346
18
19
20
21
22
23
24
25
(S)-CH2N(CH3)2
(S)-CH2NHCH2CH3
(S)-CH2NHCH2CF3
(S)-CH2NHCH2CF3
(S)-CH2NHCH3
6.85
6.15
<4.3
5.83
6.33
5.76
4ꢀpyridine
F
H
F
(S)- CH2NHCH3
(S)-CH2NHCH3
aMost values represent the mean for at least two experiments, bcLogP calculated with Biobyte software, c(h/r) human/rat.
The binding mode suggested the nitrogen in the central spacer was not essential, for instance it did not
form any hydrogen bond or salt bridge interactions. We investigated this by modifying it to an ether
spacer in compounds 18 to 25, Table 2. Compound 18 conferred a remarkable increase in potency
(pEC50 = 6.96) compared to its corresponding nitrogen version (17; pEC50 = 6.14). This promising
increase motivated us to further expand SAR on the terminal amino group as exemplified with
compounds 19-21. Different Nꢀalkyl groups were introduced unfortunately none of them displayed
greater potency and activity was only slightly lower indicating flat SAR, neither driven by an increase in
lipophilicity nor size. Interestingly, compound 22 having the appended pyridine was totally inactive
compared to the similarly substituted compound 21. This suggested that the receptor does not
accommodate substituents larger than a methyl group in this region. This is consistent with the binding
mode shown in Figure 3A, where the R1 and R2 groups penetrate deepest into the 7TM and larger
groups would clash with the surface of the binding cavity, Figure 3B.
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In the final set of experiments, a limited exploration was conducted to investigate the effects of
electronic density on the phenyl rings of the tetrahydroquinoline or distal phenyl. Compounds 23ꢀ25
bearing fluorine atoms at the phenyl ring turned out to be less active compared to the nonꢀsubstituted
analogue 18. Whilst 24 was the most active of the three (pEC50 = 6.33), analogue 25 containing two
fluorine atoms was the least active (pEC50 = 5.76). This might suggest a role for the two aryls in
stabilizing the binding mode by forming aromatic stacking interactions, or aliphatic CꢀH aromatic
interactions for instance with L6623.36c, I7565.40c, L8257.32c which are favored by electron rich rings.
Several compounds such as 9 and 11 showed higher Emax than the initial compounds such as AMN082
and 5. Furthermore, compounds 7, 9 and 11 with (S)ꢀCH3, phenyl or CF3 showed relatively high Emax
(90, 97 and 98 % respectively) and interestingly the (R)ꢀCH3 a lower Emax (69 %). Therefore, the R1
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pocket is important for functional effect. The R1 substituents are interacting with amino acids M6663.40c
,
S7635.47c and W8016.50c. These amino acids are the mGlu7 receptor equivalent of the 3 amino acids
described as the Trigger switch in mGlu2.43 They sit one turn directly above the transmission switch and
adopt different conformational behavior depending on the functional activity of the allosteric modulator.
It seems likely that the mGlu7 ligands here cause similar effects.
Receptor potency was improved quickly but metabolic stability studies in both human and rat liver
microsomes (HLM, RLM) proved more challenging (Tables 1 and 2). Most of the compounds suffered
from extensive metabolism in both species, particularly in rats. Slight improvement was observed with
the introduction of polar groups as illustrated with compound 12 and even more so for 15, suggesting a
metabolic hot spot located at the benzylic position of the bicyclic core. As anticipated, compounds 16ꢀ
21 having new potential metabolic sites did not exhibit any improvement. Reduction of electron density
on the rings, via addition of electronꢀwithdrawing groups, was not an effective strategy: compounds 23ꢀ
25, bearing fluorine atoms, showed only a subtle improvement in stability in humans.
Furthermore, as AMN082, compounds 5ꢀ25 do not affect Ca2+ responses in HEK293 cells coꢀ
expressing the human mGlu7 receptor and the promiscuous G protein Gα15 (data not shown). A same
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complex pharmacological profile of AMN082 has been described before50. While all molecules were
inactive against other mGlu receptors in this same readꢀout, we have not evaluated other receptor
responses or cell populations, assuming selectivity towards other mGlu receptor subtypes
(mGlu1,2,3,4,5,6,8) remains as good as for AMN082, given their high similarity and shared binding mode.
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Conclusion
A novel series of tetrahydroquinolines with mGlu7 receptor allosteric agonistic activity has been
described. Arising from a virtual screening hit, the molecules were rapidly optimized with the help of
modelling the binding mode into a homology model of the Glu7 receptor. Starting from hit 5, a set of
derivatives were synthesized allowing for a conclusive SAR study. Potency reached significant levels
with pEC50 close to 7 for several examples. This compared favorably to the wellꢀstudied reference
compound AMN082. Physicochemical properties were also improved, with examples such as 18 having
lower lipophilicity than compared to AMN082, with LogP of 3 for 18 vs LogP of 5.9 for AMN082.
Taken together, these results suggest promise for this tetrahydroquinolines series. However, the
challenge remains to combine potency with metabolic stability. These studies are underway and will be
the subject of future reports from our laboratories. Since it is only now that more selective mGlu7
receptor agonists and antagonists have been identified, we expect this study can contribute to a clearer
picture on the role of the mGlu7 receptor over the coming years.
