Fragment Based Optimization of Metabotropic Glutamate Receptor 2 (mGluR2) Positive Allosteric Modulators in the Absence of Structural Information

Article abstract of DOI:10.1021/acs.jmedchem.8b00161

Metabotropic glutamate receptor 2 (mGluR2) positive allosteric modulators (PAMs) have been implicated as potential pharmacotherapy for psychiatric conditions. Screening our corporate compound deck, we identified a benzotriazole fragment (4) that was rapidly optimized to a potent and metabolically stable early lead (16). The highly lipophilic character of 16, together with its limited solubility, permeability, and high protein binding, however, did not allow reaching of the proof of concept in vivo. Since further attempts on the optimization of druglike properties were unsuccessful, the original hit 4 has been revisited and was optimized following the principles of fragment based drug discovery (FBDD). Lacking structural information on the receptor-ligand complex, we implemented a group efficiency (GE) based strategy and identified a new fragment like lead (60) with more balanced profile. Significant improvement achieved on the druglike properties nominated the compound for in vivo proof of concept studies that revealed the chemotype being a promising PAM lead targeting mGluR2 receptors.

Full text of DOI:10.1021/acs.jmedchem.8b00161

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Fragment based optimization of metabotropic glutamate receptor-2 (mGluR2)
positive allosteric modulators in the absence of structural information
György Szabó, György István Túrós, Sándor Kolok, Mónika Vastag, Zsuzsanna Sánta, Miklós
Dékány, György I. Lévay, István Greiner, Minami Natsumi, Watanabe Tatsuya, and Gyorgy M. Keseru
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00161 • Publication Date (Web): 05 Mar 2018
Downloaded from http://pubs.acs.org on March 6, 2018
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Fragment based optimization of metabotropic
glutamate receptorꢀ2 (mGluR2) positive allosteric
modulators in the absence of structural information
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György Szabó,*1 György I. Túrós,1 Sándor Kolok,1 Mónika Vastag,1 Zsuzsanna Sánta,1 Miklós
Dékány,1 György I. Lévay,1 István Greiner,1 Minami Natsumi,3 Watanabe Tatsuya,3 György M.
Keserű*2
1Gedeon Richter Plc., 19ꢀ21 Gyömrői út, Budapest, 1103 Hungary
2Medicinal Chemistry Research Group, Research Centre for Natural Sciences, Hungarian
Academy of Sciences, 2 Magyar tudósok körútja, Budapest 1117 Hungary
3Mitsubishi Tanabe Pharma Corporation, 1000, Kamoshidaꢀcho, Aobaꢀku, Yokohama 227ꢀ0033,
Japan
ABSTRACT
Metabotropic glutamate receptor 2 (mGluR2) positive allosteric modulators (PAMs) have been
implicated as potential pharmacotherapy for psychiatric conditions. Screening our corporate
compound deck, we identified a benzotriazole fragment (4) that was rapidly optimized to a
potent and metabolically stable early lead (16). The highly lipophilic character of 16, together
with its limited solubility, permeability and high protein binding, however, did not allow to reach
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the proof of concept in vivo. Since further attempts on the optimization of druglike properties
were unsuccessful, the original hit 4 has been revisited and was optimized following the
principles of fragment based drug discovery (FBDD). Lacking structural information of the
receptorꢀligand complex we implemented a group efficiency (GE) based strategy and identified a
new fragment like lead (60) with more balanced profile. Significant improvement achieved on
the druglike properties nominated the compound for in vivo proof of concept studies that
revealed the chemotype being a promising PAM lead targeting mGluR2 receptors.
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INTRODUCTION
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The family of metabotropic glutamate receptors (mGlu1ꢀ8) is classified into three groups based
on structural homology, transduction mechanisms, and pharmacology.1 Group II metabotropic
receptors include mGluR2 and mGluR3 which are highly homologous (~70% at the amino acid
level) and linked to their effectors via Gi/o proteins (e.g. suppression of adenylate cyclase when
expressed in cell lines). The mGluR2/3 receptors are localized widely throughout the brain in
circuits of relevance for neuropsychiatric disorders, such as the neocortex, thalamus, striatum,
amygdala and hippocampus.2 Functionally, activation of mGluR2/3 receptors in vivo suppresses
enhanced synaptic excitability via inhibition of presynaptic glutamate release and postsynaptic
activation of potassium channels that control neuronal hyperpolarization.3 In general, mGluR2/3
receptors have a predominant extrasynaptic localization. Thus, mGluR2/3 receptors provide a
negative feedback to keep glutamate transmission within the physiological range and thus
prevent hyperexcitability from interfering with normal brain function.
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According to the glutamatergic hypothesis of schizophrenia, hyperactivity of glutamatergic
transmission in the limbic regions is the underlying mechanism in the pathogenesis of
schizophrenia. It has been postulated that agonists at mGluR2/3 receptors could decrease
schizophrenic symptoms by reducing transmission at glutamatergic synapses and thus they might
represent a novel nonꢀdopaminergic strategy for the treatment of schizophrenia.4 The
antipsychotic properties of mGluR2/3 receptor agonists are supported by broad range of
preclinical data.5 In studies using transgenic mice lacking the mGluR2 or mGluR3 receptor, it
has been shown that the antipsychotic effects of the mGluR2/3 receptor agonists in the models of
psychosis are mediated through the activation of mGluR2 and not of mGluR3 receptors.6
Moreover, the Phase II clinical trial of LY2140023 (1), a selective mGluR2/3 receptor agonist
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(Figure 1), showed efficacy on schizophrenic subjects. It has been shown to improve positive
symptoms in patients thus being comparable with olanzapine, but it has not been shown to
produce any prolactin elevation, extrapyramidal symptoms or weight gain.7 However, in 2012,
Eli Lilly announced to stop Phase III development as 1 failed to meet its primary endpoint based
on the unaltered PANSS scale.8,9
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One alternative approach to directꢀacting mGluR2 receptor agonists is the use of subtypeꢀ
selective PAMs. These ligands do not activate the mGluR2 per se but act at an allosteric binding
site to potentiate glutamateꢀinduced activation of this receptor. Since a potentiator with no
inherent agonist activity would only function in the presence of the endogenous agonist, the
receptor would not be activated continuously, avoiding receptor desensitization which often
occurs after repeated dosing of orthosteric agonists.10 In addition, the allosteric binding sites on
glutamate receptors might sufficiently be different as to make subgroup selectivity feasible.11
Over the last decade a large number of structurally different chemotypes have been disclosed
and reviewed.12 To date, two mGluR2 PAM molecules have reached the clinic (Figure 1).
AZD8529 (2) had been in phase II clinical trial at AstraZeneca in schizophrenic patients;
however further development was halted in 2011 due to a lack of efficacy.13
ADX71149 (3), also known as JNJꢀ40411813, was investigated by Addex Therapeutics and
Janssen Pharmaceuticals for the treatment of schizophrenia14 and anxious depression (major
depressive disorder with anxiety symptoms).15 In a phase IIa study 3 was well tolerated and
showed promising effects in schizophrenia patients but was not effective in patients with major
depressive disorder and anxiety.16
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Our efforts in the field of mGluR2 PAMs started with a functional screen of our corporate
compound collection. The benzyl substituted benzotriazole derivative 4 was identified from this
campaign as an attractive small molecule with low micromolar activity (mGluR2 PAM EC50 = 3
µM, Figure 2). These data confirmed that 4 is a much smaller (Nhev=16) but still potent
compound with higher ligand efficiency (LE=0.49) as compared to known mGluR2 PAMs12
(average Nhev=29+4; average LE=0.25+0.4) that validates it for followꢀup studies. Freedom to
operate analysis identified only a 5ꢀdisubstituted benzotriazole series of Merck17ꢀ19 that was
subjected to detailed evaluation. In contrast to 4 the patent describes complex drugꢀlike
compounds all having large substituents at position 5. Interestingly other positions of the core
were much less elaborated. This observation prompted us to explore the potential growing
vectors around the benzotriazole core systematically. This work has been started by the analysis
of the available pharmacophore model.20 Aligning our fragment like hit (4) to larger mGluR2
PAMs we identified the primary growing vectors and rapidly converted 4 to the biaryl derivative
5, that resulted in substantial improvement in mGluR2 PAM potency (mGluR2 PAM EC50 = 155
nM). Although incorporation of the biarylꢀring system increased the lipophilicity, we were
encouraged by the relative balance between mGlu2 PAM potency and ligand efficiency (LE =
0.44) compared to the original hit 4 (Figure 2). As 5 showed virtually the same low metabolic
stability on human, rat and mouse microsomes, our primary objective was to improve metabolic
stability, while maintaining the target potency and ligand efficiency of novel benzotriazole
derivatives. We herein report the successful optimization of benzotriazoles which led to the
identification of the advanced lead 60. This “fragmentꢀlike” molecule showed good mGluR2
receptor PAM in vitro activity, ADME and PK profile with reasonable activity in the PCPꢀ
induced hyperlocomotion and DOIꢀinduced head twitch models of psychosis.
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RESULTS
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Chemistry. The general synthesis of unsubstituted benzotriazole target compounds 5, 10ꢀ19
are shown in Scheme 1. Reaction of commercially available benzotriazole 6 with the appropriate
bromobenzyl derivatives 7 in THF using NaHMDS provided the key bromoaryl intermediates 8.
