Discovery of dihydropyrazino-benzimidazole derivatives as metabotropic glutamate receptor-2 (mGluR2) positive allosteric modulators (PAMs)

Article abstract of DOI:10.1016/j.ejmech.2019.111881

A scaffold hopping strategy converted the known 1-[(1-methyl-1H-imidazol-2-yl)methyl]-4-phenylpiperidine core (1 and 2) by cyclization to a fused [6 + 5+6] membered heterocyclic mGluR2 PAM scaffold. Pharmacophore guided structure?activity relationship (SAR) studies resulted in a series of potent and metabolically stable mGluR2 PAMs. A representative optimized compound (95) having the most balanced profile, demonstrated efficacy in the PCP-induced hyper-locomotion model in mice that revealed the new chemotype being a promising PAM lead targeting mGluR2 receptors and providing support for further translational studies.

Full text of DOI:10.1016/j.ejmech.2019.111881

Journal Pre-proof
Discovery of dihydropyrazino-benzimidazole derivatives as metabotropic glutamate
receptor-2 (mGluR2) positive allosteric modulators (PAMs)
György Szabó, Sándor Kolok, Zoltán Orgován, Mónika Vastag, Zoltán Béni, János
Kóti, Katalin Sághy, György I. Lévay, István Greiner, György M. Keserű
PII:
S0223-5234(19)31033-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.111881
EJMECH 111881
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 September 2019
Revised Date: 5 November 2019
Accepted Date: 11 November 2019
Please cite this article as: Gyö. Szabó, Sá. Kolok, Zoltá. Orgován, Mó. Vastag, Zoltá. Béni, Já. Kóti,
K. Sághy, Gyö.I. Lévay, Istvá. Greiner, Gyö.M. Keserű, Discovery of dihydropyrazino-benzimidazole
derivatives as metabotropic glutamate receptor-2 (mGluR2) positive allosteric modulators (PAMs),
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111881.
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2019 Published by Elsevier Masson SAS.

Discovery of dihydropyrazino-benzimidazole
derivatives as metabotropic glutamate receptor-2
(
mGluR2) positive allosteric modulators (PAMs)
a,
a
b
a
a
a
György Szabó *, Sándor Kolok , Zoltán Orgován , Mónika Vastag , Zoltán Béni , János Kóti ,
a
a
a
b,
Katalin Sághy , György I. Lévay , István Greiner , György M. Keserű *
a
Gedeon Richter Plc., 19-21 Gyömrői út, Budapest, 1103 Hungary
b
Medicinal Chemistry Research Group, Research Centre for Natural Sciences, Hungarian
Academy of Sciences, 2 Magyar tudósok körútja, Budapest 1117 Hungary
Corresponding author information
*
*
(Gy.Sz.) Phone: +36-1-4315180 E-mail: gy.szabo@richter.hu
(G.M.K) Phone: +36-1-3826821 E-mail: gy.keseru@mta.ttk.hu
ABSTRACT
A scaffold hopping strategy converted the known 1-[(1-methyl-1H-imidazol-2-yl)methyl]-4-
phenylpiperidine core (1 and 2) by cyclization to a fused [6+5+6] membered heterocyclic
mGluR2 PAM scaffold. Pharmacophore guided structure−activity relationship (SAR) studies
resulted in a series of potent and metabolically stable mGluR2 PAMs. A representative
optimized compound (95) having the most balanced profile, demonstrated efficacy in the PCP-
induced hyper-locomotion model in mice that revealed the new chemotype being a promising
PAM lead targeting mGluR2 receptors and providing support for further translational studies.
KEYWORDS
mGlu receptor; Positive Allosteric Modulator; Scaffold hopping; dihydropyrazino-
2
benzimidazoles; Pharmacophore based optimization
1

Abbreviations
AcOH, acetic acid; AcCN, acetonitrile; ADME, absorption, distribution, metabolism,
excretion; Boc-Gly-OH, N-(tert-Butoxycarbonyl)glycine; Clint, intrinsic clearance; Cplasma
,
plasma concentrations; Cbrain, brain concentrations; DMF, N,N-dimethylformamide; EDC, 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; EtOAc, ethyl acetate; GE, group
efficiency; GPCR, Gprotein-coupled receptor; h, hours; IBCF, isobutyl chloroformate; HOBt, 1-
hydroxybenzotriazole; LE, ligand efficiency; mGlu2, metabotropic glutamate 2; MeOH,
methanol; MW, molecular weight; NAM, negative allosteric modulator; ND, not determined;
NMM, N-methylmorpholine; NMR, nuclear magnetic resonance; PAM, positive allosteric
modulator; PCP, phencyclidine; PK, pharmacokinetics; SAR, structure−activity relationship;
SEM, standard error of mean; HPLC, high-performance liquid chromatography; SD, standard
deviation; THF, tetrahydrofuran; tPSA, topological polar surface area
1
. Introduction
Glutamate is the main excitatory neurotransmitter in brain that regulates neuronal and glial
excitability through glutamate receptors and transporters. Ionotropic receptors (NMDA, AMPA
and kainate) are cation permeable ion channels while metabotropic receptors (mGluRs) represent
class C G protein-coupled receptors signalling through G protein subunits and secondary
1
messengers such as diacylglycerol and cAMP. The eight subtypes of mGluRs (mGluR1–8) are
classified into three groups including group I (mGluR1 and 5), group II (mGluR2 and 3) and
group III (mGluR 4, 6, 7 and 8) receptors. Due to their effects on glutamatergic signalling and
more specifically on NMDA receptor function, virtually all of the mGluRs have been implicated
in psychiatric disorders. Medicinal chemistry efforts are focused developing mGluR ligands
against orthosteric and allosteric sites. The orthosteric glutamate binding site of these receptors
are located at the extracellular Venus flytrap domain that is linked to the membrane bound 7-
transmembrane (7-TM) domain. Since this site is highly conserved in all mGluRs, selective
ligands typically target the allosteric site located intrahelically in the 7-TM bundle.
Group II receptors are expressed presynaptically and they inhibit the release of
2
3
4
neurotransmitters from glutamatergic, GABAergic and dopaminergic neurons. Their
2

localization and functional profile nominate mGlu2 and 3 receptors as potential targets for novel
5
antipsychotics. In fact, the antipsychotic effect of mGluR2/3 receptor agonists are confirmed in
6
7
both preclinical and clinical settings. In 2012, however, the development of the selective
mGluR2/3 receptor agonist, LY2140023 has been stopped due to lack of efficacy found in a
8
,9
pivotal phase III trial. Although, there could be multiple reasons behind this failure, we
hypothesized that the limited subtype selectivity and the agonist character of the compound are
the major ones. Since transgenic mice studies suggested that, the antipsychotic efficacy is
1
0
mediated through the activation of mGluR2 and not of mGluR3 receptors our approach was
focused to the specific activation of the mGluR2 by PAMs. Furthermore, PAMs without inherent
agonist activity may potentiate glutamate-induced activation of the receptor and would prevent
1
1
agonist induced receptor desensitization.
As a part of our efforts developing alternative therapies for schizophrenia, an mGluR2 PAM
1
2
program was initiated. In addition to fragment based approaches our lead discovery strategy
involved the synthesis and evaluation of small libraries based on the mGluR2 PAM
1
3,14
pharmacophore.
Since the structure-activity relationship of allosteric modulators is often
1
5
steep or flat and structural modifications might switch between NAMs and PAMs, we found
1
6,17
this type of library approach specifically useful for allosteric mGluR ligands.
Our design
principle involved scaffold hopping from known mGluR2 PAM chemotypes while keeping the
key pharmacophore intact. This strategy provided several new scaffolds that were derivatized by
parallel synthesis. One of the known starting scaffolds, the 1-[(1-methyl-1H-imidazol-2-
yl)methyl]-4-phenylpiperidine was emerged from high throughput screening (HTS) hits (1) at
1
8
19
Pfizer and also identified as an HTS hit (2) at GSK (Fig. 1. A). Pfizer successfully optimized
to a representative lead compound that was in vivo active in psychosis disease models such as
1
methamphetamine-induced hyperactivity and mescaline-induced scratching in mice. Detailed
optimization of 2 resulted in selective, orally bioavailable mGluR2 positive modulators at GSK.
3