METHODS
Measurements of mGlu7 agonist activity. Modulation of forskolinꢀinduced cAMP accumulation was
measured in human mGlu7 receptorꢀexpressing CHO cells using the Lance  Ultra cAMP kit (Perkin
Elmer). Cells were seeded at 2,000 cells/well in 384ꢀwell white Optiplates (Perkin Elmer). After
addition of forskolin (2 ꢀM), cells were incubated at room temperature for 30 min, followed by addition
of Lance  Ultra cAMP reagents and additional 60 min incubation at room temperature. Lance signal
was measured on an Envision multilabel reader (Perkin Elmer). Agonist effects were picked up by a
reduction in forskolinꢀinduced cAMP production or consequently, an increase in Lance signal.
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Liver Microsomal Stability. Microsomal stability studies were conducted on a Biomek FX robotic
liquid handling workstation (Beckman Coulter, Brea, CA), which consists of a 96ꢀchannel pipet head, a
12ꢀpostion workstation deck, and a plate incubator. The test compound is incubated at a defined
substrate concentration (typically 1 ꢀM) in liver microsomes from human and rat across a time course
(typically 0, 5, 10, 20, 40, and 60 minutes). The reaction was brought to 37 °C and initiated by adding
NADPH to a final concentration of 1 mM. The reaction is terminated by addition of a suitable organic
solvent (typically, acetonitrile, methanol or DMSO). The samples are centrifuged prior to analysis by
LCꢀMS/MS analysis. The relative amount of parent compound remaining in the active incubations vs.
the control incubations (t=0 mins) for each compound is measured by peak area comparison. The
predicted hepatic clearance was derived from the intrinsic clearance value using equations from the
wellꢀstirred model (Curr. Drug Metab. 2008, 9, 940−951), where protein binding in plasma and
microsomal proteins is assumed to be similar and the blood to plasma concentration ratio is assumed to
be 1.
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Chemistry. Unless otherwise noted, all reagents and solvents were obtained from commercial
suppliers and used without further purification. Thin layer chromatography (TLC) was carried out on
silica gel 60 F254 plates (Merck). Flash column chromatography was performed on silica gel, particle
size 60 Å, mesh = 230−400 (Merck), under standard techniques. Microwave assisted reactions were
performed in a singleꢀmode reactor, Biotage Initiator Sixty microwave reactor (Biotage), or in a
multimode reactor, MicroSYNTH Labstation (Milestone, Inc.). Nuclear magnetic resonance (NMR)
spectra were recorded with either a Bruker DPXꢀ400 or a Bruker AVꢀ500 spectrometer (broaduker AG)
with standard pulse sequences NMR data operating at 400 and 500 MHz, respectively, using CDCl3 and
DMSOꢀd6 as solvents. Chemical shifts (δ) are reported in parts per million (ppm) downfield from
tetramethylsilane (δ = 0). Coupling constants are reported in hertz. Splitting patterns are defined by s
(singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), quin (quintet), sex (sextet), sep (septet),
or m (multiplet). Liquid chromatography combined with mass spectrometry (LC−MS) was performed
either on a HP 1100 HPLC system (Agilent Technologies) or Advanced Chromatography Technologies
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system composed of a quaternary or binary pump with degasser, an autosampler, a column oven, a
diode array detector (DAD), and a column as specified in the respective methods. Flow from the column
was split to a MS spectrometer. The MS detector was configured with either an electrospray ionization
source or an ESꢀCI dual ionization source (electrospray combined with atmospheric pressure chemical
ionization). Nitrogen was used as the nebulizer gas. Data acquisition was performed with MassLynxꢀ
Openlynx software or with ChemsationꢀAgilent Data Browser software. Retention time (tR) is
expressed in min. Gas chromatography combined with mass spectrometry (GC−MS) was performed
using a 6890 series gas chromatograph (Agilent Technologies) system comprising a 7683 series injector
and autosampler, a column oven, and a J&W HPꢀ5MS coupled to a 5973N MSD mass selective detector
(single quadrupole, Agilent Technologies). The MS detector was configured with an electronic impact
ionization source/chemical ionization source (EI/CI). EI lowꢀresolution mass spectra were acquired by
scanning from 50 to 550 at a rate of 14.29 scans/s. The source temperature was maintained at 230 °C.
Helium was used as the nebulizer gas. Data acquisition was performed with ChemstationꢀOpen Action
software. Melting point (mp) values are peak values and were obtained with experimental uncertainties
that are commonly associated with this analytical method and were determined in open capillary tubes
on a Mettler FP62 apparatus with a temperature gradient of 10 °C/min. Maximum temperature was 300
°C.
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Purities of all new compounds were determined by analytical reverse phase (RP) HPLC using the area
percentage method on the UV trace recorded at a wavelength of 254 nm and were found to have ≥95%
purity unless otherwise specified.
Supercritical fluid chromatography (SFC) was performed using Analytical system from Berger
instrument comprising a FCMꢀ1200 dual pump fluid control module for delivering CO2 and modifier, a
CTC Analytics automatic liquid sampler, a TMCꢀ20000 thermal control module for column heating
from room temperature to 80ºC. An Agilent 1100 UV photodiode array detector equipped with a highꢀ
pressure flow cell standing up to 400 bars was used. Flow from the column was split to a MS
spectrometer. The MS detector was configured with an atmospheric pressure ionization source. The
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following ionization parameters for the Waters ZQ mass spectrophotometer are: corona: 9ꢀa, source
tem: 140ºC, cone: 30V, probe temp 450ºC, extractor 3 V, desolvatation gas 400Lhr, cone gas 70 L/hr.
Nitrogen was used as the nebulizer gas. Data acquisition was performed with a WatersꢀMicromass
MassLynxꢀOpenlynx data system.