Target compounds 10ꢀ19 were prepared via Suzukiꢀcoupling of 8 with the corresponding boronic
acids 9. The chemical structures of 10ꢀ19 are shown in Table 1 and 3.
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Substituted benzotriazoles 24ꢀ60 were prepared following the synthesis sequence shown in
Scheme 2. Treatment of 1ꢀfluoroꢀ2ꢀnitrobenzene derivatives 20 with aminomethyl derivatives 21
afforded nitroanilines 22. Reduction of 22 in the presence of SnCl2 in MeOH/H2O yielded
dianilines 23 that were cyclized to the final benzoriazole compounds 24ꢀ60 in AcOH using
NaNO2. The chemical structures of 24ꢀ60 are shown in Table 4, 5 and 6.
Medicinal chemistry optimization of the HTS hit (4). Functional screening of our corporate
library on mGluR2 overexpressing cells resulted in a benzylꢀbenzotriazole singleton with
acceptable potency and high ligand efficiency. Structural alignment with known mGluR2 PAM
reference compounds revealed that there are two potential growing vectors available; one at
position 4 of the benzotriazole core and another at the para position of the benzyl group (Figure
2). Our first attempts were focused towards the latter vector and tried to improve the metabolic
stability of the biarylꢀbenzotriazole advanced hit 5 by exploring different substituents of the
terminal phenyl ring along vector 1 (10ꢀ19). Literature data21 suggested that 2, 4ꢀdisubstitution in
this region is beneficial for potency. A representative set of analogues to exemplify the SAR
trends identified is shown in Table 1. Overall, dual substitution at positions 2 and 4 gave
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compounds (10ꢀ15) with improved potency and metabolic stability out of which compound 11
showed the best profile. Since the highly lipophilic character of the compounds resulted in
limited solubility we next introduced ring nitrogens (16ꢀ19). Compound 16 showed reasonable
potency and metabolic stability with somewhat improved lipophilicity that nominated the
compound as an early lead.
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Compound 16 was next subjected to extended profiling that included its investigations in a
broad panel of receptors, ADME assays, in vivo pharmacokinetics and in vivo efficacy models
(Table 2).
Characterization of 16 showed that the compound has promising off target profile. Although
the kinetic solubility was still limited and plasma protein binding was considerably high, other
ADME parameters were found to be acceptable and the compound progressed to in vivo studies.
Mouse pharmacokinetics data suggested limited exposures (Cplasma: 2.0 µM, Cbrain: 1.4 µM,
Cplasma unbound: 0.015 µM) for 16 and consequently the compound did not show efficacy in the
PCPꢀinduced hyperlocomotion model (PCP HL ED50 > 100 mg/kg, i.p.). On the basis of the in
vivo data we concluded that further improvement in potency and ADME properties are needed to
realize the desired in vivo efficacy in disease models. To achieve this goal, we next tested the
effect of substituents towards the other growing vector (vector 2, Figure 2). A fluoro scan around
the benzotriazole core revealed that position 4 is beneficial for the potency that was further
optimized to compound 29 having single digit nanomolar potency and acceptable metabolic
stability (Table 3).
Unfortunately, however, the improvement in potency was associated with significant increase
in lipophilicity that turned the druglike properties of 29 less than optimal. Pharmacokinetic
investigation of this compound revealed that 29 reached improved plasma and brain levels after
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30 mg/kg oral dose in mice (Cplasma: 20 µM, Cbrain: 7.1 µM, Cplasma unbound: 0.081 µM) but these
exposures resulted in only limited improvement in the PCPꢀinduced hyperlocomotion model
(PCP HL ED50: 47 mg/kg, i.p.). Any further attempt of decreasing lipophilicity resulted in
significant drop in potency. Therefore, we decided to revisit the original hit 4 and tried its
optimization following fragment based drug discovery principles.
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Fragment based optimization of the HTS hit (4). Most of the successful fragment based
drug discovery programs rely on structural information typically from Xꢀray and/or NMR studies
of ligandꢀprotein complexes.23 Fragment optimization strategies, such as growing, merging and
linking require this information to improve the binding affinity while controlling the
physicochemical properties. Although there is an increasing number of membrane protein
structures available in PDB, mGluR1 and mGluR5, members of Group I mGluRs are the only
representatives of class C GPCRs. MGluR2 belongs to Group II and furthermore the present
PAM program requires its active conformation that is not available for any class C GPCRs.
Consequently, computational models of proteinꢀligand complexes might fail to identify the
correct binding mode and grow vectors and therefore could not be used to drive the optimization.
There is a consensus that in the lack of structural information, fragment optimizations are
challenging. Identification of growing vectors, however, can be feasible with structure activity
relationship (SAR) information. Without prior knowledge, all positions around the fragment core
have equal chance for growing or need for modifications. One of the possible strategies of using
SAR information is based on LE, suggesting that only derivatives with LE larger than 0.4 are
suitable for growing while less ligand efficient analogues the positions of substituents should be
further optimized. Tactically, one should keep or increase LE during growing with the
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simultaneous control of lipophilic measures such as LLEat or LELP.24 The other strategy is based
in the group efficiency (GE) concept.25 This methodology assumes that the molecules being
compared bind in a similar fashion and evaluates the relative contribution of structural moieties
to the binding free energy. Synthesizing and testing a number of close analogues around the
fragment core the contributions of the individual substituents can be evaluated and positions by
the lowest GE selected for subsequent modification.26 Here we implemented the latter
methodology and analyzed the GEs of compound 29 (Figure 3).
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GE analysis of 29 revealed that we should keep the benzotriazole core with the bromine
substituent at position 4. The biaryl part of the structure, however, adds less to the binding free
energy and therefore we selected this part for modification. Removal of the 2ꢀfluoroꢀ4ꢀ
chlorophenyl group in 29 to give 30 resulted in a significant reduction in potency, which was
more than compensated by the reduced size and overall resulted in an improved LE value from
0.47 to 0.59. Additionally, 30 with a bromine substituent at position 4 showed a marked increase
in potency as we compare to the original hit (4) (Table 4). It was clear from these data that the
main pharmacophore for mGluR2 PAM activity included the 4ꢀbromo benzotriazole core and we
should focus our efforts on improving the mGluR2 PAM potency and most importantly the
metabolic stability of this ring system. Our strategy was to optimize the substituents of the
benzyl part of 30 without growing the molecule substantially (Table 4).
Fluoro scan around the benzyl group suggested position 4 as the most promising. This is in line
with growth vector 1 used in the original optimization, however, compound 33 is about 10 times
more potent than 5 and more importantly its ligand efficiency and lipophilicity is much more
beneficial than that of the earlier advanced hit 5. Interestingly, unlike the case of 5 and
derivatives dual substitution at positions 2 and 4 did not improve the potency further. Next we
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tried to decrease the lipophilicity of the compounds by the insertion of nitrogens to the benzyl
ring (39ꢀ41) but the potency was substantially decreased. Continuing our efforts on the reduction
of the lipophilicity we replaced the benzyl group of 30 by a number of alkyl and cycloalkyl
substituents (Table 5).
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Both alkyl and cycloalkyl sideꢀchains decreased lipophilicity but made the compounds
metabolically vulnerable. On the other hand, cycloalkyl substituents (42ꢀ45) improved the
potency. Compound 45 showed reasonable potency with acceptable metabolic stability except
for that measured by mouse microsomes. We therefore selected this compound for further
optimization. During the final round of our medicinal chemistry program we fineꢀtuned the
substituents along vector 2 (Table 6).
To improve metabolic stability of 45 we explored position 6 by introducing a range of
substituents (51ꢀ55). Larger substituents such as trifluoromethyl (51), methoxy (55) groups and
also fluorine (54) decreased the potency as compared to other halogens (52, 53) but no
significant improvement in mouse metabolic stability was observed. Finally, we tested the effect
of different substituents at position 4 (56ꢀ60). In general, the compounds showed much lower
potency relative to the corresponding bromine derivative 45 except for the chlorine (56) and the
trifluoromethyl (60) analogue of which the latter was considered as a lead with balanced potency
and metabolic profile. Compound 60 has therefore been further profiled on extended ADME
screens that showed improved properties in solubility, permeability and plasma protein binding
as compared to 16 (Table 2). Its improved profile has been reflected in the pharmacokinetics
data, more specifically in brain levels (mouse Cplasma: 12.1 µM, Cbrain: 13.1 µM, Cplasma
unbound: 1.75 µM after 30 mg/kg oral dosing) and also in vivo results as compound 60 showed
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significant improvement in both the PCPꢀinduced hyperlocomotion and the DOIꢀinduced head
twitch models (Figure 4).
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DISCUSSION AND CONCLUSIONS
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Here we report the optimization of a mGluR2 PAM screening hit (4) by standard medicinal
chemistry and fragment based drug discovery (FBDD) principles. Since 4 was a moderately
potent compound with limited metabolic stability our first attempts were focused improving
these features. As a result, routine optimization of the screening hits resulted in a potent and
metabolically stable early lead (16). Characterization of the druglike properties of 16, however,
revealed that the compound has limited solubility and permeability and high plasma protein
binding. In line with these properties 16 showed limited exposures in the subsequent
pharmacokinetic study and it failed to demonstrate efficacy in the PCPꢀinduced hyperlocomotion
test, one of the proof of concept in vivo models. Since further attempts on the optimization of
druglike properties were unsuccessful, the chemistry program has been restarted and we decided
to optimize 4 using the principles of fragment based drug discovery. The lack of structural
information on the target, however, represented a significant challenge in the FBDD program.