Fig. 1. Scaffold hopping from 1-[(1-methyl-1H-imidazol-2-yl)methyl]-4-phenylpiperidine to
dihydropyrazino-benzimidazoles. A: Hits identified at Pfizer (1) and GSK (2). B: Design
principle for scaffold hopping. C: Chemical starting point (47) for optimization. D:
Pharmacophore alignment of 1 (green), 2 (grey) and 47 (light blue).
Using the design principle presented in Figure 1. B we arrived to the new dihidropyrazino-
benzimidazole scaffold. The syntheis and primary biological evaluation of a small library of 4-
arylpiperidine derivatives (Table 1) provided a viable submicromolar starting point (47) for our
medicinal chemistry program. Our primary objective was to improve metabolic stability while
4

maintaining the target potency of novel dihydropyrazino-benzimidazole derivatives and confirm
the in vivo antipsychotic activity of this chemotype.
Here we report the optimization of dihydropyrazino-benzimidazoles, a novel chemotype of
mGluR2 PAMs that led to the advanced lead (95) providing in vivo proof of concept for the new
scaffold in the animal models of schizophrenia.
2
. Results and Discussion
2
.1. Chemistry.
The general synthesis of dihydropyrazino-benzimidazole compounds 42-74 are shown in
Scheme 1. Reaction of ortho-nitrofluorobenzene 3 with ethanolamine 4 in DMSO at room
2
0
temperature provided 2-[(2-nitrophenyl)amino]ethanol 5. The nitro derivative 5 was then
reduced to the corresponding amine 6 by treatment with hydrazine hydrate in the presence of
Raney-Ni as catalyst. The amine 6 was cyclized using glycolic acid in the presence of HCl
solution to provide 2-(2-hydroxymethyl-benzoimidazol-1-yl)-ethanol 7 which was further
reacted to 1-(2-chloro-ethyl)-2-chloromethyl-1
H
-benzimidazole 8 using thionyl chloride in
were alkylated with dichloro benzimidazole 8 in the
to provide dihydropyrazino-benzimidazole derivatives 42-74. The chemical
structures of 42-74 are shown in Table 1, 2, 3, 4 and 5.
2
1
22,23
DMF. Finally, amine derivatives 9-41
presence of K CO
2
3
o
Scheme 1. (a) DMSO, rt, 16h; (b) EtOH, hydrazine hydrate/Raney-Ni, 40 C, 2h; (c) cc.
HCl/H O, glycolic acid, reflux, 6h; (d) CHCl , SOCl , reflux, 3h; (e) CH CN, K CO , reflux, 10h
2
3
2
3
2
3
5

Dihydropyrazino-benzimidazole-1(2H)-one derivatives 87-95 were prepared following the
synthesis sequence shown in Scheme 2. Benzimidazole carboxylic acid 75 was first reacted by
aminoethanole 4 to provide the hydroxyethyl benzimidazole derivative 76. The reaction was
carried out in the presence of EDC and HOBt at room temperature. Hydroxyethyl benzimidazole
7
6 can be cyclized in the presence of thionyl chloride in DMF to provide, in one pot, the desired
,4-dihydropyrazino[1,2-a]benzimidazol-1(2H)-one intermediate 77. Finally, 77 was alkylated
3
with bromo-derivatives 78-86 in the presence of NaH to provide the desired dihydropyrazino-
benzimidazolone derivatives 87-95. The chemical structures of 87-95 are shown in Table 5 and
6
.
o
o
2
Scheme 2. (a) DMF, EDC/HOBt, rt, 12h (b) DMF, SOCl , 150 C, 3h; (c) DMF, NaH, 100 C, 1h
The synthesis of 1,2-dihydropyrazino[1,2-a]benzimidazol-3(4H)-one derivative 102 is shown
in Scheme 3. Coupling of Boc-Gly-OH 97 with -phenylendiamine 96 gave the monoamide,
which were converted in situ to the desired heteroaromatic derivative 98 by cyclization and
o
2
4
dehydration in AcOH. Deprotection with HCl/EtOAc yielded the amino derivative 99 which
was condensed with glycolic acid via EDC/HOBt to the corresponding hydroxyl intermediate
1
00. Cyclization with Ph P/DIAD in DMF yielded the desired 1,2-dihydropyrazino[1,2-
3
a]benzimidazol-3(4H)-one intermediate 101, which was alkylated with 1-bromo-3-(3',4'-
2
5
dichlorophenoxy)propane 78 in the presence of NaH provided the desired dihydropyrazino-
benzimidazolone derivative 102.
6

o
o
Scheme 3. (a) i. NMM, IBCF, -20 C–rt, 3.5h; ii. AcOH, 65 C, 1h; (b) HCl/EtOAc, rt, 1h; (c)
glycolic acid, DMF, NMM, EDC/HOBt, rt, 2h; (d) Ph P/DIAD, DMF, rt, 16h; (e) DMF, NaH,
3
o
1
00 C, 1h
2
.2. Identification of the dihydropyrazino-benzimidazole scaffold
Following our design strategy we applied scaffold hopping on competitors’ chemotype
exemplified by the 1-[(1-methyl-1H-imidazol-2-yl)methyl]-4-phenylpiperidine scaffold. Besides
2
6
the known drivers of scaffold hopping we considered three further aspects, keeping the
druglike profile, the synthetic tractability and the feasibility for array synthesis of the designed
compounds. The scaffold hopping strategy is depicted in Fig. 1. B and centred on a cyclization at
the benzylic site to incorporate a fused [6+5+6] membered heterocycle and ring opening at the
piperidine moiety to produce the new dihydropyrazino-benzimidazole core. The optimization
potential of the dihydropyrazino-benzimidazole scaffold was first assessed by synthesizing a
small library of 4-arylpiperidine derivatives (Table 1). For our initial exploration, we explored
different substituents of the phenyl ring (42-49). Initial SAR showed that substitution at the para
position was preferred for activity, as moving the 4-fluoro substituent of compound 45 to meta-
position (44) and ortho-position (43) decreased the activity. Replacement of the fluoro
substituent by larger substituents 46-49 provided compounds with improved potency, indicating
that increased lipophilicity in that area of the molecule is advantageous. Compound 47 showed
potent mGluR2 PAM activity (EC = 0.80 µM), its activity is comparable with the Pfizer (1)
5
0
and GSK (2) hits and has reasonable calculated lipophilicity (clogP = 4.11). Interestingly,
heteroaryl moieties (51 and 52) in that area are not tolerated in terms of mGluR2 potency.
7

Table 1. Functional activity and metabolic stability data of compounds 42-52.
a
c
Cmpd Ar
clogP
4.00
3.48
mGluR2 PAM
Clint (mL/min/g liver)
human rat
b
EC50±STD (µM)
mouse
ND
4
4
2
3
5.19 ± 1.98
ND
ND
7.53 ± 0.23
2.9
4.4
3.0
4
4
4
4
4
4
5
5
5
4
5
6
7
8
9
0
1
2
3.48
3.48
3.94
4.11
3.85
3.18
4.33
2.81
3.25
4.70 ± 1.32
1.39 ± 0.68
1.83 ± 0.65
0.80 ± 0.19
2.43 ± 1.92
2.74 ± 0.55
1.2
0.7
1.0
0.7
7.2
15.7
ND
ND
ND
1.6
1.3
4.9
3.5
5.1
3.5
ND
ND
ND
1.3
0.7
0.8
2.6
7.2
1.7
ND
ND
ND
36±3%@1µM
-10±1.7%@1µM
34±22%@1µM
a
clogP calculated with ChemAxon software
b
EC values were obtained from three independent experiments
5
0
c
Intrinsic clearance (Cl ) was determined as a rate of compound consumption under first order
int
kinetic conditions using human, rat and mouse liver microsomes; ND, not determined.
2
.3. Optimization of the dihydropyrazino-benzimidazole scaffold
8

To complete the initial SAR analysis, we examined the effect of the linker length (Table 2). In
general, lengthening the linker improved the affinity significantly. Introduction of butyl and
pentyl linkers (55 and 56) led to a 12- and 40-fold increase in potency, respectively, when
compared to that of 42.
Table 2. The effect of linker length on functional activity and metabolic stability data
a
c
Comp.
n
clogP
mGluR2 PAM
EC50±STD ( M)
Cl (mL/min/g liver)
int
b
µ
human rat
mouse
4.2
5
4
5
5
5
3
2
4
5
6
1
2
3
4
5
3.05
4.00
4.44
4.89
4.67
2.7±0%@1µM
5.19 ± 1.98
1.36 ± 0.73
0.42 ± 0.23
0.13 ± 0.05
3.0
ND
ND
2.3
2.5
4.9
ND
ND
8.8
ND
ND
11.4
8.6
13.9
a
clogP calculated with ChemAxon software
b
EC50 values were obtained from three independent experiments
c
Intrinsic clearance (Cl ) was determined as a rate of compound consumption under first order
int
kinetic conditions using human, rat and mouse liver microsomes; ND, not determined.
Selection of the ideal linker has been supported by the comparative analysis of different
chemotypes in the mGluR2 pharmacophore model (Fig. 2). Reference compounds from two
2
7,28
29
30
different Pfizer scaffolds (1 and A
), Janssen (B) and AstraZeneca (C) chemotypes were
3
1
used to derive a 3D pharmacophore model by Phase. The best model consists of four
pharmacophoric features, one acceptor atom and three hydrophobic points. In some PAMs there
are further hydrophobic regions (Hy1, Hy2) which can advantageously contribute to the
1
4
activity. This analysis revealed that the four-methylene linker attached to the dihydropyrazino-
benzimidazole core at the Hy1 site allows the terminal aryl group to reach Hy2 site.
Consequently, this linker length was considered in further optimization.
9