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9
2-(benzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one (8). A mixture of 26a (0.160 g,
0.63 mmol), 27a (0.135 g, 1.26 mmol), and Et3N (0.175 mL, 35.2 mmol) in CH3CN (1.8 mL) was
stirred at room temperature for 3h and then concentrated in vacuo. The crude product was purified by
RP HPLC (Stationary phase: C18 XBridge 30 x 100 mm 5 um), Mobile phase: Gradient from 67% 0.1%
NH4CO3H/NH4OH pH 9 solution in Water, 33% CH3CN to 50% 0.1% NH4CO3H/NH4OH pH 9
solution in Water, 50% CH3CN) to give 8 as a colorless oil (0.087 g, 49%). 1H NMR (500 MHz, CDCl3)
δ 7.18ꢀ7.35 (m, 6H), 7.04ꢀ7.17 (m, 3H), 3.76 (br s, 4H), 3.56 (s, 2H), 2.70 (br t, J=6.50 Hz, 2H), 2.36 (s,
1H), 1.94 (quin, J=6.57 Hz, 2H). LCMS: m/z 281 [M + H]+, tR = 1.90 min.
10
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2-(diphenylmethylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one (9). Starting from 26a
(0.183 g, 0.72 mmol) and 27b (0.158 g, 0.87 mmol) and following the procedure described for 8,
1
compound 9 was obtained as a yellow solid (0.063 g, 25%). H NMR (500 MHz, CDCl3) δ 7.39 (br d,
J=7.22 Hz, 4H), 7.23ꢀ7.32 (m, 6H), 7.15ꢀ7.23 (m, 2H), 6.98ꢀ7.14 (m, 3H), 4.85 (br s, 1H), 3.58ꢀ3.88 (m,
2H), 3.52 (s, 2H), 2.69 (br s, 2H), 1.93 (quin, J=6.57 Hz, 2H). LCMS: m/z 357 [M + H]+, tR = 2.94 min.
(RS)-2-(1-methy-1-cyclopropyl)-benzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one (1).
Starting from 26a (0.683 g, 2.69 mmol) and 27c (0.247 g, 1.34 mmol) and following the procedure
1
described for 8, compound 10 was obtained as a yellow solid (0.075 g, 17%). H NMR (500 MHz,
CDCl3)
δ 7.19ꢀ7.36 (m, 6H), 7.01ꢀ7.13 (m, 3H), 3.69 (br s, 2H), 3.43 (q, J=15.80 Hz, 2H), 2.80 (br d,
J=8.67 Hz, 1H), 2.68 (br t, J=6.36 Hz, 2H), 1.84ꢀ2.00 (m, 2H), 1.61 (br s, 3H), 0.97ꢀ1.16 (m, 1H), 0.53ꢀ
0.69 (m, 1H), 0.29ꢀ0.46 (m, 1H), 0.13ꢀ0.26 (m, 1H). LCMS: m/z 321 [M + H]+, tR = 2.42 min.
(RS)-2-(1-methylbenzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one
(5).
Diisopropylethylamine (0.246 mL, 1.42 mmol) was added to a solution of (RS)ꢀ27d hydrochloride (0.15
g, 0.95 mmol) in 1,3ꢀdimethylꢀ3,4,5,6ꢀtetrahydroꢀ2(1H)ꢀpyrimidinone (DMPU) (3 mL) at 0ºC. The
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mixture was stirred for 5 min and NaH (0.086 mg, 2.14 mmol) was added portion wise. Then, a solution
of 26a (0.62 g, 1.9 mmol) in DMPU (2 mL) was added dropwise and the resulting mixture was
gradually warmed to room temperature and stirred for 3 days. The mixture was diluted with water and
extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The crude was purified by flash column chromatography (silica gel, EtOAc in
DCM, 0/100 to 10/90) to give 5 as a pale brown oil (0.124 g, 17%). 1H NMR (500 MHz, CDCl3) δ 7.21
(m, 6H), 7.01ꢀ7.14 (m, 3H), 3.52ꢀ4.00 (m, 3H), 3.33ꢀ3.46 (m, 2H), 2.68 (br t, J=6.50 Hz, 2H), 1.83ꢀ1.99
(m, 3H), 1.37 (d, J=6.65 Hz, 3H). LCMS: m/z 295 [M + H]+, tR = 2.13 min.
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(R)ꢀ2-(1-methylbenzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one (6). Starting from
26a (0.436 g, 1.72 mmol) and (R)ꢀ27d (0.437 mL, 3.43 mmol) and following the procedure described
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for 8, compound 6 was obtained as a colorless oil (0.215 g, 43%). H NMR (400 MHz, CDCl3) δ 7.16ꢀ
7.34 (m, 6H), 7.01ꢀ7.14 (m, 3H), 3.50ꢀ3.94 (m, 3H), 3.34ꢀ3.46 (m, 2H), 2.68 (br t, J=6.59 Hz, 2H),
2.04ꢀ2.55 (br s, 1H), 1.81ꢀ2.03 (m, 2H), 1.37 (d, J=6.70 Hz, 3H). LCMS: m/z 295 [M + H]+, tR = 2.19
min.
(S)ꢀ2-(1-methylbenzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one (7). Starting from
26a (0.450 g, 1.77 mmol) and (S)ꢀ27c (0.492 mL, 3.54 mmol) and following the procedure described for
8, compound 7 was obtained as a colorless oil (0.124 g, 24%). 1H NMR (500 MHz, CDCl3) δ 6.43ꢀ8.06
(m, 9H), 4.17 (br s, 1H), 3.38ꢀ3.99 (m, 5H), 2.66 (br s, 2H), 1.79ꢀ2.11 (m, 2H), 1.71 (br d, J=6.65 Hz,
3H). LCMS: m/z 295 [M + H]+, tR = 2.19 min.