Implementing the group efficiency based strategy22 together with several rounds of substituent
scans we were able to identify a series of analogues with reasonable potency and improved
druglike properties. This work highlighted some interesting features of the benzotriazole type
mGluR2 PAMs. Systematic exploration of the SAR around the core revealed new growing
vectors. Consequently, substituents at position 5 are not needed to achieve the target potency.
Instead, we identified multiple substitution patterns providing novel compounds with high
mGluR2 PAM efficacy. Furthermore, we demonstrated for the first time that single digit
nanomolar mGluR2 PAM potency can be realized by fragments. In due course we identified a set
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of mGluR2 PAMs with extremely high ligand efficiency indicating that the receptor can be
efficiently activated via fragments not only at the orthosteric but also at the allosteric site.
Careful fragment based optimization of the initial hit (4) finally led to 60 that still keeps the
fragment features. The balanced profile of 60 nominated this compound for in vivo
pharmacokinetic studies that revealed its significantly higher brain exposures compared to 16.
Due to its improved PK profile the compound was found to be effective in both of our proof of
concept animal models having ED50 values of 37.6 and 29.7 mg/kg in PCPꢀinduced
hyperlocomotion and DOIꢀinduced head twitch tests, respectively. The successful FBDD
strategy used for the identification of 60 provideed a suitable tool for studying the therapeutical
significance of mGluR2 PAMs and also demonstrated the feasibility of group efficiency driven
FBDD approaches in the lack of structural information.
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EXPERIMENTAL SECTION
General experimental 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 MerckꢀHitachi
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
o
flow rate was set to 1.7 mL/min and the column was maintained at 40 C. Purity of final
compounds was assessed using UV detection (Diode Arry Detector: MerckꢀHitachi Lachrom
Elite Lꢀ2450) at 215 nm; all tested compounds were ≥ 95% pure. NMR spectra were recorded at
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298K either on an Agilent (Varian) NMRSꢀ500 spectrometer (1H: 499.9 MHz; 13C: 125.7 MHz)
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equipped with a HCN 5mm PFG Triple Resonance C Enhanced Cold Probe or on an Agilent
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(Varian) NMRSꢀ400 (1H: 399.8 MHz) equipped with a Hꢀ19F/15Nꢀ31P 5mm PFG OneNMR
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Probe or on an Agilent (Varian) NMRSꢀ800 (1H: 799.7 MHz; 13C: 201.0 MHz) equipped with a
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HCN 5mm PFG Triple Resonance C Enhanced Salt Tolerant Cold Probe using DMSOꢀd6 as
solvent if not stated otherwise. Four of the samples (marked with *) were measured on a Bruker
Avance III HDX 500 spectrometer (1H: 499.9 MHz; 13C: 125.7 MHz) using DMSOꢀh6:DMSOꢀd6
1
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1:5 as solvent, therefore presaturation was used for solvent suppression. H and C chemical
shifts were referenced to TMS. 15N chemical shifts were determined from 1H,15NꢀHMBC spectra
and are referenced to nitromethane by internal autocalibration method of the software.
EIꢀMS analysis was performed on a Finnigan MAT 95 XP system. The ionization method was
EI and operated in positive ion mode. Electron energy was 70 eV and the source temperature was
set at 220°C. ESIꢀMS analyses were performed on a LTQ XL as well as a LTQ FT Ultra (Thermo
Fisher Scientific, Bremen, Germany) system. The ionization method was ESI and operated in
positive ion mode. The protonated molecular ion peaks were fragmented by CID at a normalized
collision energy of 35ꢀ45%. Data acquisition and analysis were accomplished with Xcalibur
software version 2.0 (Thermo Fisher Scientific). HRꢀESIꢀMS analyses were performed on a
Thermo Velos Pro Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) system. The
ionization method was ESI and operated in positive ion mode. The protonated molecular ion peaks
were fragmented by CID at a normalized collision energy of 35%. Data acquisition and analysis
were accomplished with Xcalibur software version 2.0 (Thermo Fisher Scientific).
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General procedure for the synthesis of unsubstituted benzotriazoles 5, 10ꢀ19 (Scheme 1).
STEP 1. 36 mmol of 1ꢀbromoꢀ4ꢀ(bromomethyl)aryl derivative (7) was added to a solution of 6 g
(50 mmol) of 1Hꢀbenzotriazole (6) in 120 mL of anhydrous tetrahydrofuran. The reaction
mixture was cooled to ꢀ30°C, and a solution of 40 mL (40 mmol) of sodiumꢀhexamethylꢀ
disilazane (1M in THF) was added dropwise over a period of 40 min. The solution was allowed
to warm up to 25°C, stirred for 2 hours, and evaporated to dryness. The residue was crystallized
from an appropriate solvent to give 1ꢀ(4ꢀbromoaryl)ꢀ1Hꢀbenzotriazole (8) in 35ꢀ40% yield.
STEP 2. 28 mmol of the corresponding 1ꢀ(4ꢀbromoaryl)ꢀ1Hꢀbenzotriazole (8) and 31 mmol of
arylboronic acid (9) in toluene/ethanol (150 mL / 50 mL) were refluxed for 2 h with 56 mmol of
Na2CO3 and 2.24 mmol (8 n/n%) of Pd(PPh3)4. After cooling, 180 mL of water was added and
the separated organic layer was evaporated to dryness. The residue was crystallized from ethanol
to give 1ꢀ(Rꢀbiarylꢀ4ꢀylmethyl)ꢀ1Hꢀbenzotriazoles (5, 10ꢀ19) in 75ꢀ90% yield.
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1
1ꢀ(biphenylꢀ4ꢀylmethyl)ꢀ1Hꢀbenzotriazole (5). H NMR (400 MHz): δ 8.07 (d, J = 8.4 Hz,
1H, H4), 7.90 (d, J = 8.4 Hz, 1H, H7), 7.59ꢀ7.68 (m, 4H), 7.52ꢀ7.58 (m, 1H), 7.38ꢀ7.48 (m, 5H),
7.32ꢀ7.38 (m, 1H), 6.04 (s, 2H, CH2N). M+=285; EIꢀMS (rel. int. %): 285(68); 256(100);
180(17); 167(42); 165(20); 152(13); 128(3); 115(3); 91(4); 77(5).
1
1ꢀ[(2',4'ꢀdifluorobiphenylꢀ4ꢀyl)methyl]ꢀ1Hꢀbenzotriazole (10). H NMR (400 MHz): δ 8.07
(d, J = 8.4 Hz, 1H, H4), 7.91 (d, J = 8.4 Hz, 1H, H7), 7.48ꢀ7.58 (m, 4H), 7.39ꢀ7.46 (m, 3H),
7.31ꢀ7.39 (m, 1H), 7.13ꢀ7.22 (m, 1H), 6.05 (s, 2H, CH2N). M+=321; EIꢀMS (rel. int. %):
321(50); 292(100); 203(38); 201(12); 183(8); 180(10); 77(4).
1ꢀ[(4'ꢀchloroꢀ2'ꢀfluorobiphenylꢀ4ꢀyl)methyl]ꢀ1Hꢀbenzotriazole (11). 1H NMR (800 MHz): δ
8.08 (d, J = 8.3 Hz, 1H, H4), 7.91 (d, J = 8.3 Hz, 1H, H7), 7.51ꢀ7.59 (m, 5H), 7.40ꢀ7.47 (m, 5H),
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7.37 (dd, J = 8.8, 1.5 Hz, 1H), 6.05 (s, 2H, CH2N). M+=337; EIꢀMS (rel. int. %): 337(43);
5
6
308(100); 272(6); 219(40); 183(32); 180(15); 91(2); 77(6).
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8
1
1ꢀ{[2'ꢀfluoroꢀ4'ꢀ(trifluoromethyl)biphenylꢀ4ꢀyl]methyl}ꢀ1Hꢀbenzotriazole (12). H NMR
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(400 MHz): δ 8.08 (d, J = 8.4 Hz, 1H, H4), 7.92 (d, J = 8.4 Hz, 1H, H7), 7.70ꢀ7.80 (m, 2H),
7.63ꢀ7.70 (m, 1H), 7.54ꢀ7.63 (m, 3H), 7.45ꢀ7.50 (m, 2H), 7.40ꢀ7.45 (m, 1H), 6.08 (s, 2H, CH2N).
M+=371; EIꢀMS (rel. int. %): 371(35); 342(100); 253(38); 183(12); 180(10); 77(3).
1ꢀ[(2'ꢀchloroꢀ4'ꢀfluorobiphenylꢀ4ꢀyl)methyl]ꢀ1Hꢀbenzotriazole (13). 1H NMR (400 MHz): δ
8.08 (d, J = 8.1 Hz, 1H, H4), 7.94 (d, J = 8.4 Hz, 1H, H7), 7.51ꢀ7.61 (m, 2H), 7.37ꢀ7.46 (m, 6H),
7.29 (td, J = 8.5, 2.7 Hz, 1H), 6.06 (s, 2H, CH2N). M+=337; EIꢀMS (rel. int. %): 337(62);
308(100); 274(20); 272(14); 219(41); 183(34); 180(18); 91(1); 77(5).