Fig. 2. mGluR2 pharmacophore model developed using competitor chemotypes represented by
compounds 1 (green), A (red), B (pink), C (yellow). Alignment of 55 (light blue) in the
pharmacophore model.
With the optimal linker length identified, a sequential SAR expansion focusing first on the effect
of substitution on the phenyl ring of 55 was conducted. A representative set of analogues is
shown in Table 3. Surprisingly, neither electron-withdrawing, nor electron-donating substituents
on the phenyl ring (57-64), had significant effect for mGluR2 potency. Beside these unexpected
results, the high metabolic turnover seen for 55 was retained for the substituted derivatives 60,
6
1.
Table 3. The effect of substituent scan on functional activity and metabolic stability data
10

a
c
Comp.
R
clogP
mGluR2 PAM
EC ±STD ( M)
Cl (mL/min/g liver)
int
b
µ
human rat
mouse
11.4
ND
50
5
5
5
5
6
6
6
6
6
5
7
8
9
0
1
2
3
4
H
4.89
4.08
0.42 ± 0.23
2.4 ± 0.53
2.3
8.8
4-CN
4-CH
ND
ND
ND
1.0
ND
ND
ND
14.6
11.1
ND
ND
ND
O
4.07
2.28 ± 0.90
0.74 ± 0.26
0.40 ± 0.16
0.34 ± 0.15
0.97 ± 0.36
0.54 ± 0.22
1.21 ± 0.19
ND
3
4-CF
O
5.66
ND
3
2,4-diCl
2,4-diF
5.44
15.3
5.1
4.51
1.1
2-F,4-CF
2-F,4-Cl
2-F,4-CH
5.25
ND
ND
ND
ND
3
4.93
ND
3
O 4.21
ND
a
clogP calculated with ChemAxon software
b
EC values were obtained from three independent experiments
5
0
c
Intrinsic clearance (Clint) was determined as a rate of compound consumption under first order
kinetic conditions using human, rat and mouse liver microsomes; ND, not determined.
This unexpected ‘flat’ aryl SAR led us to expand our investigations further around the linker by
focusing on introducing a heteroatom at the benzylic position in order to obtain compounds with
improved potency and better metabolic stability. Introduction of a sulphur heteroatom led to 65
(
EC = 0.26 µM) which was slightly more potent than 55 (EC = 0.42 µM). Aryl SAR revealed
5
0
5
0
that 3,4-disubstitution (70, EC50 = 0.09 µM) in this series is preferred over other disubstituted
69,71) and monosubstituted positions (66-68) in terms of mGluR2 potency. Along the
(
assessment of activity, thioethers (65-71) were also assessed for metabolic stability. The results
shown in Table 4 indicate that all analogues suffered from extensive metabolic degradation in rat
and mouse liver microsomes (RLM and MLM, respectively), with somewhat lower metabolic
instability in human liver microsomes (HLM). Metabolic profiling studies on 70 resulted two
main metabolites. In HLM the main metabolite was an S-oxide derivative (M1), while in RLM
11

and MLM the metabolic vulnerability could be attributed to oxidation of the piperazine moiety of
the tricyclic core (M2, Fig. 3).
Table 4. Functional activity and metabolic stability data of thioethers 65-71.
a
c
Comp.
R
clogP
mGluR2 PAM
EC ±STD ( M)
Cl (mL/min/g liver)
int
b
µ
human rat
mouse
13.8
6.8
50
6
6
6
6
6
7
7
5
6
7
8
9
0
1
H
3.71
4.32
3.86
4.48
4.00
4.00
4.92
0.26 ± 0.05
0.55 ± 0.20
0.32 ± 0.20
0.27 ± 0.07
0.35 ± 0.20
0.09 ± 0.05
0.65 ± 0.22
9.5
1.6
3.0
1.2
3.4
3.5
2.8
13.2
4-Cl
16.2
16.2
10.2
18.2
16.2
12.0
4-F
12.5
11.4
12.5
13.8
10.4
4-Br
2,4-diF
3,4-diF
2,3-diCl
a
clogP calculated with ChemAxon software
b
EC values were obtained from three independent experiments
5
0
c
Intrinsic clearance (Cl ) was determined as a rate of compound consumption under first order
int
kinetic conditions using human, rat and mouse liver microsomes; ND, not determined.
12

Fig. 3. Metabolism study of 70
Given the notable increase in potency, we turned our attention to address the high metabolic
instability of 70. To improve metabolic stability at first we replaced the sulphur by oxigen. As
seen in Table 5, a significant increase in metabolic stability was found with the ether analogue
7
2; unfortunately, this increase in stability was accompanied by a 5-fold decrease in mGluR2
potency. In this case, the decreased lipophilicity of 72 could account for the weaker activity. This
is in good correlation with the SAR trend on lipophilicity vs potency, observed with analogues
7
3 and 74. These compounds showed a significant increase in potency when compared to 72.
Fortunately, the substitution of fluorine by chlorine had little effect on metabolism of these
compounds.
To prevent hydroxylations at the tricyclic core, the corresponding amides (87, 88 and 102)
were synthesized. In case of dihydropyrazino-benzimidazole-3-one (102), the potency was
weaker, while for dihydropyrazino-benzimidazole-1-ones (87, 88), the mGluR2 affinity could be
preserve; nevertheless, metabolic stability could not be improved significantly (Table 5).
Table 5. Functional activity and metabolic stability data of ether derivatives.
13

1
a
c
Comp
.
R
R
clogP mGluR2 PAM
Cl (mL/min/g liver)
int
b
EC50±STD (µM)
human rat
mouse
1.9
7
7
7
1
8
2
3,4-diF
3.32
3.78
4.24
3.34
3.90
0.54 ± 0.16
0.17 ± 0.06
0.07 ± 0.02
30±4.3%@1µM
0.09 ± 0.02
0.6
0.9
1.2
1.3
1.0
5.4
7.7
6.0
5.3
2.4
3
3-Cl,4-F
3,4-diCl
3,4-diCl
3,4-diCl
4.3
2.1
4.3
3.5
4
02
7
8
8
3-Cl,4-F
3.44
0.18 ± 0.05
1.0
2.3
2.6
a
clogP calculated with ChemAxon software
b
EC50 values were obtained from three independent experiments
c
Intrinsic clearance (Cl ) was determined as a rate of compound consumption under first order
int
kinetic conditions using human, rat and mouse liver microsomes; ND, not determined.
The improved physicochemical profile of novel dihydropirazino-benzimidazole-1-one (88)
allowed us extending the linker (Table 6). This modification led to a remarkable improvement in
potency (89), however, it showed a worse metabolic stability profile. Metabolite identification
studies after in vitro incubation with RLM and HLM revealed that cleavage of the ether bond is
the preferred metabolic pathway for 89 (Fig. 4).
14

Fig. 4. Metabolism study of 89
Aiming for compounds with better metabolic stability led us to prepare 90-92, where the steric
effect of substituents at ortho-position were examined. Regarding metabolic stability, these
modifications were failed; however, the potencies reinforced the extension of the linker.
Electronic effect of substituents at para-position is another feasible approach to reduce the
formation of the hydroxyl metabolite (M1) and the oxidation of the phenyl ring. The introduction
of electron-withdrawing substituents on the phenyl ring (93-95) were well-tolerated in terms of
potency and resulted in significantly improved metabolic profile in all species.
Table 6. Functional activity and metabolic stability data of ether derivatives 88-95.
a
c
Comp.
n
R
clogP
mGluR2 PAM
EC ±STD ( M)
Clint (mL/min/g liver)
b
µ
human rat
mouse
2.6
50
8
8
9
9
9
9
9
9
8
9
0
1
2
3
4
1
2
2
2
2
2
2
2
3-Cl,4-F
3-Cl,4-F
3.44
3.96
3.72
3.49
4.12
3.81
4.09
4.64
0.18 ± 0.05
0.04 ± 0.01
0.09 ± 0.03
0.05 ± 0.01
0.05 ± 0.01
0.07 ± 0.03
0.04 ± 0.01
0.03 ± 0.01
1.0
1.8
3.5
0.4
1.5
0.3
0.3
0.4
2.3
5.0
8.2
4.1
10.6
0.9
0.3
0.6
7.0
2-CH
3
8.9
2,4-diF
2-Br,4-F
4-Cl
3.1
7.0
3.1
4-CF
1.6
3
5
4-CF
3
O
0.7
a
clogP calculated with ChemAxon software
b
EC values were obtained from three independent experiments
5
0
15