(RS)ꢀ2-(1-trifluoromethylbenzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one
(11).
Starting from 26a (0.272 g, 1.07 mmol) and (RS)ꢀ27e (0.113 g, 0.535 mmol) and following the
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procedure described for 5, compound 11 was obtained as a colorless oil (0.008 g, 4%). H NMR (500
MHz, CDCl3) δ 7.30ꢀ7.46 (m, 4H), 6.78ꢀ7.30 (m, 5H), 4.22 (br d, J=5.49 Hz, 1H), 3.31ꢀ4.02 (m, 4H),
2.51ꢀ2.94 (m, 3H), 1.80ꢀ2.01 (m, 2H). LCMS: m/z 349 [M + H]+, tR = 2.60 min.
(S)ꢀ2-(1-hydroxymethylbenzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one
(12).
Starting from 26a (0.256 g, 1 mmol) and (S)ꢀ27f (0.166 mL, 1.21 mmol) and following the procedure
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described for 8, compound 12 was obtained as a white solid (0.067 g, 21%). H NMR (400 MHz,
CDCl3) δ 6.51ꢀ7.65 (m, 9H), 3.24ꢀ4.11 (m, 7H), 2.22ꢀ3.21 (m, 4H), 1.80ꢀ2.08 (m, 2H). LCMS: m/z 311
[M + H]+, tR = 1.71 min.
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(S)ꢀ2-(1-hydroxymethylbenzylamino)-1-(1,2,3,4-tetrahydro-1,5-naphthyridin-1-yl)ethan-1-one
(13). Starting from 26b (0.200 g, 0.95 mmol) and (S)ꢀ27f (0.130 g, 0.95 mmol) and following the
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procedure described for 8, compound 13 was obtained as a yellow oil (0.080 g, 26%). H NMR (400
MHz, CDCl3)
δ ppm 1.86 ꢀ 2.12 (m, 2 H), 2.94 (t, J=6.78 Hz, 2 H), 3.39 ꢀ 3.95 (m, 7 H), 7.08 (dd,
J=8.03, 4.77 Hz, 1 H), 7.25 ꢀ 7.38 (m, 5 H), 8.28 (dd, J=4.77, 1.51 Hz, 1 H). LCMS: m/z 312 [M + H]+,
tR = 2.35 min.
(S)ꢀ2-(1-hydroxymethylbenzylamino)-1-(1,2,3,4-tetrahydro-1,8-naphthyridin-1-yl)ethan-1-one
(14). Starting from 26c (0.150 g, 0.712 mmol) and (S)ꢀ27f (0.085 g, 0.62 mmol) and following the
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procedure described for 8, compound 14 (0.012 g, 12%). H NMR (400 MHz, D2O)
δ 9.05 (d, J=6.62
Hz, 1H), 7.50 (d, J=7.50 Hz, 1H), 7.17ꢀ7.40 (m, 5H), 7.10 (t, J=7.28 Hz, 1H), 4.31ꢀ4.42 (m, 1H), 4.17
(s, 2H), 3.88ꢀ4.06 (m, 2H), 3.73 (t, J=5.51 Hz, 2H), 2.83 (br. s., 2H), 1.98 (d, J=5.29 Hz, 2H). LCMS:
m/z 312 [M + H]+, tR = 3.085 min.
(S)ꢀ2-(1-hydroxymethylbenzylamino)-1-(3,4-dihydro-2H-benzo[b][1,4]oxazin-1-yl)ethan-1-one
(15). Starting from 26d (0.130 g, 0.614 mmol) and (S)ꢀ27f (0.084 g, 0.61 mmol) and following the
procedure described for 8, compound 15 (0.021 g, 11%). 1H NMR (400 MHz, CDCl3)
δ ppm 3.45 ꢀ 3.60
(m, 3 H), 3.62 ꢀ 3.82 (m, 3 H), 4.17 (t, J=4.77 Hz, 3 H), 6.67 ꢀ 6.87 (m, 2 H), 6.94 ꢀ 7.05 (m, 1 H), 7.18 ꢀ
7.36 (m, 6 H). LCMS: m/z 313 [M + H]+, tR = 4.133 min.
(S)ꢀ2-(1-methoxymethylbenzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one
(16).
Starting from 26a (0.085 g, 0.334 mmol) and (S)ꢀ27g (0.061 mg, 0.401 mmol) and following the
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procedure described for 8, compound 16 was obtained as a white solid (0.045 g, 41%). H NMR (400
MHz, CDCl3)
δ
ppm 1.92 (tq, J=12.91, 6.62 Hz, 3 H) 2.67 (t, J=6.40 Hz, 3 H), 3.35 ꢀ 3.41 (m, 5 H),
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3.43 ꢀ 3.54 (m, 3 H), 3.57 ꢀ 3.84 (m, 2 H), 3.86 ꢀ 3.97 (m, 1 H), 6.97 ꢀ 7.13 (m, 1 H), 7.19 ꢀ 7.38 (m, 1
H). LCMS: m/z 325 [M + H]+, tR = 3.69 min.
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(S)ꢀ2-(1-(N-BOC-aminomethylbenzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one (28).