1
1ꢀ[(2',4'ꢀdichlorobiphenylꢀ4ꢀyl)methyl]ꢀ1Hꢀbenzotriazole (14). H NMR (400 MHz): δ 8.08
(d, J = 8.4 Hz, 1H, H4), 7.94 (d, J = 8.4 Hz, 1H, H7), 7.71 (d, J = 2.0 Hz, 1H, H3”), 7.57 (t, J =
7.6 Hz, 1H, H6), 7.46ꢀ7.52 (m, 1H, H5”), 7.38ꢀ7.46 (m, 6H, H2’, H3’, H5’, H6’, H5, H6”), 6.06
(s, 2H, CH2N). M+=353; EIꢀMS (rel. int. %): 353(55); 324(100); 290(18); 235(40); 199(8);
180(25); 165(37); 127(7); 113(3); 77(7).
1
1ꢀ{[2'ꢀchloroꢀ4'ꢀ(trifluoromethyl)biphenylꢀ4ꢀyl]methyl}ꢀ1Hꢀbenzotriazole (15). H NMR
(400 MHz): δ 8.08 (d, J = 8.5 Hz, 1H, H4), 7.97 (d, J = 0.8 Hz, 1H, H3”), 7.94 (d, J = 8.4 Hz,
1H, H7), 7.78 (d, J = 8.1 Hz, 1H, H5”), 7.54ꢀ7.65 (m, 2H, H6, H6”), 7.40ꢀ7.50 (m, 5H, H5), 6.08
(s, 2H, CH2N). M+=387; EIꢀMS (rel. int. %): 387(41); 358(100); 324(15); 269(42); 233(7);
180(19); 165(18); 77(5).
1
1ꢀ{[6ꢀ(4ꢀchloroꢀ2ꢀfluorophenyl)pyridinꢀ3ꢀyl]methyl}ꢀ1Hꢀbenzotriazole (16). H NMR (800
MHz): δ 8.80ꢀ8.83 (m, 1H, H2’), 8.07ꢀ8.10 (m, 1H, H4), 7.96ꢀ8.00 (m, 1H, H7), 7.92ꢀ7.96 (t,
1H, J = 8.5 Hz, H6”), 7.82ꢀ7.85 (m, 1H, H4’), 7.76ꢀ7.79 (m, 1H, H5’), 7.56ꢀ7.60 (m, 2H, H3”,
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H6), 7.41ꢀ7.45 (m, 2H, H5, H5”), 6.12 (s, 2H, CH2N). M+=338; EIꢀMS (rel. int. %): 338(54);
309(100); 283(10); 220(39); 193(13); 178(9); 167(12); 157(11); 77(6); 57(3).
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1ꢀ{[6ꢀ(2ꢀfluorophenyl)pyridinꢀ3ꢀyl]methyl}ꢀ1Hꢀbenzotriazole (17). H NMR (400 MHz): δ
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8.82 (d, J=2.3 Hz, 1H, H2’), 8.09 (d, J=8.4 Hz, 1H, H4), 7.98 (d, J=8.4 Hz, 1H, H7), 7.90 (dt,
J=7.9, 1.7 Hz, 1H, H6”), 7.80ꢀ7.85 (dd, J=8.2, 2.3 Hz, 1H, H4’), 7.77 (dm, J=8.2, 7.9 Hz, 1H,
H5’), 7.59 (ddd, J=8.4, 7.0, 0.9 Hz, 1H, H6), 7.46ꢀ7.52 (m, 1H, H4”), 7.43 (ddd, J=8.4, 7.0, 0.9
Hz, 1H, H5), 7.27ꢀ7.38 (m, 2H, H3”, H5”), 6.12 (s, 2H, CH2N). M+=304; EIꢀMS (rel. int. %):
304(23); 275(100); 248(16); 186(21); 159(17); 133(29).
1
1ꢀ{[6ꢀ(4ꢀchlorophenyl)pyridinꢀ3ꢀyl]methyl}ꢀ1Hꢀbenzotriazole (18). H NMR (500 MHz)*:
δ 8.76 (d, J=2.3 Hz, 1H, H2’), 8.07 (m, 2H, Ar”, H5’), 7.93ꢀ7.99 (m, 2H, H4, H7), 7.81 (dd,
J=8.2, 2.3 Hz, 1H, H4’), 7.77 (dm, J=8.2, 7.9 Hz, 1H, H5’), 7.55ꢀ7.60 (m, 1H, H6), 7.52ꢀ7.55
(m, 2H, Ar”), 7.40ꢀ7.45 (m, 1H, H5), 6.09 (s, 2H, CH2N). M+=320; EIꢀMS (rel. int. %): 320(46);
291(100); 264(14); 202(34); 179(17); 149(17); 139(28); 84(46); 66(27).
1
1ꢀ{[5ꢀ(4ꢀchloroꢀ2ꢀfluorophenyl)pyridinꢀ2ꢀyl]methyl}ꢀ1Hꢀbenzotriazole (19). H NMR (400
MHz): δ 8.64ꢀ8.68 (m, 1H, H6’), 8.05ꢀ8.10 (m, 1H, H4), 8.01 (td, J = 8.3, 1.8 Hz, 1H, H4’),
7.83ꢀ7.88 (m, 1H, H7), 7.52ꢀ7.66 (m, 3H, H6, H3”, H6”), 7.47 (d, J = 8.2 Hz, 1H, H3’), 7.40ꢀ
7.45 (m, 2H, H5, H5”), 6.17 (s, 2H, CH2N). M+=338; EIꢀMS (rel. int. %): 310(100); 283(7);
258(7); 220(31); 207(20); 193(17); 185(21); 77(7).
General procedure for the synthesis of substituted benzotriazoles 24ꢀ60 (Scheme 2). STEP
1. A mixture of 32.5 mmol of the corresponding 1ꢀfluoroꢀ2ꢀnitrobenzene (20), 35.8 mmol of
R1CH2NH2 (21) and 65 mmol of K2CO3 in 500 mL of AcCN was stirred for 18 h at room
temperature. The solid was filtered off and washed with 30 mL of AcCN. The combined organic
filtrates were evaporated in vacuo. The crude Nꢀalkylꢀnitrobenzenes (22) were obtained in 74ꢀ
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90% yield and were used in the next step without further purification. STEP 2. 50 mmol of SnCl2
was added to a mixture of 10 mmol of 22 in 50 mL of MeOH and 17 mL of cc. HCl and stirred at
50oC for 12 h. The mixture was concentrated in vacuo. The residue was taken up in EtOAc and
washed with saturated solution of NaHCO3. The combined organic layers were dried over
Na2SO4 and concentrated in vacuo. The crude was purified by flash column chromatography on
silica gel to obtain dianilines (23) in 52ꢀ65% yield. STEP 3. To a solution of 9.6 mmol of
dianilines (23) in 60 mL of AcOH was added 11.6 mmol of NaNO2. The mixture was stirred at
60°C for 3 h. The cooled mixture was evaporated in vacuo. The residue was taken up in EtOAc
and washed with saturated solution of NaHCO3. The combined organic layers were dried over
Na2SO4 and concentrated in vacuo. The crude products were purified by flash column
chromatography to give the final products 24ꢀ60 in 32ꢀ73% yield. All substituted benzotriazoles
reported were prepared by the general procedure.
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1ꢀ{[6ꢀ(4ꢀchloroꢀ2ꢀfluorophenyl)pyridinꢀ3ꢀyl]methyl}ꢀ4ꢀfluoroꢀ1Hꢀbenzotriazole (24). 1H
NMR (500 MHz): δ 8.83 (dd, J = 2.3, 0.7 Hz, 1H, H2’), 7.95 (t, J = 8.6 Hz, 1H, H6”), 7.82ꢀ7.86
(m, 2H, H7, H4’), 7.77ꢀ7.80 (m, 1H, H5’), 7.56ꢀ7.61 (m, 2H, H3”, H6), 7.41ꢀ7.44 (m, 1H, H5”),
7.24ꢀ7.30 (m, 1H, H5), 6.15 (s, 2H, CH2N). 15N NMR (50.7 MHz): δ ꢀ150 (N1), ꢀ65.5 (N1’), ꢀ0.5
(N2). [M+H]+=357; ESIꢀMSꢀMS (rel. int. %, cid=35%): 329(100); 221(2).
1ꢀ{[6ꢀ(4ꢀchloroꢀ2ꢀfluorophenyl)pyridinꢀ3ꢀyl]methyl}ꢀ5ꢀfluoroꢀ1Hꢀbenzotriazole (25). 1H
NMR (400 MHz): δ 8.83 (d, J = 1.6 Hz, 1H, H2’), 8.05 (dd, J = 8.9, 4.3 Hz, 1H, H7), 7.91ꢀ7.97
(m, 2H, H4, H6”), 7.81ꢀ7.87 (m, 1H, H4’), 7.75ꢀ7.81 (m, 1H, H5’), 7.50ꢀ7.60 (m, 2H, H6, H3”),
7.42 (dd, J = 8.5, 1.7 Hz, 1H, H5”), 6.12 (s, 2H, CH2N). MS: [M+H]+=357; ESIꢀMSꢀMS (rel. int.
%, cid=35%): 329(100); 221(9).