c
Intrinsic clearance (Clint) was determined as a rate of compound consumption under first order
kinetic conditions using human, rat and mouse liver microsomes; ND, not determined.
Due to its superior potency and metabolic profile, compound 95 was considered as an
advanced lead and was subjected to extended profiling that included its investigations in ADME
assays, in vivo pharmacokinetics, and in vivo effi cacy models (Table 7). Characterization of 95
showed that the compound has promising profile. Although the kinetic solubility was limited, the
ADME parameters were found to be acceptable, and the compound progressed to in vivo studies.
Table 7. Physicochemical properties and in vitro ADME parameters of 95.
Property
95
Physicochemical
MW (Da)
419.15
4.64
56.6
15
logD (pH 7.4, calculated)
2
tPSA (A )
aqueous solubility (
µg/mL, pH 7.4, measured)
ADME
Papp (nm/s) inward
PDR
21.1
0.9
CYP450 inhibition (IC50, µM)
1A2
9.4
2
2
2
3
C9
7
C19
D6
A4
> 10
> 10
> 10
7.7
CYP induction (MEC for PXR activation, µM)
Mouse pharmacokinetics data suggested high exposures and good brain penetration (Cplasma
3.2 M, Cbrain = 9.9 M at 1.5 h after 30 mg/kg intraperitoneal dosing) for 95, and consequently
the compound showed e cacy in the PCP-induced hyper-locomotion model in mouse (PCP HL
MED = 30 mg/kg, ip) (Fig. 5).
=
1
µ
µ
ffi
16

Fig. 5. In vivo efficacy of 95 in the PCP-induced hypermotility model in mice.
; ++; +++ : p < 0.05; p < 0.01; p < 0.001 compared to the saline group,
+
*
; **; *** : p < 0.05; p < 0.01; p < 0.001 compared to the PCP-treated group.
2
.4. Binding mode of compound 95 to mGluR2
3
2
33
Compound 95 has been docked to the homology model of the mGluR2 receptor by Glide.
Interactions formed between the best scored pose of 95 and the receptor were first evaluated. The
dihydropyrazino-benzimidazolone core was found to be embedded in a hydrophobic pocket
formed by F643, Y647, I739, I772, W773 and F776 (Fig. 6, panel A). The aromatic sidechains of
these residues form π−π interactions with the benzimidazolyl moiety while the binding mode is
further stabilized by a H-bond formed between the nitrogen acceptor of the ring accepts and the
N735 sidechain. These interactions are similar to that published for a benzimidazole type
3
4
mGluR2 PAM, JNJ-46281222 using docking and site directed mutagenesis. Furthermore, the
butoxyphenyl substituent of 95 showed similar orientation to that found for the phenylpiperazine
moiety of JNJ-46281222. The terminal triflouromethoxyphenyl group points towards the
ECL2
extracellular side of the receptors and located between F623 and H723
binding site of these compounds reinforces the success of scaffold hoping.
. The overlapping
17

A.
B.
C.
Fig. 6. The predicted binding mode of 95 (A), binding hot spot map (B) and lipophilic hot spots
C) of its binding site. The colour-coding of the binding hot spot map (B) reflects the GE of
(
18

ligands shown in Tables 1-6 and is projected on the protein atoms within 4 Å distance. The
binding pocket surface was coloured according to the maximum GE values for each protein atom
from large (red) to small (blue). Carbon, oxygen, nitrogen and fluorine atoms are coloured green,
red, blue and light blue, respectively. Lipophilic hot spots (yellow shaded surfaces on panel C)
were identified by WaterFLAP at the binding site (gray surface). Waters are colour coded to
show the energetically most disfavoured as red (>3.5 kcal/ mol), then yellow (2.2−3.5 kcal/mol),
bulk solvent as gray (−1 to 2.2 kcal/mol), and favoured waters as blue (<−1 kcal/mol). GRID
maps are contoured (transparent solid) and coloured in the following manner: CH methyl group
3
probe in gray at 1 kcal/mol defining the pocket surface and C1= probe (lipophilic) in yellow at
−
2.7 kcal/ mol. The predicted binding mode of 95 (purple) is overlaid to identify the displaced
water molecules (green circled).
The predicted binding mode of 95 was further validated by the hot spot analysis of the binding
site using SAR data available in Tables 1-6. These compounds were docked to the binding site of
9
5 and their best scored pose were used to project the calculated group efficiency (GE) on the
protein surface. This approach allowed us to analyse the contributions of different parts of the
ligands to their binding affinity and suggested two binding hot spots (Fig. 6, panel B). One of the
hot spots is occupied by the dihydropyrazino-benzimidazolone core and its carbonyl group
contributes significantly to the binding affinity. The second hot spot is located around the
terminal phenyl ring of the side-chain that is adjacent to the nearby lipophilic hot spot (Fig. 6,
panel C). Since displacement of binding site water molecules might contribute significantly to
35
allosteric binding to mGluRs water networks were investigated at the binding site of 95.
WaterFLAP calculations showed that the 95 displaces a number of water molecules in the hot
spot regions of the site. The dihydropyrazino-benzimidazolone core displaced most of the highly
disfavoured waters; however, the terminal phenyl ring and its substituents replaced energetically
disfavoured waters around position 4. These results suggest that similar to mGluR5, the
displacement of disfavoured binding site waters contributes significantly to the binding affinity
of this series of mGluR2 PAMs.
19

3
. Conclusions
In summary, the discovery and optimization of a novel series of dihydropyrazino-benzimidazoles
in search of mGlu2 receptor PAMs are reported. The scaffold hopping strategy was started from
the known 1-[(1-methyl-1H-imidazol-2-yl)methyl]-4-phenylpiperidine (1 and 2) core and led to
the identification of dihydropyrazino-benzimidazoles as a novel mGluR2 PAM chemotype.
Initial aryl SAR studies provided a viable submicromolar starting point (47) quickly with
comparable potency to that of the Pfizer and GSK hits but with high metabolic turnover in RLM
and MLM, respectively. Further optimization of mGluR2 potency was guided by a
pharmacophore model developed for known mGlu2 receptor PAM chemotypes. Subsequent
improvement of metabolic stabilities led to the identification of the lead compound 95. The
improved overall profile of 95 nominated this compound for in vivo pharmacokinetic studies that
revealed reasonable brain exposures. Due to its beneficial PK profile, the compound was found
to be effective in our proof of concept animal model, providing support for potential efficacy in
treating psychosis.
4
. Experimental Section
4
.1. Chemistry and chemical methods
All reactions were carried out under dry nitrogen. Commercially available reagents were used
without further purification. Solvents and gases were dried according to standard procedures.
Organic solvents were evaporated with reduced pressure using a rotary evaporator. Purity of final
compounds was verified using a Merck-Hitachi Lachrom Elite HPLC system. A linear gradient
using water and 0.1% TFA (solvent A) and acetonitrile and 0.1% TFA (solvent B); t = 0 min, 0%
B, t = 7.5 min, 70% B, t = 9.7 min, 90% B, t = 12.5 min, 90% B (12.5 min) was employed on
Chromolith RP18e (3 x 100 mm) column. The flow rate was set to 1.7 mL/min and the column
o
was maintained at 40 C. Purity of final compounds was assessed using UV detection (Diode
Arry Detector: Merck-Hitachi Lachrom Elite L-2450) at 215 nm; all tested compounds were
≥
9
5% pure. NMR spectra were recorded at 298K on a Bruker 500 MHz NMR spectrometer
3
equipped with a liquid helium cooled 5 mm TCI CryoProbe. Either CDCl or DMSO-d6 were
20