A mixture of 26e (0.887 g, 4.23 mmol), 27k (1 g, 4.23 mmol), and K2CO3 (1.168 g, 8.464 mmol) in
CH3CN (15 mL) was stirred at 80ºC overnight. The mixture was diluted with water and extracted with
EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The crude was purified by flash column chromatography (silica gel, petroleum ether/EtOAc 20/1
to 1/1) to give 28 (1 g, 51%). LCMS: m/z 410 [M + H]+, tR = 0.753 min.
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(S)ꢀ2-(N-BOC-1-(N-BOC-aminomethylbenzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-
one (29). A mixture of 28 (1 g, 2.44 mmol), diꢀtbutyl dicarbonate (0.559 g, 2.564 mmol), and Et3N
(0.494 g, 4.884 mmol) in CH2Cl2 (15 mL) was stirred at 50ºC overnight. The mixture was quenched
with 5% citric acid (aqueous solution). The organic layer was washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The crude was purified by flash column chromatography (silica gel,
petroleum ether/EtOAc 20/1 to 5/1) to give 29 (0.6 g, 47%). LCMS: m/z 510 [M + H]+, tR = 1.248 min.
(S)ꢀ2-(N-BOC-1-(N-mehtyl-N-BOC-aminomethylbenzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-
yl)ethan-1-one (30). To a solution of 29 (0.6 g, 1.177 mmol) in THF (12 mL) stirred at 0ºC under
Nitrogen atmosphere, NaH (0.096 g, 2.354 mmol) was portion wise added and stirred for 15 minutes.
Then methyl iodine (0.2 g, 1.41 mmol) was dropwise added and the resulting mixture was stirred at
room temperature for 16h. Then, water was carefully added. The organic layer was collected and
washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The crude was purified by
flash column chromatography (silica gel, petroleum ether/EtOAc 20/1 to 5/1) to give 30 (0.32 g, 51%).
LCMS: m/z 524 [M + H]+, tR = 1.11 min.
(S)ꢀ2-(1-methylaminomethylbenzylamino)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one (17).
A mixture of 30 (0.13 g, 0.248 mmol) in HCl 4N solution in 1,4ꢀdioxane (15 mL) was stirred at room
temperature for 2 hours. Then the mixture was concentrated in vacuo and the residue thus obtained was
dissolved in CH2Cl2, washed NaHCO3 (aqueous saturated solution) and brine, dried over anhydrous
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Na2SO4, and concentrated in vacuo. The crude was purified by flash column chromatography (silica gel,
MeOH in DCM, 0/100 to 10/90) to give 17 (0.090 g, 85%). LCMS: m/z 524 [M + H]+, tR = 1.11 min.
1H NMR (400 MHz, DMSOꢀd6) δ 10.47 (br s, 1H), 9.66 (br s, 1H), 9.11 (br s, 1H), 7.31ꢀ7.85 (m, 5H),
7.30ꢀ7.00 (m, 4H), 4.82 (br s, 1H), 4.23.7 (m, 5H), 3.16 (s, 3H), 2.53ꢀ2.85 (m, 3H), 1.49ꢀ2.11 (m, 2H).
LCMS: m/z 397 [M + H]+, tR = 0.561 min.
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(S)ꢀ2-(1-aminomethylbenzyloxy)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one (31a). To
a
solution of (S)ꢀ27l (0.3 g, 2.187 mmol) in THF (15 mL) at 0 °C was added portionwise NaH (0.131 g,
3.28 mmol). After 10 min stirring, 26e (0.550 g, 2.624 mmol) was added and the resulting mixture was
gradually warmed to room temperature and stirred for 1 day. The mixture was diluted with water and
extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The crude was purified by flash column chromatography (silica gel, MeOH in
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CH2Cl2, 0/100 to 100/0) to give 31a as a colorless oil (0.412 g, 61%). H NMR (300 MHz, CDCl3)
δ
7.42 – 7.23 (m, 5H), 7.22 – 6.98 (m, 3H), 4.51 – 4.31 (m, 1H), 4.26 (d, J = 14.0 Hz, 1H), 4.08 (d, J =
14.0 Hz, 1H), 3.88 – 3.61 (m, 2H), 3.02 – 2.79 (m, 2H), 2.70 (t, J = 6.6 Hz, 2H), 2.06 – 1.87 (m, 2H).
LCMS: m/z 311 [M + H]+, tR = 1.06 min.
(S)ꢀ2-(1-aminomethyl-4-bromobenzyloxy)-1-(6-fluoro-1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-
one (31b). Starting from 26e (0.388 g, 1.7 mmol) and (S)ꢀ27g (0.307 mg, 1.42 mmol) and following the
procedure described for 31a, compound 31b was obtained as a white solid (0.578 g, 100%).
(S)ꢀ2-(1-N-(2-nitrophenylsulfonylaminomethylbenzyloxy)-1-(1,2,3,4-tetrahydroquinolin-1-
yl)ethan-1-one (32a). 2ꢀNitroꢀNꢀmehtylꢀphenylsulfonyl chloride (0.294 g, 1.32 mmol) was dissolved in
CH2Cl2 (9 mL), and the solution was cooled to 0 °C. Diisopropylethylamine (0.329 mL, 1.991 mmol)
and 31a (0.412 g, 1.327 mmol) were added, and the resulting solution was gradually warmed to room
temperature and stirred overnight. NaHCO3 (aqueous saturated solution) was added and extracted with
CH2Cl2. The combined organic layer was dried using Na2SO4, and the solvent was removed under
reduced pressure. The residue thus obtained was purified by flash column chromatography (silica gel,
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EtOAc in Heptane, 0/100 to 100/0) to give 32a as a colorless oil (0.554 g, 84%). H NMR (300 MHz,
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CDCl3)
δ
8.09 (dd, J = 5.9, 3.3 Hz, 1H), 7.81 (dd, J = 5.9, 3.4 Hz, 1H), 7.73 – 7.58 (m, 2H), 7.37 – 7.23
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(m, 5H), 7.11 (dd, J = 9.7, 6.9 Hz, 4H), 4.60 (dd, J = 9.4, 2.9 Hz, 1H), 4.29 (d, J = 14.8 Hz, 1H), 4.00
(d, J = 14.8 Hz, 1H), 3.77 (s, 2H), 3.49 – 3.36 (m, 1H), 3.21 (dd, J = 16.1, 6.0 Hz, 1H), 2.71 (t, J = 6.6
Hz, 2H), 1.96 (q, J = 6.6 Hz, 2H). LCMS: m/z 496 [M + H]+, tR = 1.6 min.