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1ꢀ{[6ꢀ(4ꢀchloroꢀ2ꢀfluorophenyl)pyridinꢀ3ꢀyl]methyl}ꢀ6ꢀfluoroꢀ1Hꢀbenzotriazole (26). 1H
NMR (400 MHz): δ 8.83 (d, J = 1.6 Hz, 1H, H2’), 8.15 (dd, J = 9.1, 4.7 Hz, 1H, H4), 7.92ꢀ7.97
(m, 2H, H7, H6”), 7.83ꢀ7.89 (m, 1H, H4’), 7.75ꢀ7.81 (m, 1H, H5’), 7.58 (dd, J = 11.2, 2.0 Hz,
1H, H3”), 7.42 (dd, J = 8.5, 2.0 Hz, 1H, H5”), 7.34 (dt, J = 9.1, 2.3, Hz, 1H, H5), 6.07 (s, 2H,
CH2N). [M+H]+=357; ESIꢀMSꢀMS (rel. int. %, cid=35%): 329(100); 221(19).
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1ꢀ{[6ꢀ(4ꢀchloroꢀ2ꢀfluorophenyl)pyridinꢀ3ꢀyl]methyl}ꢀ1Hꢀbenzotriazoleꢀ4ꢀcarbonitrile (27).
1H NMR (400 MHz): δ 8.83ꢀ8.85 (m, 1H, H2’), 8.41 (dd, J = 8.5, 0.7 Hz, 1H, H7), 8.06 (dd, J =
7.2, 0.7 Hz, 1H, H5), 7.94 (t, J = 8.6 Hz, 1H, H6”), 7.83ꢀ7.88 (m, 1H, H4’), 7.74ꢀ7.80 (m, 2H,
H6, H5’), 7.57 (dd, J = 11.2, 2.0 Hz, 1H, H3”), 7.42 (dd, J = 8.5, 1.6 Hz, 1H, H5”), 6.20 (s, 2H,
CH2N). [M+H]+=364; ESIꢀMSꢀMS (rel. int. %, cid=35%): 336(100); 221(9).
1ꢀ{[6ꢀ(4ꢀchloroꢀ2ꢀfluorophenyl)pyridinꢀ3ꢀyl]methyl}ꢀ4ꢀ(trifluoromethyl)ꢀ1Hꢀ
1
benzotriazole (28). H NMR (800 MHz): δ 8.85ꢀ8.88 (m, 1H, H2’), 8.37 (d, J = 8.3 Hz, 1H,
H7), 7.95 (t, J = 8.7 Hz, 1H, H6”), 7.84ꢀ7.88 (m, 2H, H5, H4’), 7.75ꢀ7.81 (m, 2H, H5’, H6), 7.58
13
(dd, J = 11.2, 2.0 Hz, 1H, H3”), 7.43 (dd, J = 8.3, 2.0 Hz, 1H, H5”), 6.21 (s, 2H, CH2N). C
1
3
NMR (201 MHz): δ 159.6 (d, JF = 253 Hz, C2”), 151.3 (d, JF = 2.4 Hz, C6’), 149.4 (C2’),
3
3
140.7 (C3a), 136.7 (C4’), 134.5 (d, JF = 10.3 Hz, C4”), 133.7 (C7a), 132.0 (d, JF = 4.0 Hz,
2
C6”), 130.5 (C3’), 127.3 (C6), 125.4 (d, JF = 11.9 Hz, C1”), 125.2 (d, 4JF = 3.2 Hz, C5”), 124.1
4
1
3
2
(d, JF = 9.5 Hz, C5’), 123.3 (q, JF = 272 Hz, CF3), 122.2 (q, JF = 4.8 Hz, C5), 119.3 (q, JF =
33.5 Hz, C4), 116.9 (d, 2JF = 27.0 Hz, C3”), 115.9 (C7), 48.5 (CH2N). 15N NMR (81.1 MHz): δ ꢀ
149 (N1), ꢀ65 (N1’), 1.6 (N2). [M+H]+=407; ESIꢀMSꢀMS (rel. int. %, cid=35%): 379(100);
359(16); 339(3); 221(7).
1
1ꢀ{[6ꢀ(4ꢀchloroꢀ2ꢀfluorophenyl)pyridinꢀ3ꢀyl]methyl}ꢀ4ꢀbromoꢀ1Hꢀbenzotriazole (29). H
NMR (800 MHz): δ 8.83 (d, J = 1.6 Hz, 1H, H2’), 8.01 (d, J = 8.3 Hz, 1H, H7), 7.95 (t, J = 8.5
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Hz, 1H, H6”), 7.82ꢀ7.85 (m, 1H, H4’), 7.76ꢀ7.79 (m, 1H, H5’), 7.70 (d, J = 6.7 Hz, 1H, H5),
7.58 (dd, J = 11.0, 2.0 Hz, 1H, H3”), 7.52 (dd, J = 8.3, 7.5 Hz, 1H, H6), 7.42 (dd, J = 8.7, 2.0
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13
1
Hz, 1H, H5”), 6.13 (s, 2H, CH2N). C NMR (201 MHz): δ 159.6 (d, JF = 252 Hz, C2”), 151.3
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(d, JF = 2.4 Hz, C6’), 149.3 (C2’), 144.0 (C3a), 136.6 (C4’), 134.5 (d, JF = 10.7 Hz, C4”),
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133.8 (C7a), 132.0 (d, JF = 3.6 Hz, C6”), 130.6 (C3’), 129.0 (C6), 127.2 (C5), 125.4 (d, JF =
11.9 Hz, C1”), 125.2 (d, 4JF = 3.6 Hz, C5”), 124.1 (d, 4JF = 8.7 Hz, C5’), 116.9 (d, 2JF = 27.0 Hz,
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C3”), 111.8 (C4), 110.4 (C7), 48.7 (CH2N). N NMR (81.1 MHz): δ ꢀ149 (N1), ꢀ65 (N1’), ꢀ0.5
(N2). [M+H]+=417; ESIꢀMSꢀMS (rel. int. %, cid=35%): 389(95); 310(15); 221(10).
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1ꢀbenzylꢀ4ꢀbromoꢀ1Hꢀbenzotriazole (30). H NMR (400 MHz): δ 7.88 (d, J = 8.3 Hz, 1H,
H7), 7.66 (d, J = 7.4 Hz, 1H, H5), 7.48 (dd, J = 8.3, 7.4 Hz, 1H, H6), 7.27ꢀ7.41 (m, 5H), 6.01 (s,
2H, CH2N). M+=287; EIꢀMS (rel. int. %): 287(78); 258(61); 180(59); 152(15); 104(5); 91(100);
76(8); 65(18).
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4ꢀbromoꢀ1ꢀ(2ꢀfluorobenzyl)ꢀ1Hꢀbenzotriazole (31). H NMR (400 MHz): δ 7.87 (d, J = 8.3
Hz, 1H, H7), 7.68 (d, J = 7.4 Hz, 1H, H5), 7.49 (dd, J = 8.3, 7.4 Hz, 1H, H6), 7.36ꢀ7.44 (m, 2H),
7.19ꢀ7.27 (m, 2H), 6.06 (s, 2H, CH2N). M+=305; EIꢀMS (rel. int. %): 305(55); 276(40); 258(8);
198(28); 170(7); 155(3); 109(100).
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4ꢀbromoꢀ1ꢀ(3ꢀfluorobenzyl)ꢀ1Hꢀbenzotriazole (32). H NMR (400 MHz): δ 7.91 (dd, J =
8.3, 0.7 Hz, 1H, H7), 7.68 (dd, J = 7.5, 0.7 Hz, 1H, H5), 7.49 (dd, J = 8.3, 7.5 Hz, 1H, H6), 7.36ꢀ
7.45 (m, 1H), 7.11ꢀ7.25 (m, 3H), 6.04 (s, 2H, CH2N). M+=305; EIꢀMS (rel. int. %): 305(61);
276(35); 258(6); 198(51); 170(11); 155(4); 109(100).
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4ꢀbromoꢀ1ꢀ(4ꢀfluorobenzyl)ꢀ1Hꢀbenzotriazole (33). H NMR (400 MHz): δ 7.91 (d, J = 8.1
Hz, 1H, H7), 7.68 (d, J = 7.5 Hz, 1H, H5), 7.49 (dd, J = 8.3, 7.5 Hz, 1H, H6), 7.39ꢀ7.45 (m, 2H,
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H2’, H6’), 7.15ꢀ7.23 (m, 2H, H3’, H5’), 6.00 (s, 2H, CH2N). M+=305; EIꢀMS (rel. int. %):
305(68); 276(49); 258(4); 198(45); 170(12); 155(3); 109(100).
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4ꢀbromoꢀ1ꢀ(4ꢀchlorobenzyl)ꢀ1Hꢀbenzotriazole (34). H NMR (400 MHz): δ 7.89 (dd, J =
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8.3, 0.7 Hz, 1H, H7), 7.68 (dd, J = 7.5, 0.7 Hz, 1H, H5), 7.48 (dd, J = 8.3, 7.5 Hz, 1H, H6), 7.40ꢀ
7.45 (m, 2H, H2’, H6’), 7.33ꢀ7.39 (m, 2H, H3’, H5’), 6.01 (s, 2H, CH2N). M+=321; EIꢀMS (rel.
int. %): 321(74); 292(25); 258(55); 214(21); 179(46); 155(4); 125(100); 89(30).