used as solvent. Due to limited sample availability in some cases samples were prepared by the
dilution of two milimolar DMSO-h6 stock solutions a priori prepared for pharmacological tests
and stored for years at -20°C. In these cases presat solvent suppression technique was applied to
1
1
13
gain H NMR information. H and C chemical shifts were referenced to TMS or residual
solvent signal, while 15N chemical shifts are given on the MeNO scale using the spectrometer
2
reference routine. Data acquisition and processing were performed by Bruker Topspin 3.5 p7
software package, while for reporting ACD/Spectrus Processor 2017.1.3 was used.
Electron impact high-resolution MS measurements (EI-HRMS) were performed on a Thermo
Q-Exative GC Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The
◦
ion source temperature was set at 250 C, the applied ionization energy was 70 eV. Resolving
power of 60,000 (FWHM) at m/z 400.
Data acquisition and analysis were accomplished with Xcalibur software version 4.0 (Thermo
Fisher Scientific Inc.) in all cases.
4
4
.2. General synthesis of dihydropyrazino-benzimidazole derivatives (42-74).
.2.1. 2-(2-phenylethyl)-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (42).
STEP 1. 2-[(2-nitrophenyl)amino]ethanol (
5). To a stirred solution of 1-fluoro-2-nitrobenzene
(3, 3.60 mL, 0.030 mol) in DMSO (25 mL) ethanolamine (4, 3.62 mL, 0.060 mol) was added at
ambient temperature. The so obtained mixture was stirred for 16h at ambient temperature then a
mixture of water (50 mL) and CH Cl (50 mL) was added. The organic layer was separated and
2
2
dried over anh. MgSO4. The obtained crystalline product was used in the next step without
+
further purification. Yield: 5.2 g (95%), EI-LRMS m/z 183.1 = (M+H) .
STEP 2. 2-[(2-aminophenyl)amino]ethanol (6). To a stirred solution of 2-[(2-
nitrophenyl)amino]ethanol (5, 12.0 g, 0.066 mol) in ethanol (80 mL) hydrazine hydrate (8.26 g,
0
.165 mol) was added at ambient temperature, followed by addition of 600 mg Raney-Ni in 5
0
mL ethanol. The obtained suspension was stirred at 40 C for 2 h. The catalyst was collected by
filtration and the filtrate was evaporated in vacuo. The obtained crystalline product was used in
+
the next step without further purification. Yield: 8.75 g (87%), EI-LRMS m/z 153.2 = (M+H) .
21

STEP 3. 2-[2-(hydroxymethyl)-1H-benzimidazol-1-yl]ethanol (7). To a stirred solution of 2-
[(2-aminophenyl)amino]ethanol (6, 8.70 g, 0.058 mol) in a mixture of cc. HCl (65 mL) and water
(65 mL) glycolic acid (6.62 g, 0.087 mol) was added at ambient temperature. The reaction
mixture was refluxed for 6h. After cooling to room temperature, the pH was adjusted to 7 with
powdered NaOH. The precipitated product was collected by filtration and washed 2 times with
+
5
0 mL of water. Yield: 10.25 g (92%), EI-LRMS m/z 193.2 = (M+H) .
STEP 4. 1-(2-chloroethyl)-2-(chloromethyl)-1H-benzimidazole (8). To a stirred suspension of
2
-[2-(hydroxymethyl)-1H-benzimidazol-1-yl] (7, 10.23 g, 0.054 mol) in chloroform (180 mL)
thionyl chloride (55 mL, 0.756 mol) was added dropwise with cooling. The mixture was refluxed
for 3h then evaporated in vacuo. The residue was taken up with diethyl ether (70 mL). The
product was collected by filtration and washed 3 times with 50 mL of diethyl ether. Yield: 10.5 g
+
(
90%), EI-LRMS m/z 229.1 = (M+H) .
STEP 5. 2-(2-phenylethyl)-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (42). To a solution
of 1-(2-chloroethyl)-2-(chloromethyl)-1H-benzimidazole (8, 0.64 g, 0.00266 mol) and
phenylethylamine (9, 0.38 g, 0.00319 mol) in acetonitrile (10 mL) K CO (0.44 g, 0.00319 mol)
2
3
was added. The reaction mixture was refluxed for 10h then evaporated in vacuo. The residue was
taken up with dichloromethane (20 mL) and water (20 mL) was added. The organic layer was
separated, dried over anh. MgSO and evaporated to dryness. The residue was purified by flash
4
coloumn chromatography (silica gel, eluent: DCM:MeOH, 0-5% gradient) to yield 42 as a white
o
+
solid (43%). mp 110-111 C; ESI-HRMS calcd for C H N [M+H] : 278.16517; found:
1
8
20
3
1
2
7
1
6
78.16405; delta= -4.03 ppm.; H NMR (500 MHz, DMSO-d ) δ = 7.54 - 7.56 (1H, m, H-10),
.47 - 7.49 (1H, m, H-12), 7.28 - 7.30 (4H, m, H-17, 19, 20, 18), 7.16 - 7.23 (3H, m, H-21, 11,
3), 4.12 (2H, t, J = 5.6 Hz, H-4), 3.89 (2H, s, H-3), 3.07 (2H, t, J = 5.6 Hz, H-2), 2.85 - 2.91
1
3
(
2H, m, H-14), 2.80 - 2.85 (2H, m, H-15); C NMR (125.7 MHz, DMSO-d
42.5 (C-8), 140.1 (C-16), 134.0 (C-9), 128.7 (C-19, 20), 128.2 (C-17, 18), 125.9 (C-21), 121.7
C-13), 121.4 (C-11), 118.4 (C-10), 109.6 (C-12), 58.3 (C-14), 51.4 (C-3), 48.9 (C-2), 41.6 (C-
6
) δ = 149.4 (C-5),
1
(
4
), 32.7 (C-15).
4
.2.2. 2-(2-phenylethyl)-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (43).
Prepared according to the procedure described for 42 using 2-(2-fluorophenyl)ethanamine (10)
o
in step 5 to yield 43 as an off-white solid (51%). mp 98-100 C; ESI-HRMS calcd for
22

+
1
C
18
H
19
N
3
F [M+H] : 296.15575; found: 296.15447; delta= -4.33 ppm. H NMR (499.9 MHz,
DMSO-d6) = 7.53 - 7.58 (1H, m, H-10), 7.45 - 7.50 (1H, m, H-12), 7.40 (1H, td, J = 7.7 Hz, J =
.7 Hz, H-17), 7.23 - 7.29 (1H, m, H-21), 7.16 - 7.22 (2H, m, H-11, 13), 7.12 - 7.17 (2H, m, H-
9, 20), 4.12 (2H, t, J = 5.6 Hz, H-4), 3.90 (2H, s, H-3), 3.08 (2H, t, J = 5.4 Hz, H-2), 2.89-2.96
δ
1
1
1
3
(
2H, t, J = 6.9 Hz, H-15), 2.79 - 2.86 (2H, m, H-14); C NMR (125.7 MHz, DMSO-d ) δ =
6
1
1
5
60.6 (C-18), 149.3 (C-5), 142.5 (C-8), 134.0 (C-9), 131.3 (C-17), 128.1 (C-21), 126.6 (C-16),
24.3 (C-19), 121.7 (C-11), 121.4 (C-13), 118.4 (C-10), 115.0 (C-20), 109.6 (C-12), 56.8 (C-14),
1.4 (C-3), 48.8 (C-2), 41.5 (C-4), 26.0 (C-15).
4
.2.3. 2-[2-(3-fluorophenyl)ethyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (44).
Prepared according to the procedure described for 42 using 2-(3-fluorophenyl)ethanamine (11)
o
in step 5 to yield 44 as an off-white solid (48%). mp 86-87 C; ESI-HRMS calcd for C H N F
1
8
19
3
+
1
[
M+H] : 296.15575; found: 296.15470; delta= -3.55 ppm.; H NMR (499.9 MHz, DMSO-d ) δ =
6
7
7
.55 (1H, d, J = 7.0 Hz, H-10), 7.46 - 7.49 (1H, m, H-12), 7.16 - 7.23 (2H, m, H-11, 13), 7.12 -
.17 (2H, m, H-18, 17), 4.12 (2H, t, J = 5.6 Hz, H-4), 3.89 (2H, s, H-3), 3.07 (2H, t, J = 5.6 Hz,
1
3
H-2), 2.88 - 2.94 (2H, m, H-15), 2.82 - 2.87 (2H, m, H-14); C NMR (125.7 MHz, DMSO-d
6
) δ
=
162.2 (C-20), 149.3 (C-5), 143.2 (C-16), 142.5 (C-8), 134.0 (C-9), 130.0 (C-19), 124.9 (C-17),
1
5
21.7 (C-11), 121.4 (C-13), 118.4 (C-10), 115.4 (C-18), 112.7 (C-21), 109.6 (C-12), 57.8 (C-14),
1.4 (C-3), 48.8 (C-2), 41.5 (C-4), 32.3 (C-15).
4
.2.4. 2-[2-(4-fluorophenyl)ethyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (45).
Prepared according to the procedure described for 42 using 2-(4-fluorophenyl)ethanamine (12)
o
in step 5 to yield 45 as an off-white solid (57%). mp 128-129 C; ESI-HRMS calcd for
+
1
C
18
H
19
N
3
F [M+H] : 296.15575; found: 296.15460; delta= -3.89 ppm; H NMR (499.9 MHz,
DMSO-d6) = 7.52 - 7.59 (1H, m, H-10), 7.44 - 7.50 (1H, m, H-12), 7.28 - 7.37 (2H, m, H-17,
8), 7.15 - 7.23 (2H, m, H-11, 13), 7.07-7.13 (1H, m, H-19, 20), 4.12 (2H, t, J = 5.6 Hz, H-4),
.89 (2H, s, H-3), 3.07 (2H, t, J = 5.6 Hz, H-2), 2.85 - 2.90 (2H, m, H-15), 2.80 - 2.85 (2H, m, H-
δ
1
3
1
1
1
3
4); C NMR (125.7 MHz, DMSO-d )
δ
= 160.7 (C-21), 149.3 (C-5), 142.5 (C-8), 136.2 (C-16),
6
34.0 (C-9), 130.5 (C-17, 18), 121.7 (C-11), 121.4 (C-13), 118.4 (C-10), 114.8 (C-19, 20), 109.6
(C-12), 58.2 (C-14), 51.4 (C-3), 48.9 (C-2), 41.5 (C-4), 31.8 (C-15).
23