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(S)ꢀ2-(1-N-(2-nitrophenylsulfonylaminomethyl-4-bromobenzyloxy)-1-(6-fluoro-1,2,3,4-
tetrahydroquinolin-1-yl)ethan-1-one (32b). Starting from 31b (0.578 g, 1.42 mmol) and following
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the procedure described for 32a, compound 32b was obtained as a colourless oil (0.293 g, 35%). H
NMR (300 MHz, CDCl3) δ 7.99ꢀ8.13 (m, 1H), 7.78ꢀ7.90 (m, 1H), 7.60ꢀ7.77 (m, 2H), 7.45 (d, J=8.25
Hz, 2H), 6.76ꢀ7.23 (m, 5H), 4.62 (dd, J=2.75, 8.80 Hz, 1H), 4.27 (br d, J=14.57 Hz, 1H), 4.14 (q,
J=7.15 Hz, 2H), 3.98 (br d, J=14.57 Hz, 1H), 3.56ꢀ3.90 (m, 1H), 3.37ꢀ3.56 (m, 1H), 3.15ꢀ3.33 (m, 1H),
2.73 (br t, J=6.05 Hz, 2H), 1.97 (quin, J=6.70 Hz, 2H). LCMS: m/z 594 [M + H]+, tR = 1.68 min.
(S)ꢀ2-(1-N-(2-nitro-N-methyl-phenylsulfonylaminomethylbenzyloxy)-1-(1,2,3,4-
tetrahydroquinolin-1-yl)ethan-1-one (34a). Compound 32a (0.2 g, 0.404 mmol), Cs2CO3 (0.197 g,
0.605 mmol) and 33a (0.032 mL, 0.525 mmol) were suspended in DMF (3 mL), The mixture was stirred
at room temperature overnight. Then, water was added and extracted with EtOAc. The combined
organic layer was dried using Na2SO4, and the solvent was removed under reduced pressure. The
residue thus obtained was purified by flash column chromatography (silica gel, EtOAc in Heptane,
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0/100 to 15/85) to give 34a (0.203 g, 99%). H NMR (300 MHz, CDCl3)
δ 7.98 (dd, J = 6.0, 3.2 Hz,
1H), 7.72 – 7.54 (m, 3H), 7.32 (d, J = 11.8 Hz, 6H), 7.22 – 7.00 (m, 3H), 4.77 (t, J = 6.3 Hz, 1H), 4.17
(d, J = 13.3 Hz, 1H), 4.03 (d, J = 13.3 Hz, 1H), 3.73 (t, J = 6.3 Hz, 2H), 3.49 (d, J = 6.2 Hz, 2H), 2.96
(s, 3H), 2.70 (t, J = 6.5 Hz, 2H), 1.94 (q, J = 6.6 Hz, 2H). LCMS: m/z 510 [M + H]+, tR = 4.03 min.
(S)ꢀ2-(1-N-(2-nitro-N-ethyl-phenylsulfonylaminomethylbenzyloxy)-1-(1,2,3,4-tetrahydroquinolin-
1-yl)ethan-1-one (34b). Starting from 32a (0.128 g, 0.258 mmol) and 33b (0.027 mL, 0.336 mmol) and
following the procedure described for 32a, compound 34b was obtained (0.098 g, 72%). 1H NMR (300
MHz, CDCl3)
δ
8.09 – 8.00 (m, 1H), 7.66 – 7.54 (m, 2H), 7.32 (s, 5H), 7.11 (s, 3H), 4.73 (dd, J = 7.9,
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4.5 Hz, 1H), 4.13 (d, J = 13.0 Hz, 1H), 3.98 (d, J = 13.2 Hz, 1H), 3.77 – 3.36 (m, 6H), 2.74 – 2.56 (m,
2H), 1.93 (p, J = 6.6 Hz, 2H), 1.09 (t, J = 7.1 Hz, 3H). LCMS: m/z 524 [M + H]+, tR = 1.66 min.
(S)ꢀ2-(1-N-(2-nitro-N-ethyl-phenylsulfonylaminomethylbenzyloxy)-1-(1,2,3,4-tetrahydroquinolin-
1-yl)ethan-1-one (34c). Starting from 32a (0.224 g, 0.452 mmol) and 33c (0.159 mL, 0.678 mmol) and
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δ 7.39 – 7.23 (m, 6H), 7.21 – 7.00 (m, 3H), 4.57 (dd, J = 9.0, 2.9 Hz, 1H), 4.26 (d, J =
14.3 Hz, 1H), 4.06 (d, J = 14.3 Hz, 1H), 3.73 (d, J = 6.3 Hz, 2H), 3.35 – 3.12 (m, 2H), 3.09 – 2.95 (m,
1H), 2.82 (dd, J = 12.6, 2.9 Hz, 1H), 2.69 (t, J = 6.6 Hz, 2H), 1.94 (q, 2H). LCMS: m/z 578 [M + H]+,
tR = 4.41 min.