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4ꢀbromoꢀ1ꢀ(4ꢀbromobenzyl)ꢀ1Hꢀbenzotriazole (35). H NMR (400 MHz): δ 7.89 (d, J = 8.4
Hz, 1H, H7), 7.68 (d, J = 7.5 Hz, 1H, H5), 7.54ꢀ7.58 (m, 2H, H3’, H5’), 7.48 (dd, J = 8.4, 7.5
Hz, 1H, H6), 7.27ꢀ7.31 (m, 2H, H2’, H6’), 6.00 (s, 2H, CH2N). [M+H]+=366; ESIꢀMSꢀMS (rel.
int. %, cid=35%): 338(1); 258(2); 169(100).
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4ꢀbromoꢀ1ꢀ(2ꢀchloroꢀ4ꢀfluorobenzyl)ꢀ1Hꢀbenzotriazole (36). H NMR (400 MHz): δ 7.85
(d, J = 8.3 Hz, 1H, H7), 7.69 (d, J = 7.4 Hz, 1H, H5), 7.55 (dd, J = 8.9, 2.5 Hz, 1H, H3’), 7.49
(dd, J = 8.3, 7.4 Hz, 1H, H6), 7.40 (dd, J = 8.6, 6.2 Hz, 1H, H6’), 7.26 (td, J = 8.5, 2.5 Hz, 1H,
H5’), 6.06 (s, 2H, CH2N). M+=339; EIꢀMS (rel. int. %): 339(28); 310(21); 292(2); 276(30);
232(13); 197(34); 170(8); 155(4); 143(100); 107(19).
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4ꢀbromoꢀ1ꢀ(4ꢀchloroꢀ3ꢀfluorobenzyl)ꢀ1Hꢀbenzotriazole (37). H NMR (400 MHz): δ 7.92
(dd, J = 8.4, 0.6 Hz, 1H, H7), 7.69 (dd, J = 7.4, 0.6 Hz, 1H, H5), 7.58 (t, J = 8.1 Hz, 1H, H5’),
7.45ꢀ7.52 (m, 2H, H6, H2’), 7.12ꢀ7.17 (m, 1H, H6’), 6.03 (s, 2H, CH2N). M+=339; EIꢀMS (rel.
int. %): 339(61); 310(19); 292(4); 276(28); 232(37); 197(24); 170(7); 155(5); 143(100); 107(18).
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4ꢀbromoꢀ1ꢀ(4ꢀchloroꢀ2ꢀfluorobenzyl)ꢀ1Hꢀbenzotriazole (38). H NMR (400 MHz): δ 7.89
(d, J = 8.6 Hz, 1H, H7), 7.69 (d, J = 7.4 Hz, 1H, H5), 7.48ꢀ7.53 (m, 2H, H6, H3’), 7.42 (t, J = 8.2
Hz, 1H, H6’), 7.31 (dd, J = 8.2, 1.8 Hz, 1H, H5’), 6.05 (s, 2H, CH2N). [M+H]+=340; ESIꢀMSꢀ
MS (rel. int. %, cid=35%): 312(4); 276(2); 233(4); 143(100).
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4ꢀbromoꢀ1ꢀ[(5ꢀbromopyridinꢀ2ꢀyl)methyl]ꢀ1Hꢀbenzotriazole (39). H NMR (400 MHz): δ
8.57ꢀ8.61 (m, 1H, H6’), 8.06ꢀ8.12 (m, 1H, H4’), 7.83 (d, J = 8.3 Hz, 1H, H7), 7.68 (d, J = 7.5
Hz, 1H, H5), 7.47 (dd, J = 8.3, 7.5 Hz, 1H, H6), 7.38ꢀ7.43 (m, 1H, H3’), 6.12 (s, 2H, CH2N).
M+=366; EIꢀMS (rel. int. %): 366(1); 338(49); 259(46); 170(48); 143(17); 90(31).
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4ꢀbromoꢀ1ꢀ[(6ꢀbromopyridinꢀ3ꢀyl)methyl]ꢀ1Hꢀbenzotriazole (40). H NMR (400 MHz): δ
8.51ꢀ8.54 (m, 1H, H2’), 7.96 (dd, J = 8.3, 0.7 Hz, 1H, H7), 7.61ꢀ7.72 (m, 3H, H5, H4’, H5’),
7.50 (dd, J = 8.3, 7.5 Hz, 1H, H6), 6.05 (s, 2H, CH2N). M+=366; EIꢀMS (rel. int. %): 366(1);
337(16); 259(46); 170(54); 143(11); 90(29).
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4ꢀbromoꢀ1ꢀ[(6ꢀchloropyridinꢀ3ꢀyl)methyl]ꢀ1Hꢀbenzotriazole (41). H NMR (400 MHz): δ
8.55 (d, J = 2.5 Hz, 1H, H2’), 7.96 (d, J = 8.4 Hz, 1H, H7), 7.78 (dd, J = 8.3, 2.5 Hz, 1H, H4’),
7.69 (dd, J = 7.4, 0.6 Hz, 1H, H5), 7.47ꢀ7.54 (m, 2H, H6, H5’), 6.07 (s, 2H, CH2N). M+=322; EIꢀ
MS (rel. int. %): 322(55); 293(35); 259(29); 215(32); 179(41); 126(100).
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4ꢀbromoꢀ1ꢀ(cyclohexylmethyl)ꢀ1Hꢀbenzotriazole (42). H NMR (500 MHz): δ 7.94 (d, J =
8.3 Hz, 1H, H7), 7.66 (d, J = 7.3 Hz, 1H, H5), 7.48 (dd, J = 8.3, 7.3 Hz, 1H, H6), 4.58 (d, J = 7.3
Hz, 2H, CH2N), 1.90ꢀ2.00 (m, 1H, CH), 1.46ꢀ1.69 (m, 5H, 5xCH2eq), 0.97ꢀ1.22 (m, 5H,
5xCH2ax). 15N NMR (50.7 MHz): δ ꢀ160 (N1), ꢀ11 (N2). M+=293; EIꢀMS (rel. int. %): 293(43);
210(100); 197(11); 182(54); 169(4); 155(19); 130(4); 117(3); 103(22); 83(21); 75(14); 55(58);
41(31).
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4ꢀbromoꢀ1ꢀ(cyclopentylmethyl)ꢀ1Hꢀbenzotriazole (43). H NMR (400 MHz): δ 7.97 (d, J =
8.3 Hz, 1H, H7), 7.66 (d, J = 7.4 Hz, 1H, H5), 7.48 (dd, J = 8.3, 7.4 Hz, 1H, H6), 4.66 (d, J = 7.4
Hz, 2H, CH2N), 2.44ꢀ2.55 (m, 1H, CH), 1.47ꢀ1.67 (m, 6H), 1.23ꢀ1.34 (m, 2H). M+=279; EIꢀMS
(rel. int. %): 279(38); 210(100); 182(53); 169(7); 155(18); 130(5); 103(20); 90(5); 75(15);
69(21); 55(17); 41(39).
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4ꢀbromoꢀ1ꢀ(cyclobutylmethyl)ꢀ1Hꢀbenzotriazole (44). H NMR (500 MHz): δ 7.96 (d, J =
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6
7.8 Hz, 1H, H7), 7.66 (d, J = 6.9 Hz, 1H, H5), 7.46ꢀ7.50 (m, 1H, H6), 4.76 (d, J = 7.3 Hz, 2H,
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CH2N), 2.86ꢀ2.96 (m, 1H, CH), 1.78ꢀ2.01 (m, 6H, 3xcybCH2). N NMR (50.7 MHz): δ ꢀ160
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(N1), ꢀ13 (N2). M+=265; EIꢀMS (rel. int. %): 265(82); 237(19); 224(19); 210(99); 197(8);
182(44); 169(20); 155(25); 130(40); 117(22); 103(30); 90(16); 75(29); 55(100); 41(68).
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4ꢀbromoꢀ1ꢀ(cyclopropylmethyl)ꢀ1Hꢀbenzotriazole (45). H NMR (400 MHz): δ 7.99 (dd, J
= 8.3, 0.6 Hz, 1H, H7), 7.67 (dd, J = 7.4, 0.6 Hz, 1H, H5), 7.49 (dd, J = 8.3, 7.4 Hz, 1H, H6),
4.63 (d, J = 7.3 Hz, 2H, CH2N), 1.33ꢀ1.43 (m, 1H, CH), 0.51ꢀ0.58 (m, 2H, cypCH2x), 0.46ꢀ0.51
(m, 2H, cypCH2y). M+=251; EIꢀMS (rel. int. %): 251(64); 222(25); 210(59); 195(13); 182(30);
169(14); 155(22); 143(41); 116(35); 75(27); 55(100).
4ꢀbromoꢀ1ꢀ(2ꢀmethylpropyl)ꢀ1Hꢀbenzotriazole (46). 1H NMR (400 MHz): δ 7.95 (d, J = 8.4
Hz, 1H, H7), 7.67 (d, J = 7.4 Hz, 1H, H5), 7.48 (dd, J = 8.3, 7.4 Hz, 1H, H6), 4.56 (d, J = 7.0
Hz, 2H, CH2N), 2.28 (n, J = 7.0 Hz, 1H, CH), 0.89 (d, J = 7.0 Hz, 6H, 2xCH3). M+=253; EIꢀMS
(rel. int. %): 253(48); 210(100); 182(52); 169(13); 155(21); 130(6); 117(2); 103(22); 90(6);
75(16); 43(13); 41(14).