4
.2.5. 2-[2-(4-chlorophenyl)ethyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (46).
Prepared according to the procedure described for 42 using 2-(4-chlorophenyl)ethanamine (13)
o
in step 5 to yield 46 as an off-white solid (48%). mp 137-139 C; ESI-HRMS calcd for
+
1
C
18
H
19
N
3
Cl [M+H] : 312.12620; found: 312.12572; delta= -1.55 ppm.; H NMR (499.9 MHz,
DMSO-d6) = 7.52 - 7.58 (1H, m, H-10), 7.45 - 7.49 (1H, m, H-12), 7.28-7.35 (4H, m, H-17, H-
8,H-19, H-20), 7.18 - 7.21 (2H, m, H-11, H13), 4.11 (2H, t, J4,2 = 5.6 Hz, H-4), 3.88 (2H, s, H-
δ
1
3
1
3
), 3.06 (2H, t, J2,4 = 5.6 Hz, H-2), 2.79 - 2.90 (4H, m, H-14, 15); C NMR (125.7 MHz,
= 149.3 (C-5), 142.5 (C-8), 139.2 (C-16), 134.0 (C-9), 130.6 (C-18, 17), 130.5 (C-
1), 128.1 (C-19, 20), 121.7 (C-11), 121.4 (C-13), 118.4 (C-10), 109.6 (C-12), 57.9 (C-14), 51.4
C-3), 48.8 (C-2), 41.5 (C-4), 31.9 (C-15).
DMSO-d6)
δ
2
(
4
.2.6. 2-[2-(4-bromophenyl)ethyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (47).
Prepared according to the procedure described for 42 using 2-(4-bromophenyl)ethanamine (14)
o
in step 5 to yield 47 as an off-white solid (43%). mp 151-153 C; ESI-HRMS calcd for
+
1
C H N Br [M+H] : 356.07569; found: 356.07548; delta= -0.59 ppm; H NMR (499.9 MHz,
18
19
3
DMSO-d
6
) δ = 7.53-7.57 (1H, m, H-3), 7.44 - 7.49 (3H, m, H-20, 19, 4), 7.26 - 7.29 (2H, m, H-
1
7, 18), 7.18 - 7.21 (1H, m, H-6, 5), 4.11 (2H, t, J= 5.6 Hz, H-12), 3.88 (2H, s, H-10), 3.06 (2H,
13
t, J = 5.6 Hz, H-13), 2.79 - 2.89 (4H, m, H-14, 15); C NMR (125.7 MHz, DMSO-d )
δ
= 149.3
6
(C-8), 142.5 (C-1), 139.6 (C-16), 134.0 (C-2), 131.0 (C-17, 20, 19, 18), 121.7 (C-5), 121.4 (C-6),
1
19.0 (C-21), 118.4 (C-3), 109.6 (C-4), 57.9 (C-14), 51.4 (C-10), 48.8 (C-13), 41.5 (C-12), 31.9
(C-15).
4
.2.7. 2-[2-(4-methylphenyl)ethyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (48).
Prepared according to the procedure described for 42 using 2-(4-methylphenyl)ethanamine
o
(
15) in step 5 to yield 48 as an off-white solid (57%). mp 134-135 C; ESI-HRMS calcd for
+
1
C
19
H
22
N
3
[M+H] : 292.18082; found: 292.17965; delta= -4.00 ppm; H NMR (499.9 MHz,
= 7.54 - 7.58 (1H, m, H-10), 7.45 - 7.49 (1H, m, H-12), 7.15 - 7.23 (4H, m, H-11,
3, 17, 18), 7.06 - 7.11 (2H, m, H-19, 20), 4.11 (2H, t, J = 5.6 Hz, H-4), 3.88 (2H, s, H-3), 3.05
DMSO-d6)
δ
1
1
3
(
2H, t, J = 5.6 Hz, H-2), 2.79 - 2.84 (4H, m, H-14, 15), 2.26 (3H, s, H-22); C NMR (125.7
MHz, DMSO-d6) = 149.4 (C-5), 142.5 (C-8), 136.9 (C-16), 134.8 (C-21), 134.0 (C-9), 128.8
δ
24

(C-20, 19), 128.6 (C-17, 18), 121.7 (C-13), 121.4 (C-11), 118.4 (C-10), 109.6 (C-12), 58.4 (C-
1
4), 51.4 (C-3), 48.9 (C-2), 41.6 (C-4), 32.3 (C-15), 20.6 (C-22).
4
.2.8. 2-[2-(4-methoxyphenyl)ethyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (49).
Prepared according to the procedure described for 42 using 2-(4-methoxyphenyl)ethanamine
o
(
16) in step 5 to yield 49 as an off-white solid (52%). mp 118-119 C; ESI-HRMS calcd for
+
1
C H ON [M+H] : 308.17574; found: 308.17457; delta= -3.80 ppm; H NMR (499.9 MHz,
19
22
3
DMSO-d6)
δ = 7.53 - 7.58 (1H, m, H-10), 7.46 - 7.50 (1H, m, H-12), 7.15 - 7.23 (4H, m, H-19,
1
8, 11, 13), 6.83-6.87 (2H, m, H-20, 21), 4.12 (2H, t, J = 5.6 Hz, H-4), 3.88 (2H, s, H-3), 3.71
1
3
(
3H, s, H-24), 3.05 - 3.07 (2H, m, H-2), 2.79 - 2.81 (4H, m, H-24, 14); C NMR (125.7 MHz,
DMSO-d = 157.5 (C-22), 149.4 (C-5), 142.5 (C-8), 134.0 (C-9), 131.9 (C-17), 129.6 (C-19,
8), 121.7 (C-13), 121.4 (C-11), 118.4 (C-10), 113.7 (C-20, 21), 109.6 (C-12), 58.6 (C-14), 54.9
C-24), 51.5 (C-3), 48.9 (C-2), 41.6 (C-4), 31.8 (C-24).
6
) δ
1
(
4
.2.9. 2-[2-(naphthalen-2-yl)ethyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (50).
Prepared according to the procedure described for 42 using 2-(naphthalen-2-yl)ethanamine
o
(
17) in step 5 to yield 50 as an off-white solid (34%). mp 78-80 C; ESI-HRMS calcd for
+
C H N [M+H] : 328.18082; found: 328.17997; delta= -2.60 ppm; ESI-LRMS m/z 328 =
22
22
3
+
1
[
M+H] ; H NMR (500 MHz, DMSO-d
6
) δ = 7.82 - 7.90 (3H, m, H-21, 23, 20), 7.78 - 7.81 (1H,
m, H-25), 7.53 - 7.58 (1H, m, H-12), 7.42 - 7.50 (4H, m, H-18, 22, 9, 17), 7.13 - 7.24 (2H, m, H-
1
3
5
1
5
1, 10), 4.13 (2H, t, J = 5.6 Hz, H-3), 3.93 (2H, s, H-13), 3.11 (2H, t, J = 5.6 Hz, H-2), 3.03 -
1
3
.08 (2H, m, H-15), 2.89 - 3.00 (2H, m, H-14); C NMR (125.7 MHz, DMSO-d ) δ = 149.2 (C-
6
), 142.4 (C-7), 137.8 (C-16), 133.6 (C-8), 127.7 (C-17), 127.5 (C-20, 21, 23), 126.7 (C-25),
26.0 (C-18), 125.3 (C-22), 121.6 (C-11), 121.3 (C-10), 118.4 (C-12), 109.7 C-9), 58.2 (C-14),
1.5 (C-13), 49.0 (C-2), 41.6 (C-3), 32.9 (C-15) (from HSQC and HMBC).
4
.2.10. 2-[2-(pyridin-2-yl)ethyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (51).
Prepared according to the procedure described for 42 using commercially available 2-(pyridin-
o
2
-yl)ethanamine (18) in step 5 to yield 51 as an off-white solid (31%). mp 86-87 C; ESI-HRMS
+
1
calcd for C H N [M+H] : 279.16042; found: 279.15934; delta= -3.89 ppm; H NMR (499.9
1
7
19
4
MHz, DMSO-d
6
)
δ
= 8.47 - 8.50 (1H, m, H-20), 7.70 (1H, td, J = 7.6 Hz, J = 1.9 Hz, H-19), 7.53
25