(S)ꢀ2-(1-N-(2-nitro-N-ethyl-phenylsulfonylaminomethyl-4-bromobenzyloxy)-1-(6-fluoro-1,2,3,4-
tetrahydroquinolin-1-yl)ethan-1-one (34d). Starting from 32b (0.293 g, 0.495 mmol) and 33c (0.173
mL, 0.742 mmol) and following the procedure described for 34a, compound 34c was obtained (0.255 g,
76%). LCMS: m/z 676 [M + H]+, tR = 1.83 min.
(S)ꢀ2-(1-N-(2-nitro-N-ethyl-phenylsulfonylaminomethyl-4-pyridyl-benzyloxy)-1-(6-fluoro-1,2,3,4-
tetrahydroquinolin-1-yl)ethan-1-one (34d). To a stirred suspension of 34d (0.100g, 0.148 mmol),
pyridinylboronic acid (0.024 g, 0.178 mmol) and Na2CO3 (0.031 g, 0.297 mmol) in 1,4ꢀdioxane (5 mL)
was added PdCl2(dppf)2 (0.006 g, 0.007 mmol) and the mixture was heated at 90 °C for 16h. The
mixture was cooled to room temperature and filtered through a Celite pad. The filtrate was diluted with
water (20 mL) and extracted with EtOAc (2 × 15 mL). The organic layer was washed with brine (15
mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The crude was purified by flash column
chromatography (silica; AcOEt in heptane 0/100 to 100/0) to give the desired product 34d as a yellow
solid (0.087 g, 87%). LCMS: m/z 673 [M + H]+, tR = 1.54 min.
(S)ꢀ2-(1-ethylaminomethyl-4-pyridyl-benzyloxy)-1-(6-fluoro-1,2,3,4-tetrahydroquinolin-1-
yl)ethan-1-one (22). LiOH (0.033, 0.776 mmol) and 2ꢀmercaptoethanol (0.011 mL, 0.158 mmol) were
added to a solution of 34d (0.087 g, 0.129 mmol) in DMF (0.8 mL) stirred at 0ºC. Then, the resulting
mixture was stirred at room temperature overnight. Then, water was added and extracted with EtOAc.
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The combined organic layer was dried using Na2SO4, and the solvent was removed under reduced
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CH2Cl2, 0/100 to 15/85) to give 22 as colorless oil (0.036 g, 57%). H NMR (300 MHz, CDCl3)
δ 8.69
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(d, J=6.05 Hz, 2H), 7.65 (d, J=8.11 Hz, 2H), 7.36ꢀ7.56 (m, 4H), 6.71ꢀ6.96 (m, 3H), 5.32 (s, 1H), 4.67
(br d, J=6.46 Hz, 1H), 4.21ꢀ4.39 (m, 1H), 4.01ꢀ4.19 (m, 1H), 3.73 (br s, 2H), 3.29 (q, J=9.26 Hz, 2H),
3.01ꢀ3.19 (m, 1H), 2.89 (br d, J=12.51 Hz, 1H), 2.72 (br t, J=6.39 Hz, 2H), 1.97 (quin, J=6.56 Hz, 2H).
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(18).
Starting from 34a (0.203 g, 0.398 mmol) and following the procedure described for 22, compound 18
was obtained as colorless oil (0.054 g, 41%). 1H NMR (300 MHz, DMSOꢀd6) δ 7.23ꢀ7.61 (m, 6H), 6.91ꢀ
7.20 (m, 3H), 4.52 (br dd, J=3.64, 8.18 Hz, 1H), 4.25 (d, J=14.16 Hz, 1H), 4.06 (d, J=14.16 Hz, 1H),
3.61 (tdd, J=6.36, 12.75, 19.21 Hz, 2H), 2.55ꢀ2.97 (m, 5H), 2.34 (s, 3H), 1.85 (quin, J=6.70 Hz, 2H)
LCMS: m/z 325 [M + H]+, tR = 2.07 min
(S)ꢀ2-(1-dimethylaminomethylbenzylamino)-1-(1,2,3,4tetrahydroquinolin-1-yl)ethan-1-one (19).
NaH (0.036 mg, 0.908 mmol) was added to a solution of (S)ꢀ27h (0.152 g, 0.726 mmol) in THF (4 mL)
at 0ºC. The mixture was stirred for 10 min at 0ºC and NaH (0.086 mg, 2.14 mmol) was added portion
wise. Then, 26e (0.100 g, 0.614 mmol) was added and the resulting mixture was gradually warmed to
room temperature and stirred for 16h. The mixture was diluted with water and extracted with EtOAc.
The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The
crude was purified by flash column chromatography (silica gel, MeOH in DCM, 0/100 to 10/90) to give
19 (0.083 g, 38%). 1H NMR (300 MHz, DMSOꢀd6) δ 7.43 (br s, 1H), 7.24ꢀ7.36 (m, 5H), 7.02ꢀ7.18 (m,
3H), 4.54 (br t, J=6.19 Hz, 1H), 4.15 (d, J=13.47 Hz, 1H), 4.04 (d, J=13.47 Hz, 1H), 3.54ꢀ3.70 (m, 2H),
2.57ꢀ2.74 (m, 3H), 2.30ꢀ2.47 (m, 1H), 2.17 (s, 6H), 1.77ꢀ1.95 (m, 2H). LCMS: m/z 339 [M + H]+, tR =
2.31 min.