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4ꢀbromoꢀ1ꢀbutylꢀ1Hꢀbenzotriazole (47). H NMR (500 MHz)*: δ 7.94 (dd, J = 8.3, 0.6 Hz,
1H, H7), 7.67 (dd, J = 7.4, 0.6 Hz, 1H, H5), 7.48 (dd, J = 8.3, 7.4 Hz, 1H, H6), 4.73 (t, J=7.0 Hz,
2H, CH2N), 1.84ꢀ1.94 (m, 2H, CH22’), 1.21ꢀ1.30 (m, 2H, CH23’), 0.89 (t, J=7.4 Hz, 3H, CH3).
HRMS: [M+H]+=254.02877 (C10H13N3Br; delta=0.13); ESIꢀMSꢀMS (rel. int. %, cid=45%):
198(100).
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4ꢀbromoꢀ1ꢀpropylꢀ1Hꢀbenzotriazole (48). H NMR (800 MHz): δ 7.95 (d, J = 8.2 Hz, 1H,
H7), 7.67 (d, J = 7.4 Hz, 1H, H5), 7.49 (dd, J = 8.3, 7.4 Hz, 1H, H6), 4.70 (t, J = 7.0 Hz, 2H,
CH2N), 1.94 (sx, J = 7.4 Hz, 2H, CH2), 0.85 (t, J = 7.4 Hz, 3H, CH3). M+=239; EIꢀMS (rel. int.
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%): 239(100); 210(57); 182(50); 169(89); 155(33); 117(17); 103(25); 90(34); 75(29); 43(22);
41(23).
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4ꢀbromoꢀ1ꢀethylꢀ1Hꢀbenzotriazole (49). H NMR (500 MHz)*: δ 7.94 (dd, J = 8.3, 0.6 Hz,
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1H, H7), 7.67 (dd, J = 7.4, 0.6 Hz, 1H, H5), 7.48 (dd, J = 8.3, 7.4 Hz, 1H, H6), 4.76 (q, J = 7.3
Hz, 2H, CH2), 1.52 (t, J = 7.3 Hz, 3H, CH3). HRMS: [M+H]+=225.99756 (C8H9N3Br;
delta=0.55); ESIꢀMSꢀMS (rel. int. %, cid=45%): 198(100).
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4ꢀbromoꢀ1ꢀmethylꢀ1Hꢀbenzotriazole (50). H NMR (800 MHz): δ 7.89 (d, J = 8.4 Hz, 1H,
H7), 7.67 (d, J = 7.4 Hz, 1H, H5), 7.46ꢀ7.52 (m, 1H, H6), 4.33 (s, 3H, CH3N). M+=211; EIꢀMS
(rel. int. %): 211(93); 183(100); 168(65); 155(14); 117(6); 104(23); 88(15); 77(43); 63(29);
51(21).
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4ꢀbromoꢀ1ꢀ(cyclopropylmethyl)ꢀ6ꢀ(trifluoromethyl)ꢀ1Hꢀbenzotriazole (51). H NMR (500
MHz): δ 8.61ꢀ8.64 (m, 1H, H7), 8.02 (d, J = 0.9 Hz, 1H, H5), 4.74 (d, J = 7.4 Hz, 2H, CH2N),
1.40ꢀ1.49 (m, 1H, CH), 0.53ꢀ0.58 (m, 2H, cypCH2x), 0.48ꢀ0.53 (m, 2H, cypCH2y). M+=319; EIꢀ
MS (rel. int. %): 319(39); 290(13); 278(100); 250(33); 221(23); 184(18); 144(18); 55(65).
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4,6ꢀdibromoꢀ1ꢀ(cyclopropylmethyl)ꢀ1Hꢀbenzotriazole (52). H NMR (500 MHz): δ 8.39 (d,
J = 1.4 Hz, 1H, H7), 7.88 (d, J = 1.4 Hz, 1H, H5), 4.61 (d, J = 7.3 Hz, 2H, CH2N), 1.35ꢀ1.43 (m,
1H, CH), 0.51ꢀ0.58 (m, 2H, cypCH2x), 0.44ꢀ0.50 (m, 2H, cypCH2y). M+=329; EIꢀMS (rel. int.
%): 329(34); 288(45); 260(15); 221(13); 143(15); 115(18); 55(90).
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4ꢀbromoꢀ6ꢀchloroꢀ1ꢀ(cyclopropylmethyl)ꢀ1Hꢀbenzotriazole (53). H NMR (800 MHz): δ
8.25 (d, J = 1.6 Hz, 1H, H7), 7.79 (d, J = 1.6 Hz, 1H, H5), 4.61 (d, J = 7.4 Hz, 2H, CH2N), 1.37ꢀ
1.45 (m, 1H, cypꢀCHalpha), 0.51ꢀ0.58 (m, 2H, cypꢀCH2alpha), 0.45ꢀ0.51 (m, 2H, cypꢀCH2beta).
13C{1H} NMR (201 MHz): δ 142.9 (C3a), 133.9 (7a), 132.5 (C6), 126.9 (C5), 112.6 (C4), 110.5
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(C7), 52.5 (CH2N), 11.1 (cypꢀCH), 3.9 (cypꢀCH2). N NMR (81.1 MHz): δ ꢀ144 (N1), 2 (N2).
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M+=285; EIꢀMS (rel. int. %): 285(50); 244(79); 216(25); 177(21); 55(95).
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4-bromo-6-fluoro-1-(cyclopropylmethyl)-1H-benzotriazole (54). H NMR (500 MHz): δ 7.94
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(dd, J= 8.5, 2.1 Hz, 1H, H7), 7.72 (dd, J = 9.0, 2.1 Hz, 1H, H5), 4.58 (d, J = 7.3 Hz, 2H, CH2N),
1.33ꢀ1.43 (m, 1H, CH), 0.52ꢀ0.58 (m, 2H, cypCH2x), 0.46ꢀ0.51 (m, 2H, cypCH2y). M+=269; EIꢀ
MS (rel. int. %): 269(46); 241(10); 228(100); 200(25); 187(11); 173(17); 161(29); 134(27);
121(26); 107(19); 94(23); 55(62).
4ꢀbromoꢀ1ꢀ(cyclopropylmethyl)ꢀ6ꢀmethoxyꢀ1Hꢀbenzotriazole (55). 1H NMR (500 MHz): δ
7.42 (d, J = 2.0 Hz, 1H, H7), 7.31 (d, J = 2.0 Hz, 1H, H5), 4.55 (d, J = 7.2 Hz, 2H, CH2N), 1.33ꢀ
1.45 (m, 1H, CH), 0.51ꢀ0.58 (m, 2H, cypCH2x), 0.43ꢀ0.50 (m, 2H, cypCH2y). M+=281; EIꢀMS
(rel. int. %): 281(100); 252(11); 240(56); 212(46); 199(14); 184(17); 173(11); 159(14); 146(9);
133(18); 104(14); 90(12); 76(15); 63(15); 55(99).
4ꢀchloroꢀ1ꢀ(cyclopropylmethyl)ꢀ1Hꢀbenzotriazole (56). 1H NMR (500 MHz): δ 7.95 (dd, J
= 8.0, 1.0 Hz, 1H, H7), 7.55 (t, J = 7.5 Hz, 1H, H6), 7.52 (dd, J = 7.3, 1.0 Hz, 1H, H5), 4.65 (d, J
= 7.3 Hz, 2H, CH2N), 1.32ꢀ1.44 (m, 1H, CH), 0.52ꢀ0.61 (m, 2H, cypCH2x), 0.42ꢀ0.51 (m, 2H,
cypCH2y). M+=207; EIꢀMS (rel. int. %): 207(100); 178(43); 166(93); 151(28); 143(30);
138(52); 125(26); 111(45); 102(14); 89(17); 75(32); 63(11); 51(91).
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4ꢀfluoroꢀ1ꢀ(cyclopropylmethyl)ꢀ1Hꢀbenzotriazole (57). H NMR (800 MHz): δ 7.80 (d, J =
8.4 Hz, 1H, H7), 7.56 (td, J = 8.0, 4.6 Hz, 1H, H6), 7.24 (dd, J = 10.8, 7.7 Hz, 1H, H5), 4.64 (d,
J = 7.3 Hz, 2H, CH2N), 1.33ꢀ1.44 (m, 1H, CH), 0.55ꢀ0.58 (m, 2H, cypCH2x), 0.47ꢀ0.50 (m, 2H,
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cypCH2y). C{1H} NMR (201 MHz): δ 152.2 d JF = 255 Hz (C4), 135.8 d JF = 6.8 Hz (7a),
135.1 d 2JF = 18.7 Hz (C3a), 128.2 d 3JF = 7.2 Hz (C6), 108.3 d 2JF = 16.8 Hz (C5), 107.4 d 4JF =
4.8 Hz (C7), 52.3 (CH2N), 11.1 (cypꢀCH), 3.9 (cypꢀCH2). 15N NMR (81.1 MHz): δ ꢀ145 (N1), 0
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(N2). M+=191; EIꢀMS (rel. int. %): 191(69); 162(25); 150(100); 135(23); 122(41); 108(26);
95(52); 75(15); 55(39).