-
7.57 (1H, m, H-10), 7.44 - 7.49 (1H, m, H-12), 7.35 (1H, dt, J = 7.8 Hz, J = 1.0 Hz, H-17), 7.18
7.24 (1H, m, H-21), 7.18 - 7.21 (2H, m, H-11, 13), 4.10 (2H, t, J = 5.6 Hz, H-4), 3.89 (2H, s, H-
-
1
3
3
), 3.07 (2H, t, J = 5.6 Hz, H-2), 3.01 - 3.05 (2H, m, H-15), 2.95 - 3.00 (2H, m, H-14); C NMR
= 159.8 (C-16), 149.3 (C-5), 148.9 (C-20), 142.5 (C-8), 136.4 (C-19),
(125.7 MHz, DMSO-d
6
)
δ
1
5
34.0 (C-9), 123.2 (C-17), 121.7 (C-21), 121.4 (C-13), 121.4 (C-11), 118.4 (C-10), 109.6 (C-12),
6.5 (C-14), 51.4 (C-3), 48.9 (C-2), 41.5 (C-4), 35.1 (C-15).
4
.2.11. 2-[2-(thiophen-2-yl)ethyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (52).
Prepared according to the procedure described for 42 using 2-(thiophen-2-yl)ethanamine (19)
o
in step 5 to yield 52 as an off-white solid (28%). mp 112-113 C; ESI-HRMS calcd for
+
1
C
16
H
18
N
3
S [M+H] : 284.12159; found: 284.12095; delta= -2.28 ppm; H NMR (499.9 MHz,
DMSO-d6) = 7.53 - 7.58 (1H, m, H-3), 7.46 - 7.50 (1H, m, H-4), 7.29 - 7.32 (1H, m, H-19),
.18 - 7.21 (2H, m, H-6, 5), 6.93 - 6.96 (2H, m, H-20, 17), 4.13 (2H, t, J = 5.6 Hz, H-12), 3.90
2H, s, H-10), 3.11 (2H, t, J = 7.7 Hz, H-15), 3.08 (2H, br t, J = 5.5 Hz, H-13), 2.87 (2H, t, J =
δ
7
(
1
3
7
1
1
.7 Hz, H-14); C NMR (125.7 MHz, DMSO-d ) δ = 149.3 (C-8), 142.5 (C-1), 142.3 (C-16),
6
34.0 (C-2), 126.7 (C-20), 125.0 (C-17), 124.0 (C-19), 121.7 (C-5), 121.4 (C-6), 118.4 (C-3),
09.6 (C-4), 57.9 (C-14), 51.4 (C-10), 48.8 (C-13), 41.6 (C-12), 27.0 (C-15).
4
.2.12. 2-benzyl-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (53).
Prepared according to the procedure described for 42 using benzylamine (20) in step 5 to yield
o
+
5
2
7
3 as an off-white solid (61%). mp 114-115 C; ESI-HRMS calcd for C17
H
18
N
3
[M+H] :
= 7.54 -
1
64.14952; found: 264.14875; delta= -2.91 ppm; H NMR (499.9 MHz, DMSO-d )
δ
6
.56 (1H, m, H-10), 7.46 - 7.48 (1H, m, H-12), 7.36 - 7.42 (4H, m, H-16, 17, 18, 19), 7.28-7.33
(1H, m, H-20), 7.16 - 7.23 (2H, m, H-11, 13), 4.13 (2H, t, J = 5.6 Hz, H-4), 3.79 (2H, s, H-3),
1
3
3
.79 (2H, s, H-14), 3.01 (2H, t, J = 5.6 Hz, H-2); C NMR (125.7 MHz, DMSO-d ) δ = 149.2
6
(
C-5), 142.5 (C-8), 137.5 (C-15), 134.0 (C-9), 128.9 (C-17, 16), 128.4 (C-18, 19), 127.3 (C-20),
21.7 (C-13), 121.5 (C-11), 118.4 (C-10), 109.6 (C-12), 60.6 (C-14), 51.3 (C-3), 48.6 (C-2), 41.5
C-4).
1
(
4
.2.13. 2-(3-phenylpropyl)-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (54).
26

Prepared according to the procedure described for 42 using 3-phenylpropan-1-amine (21) in
o
step 5 to yield 54 as an off-white solid (45%). mp 115-117 C; ESI-HRMS calcd for C H N
1
9
22
3
+
1
[
M+H] : 292.18082; found: 292.18080; delta= -0.09 ppm; H NMR (499.9 MHz, DMSO-d )
δ
=
6
7
.54 - 7.58 (1H, m, H-12), 7.45 - 7.50 (1H, m, H-9), 7.26 - 7.32 (3H, m, H-19, 21), 7.23 - 7.26
2H, m, H-18, 22), 7.15 - 7.23 (1H, m, H-20, 10, 11), 4.12 (2H, t, J = 5.6 Hz, H-3), 3.81 (2H, s,
H-13), 2.98 (2H, t, J = 5.6 Hz, H-2), 2.65 (2H, t, J = 7.3 Hz, H-16), 2.58 (2H, t, J = 6.9 Hz, H-
(
1
3
1
5
1
4), 1.86 (2H, dt, J = 14.9 Hz, J = 7.5 Hz, H-15); C NMR (126 MHz, DMSO-d )
δ
= 149.4 (C-
6
), 142.5 (C-7), 141.9 (C-17), 134.0 (C-8), 128.3 (C-18, 22), 128.3 (C-19, 21), 125.7 (C-20),
21.7 (C-11), 121.4 (C-10), 118.4 (C-12), 109.6 (C-9), 56.0 (C-14), 51.5 (C-13), 48.9 (C-2), 41.6
(C-3), 32.7 (C-16).
4
.2.14. 2-(4-phenylbutyl)-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (55).
Prepared according to the procedure described for 42 using 4-phenylbutan-1-amine (22) in step
o
5
to yield 55 as an off-white solid (38%). mp 106-107 C; ESI-HRMS calcd for C20
H
24
N
3
+
1
[
M+H] : 306.19647; found: 306.19519; delta= -4.21 ppm; H NMR (499.9 MHz, DMSO-d ) δ =
6
7
.53 - 7.58 (1H, m, H-10), 7.45 - 7.49 (1H, m, H-12), 7.25 - 7.30 (2H, m, H-22, 21), 7.16 - 7.22
5H, m, H-11, 13, 19, 20, 23), 4.11 (2H, t, J = 5.6 Hz, H-4), 3.78 (2H, s, H-3), 2.96 (2H, t, J = 5.6
Hz, H-2), 2.62 (2H, t, J = 7.3 Hz, H-17), 2.59 (2H, t, J = 7.3 Hz, H-14), 1.60 - 1.68 (2H, m, H-
(
1
3
1
1
6), 1.53 - 1.60 (2H, m, H-15); C NMR (125.7 MHz, DMSO-d
6
) δ = 149.4 (C-5), 142.5 (C-8),
42.2 (C-18), 134.0 (C-9), 128.3 (C-19, 20), 128.2 (C-22, 21), 125.6 (C-23), 121.7 (C-11), 121.4
(C-13), 118.4 (C-10), 109.6 (C-12), 56.4 (C-14), 51.5 (C-3), 49.0 (C-2), 41.6 (C-4), 34.9 (C-17),
2
8.7 (C-16), 25.9 (C-15).
4
.2.15. 2-(5-phenylpentyl)-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (56).
Prepared according to the procedure described for 42 using 5-phenylpentan-1-amine (23) in
o
step 5 to yield 56 as an off-white solid (38%). mp 109-110 C; ESI-HRMS calcd for C21
H
26
N
3
+
1
[
M+H] : 320.21212; found: 320.21133; delta= -2.49 ppm; H NMR (499.9 MHz, DMSO-d )* δ
6
=
7.53 - 7.56 (1H, m, H-3), 7.45 - 7.48 (1H, m, H-4), 7.23 - 7.27 (2H, m, H-21, 20), 7.17 - 7.23
(4H, m, H-5, 6, 23, 22), 7.12 - 7.16 (1H, m, H-24), 4.10 (2H, t, J = 5.6 Hz, H-12), 3.78 (2H, s, H-
1
1
0), 2.96 (2H, t, J = 5.6 Hz, H-13), 2.59 (2H, t, J = 7.6 Hz, H-18), 2.55 (2H, t, J = 7.1 Hz, H-14),
1
3
6
.55 - 1.65 (4H, m, H-15, 17), 1.31 - 1.37 (2H, m, H-16); C NMR (125.7 MHz, DMSO-d ) δ =
27