(S)ꢀ2-(1-ethylaminomethylbenzyloxy)-1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one (20). Starting
from 34b (0.098 g, 0.452 mmol) and following the procedure described for 22, compound 20 was
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obtained (0.032 g, 43%). H NMR (300 MHz, CDCl3) δ 6.92ꢀ7.56 (m, 9H), 4.63 (dd, J=3.02, 9.07 Hz,
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1H), 4.28 (d, J=14.16 Hz, 1H), 4.12 (d, J=14.16 Hz, 1H), 3.75 (dt, J=5.84, 12.41 Hz, 1H), 2.62ꢀ3.05 (m,
5H), 2.21 (br d, J=13.33 Hz, 3H), 1.84ꢀ2.07 (m, 2H), 1.18 (t, J=7.15 Hz, 3H). LCMS: m/z 339 [M +
H]+, tR = 2.12 min.
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(21).
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Starting from 34c (0.182 g, 0.315 mmol) and following the procedure described for 22, compound 21
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was obtained (0.097 g, 77%). H NMR (300 MHz, CDCl3) δ 6.93ꢀ7.47 (m, 9H), 4.59 (dd, J=2.89, 9.07
Hz, 1H), 4.28 (d, J=14.30 Hz, 1H), 4.06 (d, J=11.13 Hz, 1H), 3.56ꢀ3.92 (m, 2H), 3.15ꢀ3.39 (m, 2H),
3.05 (dd, J=9.28, 12.44 Hz, 1H), 2.84 (dd, J=2.96, 12.58 Hz, 1H), 2.71 (t, J=6.60 Hz, 2H), 1.96 (quin,
J=6.70 Hz, 3H). LCMS: m/z 393 [M + H]+, tR = 2.47 min.
(S)ꢀ2-(1-methylaminomethyl)-4-fluorobenzylamino)1-(1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-
one (23). Starting from 26e (0.104 g, 0.497 mmol) and (S)ꢀ27i (0.070 g, 0.414 mmol) and following the
procedure described for 19, compound 23 was obtained (0.020 g, 14%). 1H NMR (300 MHz, DMSOꢀd6)
δ 8.36 (br s, 1H), 6.93ꢀ7.80 (m, 7H), 4.80 (br d, J=8.25 Hz, 1H), 4.42 (br d, J=15.26 Hz, 1H), 4.15 (br d,
J=15.40 Hz, 1H), 3.46ꢀ3.81 (m, 2H), 2.92ꢀ3.30 (m, 3H), 2.60ꢀ2.81 (m, 4H), 1.86 (quin, J=6.25 Hz, 2H).
LCMS: m/z 343 [M + H]+, tR = 2.35 min.
(S)ꢀ2-(1-methylaminomethyl)-4-benzylamino)-1-(6-fluoro-1,2,3,4-tetrahydroquinolin-1-yl)ethan-
1-one (24). Starting from 26f (0.2 g, 0.88 mmol) and (S)ꢀ27j (0.120 g, 0.8 mmol) and following the
procedure described for 19, compound 24 (0.092 g, 33%). 1H NMR (300 MHz, DMSOꢀd6) δ 7.64 – 7.41
(m, 1H), 7.41 – 7.22 (m, 5H), 7.07 – 6.88 (m, 2H), 4.47 (s, 1H), 4.21 (d, J = 14.2 Hz, 1H), 4.04 (d, J =
14.0 Hz, 1H), 3.70 – 3.50 (m, 2H), 2.77 – 2.62 (m, 3H), 2.58 – 2.51 (m, 1H), 2.27 (s, 3H), 1.92 – 1.77
(m, 2H). LCMS: m/z 343 [M + H]+, tR = 2.33 min.
(S)ꢀ2-(1-methylaminomethyl)-4-fluorobenzylamino)-1-(6-fluoro-1,2,3,4-tetrahydroquinolin-1-
yl)ethan-1-one (25). Starting from 26f (0.21 g, 0.922 mmol) and (S)ꢀ27i (0.130 g, 0.768 mmol) and
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following the procedure described for 19, compound 25 was obtained (0.070 g, 25%). H NMR (300
MHz, CDCl3) δ 6.25ꢀ8.10 (m, 7H), 4.80 (br d, J=9.49 Hz, 2H), 4.32 (br d, J=15.53 Hz, 1H), 4.06 (br s,
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1H), 3.70 (br s, 2H), 3.25 (br d, J=12.23 Hz, 1H), 2.34ꢀ3.03 (m, 6H), 1.91 (br d, J=5.50 Hz, 2H).
LCMS: m/z 361 [M + H]+, tR = 2.401 min.
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ASSOCIATED CONTENT
Supporting information
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Author Information
*Corresponding Authors.
(J.M.C.) Tel.: +34 925 245750. Eꢀmail: jcid@its.jnj.com
(A.A.T.) Tel.: +34 925 245750. Eꢀmail: atrabanc@its.jnj.com
Author Contributions
J.M.C., G.T., H.L. and A.A.T. conceived the project and designed experiments. F.T., G.T., L.P.B.,
A.A.T. and A.F. performed experiments. J.M.C., G.T., L.P.B., H.L. and A.A.T. analyzed and interpreted
results. J.M.C., G.T., H.L. and A.A.T. wrote and revised the manuscript.
Funding
This work was supported by Janssen R&D.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank members of the purification and analysis group and the in vitro metabolism team
from Janssen R&D.
References
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