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1ꢀ(cyclopropylmethyl)ꢀ1Hꢀbenzotriazoleꢀ4ꢀcarbonitrile (58). H NMR (400 MHz): δ 8.40
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(d, J = 8.5 Hz, 1H, H7), 8.04 (d, J = 7.2, 1H, H5), 7.74 (dd, J = 8.5, 7.2 Hz, 1H, H6), 4.71 (d, J =
7.3 Hz, 2H, CH2N), 1.34ꢀ1.46 (m, 1H, CH), 0.53ꢀ0.61 (m, 2H, cypCH2x), 0.45ꢀ0.53 (m, 2H,
cypCH2y). M+=198; EIꢀMS (rel. int. %): 198(29); 169(25); 157(100); 149(10); 142(19); 129(38);
116(18); 102(28); 75(8); 55(37).
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1ꢀ(cyclopropylmethyl)ꢀ4ꢀmethoxyꢀ1Hꢀbenzotriazole (59). H NMR (500 MHz)*: δ 7.40ꢀ
7.46 (m, 2H, H6, H7), 6.81 (dd, J = 6.9, 1.5 Hz, 1H, H5), 4.55 (d, J = 7.2 Hz, 2H, CH2N), 4.02
(s, 3H, OMe), 0.82ꢀ0.88 (m, 1H, CH), 0.51ꢀ0.57 (m, 2H, cypCH2x), 0.42ꢀ0.47 (m, 2H,
cypCH2y). M+=203; EIꢀMS (rel. int. %): 203(100); 202(67); 189(29); 174(72); 163(50); 151(52);
145(32); 121(31); 117(40); 91(42); 66(80).
1
1ꢀ(cyclopropylmethyl)ꢀ4ꢀ(trifluoromethyl)ꢀ1Hꢀbenzotriazole (60). H NMR (800 MHz): δ
8.33 (d, J = 8.4 Hz, 1H, H7), 7.82 (d, J = 7.2 Hz, 1H, H5), 7.72ꢀ7.75 (m, 1H, H6), 4.71 (d, J =
7.3 Hz, 2H, CH2N), 1.38ꢀ1.44 (m, 1H, CH), 0.54ꢀ0.58 (m, 2H, cypCH2x), 0.49ꢀ0.53 (m, 2H,
3
cypCH2y). 13C{1H} NMR (100.5 MHz): δ 140.5 (C3a), 133.7 (7a), 126.7 (C6), 121.9 (q, JCF=5
1
2
Hz, C5), 123.1 (q, JCF=272 Hz, CF3), 119.0 (q, JCF=33.5 Hz, C4), 116.0 (C7), 52.3 (CH2N),
11.2 (cypꢀCH), 3.9 (cypꢀCH2). HRMS: [M+H]+=242.08966 (C11H11N3F3; delta=ꢀ1.2); ESIꢀMSꢀ
MS (rel. int. %, cid=35%): 240(2); 222(6); 188(100); 168(3).
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Biological evaluation. Human mGluR2 in vitro functional assay. Chemꢀ1 cells stably
expressing human mGlu2 receptors were cultured in the medium detailed above. Fluorometric
measurements of cytosolic Ca2+ ion concentration ([Ca2+]i) were carried out using cells seeded in
standard 96ꢀwell microplates at a density of 30000 cells/well and maintained overnight in a
tissue culture incubator at 37 °C under an atmosphere of 95 % air/5 % CO2. Composition of the
plating medium was: DMEM, 10 % FBS, 1× MEM nonꢀessential amino acids (Sigma M 7145),
1× PSA. The growth medium was removed and the cells were washed with assay buffer (without
GPT/pyruvate), both actions were performed with a cell washer (BioTek Elx405UCVWS). At
the end of the washing step 50 ꢁl/well assay buffer (without GPT/pyruvate) was left in the wells,
and 50 ꢁl/well Calcium 5 kit diluted 4ꢀfold in assay buffer (with 6 ꢁg/ml GPT/4 mM pyruvate)
was added manually using an 8ꢀchannel pipette. After an incubation period (30 min, 37 °C) the
dye was removed and the cells were washed with the cell washer using the same protocol as
above. After washing the plate was incubated for 10 min at 37 °C. Then 50 ꢁl/well assay buffer
(with 6 ꢁg/ml GPT/4 mM pyruvate) containing vehicle (DMSO) or the test compounds or
reference mGluR2 positive modulators was added manually and the cells were incubated for an
additional 30 min at 37 °C. Baseline and agonistꢀevoked [Ca2+]iꢀchanges were monitored with
FlexStation II (Molecular Devices, Sunnyvale, CA), a plate reader fluorometer with integrated 8ꢀ
channel fluid addition capability. Fluorescence measurements were carried out at 37 °C. The dye
was excited at 485 nm, emission was sampled at 525 nm at 1.4ꢀs intervals. Baseline was
recorded for 20 s followed by agonist stimulation. The 50 µl 3× concentrated agonist solution
was added to the cells using the pipettor of FlexStation II and fluorescence was monitored for an
additional 40 s. Agonist was glutamate administered at an EC5ꢀEC20 concentration. Final DMSO
concentration was 1 % for all treatments. To achieve this a series of DMSO stock solutions were
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prepared from all test compounds. These stocks were stored under 0 °C and were further diluted
in assay buffer without GPT/pyruvate to obtain the desired final concentration immediately
before the measurement. Results were expressed as ꢂF/F values using SoftMax Pro software
(Molecular Devices), where F was the resting fluorescence preceding agonist application and ꢂF
was the increase in fluorescence at a given time (ꢂF = maximum fluorescence intensity values
after stimulation minus average fluorescence intensity values before stimulation). In all
experiments, all treatments were measured in multiple wells in parallel, and the mean ꢂF/F
values were used for analysis. Where appropiate, sigmoid curves were fitted to the
concentration–response data to obtain EC50 values.
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Human, rat and mouse liver microsomal stability assay. In vitro metabolic stability was
assessed using human (Xenotech, USA), Wistar rat (In Vitro Metabolism Research, Gedeon
Richter Plc, Hungary) and NMRI mice (In Vitro Metabolism Research, Gedeon Richter Plc,
Hungary) liver microsomes. Test compounds at 2.5 ꢁM initial test concentration were incubated
for various lengths of time with the liver microsomes (0.5 mg/mL). In vitro intrinsic clearance
(Clint mL/min·g liver) was calculated using the basic concept of clearance prediction34 according
to the following equations: Clint = Vmax/KM or, if S ≪ KM, Clint = V/S; Vmax = maximal rate of
enzyme reaction; KM = affinity constant of substrate concentration; V = actual rate of enzyme
reaction under first order conditions.
Animals. Male mice of ddY strains (6 weeks old) were purchased from Japan SLC (Shizuoka,
Japan). All animal experiments were performed under experimental protocols approved by the
Institutional Animal Care and Use Committee, Mitsubishi Tanabe Pharma Corporation.
PCPꢀinduced hyperlocomotion. Locomotor activity was measured using the passive infrared
sensor system (Supermex®; Muromachi Kikai, Japan). Mice acclimated to the test cage for 60
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min, and were injected intraperitoneally with the test compounds. Immediately, mice were
injected with PCP (2.5 mg/kg, s.c.), then were placed back in the test cage for another 60 min,
during which locomotor activity was recorded.
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DOIꢀinduced head twitches. The test compounds were injected intraperitoneally 20 min prior
to injection of DOI (0.75 mg/kg, s.c.). The number of head twitches were counted in a
transparent cylinder (height: 25.5 cm, diameter: 19 cm) for the period between 10 and 18 min (8
min) after the DOI injection.
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ANCILLARY INFORMATION
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Supporting Information
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Molecular Formula Strings
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Corresponding author information
*(Gy.Sz.) Phone: +36ꢀ1ꢀ4315180 Eꢀmail: gy.szabo@richter.hu
*(G.M.K) Phone: +36ꢀ1ꢀ3826821 Eꢀmail: gy.keseru@mta.ttk.hu
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Abbreviations used
AcOH, acetic acid; AcCN, acetonitrile; ADME, absorption, distribution, metabolism,
excretion; Clint, intrinsic clearance; Cplasma, plasma concentrations; Cplasma unbound, unbound plasma
concentrations; Cbrain, brain concentrations; DMF, N,Nꢀdimethylformamide; DOI, 2,5ꢀ
dimethoxyꢀ4ꢀiodoamphetamine; EtOAc, ethyl acetate; FBDD, fragment based drug discovery;
GE, group efficiency; GPCR, Gproteinꢀcoupled receptor; h, hours; HTS, highꢀthroughput
screening; LE, ligand efficiency; mGlu2, metabotropic glutamate 2; MeOH, methanol; MW,
molecular weight; NaHMDS, sodium bis(trimethylsilyl)amide; ND, not determined; NMR,
nuclear magnetic resonance; PAM, positive allosteric modulator; PANSS, positive and negative
syndrome scale; PCP, phencyclidine; PK, pharmacokinetics; SAR, structure−activity
relationship; SEM, standard error of mean; HPLC, highꢀperformance liquid chromatography;
SD, standard deviation; THF, tetrahydrofuran; TPSA, topological polar surface area
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