1
49.4 (C-8), 142.5 (C-1), 142.2 (C-19), 134.0 (C-2), 128.3 (C-23, 22), 128.2 (C-21, 20), 125.6
(C-24), 121.6 (C-5), 121.4 (C-6), 118.4 (C-3), 109.6 (C-4), 56.6 (C-14), 51.5 (C-10), 49.0 (C-
1
3), 41.6 (C-12), 35.1 (C-18), 30.8 (C-17), 26.4 (C-16), 26.1 (C-15).
4
.2.16. 4-[4-(3,4-dihydropyrazino[1,2-a]benzimidazol-2(1H)-yl)butyl]benzonitrile (57).
Prepared according to the procedure described for 42 using 4-(4-aminobutyl)benzonitrile (24)
o
in step 5 to yield 57 as an off-white solid (32%). mp 131-132 C; ESI-HRMS calcd for C H N
2
1
23
4
+
1
[
M+H] : 331.19172; found: 331.19121; delta= -1.55 ppm; H NMR (499.9 MHz, DMSO-d )
δ
=
6
7
.70 - 7.76 (2H, m, H-21, 22), 7.52 - 7.57 (1H, m, H-10), 7.45 - 7.49 (1H, m, H-12), 7.42 - 7.45
(2H, m, H-19, 20), 7.18 - 7.20 (1H, m, H-13), 7.15 - 7.20 (1H, m, H-11), 4.10 (2H, t, J = 5.6 Hz,
H-4), 3.78 (2H, s, H-3), 2.96 (2H, t, J = 5.6 Hz, H-2), 2.71 (2H, t, J = 7.5 Hz, H-17), 2.59 (2H, t,
1
3
J = 7.1 Hz, H-14), 1.62 - 1.68 (2H, m, H-16), 1.53 - 1.59 (2H, m, H-15); C NMR (125.7 MHz,
DMSO-d6) = 149.4 (C-5), 148.4 (C-18), 142.5 (C-8), 134.0 (C-9), 132.2 (C-21, 22), 129.4 (C-
9, 20), 121.6 (C-11), 121.4 (C-13), 119.0 (C-24), 118.4 (C-10), 109.6 (C-12), 108.5 (C-23),
δ
1
5
6.2 (C-14), 51.5 (C-3), 48.9 (C-2), 41.5 (C-4), 34.9 (C-17), 34.8, 28.1 (C-16), 27.8, 25.8 (C-15).
4
.2.17. 2-[4-(4-methoxyphenyl)butyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (58).
Prepared according to the procedure described for 42 using 4-(4-methoxyphenyl)butan-1-
o
amine (25) in step 5 to yield 58 as an off-white solid (44%). mp 119-120 C; ESI-HRMS calcd
+
1
for C H ON [M+H] : 336.20704; found: 336.20621; delta= -2.48 ppm; H NMR (500 MHz,
2
1
26
3
DMSO-d
6
)
δ
= 7.50-7.55 (1H, m, H-3), 7.45 - 7.49 (1H, m, H-4), 7.16 - 7.22 (2H, m, H-5, 6),
7
.10 - 7.13 (2H, m, H-20, 19), 6.81 - 6.85 (2H, m, H-21, 22), 4.10 (2H, t, J= 5.6 Hz, H-12), 3.77
(2H, s, H-10), 3.71 (3H, s, H-25), 2.96 (2H, t, J = 5.6 Hz, H-13), 2.51 - 2.61 (4H, m, H-17, 14),
1
3
1
1
1
3
6
.51 - 1.63 (4H, m, H-16, 15); C NMR (126 MHz, DMSO-d ) δ = 157.3 (C-23), 149.4 (C-8),
42.5 (C-1), 134.0 (C-2), 134.0 (C-18), 129.2 (C-20, 19), 121.6 (C-5), 121.4 (C-6), 118.4 (C-3),
13.6 (C-21, 22), 109.6 (C-4), 56.4 (C-14), 54.9 (C-25), 51.5 (C-10), 49.0 (C-13), 41.6 (C-12),
4.0 (C-17), 28.9 (C-16), 25.8 (C-15).
4
.2.18. 2-{4-[4-(trifluoromethoxy)phenyl]butyl}-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole
(
59).
28

Prepared according to the procedure described for 42 using 4-[4-
(trifluoromethoxy)phenyl]butan-1-amine (26) in step 5 to yield 59 as an off-white solid (37%).
o
+
mp 106-108 C; ESI-HRMS calcd for C H ON F [M+H] : 390.17877; found: 390.17747;
2
1
23
3 3
1
delta= -3.34 ppm; H NMR (499.9 MHz, DMSO-d
1H, m, H-4), 7.31 - 7.36 (2H, m, H-20, 19), 7.24 - 7.29 (2H, m, H-22, 21), 7.17 - 7.23 (2H, m,
H-6, 5), 4.13 (1H, t, J = 5.8 Hz, H-12), 3.85 (2H, br s, H-10), 3.02 (2H, br s, H-13), 2.62-2.68
6
) δ = 7.54 - 7.58 (1H, m, H-3), 7.46 - 7.51
(
1
3
(
(
(
4H, m, H-17, 14), 1.50 - 1.72 (4H, m, H-16, 15); C NMR (125.7 MHz, DMSO-d ) δ = 146.4
6
C-23), 142.2 (C-1), 141.7 (C-18), 133.9 (C-2), 130.0 (C-20, 19), 121.8 (C-5), 121.5 (C-6), 120.8
C-22, 21), 118.3 (C-3), 109.7 (C-4), 56.2 (C-14), 51.3 (C-10), 48.8 (C-13), 41.4 (C-12), 34.1 (C-
1
7), 28.5 (C-16), 25.7 (C-15).
4
.2.19. 2-[4-(2,4-dichlorophenyl)butyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (60).
Prepared according to the procedure described for 42 using 4-(2,4-dichlorophenyl)butan-1-
22 3 2
amine (27) in step 5 to yield 60 as an off-white solid (52%). ESI-HRMS calcd for C20H N Cl
+
[
M+H] : 374.11853; found: 374.11812; delta= -1.09 ppm;
1
3
H NMR (399.8 MHz, CDCl ) δ = 7.62 - 7.73 (1H, m, H-3), 7.35 - 7.37 (2H, m, H-22, 4),
7
.24- 7.29 (2H, m, H-6, 5), 7.11 - 7.18 (2H, m, H-21, 20), 4.14 (2H, t, J = 5.6 Hz, H-12), 3.91
(2H, s, H-10, 10), 3.02 (2H, t, J = 5.6 Hz, H-13), 2.75 (2H, t, J = 7.3 Hz, H-17), 2.66 (2H, t, J =
1
3
6
1
1
3
.8 Hz, H-14), 1.60 - 1.71 (4H, m, H-16, 15); C NMR (101 MHz, CDCl ) δ = 149.2 (C-8),
42.2 (C-1), 138.2 (C-18), 134.4 (C-19), 133.8 (C-2), 132.1 (C-23), 131.0 (C-20), 129.1 (C-22),
27.2, 126.9 (C-21), 122.5 (C-6), 122.5, 122.2 (C-5), 118.8 (C-3), 108.8 (C-4), 57.2 (C-14), 51.7
(C-10), 49.6 (C-13), 41.8 (C-12), 32.7 (C-17), 27.2 (C-15), 26.5 (C-16).
4
.2.20. 2-[4-(2,4-difluorophenyl)butyl]-1,2,3,4-tetrahydropyrazino[1,2-a]benzimidazole (61).
Prepared according to the procedure described for 42 using 4-(2,4-difluorophenyl)butan-1-
22 3 2
amine (28) in step 5 to yield 61 as an off-white solid (49%). ESI-HRMS calcd for C20H N F
+
1
[
M+H] : 342.17763; found: 342.17708; delta= -1.60 ppm; H NMR (499.9 MHz, DMSO-d )* δ
6
=
7.53 - 7.57 (1H, m, H-12), 7.45 - 7.49 (1H, m, H-9), 7.32 - 7.40 (1H, m, H-22), 7.17 - 7.21
2H, m, H-10, 11), 7.13 - 7.17 (1H, m, H-23), 6.97 - 7.05 (1H, m, H-20), 4.11 (2H, t, J = 5.5 Hz,
H-3), 3.79 (2H, s, H-13), 2.97 (2H, t, J = 5.5 Hz, H-2), 1.50 - 1.65 (4H, m, H-16, 15).
(
29